Home » Articles posted by tbpatil

Author Archives: tbpatil

Want to Get Involved In Research?

[su_heading size="15" margin="0"]The BioInnovation Group is an undergraduate-run research organization aimed at increasing undergraduate access to research opportunities. We have many programs ranging from research project teams to skills training (BIG-RT) and Journal Club.

If you are an undergraduate interested in gaining research experience and skills training, check out our website (https://bigucd.com/) to see what programs and opportunities we have to offer. In order to stay up to date on our events and offerings, you can sign up for our newsletter. We look forward to having you join us![/su_heading]

Newest Posts

Disparities in Reproductive and Sexual Healthcare of Women with Disabilities

By Manasvini Pochimireddy.
Author’s Note
: I wrote this piece for a general academic audience of my peers. I specifically chose this topic because disabled women have been a historically marginalized group, with inadequate resources in the healthcare system to lead healthy lives. In terms of their reproductive and sexual healthcare, these women face a public stigma and are never made aware of any risks. While much more research is needed in order to implement lasting changes in the healthcare of this population, I wanted this paper to simply introduce people to this topic and raise awareness that this is a sector of healthcare requiring attention.

Abstract: 

Women with disabilities face many challenges in the healthcare system, as there has been a long-standing stigma around disabilities. Specifically, these women have been met with disregard and insensitivity, and sometimes they are even denied access to sexual and reproductive healthcare. Despite the large number of individuals with disabilities in the general population, there is a lack of research on the various issues faced specifically by women with disabilities. This review of literature will investigate the question: “What does current scholarship tell us about the disparities in reproductive and sexual healthcare faced by women with disabilities?” To provide a more complete understanding of why this disparity exists and how to effectively address it, more studies need to be conducted about changes that need to occur in this field.

Introduction: 

The term “disabilities” encompasses a variety of conditions that can result in lack of function, either physically (impairment in bodily activity) or cognitively (impairment in mental well-being) [1, 2]. Currently, individuals with some form of disability compose approximately 15% of the world population. However, when considering the amount of disabled individuals by gender, 19.2% of all women are affected globally, whereas only 12% of all men are affected [2]. This 7% difference would mean that there are approximately 250 million more women than men experiencing disability in our current global population [2].This has been especially prevalent for women, as their reproductive and sexual healthcare is not seen as a priority [3, 4]. Reproductive and sexual healthcare refers to treatment of women before and after conception, as well as gender specific care.  This review will focus on the disparities faced by disabled cis-gendered women in the United States, specifically in terms of their reproductive and sexual healthcare. Furthermore, when referring to “women” in this paper, it will be focusing on cis-gendered women. This is not to say transgender women do not face similar difficulties.

Women with disabilities face a multitude of challenges in the healthcare system, including a lack of proper health education, stigma concerning their reproductive health, and greater risk factors for specific ethnic groups [2, 3, 5-8]. In terms of education, women with disabilities are not provided with enough knowledge to make informed decisions regarding their well-being in terms of reproductive and sexual health [3, 7]. Healthcare practitioners and caretakers are equally uneducated on providing resources and knowledge to these women, leading to unanswered questions and uncomfortableness on both ends of these conversations [2].  This stems from lack of discussion in school systems regarding people with disabilities in the context of reproductive and sexual health [9].  Furthermore, many healthcare practitioners and family members hold a stigma against women with disabilities and their reproductive health, leading to poor quality of care in the few appointments that do occur [4, 8]. Many women with disabilities are further subjected to increased preconception risks (obesity, stress, medications) and marginalized because of their race [5, 6]. 

While we have made significant advances in many aspects of healthcare, resources to support these women have been at a standstill [3, 4]. The education about disabled women’s needs has also remained both inadequate and stagnant. While research has been done to establish that this is an issue, no further steps have been taken to improve the state of this problem. This review of literature will investigate the different factors that exacerbate the disparity in reproductive and sexual healthcare for women with disabilities. 

Reproductive and Sexual Health Education 

Inadequate education in terms of sexual health has remained a contributing factor to the lack of healthcare resources in preconception health for disabled women. Preconception health represents the general well-being of a woman during her reproductive/fertile years (between puberty and menopause) in terms of her overall health, including pre-existing conditions. Despite women with and without disabilities both having similar pregnancy rates, there was a significant lack of knowledge among disabled women regarding their own well-being and personal expectations for pregnancy [3, 7]. From 2018 to 2022, multiple independent studies confirmed there were higher rates of unplanned pregnancies reported amongst the disabled womens’ population when compared to women without disability [3, 7, 10]. For example, one study found among pregnancies in women with disabilities, 53% were unwanted vs. 36% being unwanted in non-disabled women [7]. Along with high rates of unplanned pregnancies, a similar trend exists in the use of birth control. A survey identifying rates of birth control use found 19.7% of sexually active disabled women among the ages of 15-24 were not using any method of birth control, compared to 10.6% of non-disabled women in the same age group [10]. This means that nearly double the amount of disabled women are at risk for unplanned pregnancies when compared to non-disabled women [10]. These higher rates of birth control usage and unplanned pregnancies in the disabled womens’ population stem from the lack of sexual and reproductive health education being taught during their formative years [3, 9, 10]. Current studies examine the lack of sexual health education for women with disabilities, but more studies should be conducted on how and when to provide this type of education to ensure that these women are informed about their health. Moreover, this discussion needs to be normalized to create amore inclusive environment when educating about disabilities in the context of reproductive and sexual health.

Not only is there a lack of sexual and reproductive health education for women with disabilities, but there is also a lack of this same education on the part of healthcare practitioners and caretakers in this field [1-3, 8]. It is vital for people in these positions to understand the needs of a woman with a disability in order to prepare them for what to expect, along with the fact that their needs may be separate from the needs of a woman without a disability [2, 3]. Kalpakijan et al. conducted a study among 81 self-reportededly disabled women who explained via group and individual interviews regarding their reproductive healthcare experiences. Kalpakijan et al. identified five major themes that informed a framework to be implemented across institutions. These themes were knowledge about reproductive health, communication about reproductive health, relationships, the reproductive health care environment, and self-advocacy/identity [11]. To provide adequate, quality care and be responsive to the questions of disabled women regarding their sexual health, changes need to be made to include sensitivity training, specifically in properly and respectfully communicating differences in needs for disabled women during pregnancy [2, 3, 8]. Further research needs to be conducted on how healthcare providers and caretakers can be further educated on the needs of disabled women in terms of sexual and reproductive health. 

Effects of Ethnicity on Healthcare Disparities of Disabled Women 

Ethnicity plays a role in exacerbating the differences in reproductive and sexual healthcare between disabled and non-disabled women. The systemic differences associated with different racial groups that result in unequal access to a variety of health resources extend to women with disabilities and their access to reproductive health resources as well [6]. There are much higher rates of reproductive-related complications, such as post-conception/postpartum complications, or issues during the pregnancy itself among disabled women when compared to non-disabled women. However, upon further examination, an unequal distribution of these complications across different ethnicities is found [5, 6].The combination of having a disability and being an ethnic minority seems to have an additive effect on the disparity in accessing adequate reproductive healthcare resources for women [5].  

There are certain preconception health risks (obesity, stress, medications) that are more severe in certain ethnic communities of disabled women, such as obesity in disabled black women [5]. There are also higher rates of gestational diabetes and gestational hypertension (preeclampsia) found among disabled women of Hispanic and African-American origin, with multiple possible causes, including high mental distress, lack of access to proper nutrition, and low levels of exercise [4, 5]. Most, if not all of these adverse preganancy conditions are rooted in structural racism resulting in restricted access to determinants of health [5].  Further studies should be done on how to lessen the impact of the combination of ethnicity and disability on access to healthcare, and to isolate why specific conditions and complications are more prevalent among specific ethnic communities of disabled women. 

Social Attitudes Towards Women with Disabilities 

Public stigma of women with disabilities severely limits their access to adequate resources in healthcare, specifically when concerning pregnancy and reproductive health. Firstly, women with disability are seen as less sexually active, despite many studies showing equal rates of sexual activity amongst women with disability and women without disability [9, 12, 13]. 

With these false expectations, many healthcare providers do not think to offer the same resources concerning reproductive and sexual healthcare to women with disabilities [3, 9]. Many disabled women are unaware of the fact that they could have children, due to the lack of information from their healthcare providers. As a result, they are often surprised by unforeseen pregnancies [3] In a survey from 2022 asking women about their healthcare provider visit, when disabled women opted to discuss their reproductive and sexual healthcare, they often felt insensitivity from the provider [3, 8]. They also felt uncomfortable asking further questions about their health [3, 8, 9] Not only did women feel this sense of embarrassment, their family members also felt this due to the public stigma of women with disabilities engaging in sexual activities [8].

This stigma resulted in a decrease in quality of care provided to women with disabilities [4]. When evaluating the number of visits by expectant mothers during pregnancy, it was found that there was a significantly lower number of overall visits amongst women with disabilities when compared to women without disabilities, especially in the first trimester [4]. Moreover, when comparing rates of miscarriage among women with disabilities vs without, it was found that disabled women experience miscarriages at a rate of 31.63 % versus women without disabilities experience miscarriages at a rate of 21.83% [14]. Many women also experience fear for their offspring inheriting their disability, even when there is no genetic link, due to the inadequate explanations provided by healthcare providers [2, 8]. These concerns should be properly addressed and answered without judgment, but instead are sidelined or in some cases, not even received [2]. The existing stigma can be combated through open communication between individuals with disabilities regarding their needs and people in positions who can implement changes to diminish the existing disparity. In addition, a more inclusive curriculum in sexual health should be implemented in primary school systems so that more individuals, as well as future healthcare professionals, can be exposed to this subject from an early age. This would allow for discussion about disabilities in context to reproductive health to increase, and to become more normalized in society. 

Conclusion: 

Overall, a variety of factors aggravate the disparity present in the reproductive and sexual healthcare of disabled women. The lack of sexual health education for disabled women remains, along with the lack of training and knowledge on the part of healthcare practitioners and caretakers attending to the needs of disabled women [1, 3, 7, 9]. This causes difficulties during family planning stages and during treatment, when these women haven’t been properly informed of the necessary measures [1, 15]. The current healthcare system has resources to support women without disabilities who face these issues, but when disabled women undergo similar circumstances, they find themselves with few places to turn to that can accommodate their needs [15]. Further studies also need to be conducted to find out why certain preconception health risks are more severe among certain ethnic groups, as there is evidence that specific medical conditions are more prevalent in specific communities [5, 10]. A systemic change to tackle the disparities faced by women with disabilities who are also ethnic minorities needs to be taken. To this day, disabled women who pursue reproductive health measures face negative attitudes from healthcare professionals [2, 11]. These attitudes can be dissipated through changes in the current sexual education curriculum, which would allow for both the disabled community and non-disabled community to learn about the needs of disabled women. Another vital part of reducing these disparities includes encouraging conversations about mental and physical differences that clearly exist and acknowledging them, so that more individuals are aware of the challenges faced by disabled women in the healthcare system. In the literature reviewed, there was a great discussion and evidence of the existence of this problem, but rarely were any solutions offered. There were a couple of suggested frameworks, but none that looked at the efficacy of its implementation. Moving forward, there need to be studies done regarding possible methods that can truly alleviate the disparity faced by disabled women.

References: 

  1. Crabb C, Owen R, Heller T. Female medicaid enrollees with disabilities and discussions with health care providers about contraception/family planning and sexually transmitted infections. Sex Disabil. 2019; 38: 299-312 doi: 10.1007/s11195-019-09599-y 
  2. Nguyen TV, King J, Edwards N, Dunne MP. “Nothing suitable for us”: Experiences of women with physical disabilities in accessing maternal healthcare services in northern Vietnam. Disability and Rehabilitation. 2020;44(4):573-581. doi:10.1080/09638288.2020.1773548
  3. O’Connor-Terry C, Harris, Pregnancy decision-making in women with physical disabilities. Disabil Health J. 2022;15(1): 1-5 doi: 10.1016/j.dhjo.2021.101176 7. 
  4. Horner-Johnson W, Biel FM, Caughey AB, Darney BG. Differences in prenatal care by presence and type of maternal disability. American Journal of Preventive Medicine. 2019;56(3):376-382. doi:10.1016/j.amepre.2018.10.021 
  5. Horner-Johnson W, Akobirshoev I, Amutah-Onukagha NN, Slaughter-Acey JC, Mitra M. Preconception health risks among U.S. women: Disparities at the intersection of disability and race or ethnicity. Womens Health Issues. 2021;31(1):65-74 doi:10.1016/j.whi.2020.10.001 
  6. Alhusen JL, Bloom T, Laughon K, Behan L, Hughes RB. Perceptions of barriers to effective family planning services among women with disabilities. Disability and Health Journal. 2021;14(3):1-6. doi:10.1016/j.dhjo.2020.101055 
  7. Horner‐Johnson W, Dissanayake M, Wu JP, Caughey AB, Darney BG. Pregnancy intendedness by maternal disability status and type in the United States. Perspectives on Sexual and Reproductive Health. 2020;52(1):31-38. doi:10.1363/psrh.12130 
  8. Nguyen TV, King J, Edwards N, Pham CT, Dunne M. Maternal healthcare experiences of and challenges for women with physical disabilities in low and middle-income countries: A review of qualitative evidence. Sexuality and Disability. 2019;37(2):175-201. doi:10.1007/s11195-019-09564-9
  9. Namkung EH, Valentine A, Warner L, Mitra M. Contraceptive use at first sexual intercourse among adolescent and young adult women with disabilities: The role of formal sex education. Contraception. 2021;103(3): 178-184. doi:10.1016/j.contraception.2020.12.007 
  10. Mosher W, Hughes RB, Bloom T, Horton L, Mojtabai R, Alhusen JL. Contraceptive use by disability status: New National Estimates from the National Survey of Family Growth. Contraception. 2018;97(6):552-558. doi:10.1016/j.contraception.2018.03.031 
  11. Kalpakjian CZ, Kreshcmer JM, Slavin MD, Kisala PA, Quint E, Chiaravalloti ND, Jenkins N, Bushnik T, Amtmann D, Tulsky DS, Madrid R, Parten R, Evitts M, Grawi CL. Reproductive health in women with physical disability: A conceptual framework for the development of new patient-reported outcome measures. J Womens Health. 2020;29(11):1427-1436 doi:10.1089/jwh.2019.8174 
  12. Shandra C. L. & Chowdhury A. R. “The First Sexual Experience Among Adolescent Girls With and Without Disabilities.” 2012. Journal of Youth and Adolescence. 41:515–532 https://doi.org/10.1007/s10964-011-9668-0 
  13. Kah, N.F. & Halpern, C.T. Experiences of Vaginal, Oral, and Anal Sex From Adolescence to Early Adulthood in Populations With Physical Disabilities. 2018. Journal of Adolescent Health. 62(3):294-302. https://doi.org/10.1016/j.jadohealth.2017.08.003 
  14. Dissanayake, M. V., Darney, B. G., Caughey, A. B., & Horner-Johnson, W. (2020). Miscarriage Occurrence and Prevention Efforts by Disability Status and Type in the United States. Journal of women’s health (2002), 29(3), 345–352. doi: /10.1089/jwh.2019.7880
  15. Collins B, Hall J, Hundley V, Ireland J. Effective communication: Core to promoting respectful maternity care for disabled women. Midwifery. 2022:1-27. doi:10.1016/j.midw.2022.103525

Most Endangered Whale on Earth is America’s–and you’ve never heard of it

By David Kwon.
In 2021, a joint team of researchers led by the National Marine Fisheries Service (NMFS) revealed the discovery of a new species of baleen whale: the Rice’s whale. Baleen whales (mysticetes) are whales that don’t have teeth. Instead, they have bristle-like structures called
baleen that are perfected for filter-feeding. Unlike most new species, which are often small critters hidden in corners seldom disturbed by humans, this cetacean was hiding in plain sight all along. Long as a school bus, it inhabits the northern Gulf of Mexico (GOMx), not far from United States shores [1]. In fact, if you hitch a seaplane in Pensacola, Florida and fly a 40-minute trip about 60 miles south) to the offshore De Soto Canyon (roughly the distance from Davis to Berkeley), you’ll have reached the area these whales call home. Perhaps you’ll even spot a surfacing individual! But alongside the distinction as its own species, this baleen whale had eluded another, more dire secret–it’s on the very brink of extinction with only about 51 whales remaining [2]. It is already the second most endangered marine mammal in the world.

 

A new species is, whale, very hard to name

Figure 1. Maximum size of a Rice’s whale scaled to a human and a school bus with the whale’s diagnostic features labeled

 

How have we missed a giant whale swimming along our doorstep? Actually, we have known a population of baleen whales has existed in the GOMx since at least 1965 [3], and historical records suggest that local whalers attempted to hunt them since at least the late 1700s [4]. However, scientists initially believed that they were members of another wide-ranging tropical species called the Bryde’s whale, which frequents the neighboring Atlantic Ocean and Caribbean Sea. This is easily forgivable, as the outward appearance of these GOMx whales are virtually indistinguishable. Both have essentially the same streamlined, sleek bodies of uniform dark charcoal gray or brown coloration on top, and a mix of pale and pink underneath. A large, hook-shaped dorsal fin is placed around two-thirds down from the tip of the snout. The snouts themselves are spear-shaped with three distinct prominent ridges on top (a thick central one extending from snout end to blowhole, and two slightly smaller ones flanking both sides) (Figure 1) [1]. It was not until the 1990s [3] when scientists realized that the GOMx whales inhabits an extremely narrow and isolated area. Nearly all reside within the scientist-dubbed “core habitat”—a thin deep-water stretch off the western coastlines of Florida with seafloors between 150-410 meters deep—while a few exist in even narrower secondary habitats of similar seafloor depths extending to Texas (Figure 2) [1]. This hinted that they may be set apart from the Atlantic Bryde’s whales.

Figure 2. Map of the Rice’s Whale habitat within the GOMx

 

The possibility that populations thought to be an existing known species are actually new species is nothing new with cetaceans. Mobile marine mammals that seek refuge below the surface can be difficult to study, and granting any divergent population its own scientific name requires (1) substantial morphological and genetic evidence from bone and tissue samples to prove it’s not simply a variant of an existing species, and (2) a skull to serve as a defining specimen, or holotype. In other words, we need good DNA and dead whales. As a result, many potential species are left undescribed as scientists wait for a beached individual to obtain a cetacean cadaver without needing to harm the same whales they seek to study and save [1].

 

This was the case for the GOMx whales when Rosel & Wilcox (2014), two NMFS geneticists, compared the genetic sequences of the mitochondrial DNA control region (mtDNA CR) collected from beachings and biopsies with other Bryde’s whales. The mtDNA CR is often used by geneticists to study recent evolutionary histories due to its exceptionally fast mutation rate, capturing even the most subtle of population diversity [5][6]. The geneticists found that the GOMx whales were actually most closely related to a similar Asian species called the Eden’s whale–also previously thought to be a Bryde’s whale–with a ~10% net nucleotide divergence. This was far above the 2% divergence threshold for a distinct species, establishing that a new species likely exists [7]. A multi-year hunt for a holotype followed. First, the NMFS visited museums that held skeletons of GOMx Bryde’s whales, but none had a complete skull. Then, they attempted to recover the skeleton of a whale that washed up in Tampa Bay, Florida after colliding with a ship in 2009 and was subsequently buried in a nearby park [1] as standard disposal practice to prevent spreading infectious diseases or attracting scavengers [8]. Unfortunately, upon excavation in 2018, they found that seeping tides had damaged much of the specimen over its years of burial. Finally, a stroke of luck occurred when in 2019 another individual washed up in Everglades National Park, which had bled to death due to a laceration in its stomach caused by a tiny piece of plastic it swallowed. The NMFS and National Park Service quickly genetically sampled and buried the whale in a more secure location, exhumed the skeleton, and safely transported it to the Smithsonian’s Whale Collections. There, Rosel et al. (2021) had the holotype and morphological evidence, in the form of a unique cranial and nasal structure, needed to formally describe a new species, giving it the scientific name Balaenoptera ricei [1].

 

Second most endangered in the world

 

Though identical in external appearance, the Rice’s and Bryde’s whales are otherwise very different. A molecular clock shows that they diverged at least 4-8 million years ago [1, 9], and the few hints we have so far suggest a unique biology for the former. But as further study of the new species unfolds, understanding how exactly it stands apart continues to develop [1, 2, 10, 11]. We know that it’s the only baleen whale native to the GOMx. While the basin receives occasional fin, blue, sei, minke, and right whale visitors, they never stay for long and have no influence on the local ecology [1, 12]. Rice’s whales are also entirely resident, meaning the species stays in the same area year-round and doesn’t migrate. This is the only instance we know of that an entire species of mysticete is tied to a tiny piece of the ocean. While other baleen whales hold populations and subspecies that have settled into areas of reliable year-round productivity, such as the Arabian Sea humpback whales [13] and Gulf of California fin whales [14], their species still include wide-wandering members that will endure when one population tragically dies out. To lose the Rice’s whales is to lose a special diversity, endemism, and ecological role of an entire classification of animals in the GOMx.

 

This newest species of marine mammal is, ironically, also one of the closest to extinction. The most recent population study from a 2017-2018 survey within US waters numbers 51 individuals left in existence [2], with only 26 of them being mature individuals capable of reproduction [15]. This makes it the second most endangered marine mammal species and arguably the fourth most endangered mammal species in the world. Only the Sumatran rhinoceros, with a slightly lower population of 34-47 individuals [16], Hanian gibbon, with at least 35 individuals [17], and vaquita, with merely 10 individuals remaining [18], inch closer to extinction. As is often the consequence of such a low population, the Rice’s whale also suffers from extremely low genetic diversity. Only two haplotypes, inherited chromosomal DNA variants, were identified from a sample of 36 individuals. Tests for Hardy-Weinberg equilibrium yielded a mean observed heterozygosity frequency of 0.256, meaning that on average, over 74% of the samples’ gene pool were homozygous genotypes that carried only one allele each. Previous studies have shown that healthy Bryde’s whale populations can display an average of 3.6 to 9.3 alleles per loci, or different genes within fixed positions on chromosomes. But examination of 42 loci within the Rice’s whale population showed an average of 2.5 alleles per locus. This lack of diversity underlines the genetic threat Rice’s whales face in restoring populations [5]. With less available alleles, a population has less variety of traits that support adaptability. In addition, increased homozygosity decreases the ability to suppress recessive genes that are deleterious, or cause genetic diseases. Such a low genetic diversity further puts the Rice’s whale at risk of inbreeding depression, which can cause the accumulation of deleterious genes that will further weaken the population [1, 5, 12]. At worst, pessimistic ecologists expect the species to be the first great whale to go extinct in over 300 years and largest victim of the Anthropocene extinction to date [19, 20].

 

Things look grim for the Rice’s whale, but there is hope: rebounding from the spiral toward extinction is possible. For this, ecologists recommend the 50/500 rule, which postulates that a minimum of 50 viable individuals are required to escape inbreeding depression and 500 individuals to reduce harmful genetic drift [12]. It has been projected that a population of 35 Rice’s whales could achieve the 500 threshold within 68 years if conservation efforts can successfully sustain a recovery trend [12, 21].

 

Demystifying Gulf of Mexico’s only native mysticete

 

Setting the stage for this recovery requires better understanding of the ecological dynamics of the Rice’s whale and how it intersects with potential environmental threats. Although the existence of its population was acknowledged for decades, they remained unstudied [4] until the mid 2010s, meaning research of the whale is novel. To unravel the secrets mystifying a new mysticete, scientists face the challenge of finding a starting point to begin at. For the Rice’s whale, this was identification. Because Bryde’s whales were never known in the GOMx, one could infer that any similar baleen whale spotted in the area is likely a Rice’s whale. But since the two can’t be visually distinguished, this geographic inference isn’t an objectively rigorous method. Genetically testing biopsy samples, while conclusive, isn’t feasible for every encounter. Instead, scientists are adopting acoustic measurements as a method of differentiation that is both efficient and readily available [2, 22, 23]. By sticking microphones underwater during ship expeditions, in a procedure called passive acoustic monitoring (PAM), scientists can collect a soundscape of the surrounding ocean environment. While the soundscape will contain ambient noises of both random water sounds and the ship’s own propeller, it may also pick up vocalizations of marine animals that are stereotyped, or unique to them. By identifying these stereotypes from ambient noise, scientists can identify the animal producing them. The possibility of identifying Rice’s whales through their vocalizations was recognized in literature as early as 2014 [22, 23], but it took years of PAMs, survey expeditions, and painstaking data analysis to connect beyond reasonable doubt each potential whale vocalization to the species. It was eventually concluded by 2022 that Rice’s whales have a vocal repertoire of at least three stereotyped call types, the most common and diagnostic being dubbed the long-moans. Sounding like a faraway airplane’s takeoff but slowed-down and muffled, this call was especially easy to identify for its unusually long duration of about 22 seconds on average [2].

 

Scientists then studied the whale’s feeding behavior. In 2015 and 2018, scientists tagged two Rice’s whales. The suction-cup tags, which contained kinematic recorders, collectively provided nearly 90 hours of data of the individuals’ exact positions and orientations in a 3D space. This revealed their quarter-week routines. With a big gulp of air, the whales descended to the twilight zone and foraged near the seafloor for up to 10 minutes at a time. There, the whales swam large circles around their prey before capturing them with a powerful lunge or two. They spent the entire day cycling between entering deep water to feed (up to 271 meters below the surface) and surfacing to breathe. At nightfall, the whales ascended and hung around the surface, spending 85% of the night within 15 meters of the surface, and descended again at daybreak. [2, 11, 24] This pattern of oscillating between deep and shallow water through day and night is a typical example of diel vertical diving [10]. If the two samples are representative for the Rice’s whale, it’s an unusual strategy, as most baleen whales like the Bryde’s whale will typically feed at the surface or within the sunlight zone [25].

 

A 2023 study investigated the deep-water diet behind this peculiar behavior using the emerging techniques of stable isotope models. Each animal carries a distinctive signature of stable isotope ratios that broadly reflect that animal’s ecology, most significantly its diet. True to the phrase “you are what you eat,” when a predator eats another animal, the prey’s isotope signatures are integrated into the predator’s tissue. How the predator’s resulting signature is made up depends on what prey it ate and how much of it, creating a sort of chemical food diary. By comparing the stable isotope ratios of tissue samples of the predator and suspected prey and using a set of probabilistic models that addresses the abundance and biomass of suspected prey relative to its ecosystem, one can reconstruct and quantify the entire food web of the predator, including what it specifically targets as its favorite food. This is all without cutting open a single stomach [26, 27]. The scientists measured the stable isotope ratios of carbon (δ13C) and nitrogen (δ15N), the most informative in revealing diet, in skin and blubber biopsy samples of Rice’s whales collected during survey expeditions. They then compared with the ratios found in potential prey species caught during a July 2019 trawling survey in the core habitat, whose relative abundances and biomasses were also determined. The resulting analysis found that the Rice’s whales are picky eaters. Their diet is overwhelmingly composed of a single prey–a stumpy sardine-like schooling fish called the silver-rag driftfish (Ariomma bondi)–which made up over two-thirds of Rice’s whale diet. Interestingly, Rice’s whales do not appear to target the extremely common Atlantic pearlside (Maurolicus weitzmani), a similar schooling fish that made up over 88% of the core habitat’s ecological community by abundance. By contrast, the silver-rag only comprised 1.21% of community abundance. Why bother the effort to seek out a far rarer species? It turns out that the energy content of silver-rags surpasses all other prey by a wide margin. Every pound in wet weight of silver-rag contains on average over 36% more calories than the next animal on the menu. Rice’s whales seem to actively select their prey based on nutritional quality rather than abundance, singling out the silver-rag as the best bang for buck [26].

 

These feeding preferences also demonstrate an ecological importance of Rice’s whales: as ecosystem engineers. Whales feeding at the seafloor consume nutrients essential for photosynthesis, like iron and phosphorus, and then release them at the surface in massive fecal plumes. Through their diel vertical behavior, Rice’s whales are essentially pumps, cycling nutrients tucked away hundreds of meters deep up into sunlight-rich waters where they stimulate primary productivity. The amount of biomass this can produce is immense; one 2023 study suggests that a population of 2,500 minke whales, the smallest of the baleen whales, can cycle enough phosphorus to support over 70 metric tons of carbon biomass per day [28]. If Rice’s whales achieved a similar population in the past, they would have been no different.

 

Collision course with an industrialized Gulf

 

We do not yet know how the Rice’s whales came to be so endangered. Although historical records suggest attempted hunting during the 1700s and 1800s, the NMFS ruled out historical whaling as a likely factor for population decline. None of these records included successful kills, and the population evidently never experienced the dramatic rebound characteristic of other whales hunted to near-extinction in the past once overhunting by humans stopped [29]. But we know what threatens the species today is industrialization. Both the core habitat and secondary western habitats of the Rice’s whale are situated right within the intersection of the most commercially and industrially active waters in North America [10, 12, 29]. This is a place home to ten of the fifteen largest ports in the US and holds vast networks of shipping lanes with hundreds to thousands of giant commercial ships that carry nearly half America’s sea cargo by weight per year [12]. This places Rice’s whales at high risk of a literal collision course with vessel strikes that are prone to going unreported. Because of the species’ diel vertical behavior, risk of collision is especially high during nighttime, when the whales reside at the surface while visibility is reduced [10]. At least two instances of vessel strikes are already documented: the 2009 Tampa Bay beaching and a survivor left with a deformed spine [30]. Using a model for unreported collisions, the NMFS estimated that up to 20 ship strikes already occurred between 2002 and 2018. They further extrapolated that strikes may kill or seriously injure up to 17 Rice’s whales, over 30 percent of the current population, in the next 50 years without increased vessel restrictions [31]. Ships that don’t run over whales still produce voluminous quantities of underwater noise that muffle out vocal communication between whales and cause heavy stress that adversely alter behavior [12, 29].

 

Figure 3. Diagram illustrating Rice’s whale foraging behavior and its risks by human activity

 

The northern GOMx is also one of the US’ most active sites for offshore oil exploitation. Since the first drillings in 1942, over 6,000 oil rigs have been installed, with 3,200 active rigs still extracting fossil fuel from the seafloor, and oil companies continuing to explore new marine reservoirs to tap [32]. Exploration involves the frequent use of powerful airguns to blast ground-penetrating sound waves with intensities of up to 260 dB, which easily rupture cetacean eardrums. These airguns fire every few seconds in many parts of the GOMx [12, 29]. But the greatest threat to the Rice’s whales is the oil itself. With so many active oil rigs, the GOMx is a site for frequent spills of this environmentally toxic substance, which ocean currents can carry eastward into the core habitat. While the majority of spills are rather small-scale [33], a combination of wavering environmental regulations and susceptibility of oil companies to skirt those regulations set the groundwork for a catastrophe. We know this because it has already happened. In 2010, an explosion of the BP-operated rig Deepwater Horizon off the coast of Louisiana leaked over 4 million barrels of oil into water. Federal litigation ruled that this would have been preventable if regulations were followed, but BP chose to ignore them [34]. Policy scholars also cited those regulations as too lax to enforce proper accountability anyways [35, 36]. The result was the worst environmental disaster in North American history. Caught in the center were the Rice’s whales. Their core habitat was near the oil rig, and as a result nearly half of this critical refuge was smothered in oil (Figure 2). The consequences on the population are staggering. A 2015 federal investigation found that the event directly killed 17 percent of the population and caused reproductive failure in 22 percent of the female population, in total single-handedly reducing the Rice’s whale population by 22 percent. Take a moment to let that sink in. One-fifth of an entire species was wiped out by a single company’s negligence. Even of the survivors, 18 percent will live on with potentially lifelong illnesses [21]. We were never able to clean up all of the oil, which still remains on the seafloor or as marine snow and other bioaccumulating derivatives that will continue to affect Rice’s whale health [37].

 

Road to recovery: disentangling environmental policy and politics

 

Despite the odds against the Rice’s whales, it’s not too late to bring them back from the brink. As emphasized in a 2022 open letter signed by over a hundred marine and wildlife scientists, Rice’s whales are still reproducing [38]. We can still secure a rebound through efforts to eliminate their threats. With 51 individuals balancing on a knife’s edge, the fate of this unique whale lies within the hands of our immediate collective action–or inaction. This is so delicate that there was even a debate over whether an alternate common name “Gulf of Mexico whale” is more effective in inspiring action [20]. Saving an endangered species is no cakewalk, but the road to recovery for Rice’s whales will be especially challenging. Its habitat lies at the intersection of powerful industries, bringing the financial interests of the energy, shipping, and resource sectors, and even the military’s defense interests to the forefront of conservation decisions. 

 

Rice’s whales are primarily protected under two major environmental laws. The Marine Mammal Protection Act (MMPA) bans any killing, capture, or harassment of a marine mammal. The latter refers to direct actions with the potential to injure or disrupt its behavior (i.e. causing stress). The NMFS bears responsibility of translating the MMPA into specific regulations [39] and has interpreted harassment as to include harmful activities within proximity of a whale. For example, one MMPA-justified policy requires oil exploration vessels to cease all airgun activities within 500 meters of a whale sighted by onboard protected species observers in GOMx waters east of Mobile, Alabama or within seafloors deeper than 200 meters [40].

 

The second law is the Endangered Species Act (ESA), of which the species was listed as “Endangered” as a then-unnamed Bryde’s whale subspecies in 2019. The listing was the fruit of years of fierce efforts, beginning with a petition by the non-governmental organization (NGO) Natural Resources Defense Council (NRDC) within two months of Rosel & Wilcox (2014)’s publication. The agency subsequently assembled a research team which published their concurring investigation in 2016. The final decision to list was made after additional years of deliberation despite opposition by industry interests and following a mandatory public notice and comment session that received nearly a thousand comments and letters with over 115,000 combined signatures [29].

 

The ESA mandates the federal government to (1) establish a “critical habitat” zone for a listed species, (2) form a recovery plan that includes a delisting criteria, and (3) execute the plan until the species is delisted [41]. These duties for Rice’s whales are delegated to the NMFS, which is awaiting public comments for a proposed critical habitat uniting the core and secondary habitats until September 2023 [42] and is working on the recovery plan. As they construct the plan, the agency must navigate through environmental and political webs to finalize something that is both effective and legally sound. In 2021, the NMFS hosted a workshop involving 45 scientists, environmental specialists, NGO leaders, and industry representatives to contextualize the focuses of the recovery plan. A Word Cloud illustrated in order the four greatest challenges to Rice’s whale recovery according to participants: small population, vessel strikes, energy exploitation, and insufficient regulation [43].

 

While the critical habitat remains in progress, measures are being taken to secure the core habitat as a surrogate. In a 2020 biological opinion regarding an expansion to leasing parts of the GOMx for oil and gas, the NMFS proposed a 10-knot daytime speed limit and entry ban during nighttime or low-visibility conditions within the area for oil and gas exploration vessels. Ships traveling at 10 knots or less were significantly less likely to hit large whales than faster speeds. When collisions do occur, the worst would be minor injuries [31]. These recommendations have since been adopted by the Bureau of Ocean Energy Management (BOEM), the agency managing offshore energy leases, as conditions for authorizing oil and gas activities [44]. However, Rice’s whales are still at risk of lethal high-speed collisions by unaffiliated vessels, such as cargo traffic from Florida’s western ports. Recognizing this, a team of six organizations led by the NRDC submitted a petition the following year to expand the NMFS’ proposal as legal policy for all vessels within the core habitat. They pointed out the successes of universal slow-zones for critically endangered North Atlantic Right whales [30], estimated to have reduced the risk of lethal strikes by up to 90 percent [45]. The proposal is not without stakeholder concerns: local fishing communities that rely on hotspots within the core habitat fear paralysis of their main sources of income [46], while the Florida Ports Council, an organization representing the state’s ports, argues it jeopardizes the western ports [467. Nevertheless, the NMFS is now considering the petition and requested public comments until July 2023 [30].

 

Another victory came in 2023 when the NMFS denied permission for the Florida-based Eglin Air Force Base to test weapons within the core habitat during an authorization renewal. The base frequently conducts tests in the northeastern GOMx with the potential to inflict considerable harm to nearby cetaceans [48]. The NMFS is mandated to review the military’s plans to ensure minimal damage to marine mammals without seriously hampering national defense capabilities. Sometimes accidental harm can’t be avoided, so the NMFS can invoke exemptions in the MMPA and ESA if necessary [39, 48]. By choosing to safeguard the Rice’s whales, the NMFS set an important precedent on how valuable protecting the endangered species is. But this is vulnerable to shifting political landscapes. Opposing the decision was far-right US congressman Matt Gaetz, who tried to see Rice’s whale protections rolled back. Claiming that 51 poorly-known whales should not stand in the way of conflict readiness [49], he went as far as to personally introduce legislation to exempt the species from the MMPA [50]. While Gaetz’s efforts ultimately failed, this demonstrates the political dangers entangling the Rice’s whales.

 

Towards a future for the whales and us

 

While successes occur in core habitat, none of these measures cover the western secondary habitats. The secondary isn’t as formally defined as the core yet, but another reason why the NMFS can act swiftly in the core habitat may be because the area sees comparatively little economic pursuits. This is in part due to a congressional moratorium on offshore drilling in the eastern GOMx recently extended by President Trump until 2032 [51]. On the other hand, the majority of oil rigs and vessel activity operate in the western and central GOMx (Figure 2). The Deepwater Horizon spill must remind us of the ease of spillovers into the core habitat. Additionally, whales that venture westward (as confirmed by an acoustic survey [52]) face the brunt of marine industrialization. Any resulting deaths are detrimental to the 51 remaining whales. Protecting these corridors are therefore as important as protecting the core habitat.

 

The unfortunate reality is that Americans still rely heavily on GOMx oil [53], so an immediate prohibition isn’t feasible. Instead, we must take steps to reduce the impact of existing oil activities while progressively phasing out the industry. In the short term, we ought to demand better government oversight on oil regulations to hold the accountability necessary to prevent negligent spills. In the meantime, the 2021 NMFS workshop also recommended a clean-up: decommissioning the over 14,000 defunct oil wells at risk of spills [43, 54]. While President Biden has since signed into law the bipartisan Infrastructure Investment and Jobs Act that funds such efforts, the $4.7 billion set aside [55] is a small fry to the estimated $30 billion needed to neutralize all disused GOMx wells [54]. Raising this requires joint political action and/or grassroots fundraising. In the long term, we must collectively discourage oil expansion by supporting renewable energy. This isn’t just innovation; it also means bottom-up environmental justice that opens accessibility to marginalized groups least likely to afford clean technologies. But as we develop renewables, the NMFS workshop emphasized mindfulness to prevent potential harms to the Rice’s whales: poorly-placed marine infrastructure can introduce debris hazards and exacerbate shipping traffic and underwater noise, avoidable through robust spatial planning [43].

 

Long-term change towards a green future also addresses another fundamental challenge to the Rice’s whales’ future: climate change. With their reliance on a single prey, Rice’s whales are especially vulnerable to the unraveling crisis. The core habitat’s location is no coincidence: it’s within one of the most silver-rag-abundant regions in the northern GOMx [56]. This is thanks to the area’s unique position between the fresh nutrient-rich Mississippi River Delta, the warm Loop Current, and upwelling from the De Soto Canyon, mixing the three waters to produce a highly productive environment [11, 57]. The Loop Current is part of the thermohaline circulation (THC), earth’s global ocean conveyor belt, powered by optimal water temperature and salinity. But as the planet warms, the THC weakens. This diminishes the Loop Current [58], threatening the water-mixing that enriches the core habitat and therefore the abundance of silver-rags that sustain the Rice’s whales. This isn’t an isolated relationship: countless animals globally rely on few resources that may be lost due to climate-induced habitat disruption [59]. By acting to protect and recover the Rice’s whales with a climate mindset addressing big oil and other industrializers of the Gulf, we are not just “saving the whales”: we are also setting a precedent for tackling the looming global catastrophe faced by everyone everywhere.

 

We have power in the word

 

A most important component to recovering the Rice’s whale–but one that lags behind–is public awareness. Many Americans remain unaware of the Rice’s whale’s existence. While much can be done through policymaking by the NMFS and other government agencies, their top-down authority alone can only go so far when fundamental long-term and climate-allied change is ultimately necessary to truly pull back the critically endangered species. Rice’s whales are under direct protection by some of the most powerful environmental laws in the world, but the effectiveness of this advantage depends on the willingness of the nation’s citizens to push for lasting change in the GOMx.

 

Public awareness positively shapes the collective perspective. It combats disinformation [60], such as the false narrative that the new species’ recognition was arbitrarily crafted by “some scientist” [47, 49] to push an agenda, and empowers stakeholders like local boaters to participate in policy-making. This removes barriers to the synthesis of valuable community experience with scientific knowledge into policies that both protects the Rice’s whale and respects community needs. When people learn about species on the brink, it builds sympathy and determination to do what can be done to save them, inspiring political willpower to influence both citizen and government decisions from the bottom-up [61].

 

Rice’s whales hold strength through their potential to be a charismatic species: their enormous size to man’s eyes captures the icon of a majestic gentle giant. Through its direct relationship with oil, the Rice’s whale can be a flagship for what is preserved–or destroyed–by our choice on climate. Finally, the Rice’s whale is all-American: no other nation can be responsible for its extinction; its fate is the reflection of how we as Americans treat the environment. It’s why senior marine mammalogist Peter Corkeron of the New England Aquarium, one of the leading figures in the species’ conservation effort, suggested Balaenoptera ricei is better called the “American whale” [62].

 

Informing the people about the Rice’s whale, what our industrial actions are doing to them, and piquing a vision of a brighter future that could be–a healthy Gulf of Mexico echoing with year-round songs of America’s baleen whale, teeming with fish, dolphins, birds, and sea turtles fated not to an oily death but flourishing at a diversity imperceivable today alongside the whale that helped engineer its possibility–is enough to inspire support for the cause.

 

The road ahead is hard, but there is power for wonderful change in you, the reader, through a simple action. Tell another around you. Let them know: a great American whale is on the brink of extinction. Spread the word.

References

  1. Rosel, et al. 2021. Mar Mamm Sci. 37(2):577-610.
  2. Soldevilla, et al. 2022a. J Acoust Soc Am. 151(6):4264–4278.
  3. NOAA Fisheries. 2019. NOAA Lists Gulf of Mexico Bryde’s Whales as Endangered. Accessed 2023.
  4. Reeves, et al. 2011. Gulf Mex Sci. 29(1):41-67.
  5. Rosel PE & Wilcox LA. 2014. Endanger Species Res. 25:19-34.
  6. Bronstein, et al. 2018. BMC Evol Biol. 18(1):1-15.
  7. Rosel, et al. 2017. Mar Mamm Sci. 33(S1):76-100.
  8. Marine Mammal Health and Stranding Response Program (U.S.). Marine Mammal Carcass Disposal Best Practices. Accessed 2023.
  9. Sasaki, et al. 2006. Mol Phylogenet Evol. 41(1):40-52.
  10. Soldevilla, et al. 2017. Endanger Species Res. 32:533-550.
  11. Garrison, et al. 2021. The Trophic Ecology and Habitat of the Gulf of Mexico Bryde’s Whale (Balaenoptera edeni). Accessed 2023.
  12. Rosel, et al. 2016. NOAA Technical Memorandum NMFS-SEFSC-692.
  13. Pomilla, et al. 2014. PLOS ONE. 9(12):e114162.
  14. Jiménez López, et al. 2019. PLOS ONE. 14(1):e0209324.
  15. Rosel, et al. 2022. IUCN Red List Threat Species. 2022:e.T215823373A208496244.
  16. Save the Rhino International. Rhino populations. Accessed 2023.
  17. Liu, et al. 2022. Int J Primatol. 43:932-945.
  18. Robinson, et al. 2022. Science. 376(6593):635-639.
  19. Corkeron P & Kraus SD. Nature. 554: 169.
  20. Corkeron, et al. 2022. Mar Mamm Sci. 38(2):847-849.
  21. DWH MMIQT. 2015. 
  22. Rice, et al. 2014. J Acoust Soc Am. 135(5):3066-3076.
  23. Sirovic, et al. 2014. Mar Mamm Sci. 30(1):399-409.
  24. Kok, et al. 2023. Sci Rep. 13:8996.
  25. Tanaka Y. 2022. Royal Soc Open Sci. 9(11):221353.
  26. Kiszka, et al. 2023. Sci Rep. 12:6710.
  27. Zimmo, et al. 2012. Nat Educ Knowl. 3(12):3.
  28. Freitas, et al. 2023. Prog Oceanogr. 210:102927.
  29. 84 FR 15446
  30. 88 FR 20846
  31. FPR-2017-9234
  32. NCEI. Gulf of Mexico Data Atlas. Accessed 2023.
  33. Ellis EG. 2016. Thousands of Invisible Oil Spills Are Destroying the Gulf. Accessed 2023.
  34. United States v. BP Exploration & Prod., Inc. 21 F. Supp. 3d at 657.
  35. Davis M. 2012. Wash Lee J Energy Clim Env. 3(2):155-175.
  36. Glicksman RL. 2010. GW Law Fac Publ Other Works. 608.
  37. Farrington, et al. 2021. Oceanography. 34(1):76-97.
  38. Corkeron, et al. to Biden Administration. October 2022.
  39. Pub. L. 92-522.
  40. BOEM NTL No. 2016-G02.
  41. Pub. L. 93-205
  42. 88 FR 47453
  43. NOAA Fisheries. 2021. Rice’s Whale Recovery Planning Workshop. Accessed 2023.
  44. Christensen L to Herbert B. New Orleans, LA. November 22, 2022.
  45. Conn PH & Silber GK. 2013. Ecosphere. 4(4):1-16.
  46. Bestor C. 2023. Okaloosa County Commission wary of proposed Rice’s whale conservation plan. Accessed 2023.
  47. Florida Ports Council. 2023. President’s Message: June 2023. Accessed 2023
  48. 88 FR 24058.
  49. McLaughlin T. 2023. There are only 51 Rice’s Whales left in the Gulf. Matt Gaetz wants to lift their protections. Accessed 2023.
  50. Lower Energy Costs Act. H.R. 1, 118th Cong. (2023).
  51. Memorandum on the Withdrawal of Certain Areas of the United States Outer Continental Shelf from Leasing Disposition. (September 8, 2020).
  52. Soldevilla, et al. 2022b. Endanger Species Res. 48:155-174.
  53. Nelson JR & Grubesic TH. 2018. J Mar Sci Eng. 6(2): 30.
  54. Agerton, et al. 2023. Nat Energy. 8:536-547.
  55. NCSL. Infrastructure Investment and Jobs Act: Implementation and Key Resources. Accessed 2023.
  56. Lamkin J. 1997. Bull Mar Sci. 60(3): 950-959.
  57. Ocean Explorer. Geology: Deep Scope Sites in the Gulf of Mexico. Accessed 2023.
  58. Liu, et al. 2012. J Geophys Res Oceans. 117(C5):1-8
  59. NPS. Climate Change Endangers Wildlife. Accessed 2023.
  60. Miller, et al. 2021. Ecol Soc. 26(3):11.
  61. DeYoung B. 2021. Thar she blows! Whales sighted at Clearwater Marine Aquarium. Accessed 2023.
  62. Roman J. 2021. America’s New Whale Is Now at Extinction’s Doorstep. Accessed 2023.

How Toxoplasma gondii, a protozoa parasite, performs its parasitic behavior and how the infection would influence the intermediate hosts’ health and behavior.

By Chengyu Sun, Evolution, Ecology, and Biodiversity, ’25

Author’s Note:

  This review article was written for the assignment of Dr. Brenda Rinard’s UWP 102B. I chose the topic of discussing the behavior and uniqueness of Toxoplasma gondii because parasitic behavior really fascinates me and I would want to further study this area in the future. Initially I wanted to discuss multiple parasite’s behavior and the similarities but was told by Dr. Rinard to narrow down my topic to one species and I chose Toxoplasma gondii. Although the protozoan parasite does not cause much of a health problem in immunocompetent people, it does accomplish infecting more than one third of the entire world population. This review article aims not only to inform the scientific scholars of new approaches in studying this parasite or studying different species, but also could function as a general education article to let more people know the disease and understand its functions. 

Abstract

    Infection of Toxoplasma gondii within intermediate hosts is not virulently fatal but cognitively concerning. They usually occur within muscle cells and the central nervous system, which could be the explanation for multiple behavior alterations observed in hosts. Through experiments done on infected lab rats, scientists found that innate behaviors of neophobia and escaping from predators were transformed by a single infection. Infections in humans are commonly described as flu-like symptoms and are not lethal unless the patient is immunodeficient or the parasite was transmitted from mother to child during pregnancy (McAuley 2014; Martorelli Di Genova et al. 2019). Infected human patients have shown more outgoing or violent personality changes depending on one’s sex (Flegr and Hrdý 1994). Flegr and Hrdý showed that the parasite causes infection, and infection results in abnormal personality changes associated with mental illness such as schizophrenia and bipolar disease. The same antipsychotic medication and mood stabilizer used to treat schizophrenia and bipolar disease had slowed the infection of T.gondii

Introduction

    Toxoplasma gondii is a protozoan parasite that evolved to infect cat hosts but has the capability of being transmitted to all mammalian animals and making them their intermediate hosts (Dubey et al. 2012). In fact, at least one-third of the world’s human population has been infected by this protozoan. In some areas like Brazil, at least 50% of the population have antibodies toward this parasite due to the highly contaminated environment containing significant levels of oocysts, a stage of the parasite that is often found in host feces and produces infectious spores if ingested. (Dubey et al. 2012; Attias et al. 2020). As a model of the intermediate host, infected rodents are surprisingly prone to the odor of cat urine and identify the smell as “attractive”, while uninfected rodents would always escape from an area with cat smells. This difference in behavior makes the infected more likely to be eaten by cats thus helping the parasites to enter into cats’ intestines (Webster 2007). 

Humans are considered to be dead-end hosts due to the low probability of someone being eaten by any feline species, however, the parasite infection would be concentrated mostly in the central nervous system where they could alter the neurotransmitter secretion concentration, mainly dopamine, further leading to obvious personality change. The personality change often accompanies risky behavior or outgoing characteristics and an increase in the probability of congenital transmission to infants (Attias et al. 2020)

Even though research on T.gondii has just begun in the past hundred years, this parasite has coevolved with nature since domestic cats first appeared in history—their DNA even appearing in the mummies of ancient Egypt (Khairat et al. 2013). These thousands of years of coevolution with all its hosts provided this parasite with the ability to manipulate each of them. There is certainly far more knowledge of this parasite that is unknown to us. In this review, we will see how current science unravels the mystery of how T.gondii manipulates host behavior. 

Intestinal 

Figure 1: Transmission of T.gondii could involve multiple intermediate hosts primarily through ingesting contaminated food sources. 

Delta-6-desaturase: why cats?

We first must address the elephant in the room, that is why cats? Why did T. gondii choose the feline as its definitive host when it has the ability to infect almost all other mammals? It turns out that linoleic acid secretion is the key.

As it turns out, cats are the only mammal that secretes linoleic acid in their body since they don’t carry the enzyme delta-6-desaturase in its small intestine. This enzyme decomposes intestinal linoleic acid in all other species, but since cats don’t have the enzyme, their linoleic acid levels remain very high. (Martorelli et al. 2019). Martorelli et al. (2019) mimicked the intestinal environment of a cat to determine whether this difference makes cats ideal for sexual reproduction. The scientists shredded cat intestine epithelial cells onto glass coverslips to best replicate the favorable environments for parasite reproduction rather than in vivo experiments. The experiment utilizes two cat intestine models; the control group has no amino acids attached while the test group intestine model has linoleic acid and oleic acid supplements which have almost identical molecular structure but one double bond difference. To detect T. gondii, they used merozoite markers, a detection method that finds amoeboids capable of reproduction. They found that the man-made intestine model with a large amount of linoleic acid successfully cultured T. gondii while the control groups did not show signs of sexual reproduction. 

With this discovery, they proposed another hypothesis: if the delta-6-desaturase pathway in infected mice is shut down using a chemical named SC-26196, would the mice obtain a high level of linoleic acid and thus induce sexual reproduction of T. gondii in their intestine? A followup experiment found that merely SC-26196 alone couldn’t help the parasite reach the reproductive stage in mice. Instead, both the chemical and additional linoleic acid were needed, and even then the generated oocysts could not infect other live mice well. This means that T. gondii also needs optimal physiological conditions such as a stable body temperature, and ideal microbiome inside the intestines to enable access to the linoleic acid. The study also indicated that the absence of delta-6-desaturase doesn’t always make the ideal environment for sexual reproduction: extremely high concentrations of linoleic acid is also toxic for tachyzoites (T. gondii’s rapid replication phase during early infection).

Lastly, Martorelli et al. found that linoleic acid is likely to function as a signaling molecule instead of nutrition for the parasite because of its similarity to oleic acid with only one double bond difference, making it less likely a reaction material. The species barrier for the sexual development of T. gondii could be broken but there is no evidence that the parasite would alter the intermediate hosts’ intestine culture, at least so far. 

Infection in rodents—behavior manipulation towards fearless action

We’ve now established that T.gondii can’t accomplish sexual reproduction in mice, so they have to make their way out of the rodent and into the body of a cat. According to the manipulation hypothesis, parasites increase the transmission rate of themselves by controlling the intermediate host’s behavior to have a higher chance of being eaten by the definitive host. T. gondii achieved this goal with three distinct actions, (1) increase in infected species movement and exploring activity (less periods of stillness resulting in decreased surveillance behaviors), (2)  decrease in neophobia (fear of novelty), and (3) impairment of cognitive perception in face of predation risk. However, scientists still don’t know the pathological origin of these abnormal behaviors (Berdoy et al. 2000; Webster 2007). Berdoy et al. (2000)’s experiment includes lab rats, hundreds of generations without getting in contact with cats. The scientists placed them in maze-like pens with 16 cells, presenting multiple odors including control groups and cat’s urine odor. The T. gondii-infected rats showed great attraction to the cat odor and spent most of their time exploring the cells and staying active during the night while uninfected rats performed great aversion towards its natural predator’s scent. Their responses towards other smells were evidently similar with uninfected rats, indicating that the parasites do not impair their olfactory faculties but instead control their evolutionary behavior against predators. At the same time, similar “fearless” actions in rats are found in the ones given anti-anxiety drugs, proposing a similar pathway between manipulation and anxiety control mechanisms (Berdoy et al. 2000).

Another study on T.gondii’s influence on rodent behavior noted that even though the high infection rate in their subjects’ brains contributes to this phenomenon (reduction in neophobia and increased fearlessness towards predators), whether the specific alternation happens neurologically or immunologically is still unknown (Webster 2007). By reviewing research in human schizophrenia drug tests and Webster’s own previous research, she proposed another possible pathway for control that is similar to the anxiety-repressing technique. By blocking the host’s anxiogenic N-methyl-D-aspartic acid receptors in the amygdala and providing serotonin antagonists that could make the rats disregard danger, Webster found a possible connection with schizophrenia’s roots, pointing out a new possible direction in treating the disease (2007). This specific operation in neurological and psychological behavior could be significantly observed in human hosts.  

Infection in humans—health risk and personality alternation towards more interactions

Since human beings are also part of the infection cycle of T.gondii, Flegr and Hrdý (1994) believe the alternation of behavior seen in rodents and other species of hosts could also be seen in humans, especially personality changes towards exposure to danger. By obtaining data from 195 male and 143 female subjects experiencing chronic infection of T. gondii over a period of more than a year and using Cattell’s sixteen factor questionnaire (16PF) to generate the results, they discovered more significant personality differences in infected and healthy male subjects. In particular the results showed infected men are more schizothymic (detached and critical), have lower superego strength (disregards rules) while self-sentiment strength is low and experience more pretension (suspecting and jealousy). However the data collection might have been biased since the tested subjects were all collected from Charles’ University’s Faculty of Science (Flegr and Hrdý 1994). Among the infected male subjects, one could deduce that several factors are interrelated like schizothymia and low self-sentiment, and are thus likely caused by pretension and low superego rather than the parasite. Thus, the link between T. gondii and personality alteration still requires more research. 

Flegr conducted another study in 2007 that examines broader human behaviors. This time, Flegr had his participants go through 16PF and Cloninger’s Temperament and Character Inventory (TCI) personality test. This research is more reliable than the previous study since it includes more diverse samples from not only the university departments but also military personnel and blood donors, making the result more widely applicable. For the results, infected men tended to be more violent and more likely to ignore rules. Infected women showed a higher superego, becoming more outgoing and warmhearted and having higher apprehension compared with their healthy counterparts. Latently infected individuals had significantly longer response times to approaching danger and more easily lost their attention, making them 2.65 times more likely to have a traffic accident either as the pedestrian or the driver (Flegr 2007). Just like in rodents, we see that T. gondii impairs motor performance in humans as well.

Symptoms in infected patients are not the only problems worth concerning; T. gondii is also capable of infecting the fetus of a pregnant mother. In an earlier 2005 study, Flegr et al. examined the health impacts of T. gondii on pregnant women, using 16-week pregnancy individuals and discarding high-antibody subjects due to insufficient infection period. They found that early infection decreases the body weight of pregnant women and slows or stunts fetus development.  Ultrasonography tests at 16-weeks pregnancy may underestimate the severity T.gondii has on the weight of pregnant women. 

Psychological illnesses, their relationship to T. gondii infection, and possibly effective treatment:

T.gondii infection and personality and behavior change can be further linked to symptoms of neurological disorders such as bipolar disorder and the aforementioned schizophrenia, as the infection could do damage to serotonin secretion and lead to such psychological illnesses. But how can we distinguish parasite-induced mental illnesses from regular psychological abnormalities? In order to find out the solution to this, scientists looked back at the rodents to find some possible treatments. Webster et al. (2006), for example, recycled their neophobic mice experiment setup for another study on whether three medications, HAL (an antipsychotic), VAL (a mood-stabilizer), and an anti-parasite medication called PD, would affect treated subjects. They tested all three treatments on both infected rats and non-infected rats separately (with non-treated infected and uninfected rats as control groups) to test their activity level, feline attraction, and alertness. 

Figure 2: Drugs treating psychotic diseases of humans used on rodents show alleviation of infection, but adverse effects appear when these drugs are used to treat uninfected rodents, suggesting careful drug use and correct diagnosis before treatment. 

The results showed that all three treatments significantly decreased the rats’ attraction to cat odor and decreased their likelihood of entering the cat area by almost 200% compared to the control groups.  But the duration of their stay in the cat area decreased insignificantly. HAL and VAL treatments significantly reduced the time spent on exposure to cat smells and staying still in the cat area by 75% and 50% respectively, while HAL and PD significantly reduced the time spent on grooming by 60%.  However, the medication used on uninfected rats made them more likely to enter the cat area and increased their duration of stays there, suggesting caution is necessary when assigning these medications to humans in order to avoid negative effects making the infection worse. 

Finally, the main focus of medication should be aimed at the treatment of T. gondii infection in humans. Jones et al. (2003) tried to explore how antipsychotics and mood stabilizers would contribute to the inhibition of T.gondii infection as multiple antipsychotic medications have been found to have a certain level of antiprotozoal activity in patients. 

They tested the effect of 8 antipsychotics and 4 mood stabilizers on both parasite and host cells. Each medication was ranked using a  therapeutic index (TI)—the higher the value, the safer and better the drug. For example, the 12.1 TI value for trimethoprim, a commonly used drug for the treatment of T.gondii in humans, is similar to the 13.9 TI value of valproic acid. When these two drugs were tested together on their effect against T. gondii, the TI reached 39.5, while the concentration of trimethoprim and valproic acid alone in this treatment were much lower than the prescription level one patient would receive from their doctor , suggesting  significant synergistic effects. The findings also show  that the normal treatment of valproic acid for schizophrenic and bipolar patients is way above the tested threshold that the medication would be effective in inhibiting T.gondii infection (Jones-Brando 2003). However, as Webster (2006) observed uninfected mice developing infected-like behavior under treatment, antipsychotics and mood stabilizers should nevertheless be prescribed with great caution. 

Conclusion

The protozoan parasite Toxoplasma gondii was found around 100 years ago. Although we have reliable evidence on how the parasite infects a host and the possible consequences for different hosts including rodents and humans, there are still lots of unknown aspects of this parasite such as personality manipulation (Webster, 2007). As human development over the past hundreds years was profoundly significant, determining when the parasite gained the ability to infect humans is also an important component of unlocking this protozoan’s learning mechanism to help tackle worldwide infections. 

Works Cited

  1. Attias M, Teixeira DE, Benchimol M, Vommaro RC, Crepaldi PH, De Souza W. 2020. The life-cycle of Toxoplasma gondii reviewed using animations. Parasites Vectors. 13(1):588. doi:10.1186/s13071-020-04445-z.
  2. Berdoy M, Webster JP, Macdonald DW. 2000. Fatal attraction in rats infected with Toxoplasma gondii. Proc R Soc Lond B. 267(1452):1591–1594. doi:10.1098/rspb.2000.1182.
  3. Dubey JP, Lago EG, Gennari SM, Su C, Jones JL. 2012. Toxoplasmosis in humans and animals in Brazil: high prevalence, high burden of disease, and epidemiology. Parasitology. 139(11):1375–1424. doi:10.1017/S0031182012000765.
  4. Dubey JP, Lindsay DS, Speer CA. 1998. Structures of Toxoplasma gondii Tachyzoites, Bradyzoites, and Sporozoites and Biology and Development of Tissue Cysts. Clin Microbiol Rev. 11(2):267–299. doi:10.1128/CMR.11.2.267.
  5. Flegr J. 2007. Effects of Toxoplasma on Human Behavior. Schizophrenia Bulletin. 33(3):757–760. doi:10.1093/schbul/sbl074.
  6. Flegr J, Hrdá Š, Kodym P. 2005. Influence of latent “asymptomatic” toxoplasmosis on body weight of pregnant women. FOLIA PARASIT. 52(3):199–204. doi:10.14411/fp.2005.026.
  7. Flegr J, Hrdý I. 1994. Influence of chronic toxoplasmosis on some human personality factors. Folia Parasitol (Praha). 41(2):122–126.
  8. Jones-Brando L. 2003. Drugs used in the treatment of schizophrenia and bipolar disorder inhibit the replication of Toxoplasma gondii. Schizophrenia Research. 62(3):237–244. doi:10.1016/S0920-9964(02)00357-2.
  9. Khairat R, Ball M, Chang C-CH, Bianucci R, Nerlich AG, Trautmann M, Ismail S, Shanab GML, Karim AM, Gad YZ, et al. 2013. First insights into the metagenome of Egyptian mummies using next-generation sequencing. J Appl Genetics. 54(3):309–325. doi:10.1007/s13353-013-0145-1.
  10. Martorelli Di Genova B, Wilson SK, Dubey JP, Knoll LJ. 2019. Intestinal delta-6-desaturase activity determines host range for Toxoplasma sexual reproduction. Striepen B, editor. PLoS Biol. 17(8):e3000364. doi:10.1371/journal.pbio.3000364.
  11. McAuley JB. 2014. Congenital Toxoplasmosis. Journal of the Pediatric Infectious Diseases Society. 3(suppl_1):S30–S35. doi:10.1093/jpids/piu077.
  12. Webster JP. 2007. The Effect of Toxoplasma gondii on Animal Behavior: Playing Cat and Mouse. Schizophrenia Bulletin. 33(3):752–756. doi:10.1093/schbul/sbl073.
  13. Webster JP, Lamberton PHL, Donnelly CA, Torrey EF. 2006. Parasites as causative agents of human affective disorders? The impact of anti-psychotic, mood-stabilizer and anti-parasite medication on Toxoplasma gondii ’s ability to alter host behaviour. Proc R Soc B. 273(1589):1023–1030. doi:10.1098/rspb.2005.3413.

Impact of Perception on Animal Conservation Efforts and Biodiversity

By Adyasha Padhi, Biochemistry and Molecular Biology and Sociocultural Anthropology ’25

Humanity has impacted our planet’s biodiversity in extensive ways, both deliberately and accidentally. According to the World Wildlife Fund, there has been an average of a 69% decline in worldwide biodiversity since 1970 [1]. However, some geographical areas and ecosystems are disproportionally affected. Biodiversity in the Caribbean and Latin America has dropped 94% and their freshwater populations are experiencing an 83% decrease in biodiversity [1]. The four most significant drivers of biodiversity loss are land use change (30%), overexploitation (20%), climate change (14%), and invasive species (11%), all of which are associated with human activity [2]. Overall, the impact of all biodiversity drivers is increasing and so is the rate of biodiversity decline. 

One of the major ways that we attempt to repair the damage that we have done to our planet is through wildlife conservation efforts. However, examining conservation efforts reveals startling trends in which species receive focused protection, policy efforts, research, and funding. These inequities call into question the efficacy of many widespread conservation programs. 

What is biodiversity and why is it important: 

Biodiversity refers to the variety of life on Earth at all levels, from genetic diversity in a population to organism diversity within an ecosystem. This encompasses both the sum of total life forms across an area and the range of differences between those forms. While the biodiversity of some organisms is widely valued instinctively, some are not, such as microbiota, insects, and plants. Nevertheless, these undervalued organisms perform functions that are essential to maintaining stable ecosystems. Biodiversity supports healthy life on Earth– a lack of diversity at any level of life can lead to ecosystem collapse. This is because greater biodiversity in individuals, species, and ecosystems leads to greater ecosystem stability, and the populations within them are more likely to be able to withstand a range of disturbances, such as disease outbreaks and climate change. 

Biodiversity is usually explored at three levels: genetic, species, and ecosystem. For the purposes of conservation, species diversity is most often the focus. While genetic and ecosystem diversity are both extremely important, species diversity can offer insight into the health of both of these while being more manageable in scope. If species diversity is low in an ecosystem, that has implications on both the genetic diversity of the species that make up that ecosystem and that ecosystem as a whole in relation to other ecosystems. One way to measure species biodiversity is with a biodiversity index. A simple biodiversity index is calculated by dividing species richness (the number of species in an area) by species evenness (the total number of individuals in the same area) [7]. 

By using metrics such as the biodiversity index, researchers have found that biodiversity has been decreasing at an unprecedented rate [3]. Furthermore, certain areas around the equator known as biodiversity hotspots are experiencing biodiversity loss at even higher rates than other areas [3]. Human activity is contributing to all of the major driving factors for biodiversity decline, such as climate change, the effects of which are accelerated by the continued loss of biodiversity in ecosystems worldwide, creating a cycle of damage and loss. 

Conservation: Who gets chosen, and why? 

One way to mitigate the negative impacts humans have had on the planet is through conservation efforts, or protecting species and their habitats to restore populations and ecosystems. Conservation efforts typically fall into one of two categories: preventing further destructive forces from acting on ecosystems, such as stopping deforestation or stimulating populations through breeding and protection, in order to revitalize injured populations. These efforts are typically in-situ (conservation of habitats, species, and ecosystems where they naturally occur) or ex-situ (conservation of elements of biodiversity out of the context of their natural habitats, such as zoos, seed banks, and botanical gardens). 

Financial, land, and labor resources are limited, so the conservation of certain areas or species is forced to be prioritized over others. A common approach for selecting these priority areas for conservation is to focus on hotspots of diversity. These “hotspots” are regions of high conservation priority due to their high levels of richness in species, rates of endemism (uniqueness to that certain region), and threats to survival. Currently, there are 36 designated hotspots worldwide. These biogeographic regions are most commonly found around the equator and hotspots have lost around 85% of their habitat area [3]. 

Another metric used to determine the status of certain species is the International Union for Conservation of Nature (IUCN) Red List. The IUCN is an international organization for nature conservation and the sustainable use of natural resources. It is most well known for its Red List, which is regarded as the world’s most comprehensive inventory of the global conservation status of biological species. They report that they have assessed 42,100 species at risk of extinction, but a 2019 United Nations report estimates that of all existing species, a million are at risk of extinction in total. This disparity reflects how few existing species have been assessed by the IUCN. Prioritizing certain species and ecosystems is needed due to the limited resources that conservation organizations have, and the increasingly complex social, cultural, and political factors that further inhibit conservation efforts. 

Conservation efforts are often focused on a single species, and these species are often categorized into three categories that define their relation to their ecosystem: keystone, indicator, or flagship, with overlap between the categories. Keystone species play an essential role in the structure, function, or productivity of a habitat or ecosystem and the disappearance or diminishment of one of these species may lead to significant ecosystem change and dysfunction. One famous example of a keystone species is the Grey wolf in Yellowstone. As predators that regulate prey populations, the wolves enabled many species of plants and animals to flourish. After their disappearance in 1926, their reintroduction into Yellowstone in 1995 was a conservation effort that had highly successful effects on the entire food structure that defines the Northern Rockies ecosystem [12]. An example of a keystone species acting as ecosystem engineers are beavers, who regulate tree growth in the river ecosystems and also divert rivers with their dams, creating wetlands and ponds which support a wide range of organisms, which would not be able to survive in the more aggressive environment of a river. Despite their small size, bees are also a keystone species due to their integral role in pollination [13]. Plants too can be keystone species, such as Mangrove trees, which play an essential role in many coastal ecosystems by protecting coastlines from the impacts of waves and reducing erosion, while also providing a safe haven for small marine organisms to survive, which in turn has wider impacts throughout the food web.

Indicator species are species or groups of species chosen as an indicator or proxy for the state of an ecosystem. An example of this is crayfish as indicators of freshwater quality and peregrine falcons as an indicator of pesticide loads. Flagship species are selected to act as a symbol for a defined habitat or environmental cause due to their “charismatic” nature, especially in Western cultures, which is often where the funding for many conservation campaigns comes from [5]. These flagship species are typically large mammals that may or may not be keystone species and are not necessarily good indicators of biological processes in their ecosystems or of the conversation status of their ecosystem. Examples include tigers, kangaroos, elephants, and pandas [11]. 

Flagship species campaigns centered around these “charismatic” animals have been found to be more effective than other types of conservation campaigns [4]. Researchers have found that public donors were more likely to donate to a flagship charismatic species, regardless of the endangered status of the species [4]. Features of charismatic species include colorful coats or forward-facing eyes [6] because these appeal most to the broadest audience and these species are often featured in media & spaces where the public can interact with them, such as in movies or in zoos. The Similar Principle Theory finds that humans have a preference for animal species that are more similar to them. This is in both physical characteristics, such as forward-facing eyes and expressive faces, and social characteristics. As such, some researchers posit that the chances of survival for many species depend as much on human preferences as on existing biotic and abiotic factors, if not more [4]. 

Paradoxically, many of the animals regarded as the most charismatic are at high risk of imminent extinction, which some researchers attribute to the biased perception of the abundance of these animals since they are commonly overrepresented in our culture, such as pandas, tigers, and lions being common in our zoos, media, and educational material [6]. While some have called out this focus on animal charisma as being too narrow and excluding many species, charismatic flagship species conservation campaigns have been more successful in securing funding than campaigns that do not feature a flagship species [4]. However the status of many other species which share their habitat – or are vulnerable to the same threats – may also be improved by campaigns that target charismatic species. This is due in part to the large habitats that charismatic animals tend to live in due to their size and their position often being higher up on the food chain. Ultimately, researchers are calling for the shift away from campaigns centered around charismatic species and towards ones that focus more on the health of the ecosystem as a whole, even if this is less visible [6]. 

Conclusion

Biodiversity on all levels is an essential aspect of global health as it supports ecosystem health and stability via pollination, water purification, climate regulation, seed dispersal, agricultural pest control, and nutrient cycling. As such, all organisms, biotic, and abiotic factors that make up an ecosystem are extremely important in their own ways, and this means that even organisms that we don’t intrinsically value or find charismatic are important. Protecting all species is integral to protecting the health of the world as a whole and combating climate change. 

References

[1] “69% average decline in wildlife populations since 1970, says new WWF report,” World Wildlife Fund, 2022. https://www.worldwildlife.org/press-releases/69-average-decline-in-wildlife-populations-since-1970-says-new-wwf-report

[2] The Royal Society, “What is the human impact on biodiversity? | Royal Society,” royalsociety.org, 2022. https://royalsociety.org/topics-policy/projects/biodiversity/human-impact-on-biodiversity

[3]N. Myers, R. A. Mittermeier, C. G. Mittermeier, G. A. B. da Fonseca, and J. Kent, “Biodiversity hotspots for conservation priorities,” Nature, vol. 403, no. 6772, pp. 853–858, Feb. 2000, doi: https://doi.org/10.1038/35002501.

[4]A. Colléony, S. Clayton, D. Couvet, M. Saint Jalme, and A.-C. Prévot, “Human preferences for species conservation: Animal charisma trumps endangered status,” Biological Conservation, vol. 206, pp. 263–269, Feb. 2017, doi: https://doi.org/10.1016/j.biocon.2016.11.035.

[5]C. Mazzoldi et al., “From sea monsters to charismatic megafauna: Changes in perception and use of large marine animals,” PLOS ONE, vol. 14, no. 12, p. e0226810, Dec. 2019, doi: https://doi.org/10.1371/journal.pone.0226810.

[6]F. Courchamp, I. Jaric, C. Albert, Y. Meinard, W. J. Ripple, and G. Chapron, “The paradoxical extinction of the most charismatic animals,” PLOS Biology, vol. 16, no. 4, p. e2003997, Apr. 2018, doi: https://doi.org/10.1371/journal.pbio.2003997.

[7]“Guide on biodiversity measurement approaches (2nd edition),” Finance for Biodiversity Pledge. 2022. https://www.financeforbiodiversity.org/publications/guide-on-biodiversity-measurement-approaches/

[8]K. Whiting, “6 charts that show the state of biodiversity and nature loss – and how we can go nature positive,” World Economic Forum, Oct. 17, 2022. https://www.weforum.org/agenda/2022/10/nature-loss-biodiversity-wwf/

[9]J. H. Lawton and K. J. Gaston, “Indicator Species,” ScienceDirect, Jan. 01, 2001. https://reader.elsevier.com/reader/sd/pii/B9780123847195000745?token=68CA7F85D7A3FD42B151DC0DDD681D1E44822EC03A422ABFA252CF19E4E9E70F88BD7C79CB9E562C1AD10E92193926FA&originRegion=us-east-1&originCreation=20230411171712 

[10]M. C. Horner-Devine, K. M. Carney, and B. J. M. Bohannan, “An ecological perspective on bacterial biodiversity,” Proceedings of the Royal Society of London. Series B: Biological Sciences, vol. 271, no. 1535, pp. 113–122, Jan. 2004, doi: https://doi.org/10.1098/rspb.2003.2549.

[11]J. Qian and H. Zhuang, “Selecting flagship species to solve a biodiversity conservation conundrum,” Plant Diversity, Jan. 2021, doi: https://doi.org/10.1016/j.pld.2021.01.004.

[12]A. P. Dobson, “Yellowstone Wolves and the Forces That Structure Natural Systems,” PLoS Biology, vol. 12, no. 12, p. e1002025, Dec. 2014, doi: https://doi.org/10.1371/journal.pbio.1002025.

[13]A. Easton-Calabria, K. C. Demary, and N. J. Oner, “Beyond Pollination: Honey Bees (Apis mellifera) as Zootherapy Keystone Species,” Frontiers in Ecology and Evolution, vol. 6, Feb. 2019, doi: https://doi.org/10.3389/fevo.2018.00161.

Lung Cancer Vaccines: An Investigation of Potential Targets for a Novel Immunotherapy

By Rhea Bains.

Abstract:

This review comprehensively synthesizes research published within the last five years about a novel immunotherapy for lung cancer, known as a cancer vaccine in situ or intratumoral vaccination. The treatment involves an injection administered directly to the site of the tumor to trigger an immune response in the body for cancer treatment, thereby having the potential to extend the efficacy of other commonly used immunotherapies for lung cancer. Current immunotherapies have been found to not work in all patients and, given that lung cancer is known for high mortality rates, this treatment could potentially save countless lives. The scope of the research reviewed is limited to the two primary subsets of lung cancer vaccines in situ: vaccines containing genetically modified whole cells and vaccines containing modified viruses. It was found that most whole-cell vaccines can be optimized with the concurrent use of existing immunotherapies, but these vaccines have not been proven to improve patient prognoses. This subsequently contributed to the rise of research in the area of virus-based vaccines, which may also require simultaneous use with current immunotherapies to prevent immune-related adverse events. Although each method explored shows promise respectively, it remains unclear which method would be most valuable to isolate as a potential future lung cancer treatment. Many of the trials that show promise were conducted on mouse models, so further human trials of both whole-cell and virus-based vaccines would be beneficial in the quest to expand upon the current capabilities of modern cancer treatment.

Introduction: 

Lung cancer is best known for its high rates of mortality, making up for 23% of cancer-related deaths in 2020 alone [1]. Lung cancer’s two main subsets include non-small cell lung cancer and small cell lung cancer, the latter being rarer (15% of all cases) than the former (85% of all cases) [2]. Studied intensively over the past several decades, lung cancer in both its forms has proven a difficult ailment to combat, but extensive progress in treatment has been made. Immunotherapy is considered to be a rising field in potential treatments for advanced cancers. Using the body’s own immune mechanisms to produce an antitumor response is an effective way to improve patients’ prognoses [3]. Leading research institutions work on innovative immunotherapy solutions in the hopes of extending the efficacy of current treatments, which include chemotherapy, radiation, and surgery. Ideally, a new cancer immunotherapy treatment, a vaccine, would help mitigate the common complications brought on by traditional therapies and minimize healthy tissue damage [4]. 

Recently, there has been a push for the implementation of a cancer vaccine as a vector for future immunotherapies. Because of cancer’s innate ability to evade immune system detection through downregulating antigen (binding molecules) presentation on its cells [5], scientists are looking into a variety of approaches to either mount an immune system response against the tumors actively, using added antigens, or passively, using a nonspecific reaction against the area the tumor cells are located [2-4, 6, 7-9]. Similar to how vaccines evoke a response from the body to fight off infectious diseases, a cancer vaccine, or an intratumoral immunotherapy can be administered at the tumor site to invoke immune mechanisms [10]. An ideal combination of chemical and biological compounds would be administered via an intratumoral immunotherapy injection delivered locally to the site of the tumor (“in situ”), eliciting an immune response. Two components being investigated now fall into the categories of genetically modified virus-based and cell-based vaccines both surmounting an immune response to the tumor [2-4, 6, 7-9]. This review will discuss current research being conducted to evaluate modified viruses and modified whole cells for use in intratumoral immunotherapy, a lung cancer vaccine in situ. Used prior to (neoadjuvant) or after (adjuvant) traditional therapies, advancements in the targets and components of this vaccine could revolutionize lung cancer immunotherapy treatments. 

Figure 1: Whole cell and oncolytic virus injections in-situ induce immune response to tumor cells.

Tumor Evasion of Immune System Responses and The Rise of the Cancer Vaccine: 

Checkpoint inhibition is a common immunotherapy where patients are administered a compound that commonly blocks interaction between receptors, or specific antigens that bind to the antigens of other cells, found on immune system-derived T-cells and tumor cells, such as the PD-1 and PD-L1 interaction [11]. Normal interaction between the PD-1 and PD-L1 receptors inhibits the T-cell killing of tumor cells in an immune response, which occurs by the interaction of a different pair of cell receptors. An inhibitor of the PD-1 and PD-L1 interaction is known to promote T-cell killing of tumor cells. However, this first-line therapy has been found to only produce a response rate in only 20% of patients [6, 7]. It is unclear why 80% of patients are unable to produce an efficient immune response, but it has been theorized by Lee et al. that these tumors experience under-expressed antigens which limit the response the immune system can produce. 

Figure 2: Mechanistic view of T-cell inhibition through PD-1 and PD-L1 interaction.

It is known that some tumors downregulate antigen processing because of low levels of mRNA genes that are essential to signaling the cell to express certain intracellular proteins as antigens. This lowers the amount of antigen expression on the surface of their cells [5]. The human body’s immune cells (including T-cells, dendritic cells, and others) require antigens to effectively recognize tumor cells to bind, and a lack of PD-L1 expression in particular can make tumor cells immune to traditional checkpoint inhibition therapy [6]. Restifo et al. note that while one way to combat this is through administering drugs such as interferon gamma to enhance expression of the low mRNA, this treatment may interfere with the proliferation of T-cells and other immune cells which would counteract effective immune response. Therefore, another method being researched to combat this is using intratumoral immunotherapy to create an immune response that enhances antigen presentation of tumors [4, 6, 9], recruits immune cells [3, 8], and/or induces an inflammatory immune response [7]. 

Using either cell-based vaccines or virus-based treatments, current research focuses on achieving one or more of the aforementioned results to effectively treat patients with advanced stages of lung cancer. Those who have developed resistance to prior immunotherapeutic treatments or do not initially respond stand to benefit most from vaccination in situ. In combination with other immunotherapies and conventional treatments, this therapy would reduce the recurrence of tumors by building a memory within the immune system. This would transcend the current abilities of treatments like chemotherapy, radiation, and surgery [2], which require multiple treatments in cases of tumor recurrence, each with its own undesirable side effects. 

Mechanisms of Modified Whole Cells in Intratumoral Immunotherapy: 

The use of modified whole cells in cancer vaccines can be further subdivided into immune cell, tumor cell, and bacterial cell vaccines. The dendritic cell studies mentioned here were conducted in humans while the tumoral and bacterial vaccine studies are mouse-model (murine) based. Each study demonstrated technical success, but there are limitations presented by each of the research groups. Using modified dendritic immune cells to produce an immune response can help improve patient survival rates through increasing antigen presentation for evoked immune responses [6]. Locally injecting a modified dendritic cell injection containing tumor antigens twice and then collecting a tumor biopsy after a seven-day period yielded raised T-cell counts in 8 of 16 patients, so future use of gene-modified dendritic cells along with PD-1 and PD-L1 inhibition could be a promising avenue of therapy. Lee et al. state that tumor cells exhibit low antigen expression, corroborated by Restifo et al., which is why this treatment in concurrence with checkpoint inhibition is ideal to ensure that the best results are achieved. Increased antigen presentation from intratumoral therapy would be used for immune system response to the tumor, and any inhibitory antigens that present could be countered through checkpoint inhibition. Dendritic cells can also be modified to attract other immune cells. These administered whole cells combat cancer’s ability to suppress immune cell activity by recruiting the body’s T-cells to fight off malignancies. Shahrouki et al. conducted a feasibility study and found using dendritic cells in intratumoral immunotherapy to be potentially practical for widespread use, with technical success and minimal complications. However, Shahrouki et al. concurs with the assessment of Lee et al. that the best practice of intratumoral immunotherapy with modified dendritic cells is used in combination with checkpoint inhibitors. 

Inactivated whole tumor cells in an intratumoral vaccine can be used to increase antigen expression, primarily when exposed to radiation. Radiation was used in prior studies to inactivate the cells, but Luo et al. explore the possibility that irradiating tumor cells for use in cancer vaccines releases tumor antigens, allowing them to be more easily recognized by the immune cells of the body. The study yielded that irradiated tumor cells produced a substantial immune response in mice, but it is acknowledged that a human trial would be more telling of the efficacy of this treatment. 

Bacterial cells can also be used to stimulate an immune reaction, particularly modified E. coli. This bacterium produces a stimulator of interferon genes, which produce interferon gamma, through the STING pathway, activating a cascade of dendritic and T-cells through increased antigen presentation [5, 8]. However, again, this pathway works best when used in combination with a checkpoint inhibitor (atezolizumab), analogous to the findings of Lee et al. and Shahrouki et al, and only 2 of 23 patients experienced stable disease post-treatment. Thus, the markers of increased antigen presentation and immune response in each study are promising, but further human trials need to be conducted with each compound adjacent to checkpoint inhibition to fully understand the efficacy of the treatment. 

Mechanisms of Viruses in Intratumoral Immunotherapy: 

Discourse in the scientific community suggests that whole-cell vaccines have limited efficacy. Whole cells have shown some success in clinical trials, but further improvements need to be made to enhance it for use in patients [9], and many whole-cell vaccines do not improve overall survival in studies [2]. In fact, although Lee et al. found increased immune response in lung cancer patients, the median survival of the research participants was a mere 3.9 months. As a result of these challenges, some studies have pursued a different method entirely as a result, using select viruses that are injected into tumors via an intratumoral vaccination rather than whole cells. 

Using a strategy called gene-mediated cytotoxic immunotherapy (GMCI), a respiratory virus (adenovirus) is administered locally to a tumor site, prior to tumor resection surgery [7]. The oncolytic virus in the form of adenoviruses, herpes simplex viruses, and others only replicate within tumor cells, killing only the cancerous cells in the body [2]. Predina et al. Used an oncolytic virus to selectively infect and kill tumor cells when a secondary drug was added to activate the virus (valacyclovir). This trained the immune system to recognize and kill cancerous cells during a secondary immune response triggered by the inflammation the killing of the tumor cells induces, shrinking the tumor. Post cell death, the inflamed and infected area released tumor antigens that were targeted by the immune system. Predina et al. found that the method elicited increased T-cell activation and more surface antigens, but few patients showed evidence of antibody responses. They hypothesize that the antigen upregulation increased the expression of PD-L1, causing more inhibitory interactions between cancer and T-cells, which could reduce the efficacy of the treatment. Future studies might use this method with checkpoint inhibition to counter the uptake in PD-L1 expression that occurs from increased tumor antigen expression, just as Riese et al., Shahrouki et al., and Lee et al. found in whole-cell vaccine models. 

Wang et al. found that although the adenovirus method is effective, two viral injections achieved the best results in a mouse model, rather than just one. The virus would be injected in two parts: the first injection (not intratumoral) would hold whole inactivated tumor cells infected with a modified Newcastle virus, and the second injection would be an intratumoral injection holding just the virus. This virus not only killed tumor cells and upregulated T-cell activation but also increased inflammation and anti-inflammatory responses [9]. Again, this method shows promise but requires further human studies to confirm efficacy; however, Wang et al. provides a solid foundation for future studies on oncolytic viruses in potential intratumoral immunotherapies. 

Conclusion: 

Intratumoral immunotherapy is a rising field in potential treatment for advanced lung cancer, but there is a wide array of differing opinions about the best mechanism to use to target cancer. While modified cells and viruses have each shown some progress, there are still challenges to be overcome in both areas of research, and consensus has not yet been reached about which method holds the most merit. Both methods may require use with other immunotherapies to maximize success, so current therapies cannot be replaced entirely. There is an array of different types of both cells and viruses that have shown some potential in their own way, so no one method shows significant benefit over the other. In addition, Truong et al. suggest that both methodologies do not account for the typically lower immune system of older patients, who typically make up the demographic suffering from lung cancer. However, current research suggests that in patients with active immune systems (when used with checkpoint inhibitors) cell-based intratumoral vaccines and virus-based vaccines each have the potential to combat low antigen presentation by malignant cells and produce an autologous immune response in patients. With future efforts, perhaps one method of treatment can be isolated and then personalized for individual treatment.

Works Cited:

  1. Centers for Disease Control and Prevention. An Update on Cancer Deaths in the United States. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control and Prevention, Division of Cancer Prevention and Control; 2022. 
  2. https://www.cdc.gov/cancer/dcpc/research/update-on-cancer-deaths/index.htm. 
  3. Truong, Cao-Sang, and So Young Yoo. “Oncolytic Vaccinia Virus in Lung Cancer Vaccines.” Vaccines vol. 10,2 240. 4 Feb. 2022, https://doi.org/10.3390/vaccines10020240. 
  4. Shahrouki, Puja, et al. “Technical Feasibility and Safety of Repeated Computed Tomography–Guided Transthoracic Intratumoral Injection of Gene-Modified Cellular Immunotherapy in Metastatic NSCLC.” JTO Clinical and Research Reports, vol. 2, no. 11, 2021, pp. 100242–100242, https://doi.org/10.1016/j.jtocrr.2021.100242. 
  5. Luo, Lumeng, et al. “Irradiation Increases the Immunogenicity of Lung Cancer Cells and Irradiation-Based Tumor Cell Vaccine Elicits Tumor-Specific T Cell Responses in Vivo.” OncoTargets and Therapy, vol. 12, 2019, pp. 3805–15, 
  6. https://doi.org/10.2147/OTT.S197516.
  7. Restifo, N. P., et al. “Identification of Human Cancers Deficient in Antigen Processing.” The Journal of Experimental Medicine, vol. 177, no. 2, 1993, pp. 265–72, https://doi.org/10.1084/jem.177.2.265. 
  8. Lee, Jay M., et al. “Phase I Trial of Intratumoral Injection of CCL21 Gene-Modified Dendritic Cells in Lung Cancer Elicits Tumor-Specific Immune Responses and CD8 + T-Cell Infiltration.” Clinical Cancer Research, vol. 23, no. 16, 2017, pp. 4556–68, https://doi.org/10.1158/1078-0432.CCR-16-2821. 
  9. Predina, Jarrod D., et al. “Neoadjuvant Gene-Mediated Cytotoxic Immunotherapy for Non-Small-Cell Lung Cancer: Safety and Immunologic Activity.” Molecular Therapy, vol. 29, no. 2, 2021, pp. 658–70, https://doi.org/10.1016/j.ymthe.2020.11.001. 
  10. Riese, Richard, et al. “500 SYNB1891, a Bacterium Engineered to Produce a STING Agonist, Demonstrates Target Engagement in Humans Following Intratumoral Injection.” Journal for Immunotherapy of Cancer, vol. 9, no. Suppl 2, 2021, pp. A532–A532, https://doi.org/10.1136/jitc-2021-SITC2021.500. 
  11. Wang, Hui, et al. “Tumor Cell Vaccine Combined with Newcastle Disease Virus Promote Immunotherapy of Lung Cancer.” Journal of Medical Virology, 2023. https://doi.org/10.1002/jmv.28554.
  12. Hammerich, Linda, et al. “In situ vaccination: Cancer immunotherapy both personalized and off-the-shelf.” Molecular oncology vol. 9,10 (2015): 1966-81. 
  13. https://doi.org/10.1016/j.molonc.2015.10.016. 
  14. Li, Rui, et al. “Inhibition of Granulocytic Myeloid-Derived Suppressor Cells Overcomes Resistance to Immune Checkpoint Inhibition in LKB1-Deficient Non-Small Cell Lung Cancer.” Cancer Research (Chicago, Ill.), vol. 81, no. 12, 2021, pp. 3295–308, https://doi.org/10.1158/0008-5472.CAN-20-3564. 

Vesicles in the Study of Flaviviruses

By Nick Puso, Biochemistry & Molecular Biology ’23

Author’s Note: Nick Puso is a biochemistry & molecular biology graduate of the class of 2023. He wrote this review because, in his own words, he “really loves vesicles”. Nick found this topic particularly exciting to write on because it combines biochemistry, structural biology, genetics, drug design, and lipidomics. He is currently applying to medical school and hopes to become an ER doctor so he can put his love of the biological sciences to good use. Academics aside, Nick loves weightlifting, cycling, backpacking, and pushing wheelchairs at the VA where he volunteers. If you’re looking for him, your best bet is to comb the Sierra Nevadas in a helicopter.

INTRODUCTION 

Flaviviruses, including ZIKA, Dengue virus, and others infect over 400 million people annually [1], causing serious neurological symptoms, hemorrhaging, and birth defects. This issue is exacerbated by the failure of past research to produce any antivirals that prove safe and effective in early clinical trials [2]. An effective antiviral would significantly reduce the global mortality and morbidity of flaviviruses, but development is long and difficult. The process of producing an antiviral relies on knowledge of a critical mechanism in a virus’s infectious biology. In the case that this knowledge applies to more than one virus, the potential exists to develop an antiviral with broad efficacy- saving essential time and money. Flaviviruses as a genus share many fundamental attributes in their infectious biology, particularly concerning their use of vesicles. These conserved features could be exploited to create antivirals with broad efficacy within the genus. The flaviviruses referred to here include Zika (ZIKV), Dengue virus (DENV), Langat virus (LGTV), West Nile virus (WNV), and Powassan virus (POWV). This review will discuss recent advancements in our understanding of the role vesicles play in the infectious biology of flaviviruses. This will include release from endosomes after entry into the cell, viral replication within the unique “vesicle packet” organelle, and a novel exosome-based mechanism of infection. Furthermore, we will explore how this knowledge could inform drug development. 

Background 

Flaviviruses are a genus of enveloped viruses, meaning that their genetic material is contained within a lipid bilayer. Embedded in this bilayer are the Envelope (E) protein (sometimes called E-glycoprotein) and Membrane (M) protein. E-Protein is responsible for attaching naked viral particles (virions) to target cell membranes, upon which they enter the cell via clathrin-mediated endocytosis [3]. The clathrin coated proteins mold a small bubble of host membrane material–an endosomal vesicle–which carries the virions into the cell.

For the viral genome to escape the endosome, and enter the cytoplasm, the viral envelope must fuse with the membrane of the endosome. Fusion is initiated by the acidic pH of endosomes, which drives a conformational shift in viral E-protein that merges the viral envelope with the endosome [3]. This mechanism is conserved in all flaviviruses. 

Flaviviruses also utilize a unique replication organelle, derived from the membrane of the endoplasmic reticulum (ER), called the vesicle packet. The vesicle packet is an invagination into the ER membrane in which the machinery of viral replication is contained, and likely protected from host cell innate-immune factors. A combination of host factors and viral proteins, including non-structural protein 1 (NS1) conserved in flaviviruses, work together to construct the vesicle. [4]. However, the specific host factors involved in vesicle packet formation were largely unknown until recently. 

Once translated and assembled, virions exit the cell via the secretory pathway, including the trans-Golgi-network (TGN). Like the endosome earlier in the life cycle, the TGN has an acidic pH. M-protein requires this acidic pH to mature. Specifically, prM, the precursor to M-protein, undergoes an acid-driven conformational shift which exposes cleavage sites for Furin-protease, a host protease [5]. Cleavage releases mature M-protein from ‘prM’. The ‘pr’ element itself is responsible for preventing premature fusion of the virion envelope with host membranes, so it remains associated with the virion until secretion [5]. The dissociation of ‘pr’ after cleavage allows the mature virion to later undergo acid-driven fusion with endosomes in a new cell, completing the cycle [5]. 

Endosomal release and niclosamide’s antiviral potential 

A compound that safely inhibits endosomal and secretory pathway acidification could represent a pan-flavivirus antiviral. This is because attenuation of acidic pH would prevent the conformational shift in E-protein necessary for the fusion of the virion with the endosome. Recently, researchers determined that niclosamide–an FDA-approved antiparasitic drug previously known to inhibit ZIKV infection–did exactly this in DENV-infected cells [6]. Niclosamide is a protonophore;a compound that can exchange protons across membranes, bypassing the proton pumps responsible for endosomal acidification. Treatment with niclosamide significantly reduced viral load in baby-hamster-kidney (BHK21) cultures infected with DENV. Using pH-sensitive dye A0, which turns from clear to pink at pH 6, the mechanism was confirmed to be attenuation of endosomal acidification. Additionally, a western blot of DENV protein-E showed that the acid-driven confirmation shift in protein-E necessary for endosomal fusion did not take place. This western blot relied on the fact that E-protein’s conformational shift results in its degradation. An antibody could visualize E-protein in the SDS-page (electrophoresis which separates proteins by mass) of the treatment group, meaning the degradation did not take place. This indicates that the conformational shift was blocked by niclosamide as hypothesized. Because no effects were noted on viral genome replication or endocytosis independently of endosomal release, the authors do not put forward any additional mechanisms of action. 

However, another study found that niclosamide also prevents the maturation of DENV in human (Huh7) cells via deacidification of the TGN [7]. Researchers determined that deacidification inhibited cleavage of DENV/ZIKV prM protein by preventing the exposure of furan-protease cleavage sites. Compared to a control, Western blot of the treatment grouprevealed uncleaved prM when niclosamide was present. Virions with immature prM protein due to niclosamide treatment would be unable to fuse with endosomes upon infection of a new cell and are thus dead. The authors suggest discrepancies from Kao et al [6] arrose because they used different time points in their analysis of niclosamide’s effect on genome replication and virion maturation. 

Niclosamide’s ability to de-acidify endosomes and the TGNdemonstrates its potential as a pan-flavivirus antiviral, given that E-protein and prM are conserved and essential in all flaviviruses. Both authors argue that niclosamide’s safety as an antiparasitic drug and its effect on ZIKA and DENV warrant investigation into antiviral applications. Additionally, both studies put forward inhibition of endosomal release as a viable strategy in anti-flavivirus drug development overall. Jung et al also recommend inhibition of prM cleavage for therapeutic investigation.

Targeting host factors in ‘Vesicle packet’ formation 

Replication inside the vesicle packet is another potential target for pan-flavivirus antiviral development because it is conserved in the genus. Knowledge of the involved host factors–proteins in the host that the virus manipulates to its own ends–could allow for antivirals that knockout vesicle packet formation, inhibiting viral replication. Recently, a class of host ER-shaping proteins called Atlastins were found to play a role in the formation of the vesicle packet [8]. Researchers used small-hairpin RNAs (shRNAs), which are processed into silencing siRNA to knock out various atlastins in A529 cells exposed to ZIVK, DENV, and WNV. Transmission electron microscopy, which uses electrons to visualize microscopic structures, revealed that Atlastin-2 (ATL2) knockout shrunk both the size and number of vesicle packets, and disrupted their localization. Immunofluorescence, which visualizes the location of small molecules using a fluorescent antibody, then showed that the localization of viral double-stranded RNA (dsRNA) was changed. This indicates that the coordination of vesicle packets with the replicating viral genome was disrupted. Also, the overall viral load of ZIKV, DENV, and WNV was reduced by Atlastin-2 knockout. In the case of ZIKV, this reduction was potentially as much as 16-fold. While the authors make no specific recommendations, future research should explore whether these factors inhibit flavivirus replication outside of DENV, ZIKV, and WNV. Given that all flaviviruses form a vesicle packet for replication, the host factors they manipulate to do so may be the same. If this is the case, disruption of flavivirus/Atlastin interaction may be a powerful therapeutic approach. 

A host protein called Receptor of Activated C Kinase (RACK1)has also been identified as essential to vesicle packet formation for flaviviruses WNV, DENV, POWV, and LGTV [9]. Researchers used a similar approach to Neufeldt et al, employing small ‘interfering’ RNAs (siRNAs) to silence host factors and measure the effect on viral load. Specifically, a CRISPR-based genome-wide knockout screen using LentiCRISPRv2-GecKO (an siRNA library) provided siRNAs to human Huh7.5 cells. RACK1 knockout produced the strongest reduction in viral load out of all targets. To determine the mechanism, researchers analyzed the time dependency of viral load reduction on RACK1 knockout. The results indicated that RACK1 did not act on viral entry or translation, but rather on replication, which brought attention to the vesicle packet. Transmission electron microscopy then demonstrated a significant reduction in vesicle packets after RACK1 knockout. Immunofluorescence found that viral NS1, a known critical factor in vesicle packet formation, failed to localize to the ER. The failure of NS1 to localize to the ER during RACK1 knockout would suggest that an interaction between NS1 and RACK1 is necessary for vesicle packet formation. Co-expression of RACK1 and NS1 gave strong evidence for their co-localization. To confirm the interaction, a pulldown assay- which measures binding affinity between two molecules- was performed and RACK1 was proven to bind NS1. The authors thus posit that RACK1 is responsible for localizing NS1 and that RACK1 knockout disrupts vesicle packet formation by the failure of NS1 to localize. 

The discovery of ATL2 and RACK1 as host factors necessary to the formation of the vesicle packet offers new targets for disrupting flavivirus replication. The respective authors argue that dependence on RACK1 and Atlastin-2 for vesicle packet formation is likely conserved in all flaviviruses. Shue et al thus argue that RACK1 is a promising target for antiviral development, and recommend further research into RACK1 knockout as a therapeutic strategy. ATL2’s importance to vesicle packet formation warrants exploration into its potential as a pan-flavivirus antiviral target. 

Exosome-based infection: SMase and Tsp29Fb as drug targets 

Recently, three studies have identified a novel exosome-based mechanism of flavivirus infection and identified potential targets for antiviral development. Zhou et al, (2018) found that LGTV uses extracellular vesicles (exosomes) from its tick host to infect mammalian cells, and that infected mammalian brain-endothelial cells, which make up the blood-brain barrier, produce exosomes capable of infecting neuronal cells [10]. In a follow-up to their 2018 study, Zhou et al (2019) found that ZIKA-infected, neuronal-cell-derived exosomes readily infected neurons of the cortex [11]. A third study, Vora et al, demonstrated that exosomes derived from DENV-infected mosquitoes were infectious to human blood-endothelial cells [12]. The ability of flaviviruses to use exosomes, derived from host cells, to infect other cells was previously unknown and represents a significant advancement in our understanding of their infectious biology as a genus. 

Additionally, GW4869, an exosome release inhibitor, proved to reduce viral load in cell cultures infected with their respective flavivirus in all cases. GW4869 works by inhibiting sphingomyelinase (SMase), an enzyme 

that produces lipids necessary for exosome formation. The effect of GW4869 on viral load confirms that exosomes provide significant infectious potential, in a novel mechanism, for 3 prominent flaviviruses: LGTV, ZIKV, and DENV. GW4869’s ability to inhibit this mechanism in all three suggests that an antiviral targeting exosome release may have efficacy across the genus. All three studies thus cite GW4869 as a promising target for drug development to disrupt flavivirus infection via exosomes. GW4869’s efficacy also suggests the anti-flaviviral potential of SMase inhibition more broadly. 

All three studies used immunoblotting and quantitative real-time PCR to show the enrichment of viral RNA and proteins in infectious exosomes.

All three also confirmed that these viral components are fully inside the exosomes. To do this, they exposed these exosomes to RNase and flavivirus envelope protein antibodies, which neutralize the infectious potential of naked virions by degrading exposed viral RNA and protein. The infectious potential of exosomes was unaffected by RNase or antibody treatment. All three studies thus argue that the exosome protects viral RNA and protein from antibodies and RNase. 

Vora et al also found that Tsp29Fb–an ortholog of high similarity to human exosome marker CD63–was enriched on infected extracellular vesicles [12]. Specifically, qRT-PCR, which tracks the expression of RNA over time, found that Tsp29Fb was overexpressed during infection. Additionally, the CD63 antibody (which is highly cross-reactive with Tsp29Fb) was used in immunoblotting to show that Tsp29Fb is enriched on the exosomes. Researchers then used immunoprecipitation, which removes a target from solution using an antibody, to find that Tsp29Fb binds DENV envelope protein. This binding interaction indicates Tsp29Fb may play a role in DENV’s ability to use exosomes for infection. To test this, researchers silenced Tsp29Fb with siRNA and found drastically reduced viral protein in the resulting exosomes. The researchers posit that Tsp29Fb thus plays a role in loading protein into secretory vesicles post-translation, which then exit the cell as infectious exosomes. For that reason, the authors put Tsp29Fb silencing forward as an antiviral development tactic.

These three studies demonstrate that extracellular vesicles provide infectious potential to three flaviviruses. Additionally, two promising candidates for antiviral research have been put forward to disrupt viral exosomes. Namely, GW4869 targeting SMase, and Tsp29Fb as a target itself. 

CONCLUSION

Flaviviruses as a genus have great similarities in their infectious biology concerning their manipulation of host membranes and vesicles. Post-entry endosomal release and the formation of the vesicle packet are shared features of the flavivirus infection cycle. In a completely new infectious mechanism, LGTV, ZIKV, and DENV were all found to invade new cells using exosomes. All three discussed uses of vesicles have been proven essential to infection and are present in multiple flaviviruses. Furthermore, workable targets for the inhibition of these faculties have been identified, which subsequently reduce viral load in vitro. Multiple promising candidates for drug development have thus emerged from these targets. Future research should focus on whether an exosome-based infection is present in other flaviviruses and establish the relevance of SMase and Tsp29Fb to the broader genus. If exosome-based infection proves critical to flaviviruses more broadly, SMase may be a viable target, and GW4869 might have potential as a pan-flavivirus antiviral. With respect to targeting replication in the vesicle packet, RACK1 and ATL2 knockout should be investigated for safety and efficacy. Niclosamide’s ability to inhibit post-entry edosomal release must also be established in the genus as a whole, and the therapeutic dose already approved by the FDA for other conditions should be tested for efficacy against flavivirus.

REFERENCES

  1. Pierson, T.C., Diamond, M.S. The continued threat of emerging flaviviruses. Nat Microbiol 5, 796–812 (2020). https://doi.org/10.1038/s41564-020-0714-0
  2. Qian, Qi. 2022. Mosquito-Borne Flaviviruses and Current Therapeutic Advances. Viruses. 2022, 14(6), 1226; https://doi.org/10.3390/v14061226
  3. Modis Y, Nayak V. Molecular Mechanisms of Flaviviral Membrane Fusion. J Virol. 2009:265–86. doi: https://doi.org/10.1007%2F978-0-387-79840-0_12 
  4. Chatel-Chait, Bartesenschlager. 2014. Dengue Virus- and Hepatitis C Virus-Induced Replication and Assembly Compartments: the Enemy Inside—Caught in the Web. Virology. 2014; 88(11). doi: https://doi.org/10.1128/jvi.03404-13
  5. Yu et al. 2009. Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion. Virology. 2009 Dec; 83(23): 12101–12107. doi: 10.1128/JVI.01637-09 
  6. Kao JC et al. 2018. The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR. PLoS Negl Trop Dis. 2018 Aug 20;12(8):e0006715. doi: https://doi.org/10.1371%2Fjournal.pntd.0006715 
  7. Jung, E. et al. 2019. Neutralization of Acidic Intracellular Vesicles by Niclosamide Inhibits Multiple Steps of the Dengue Virus Life Cycle In Vitro. Sci Rep 9, 8682 (2019). https://doi.org/10.1038/s41598-019-45095-1
  8. Neufeldt et al. 2019. ER-Shaping Atlastin proteins act as central hubs to promote flavivirus replication and virion assembly. Nat Microbiol. 2019 Dec; 4(12): 2416–2429. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881184/
  9. Shue et al. 2021. Genome-wide CRISPR screen identifies RACK1 as a critical host factor for flavivirus replication. Virology. 2021; 95(24). doi: https://doi.org/10.1128/JVI.00596-21
  10. Zhou et al. 2018. Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells. PLoS Pathog. 2018 Jan; 14(1): e1006764. doi: 10.1371/journal.ppat.1006764 
  11. Zhou et al. 2019. Exosomes mediate ZIKA virus transmission through SMPD3 neutral sphingomyelinase in cortical neurons. Emerging Microbes and Infections. PLoS Pathog.2019; 8(1): 307–326. doi:1080/22221751.2019.1578188
  12. Vora et al. 2018. Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb. Proc Natl Acad Sci USA. 2018 Jul 10; 115(28): E6604–E6613.https://doi.org/10.1073%2Fpnas.1720125115

Neonatal Isoerythrolysis in Equines, Felines, and Other Species

By Sara Su, Animal Science and English ’24

Neonatal Isoerythrolysis: An Overview

Neonatal isoerythrolysis (NI) is an alloimmune disease, an immune response against non-self-antigens from the same species. A non-self antigen can be any foreign substance that can trigger the host’s immune system. Hemolytic anemia is the most common symptom of NI, where a newborn’s red blood cells are the non-self-antigens, and are targeted and destroyed by maternal antibodies [1, 2, 3]. These antibodies are absorbed when the newborn ingests colostrum, otherwise known as “first milk” [1, 2]. In essence, neonatal isoerythrolysis occurs when an offspring inherits the father’s blood type while the mother’s blood type contains antibodies against it. Antibodies to red blood cells are commonly referred to as anti-erythrocyte antibodies, and if they are passed to another animal via ingestion or transfusion, they will attack the host’s own red blood cells at a rate at which they cannot be replaced. If left untreated, NI is fatal. Neonatal isoerythrolysis is most common in cats and horses, and is rarely observed in other species [1,2]. 

Colostrum is highly important because neonates (newborns) ingest the mother’s antibodies to gain passive immunity, the first layer of protection against pathogens after birth. For the first 24 hours of life, neonates are able to absorb antibodies in the gut. After this window, the gut begins to close and absorption decreases dramatically. Because of this, it is important to ingest colostrum as soon as possible after birth [2]. Isoerythrolysis occurs when those maternal antibodies become alloantibodies, which specifically target non-self red blood cells–in this case, the foal’s– and causes hemolysis [4]. 

The most common symptoms are weakness, pale mucous membranes, icterus (jaundice), tachycardia (rapid heartbeat), and tachypnoea (rapid breathing) [1, 5, 6, 7]. Symptoms can occur hours to a week within ingestion of colostrum, and death will occur if left untreated. Across all species, offspring that are positive for NI should not drink colostrum from their biological mothers. Preventing ingestion of colostrum containing alloantibodies can be done by muzzling the offspring (in the case of foals) or removing them from the mother (in the case of kittens). The mother must be milked periodically to eliminate any remaining colostrum, so that she can later produce milk that is harmless. In the meantime, the newborn(s) should be fed colostrum from a different animal.

Because neonatal isoerythrolysis results in hemolytic anemia, blood transfusions are a common treatment. Similar to human blood transfusions, horses and cats can receive transfusions from any individual with the same blood type. However, in horses it is highly recommended to use the blood from the mother after it is washed with saline and centrifuged to get rid of maternal alloantibodies [1]. 

To the untrained eye, the various symptoms of NI might appear to be the result of a contagious pathogen rather than an alloimmune disease. Nonetheless, it is very easy to prevent–conscientious breeders should preemptively blood test breeding pair, and test the pregnant mother again two weeks before parturition (birth) [5]. This would serve to inform the breeders about potentially conflicting blood types and prevent the conception of a foal that could become a victim of NI. Additionally, care should be taken when breeding females that have already given birth, or have received blood transfusions or certain vaccinations. Ultimately, the best method of combating NI is through prevention: a simple blood test of a breeding pair will be able to predict whether the neonate will develop this condition or not [1, 5].

Figure 1. The red blood cells of a neonate carrying the father’s blood type is at risk of being targeted by anti-erythrocyte antibodies contained in the mother’s colostrum

Neonatal Isoerythrolysis in Equines

Neonatal isoerythrolysis is most common in horses and mules, although it is also possible in other equids such as zebras. There are 16 blood types for horses; of these types, Aa and Qa are the most likely to become antigenic and cause NI [1, 2, 3]. Reactions to other blood types, such as Qc and Db, have also been observed [6]. When a mare is exposed to these antigens from a pregnancy or blood transfusion, she will produce alloantibodies that will attack the red blood cells of her future foal. This becomes complicated when a mare that has previously given birth to unaffected foals is introduced to an antigen, putting her future foals at risk for NI [1, 2].

Furthermore, there is something called a “donkey factor,” which is a type of red blood cell antigen specific to donkeys [8]. Thus, horses cannot receive blood transfusions from donkeys, but donkeys can receive blood transfusions from horses [1, 8, 9]. The donkey factor means that there is a much higher chance of Neonatal Isoerythrolysis occurring in mares pregnant with mule foals–10% in horse x donkey crosses compared to 1% when breeding horses. However, this is only a problem for mares that have carried more than one mule foal [7, 9].

In horses, NI is diagnosed with a Coomb’s test, which detects hemolytic anemia by cross-matching the mare’s blood serum with her foal’s red blood cells. If the result is positive, agglutination (clumping in the test tube) will occur [5, 10]. While the Coomb’s test is a definitive diagnosis that evaluates hemolysis, NI can also be prevented by conducting a jaundiced foal agglutination test (JFAT), which is a cruder method utilizing the mare’s colostrum and her foal’s erythrocytes. As with the Coomb’s test, if agglutination occurs there is a high risk of NI and the foal should be prevented from nursing [2, 5]. Thus, it is important to run these tests as soon as possible to prevent the foal from nursing from its biological mother [11]. Colostrum from an unrelated donor should be fed to the foal, and the biological mother should be milked periodically. Fortunately, antibody absorption drops dramatically after the first 24 hours of life, so this solution need not be implemented long-term. However, for foals that have already ingested colostrum and become severely anemic to the point of not wanting to nurse, blood transfusions are recommended. The blood should be cross-matched, and if that is not possible it is recommended to transfuse blood from an Aa Qa blood type negative gelding(a castrated male horse) [7]. In all cases, it is not recommended to separate a foal from its mother due to separation anxiety; thus, the foal should be muzzled. 

Figure 2. The Coomb’s test detects hemolytic anemia, which is a common symptom of Neonatal Isoerythrolysis, by cross-matching the mare’s blood serum with her foal’s red blood cells

Neonatal Isoerythrolysis in Felines

Neonatal Isoerythrolysis may also occur in cats, and although this condition is much rarer in felines compared to equines, the mortality rate is still very high [12]. There are 3 blood types in cats: A, B, and AB [12, 13]. Type A is the most common by far, and it is also dominant to type B [14]. Type AB is not well-documented, but it is known that it is the rarest blood type. It is dominant to type B, but recessive to type A[12]. If the queen (mother cat) has type B, she naturally produces alloantibodies to blood type A. Neonatal Isoerythrolysis will occur if a Type B female is bred with a Type A tomcat so that the kittens are type A/B. In the United States, Type A is the most common blood type among cats, while Type B is rare [12]. It should be noted that wild felines share the same AB blood-grouping, so diagnosis and treatment may be applied to zoos in addition to domestic homes [13]. 

 In cats, diagnosis is based on clinical signs of the kittens and blood typing. Clinical signs include weak nursing, pale or jaundiced mucous membranes, and dark red-brown urine [12]. If blood work is done, it will show anemia. Because feline litter sizes are large, it is common to use placental blood for testing. It should be noted that kittens in a litter may show varying levels of symptom severity; researchers theorize that this is due to different levels of colostrum intake. Unlike equines, felines can be directly removed from their mother and housed with another queen as soon as symptoms appear. 

Neonatal Isoerythrolysis in Other Species

Although these instances are much rarer compared to cats and horses, neonatal isoerythrolysis has also been observed in cattle, dogs, and pigs [15, 16, 17]. Interestingly, NI is not a natural phenomenon in cattle–it was first observed in 1960 and was caused by homologous blood vaccines administered by veterinarians [17]. The threshold of response to these vaccines was variable so that the number of injections that would induce symptoms differed between every individual.

Hemolytic diseases similar to Neonatal Isoerythrolysis exist in humans as erythroblastosis fetalis or Rh disease [18]. It is termed differently because alloantibodies can cross the placenta during human pregnancy, but can’t in other mammals–offspring would only come into contact with these antibodies after birth via colostrum. In human blood typing, the “+” and “-” signs refer to the Rh factor. If an individual is positive, the Rh protein is present on the surface of their red blood cells, and if the Rh protein is not present then the individual tests negative. Rh disease occurs when one develops a hypersensitive immune response, developing anti-Rh factor antibodies that kill healthy Rh-containing blood cells. However, Rh disease can only occur within a specific circumstance: if the father is positive for Rh factor and the mother is negative, yielding an Rh-positive offspring. Even under these conditions, only the second child would be in danger of Rh disease–the mother builds antibodies against the Rh factor during her first pregnancy but the disease itself doesn’t manifest. To prevent Rh disease, if a mother in her second pregnancy has a fetus of opposite sign, RhIg is administered–an anti-Rh factor antibody that inhibits immune response [18, 19].

Conclusion

To conclude, neonatal isoerythrolysis can quickly become fatal to the neonate, but is perfectly preventable and treatable. Measures of prevention are particularly important, because neonates may begin nursing and absorbing antibodies that destroy red blood cells within hours of birth. Because the clinical signs of this disease are so severe, early detection is key to neonate survival. However, it is extremely easy to prevent this disease–a simple blood-type cross-match between a prospective father and mother will suffice. In the end, this is an issue of responsible breeding, thus conscientious breeders should not encounter Neonatal Isoerythrolysis at all.

References

  1. Jamieson CA, Baillie SL, Johnson JP. 2022. Blood Transfusion in Equids-A Practical Approach and Review. Animals (Basel). 12(17):2162. doi: 10.3390/ani12172162.
  2. Kähn W, Vaala W, Palmer J. 1991. Die neonatale Isoerythrolyse bei neugeborenen Fohlen [Neonatal isoerythrolysis in newborn foals]. Tierarztl Prax. 19(5):521-9. 
  3. Becht JL, Semrad SD. 1985. Hematology, blood typing, and immunology of the neonatal foal. Vet Clin North Am Equine Pract. 1(1):91-116. Doi: 10.1016/s0749-0739(17)30771-x. PMID: 3907769.
  4. Proverbio D, Perego R, Baggiani L, Ferrucci F, Zucca E, Nobile F, Spada E. 2020. Prevalence of Ca Blood Type and Alloantibodies in a Population of Horses from Italy. Animals (Basel). 10(7):1179. doi: 10.3390/ani10071179. 
  5. Felippe, J.B. Equine Neonatal Isoerythrolysis. 2017. In Interpretation of Equine Laboratory Diagnostics; John Wiley & Sons, Ltd. pp. 251–255. 
  6. MacLeay JM. 2001. Neonatal isoerythrolysis involving the Qc and Db antigens in a foal. J Am Vet Med Assoc. 219(1):79-81, 50. doi: 10.2460/javma.2001.219.79. 
  7. Carr EA. 2014. Field triage of the neonatal foal. Vet Clin North Am Equine Pract. 30(2):283-300, vii. doi: 10.1016/j.cveq.2014.05.001.
  8. McClure JJ, Koch C, Traub-Dargatz J. 1994. Characterization of a red blood cell antigen in donkeys and mules associated with neonatal isoerythrolysis. Anim Genet. 25(2):119-20. doi: 10.1111/j.1365-2052.1994.tb00091.x. 
  9. Blackmer, J.M. 2010. Strategies for prevention of neonatal isoerythrolysis in horses and mules. Equine Vet. Educ. 15, 6–10. 
  10. Wardrop KJ. 2005. The Coombs’ test in veterinary medicine: past, present, future. Vet Clin Pathol. 34(4):325-34. doi: 10.1111/j.1939-165x.2005.tb00057.x.
  11. Becht JL, Page EH, Morter RL, Boon GD, Thacker HL. 1983. Evaluation of a series of testing procedures to predict neonatal isoerythrolysis in the foal. Cornell Vet. 73(4):390-402. 
  12. Silvestre-Ferreira AC, Pastor J. 2010. Feline neonatal isoerythrolysis and the importance of feline blood types. Vet Med Int. 2010:753726. doi: 10.4061/2010/753726. 
  13. Silvestre-Ferreira A, Pastor J. 2021. Wild Felids Blood Group System. Animals (Basel). 11(12):3533. doi: 10.3390/ani11123533.
  14. Giger U, Kilrain CG, Filippich LJ, Bell K. 1989. Frequencies of feline blood groups in the United States. J Am Vet Med Assoc. 195(9):1230-2. 
  15. Stormont C. 1975. Neonatal isoerythrolysis in domestic animals: a comparative review. Adv Vet Sci Comp Med. 19:23-45.
  16. Cohn LA, Kaplan-Zattler AJ, Lee JA. 2022. Fluid Therapy for Pediatric Patients. Vet Clin North Am Small Anim Pract. 52(3):707-718. doi: 10.1016/j.cvsm.2022.01.007. 
  17. Stormont, C. 1977. The Etiology of Bovine Neonatal Isoerythrolysis. The Bovine Practitioner. 22–28. https://doi.org/10.21423/bovine-vol1977no12p22-28
  18. Vossoughi S, Spitalnik SL. 2019. Conquering erythroblastosis fetalis: 50 years of RhIG. Transfusion. 59(7):2195-2196. doi: 10.1111/trf.15307. 
  19. Webb J, Delaney M. 2018. Red Blood Cell Alloimmunization in the Pregnant Patient. Transfus Med Rev. 32(4):213-219. doi: 10.1016/j.tmrv.2018.07.002. 

Interview: Exploring the Impact of Biology Courses on Student Understanding of Biological Diversity

By Adyasha Padhi, Biochemistry & Molecular Biology and Sociocultural Anthropology ’25

Introduction: 

Hannah Higuera, a Ph.D. student in the Department of Evolution & Ecology, and Dr. Laci Gerhart, an Assistant Professor of Teaching in the Department of Evolution & Ecology, have been working together to understand how college students think about animal diversity. The first biology course I took here at UC Davis was BIS 2B with Dr. Gerhart and Hannah and it was my experience in that class that inspired me to look deeper into our perceptions of animals, specifically as it relates to conservation. I sat down with them to discuss their thoughts on the topic and hear more about their research. 

Note: This interview has been edited slightly for clarity and cohesion but care was taken to preserve the interviewee’s original statements.

Adyasha Padhi: Could you introduce yourselves? 

Hannah Higuera: Hi, my name is Hannah Higuera. I am a Ph.D. student in the Evolution & Ecology department [at the University of California, Davis] and really excited to be working on this project with Laci. So both of us have been teaching BIS 2B, her as an instructor and me as a Teacher’s Assistant. I’ve been teaching BIS 2B for the last several years and I’ve noticed that students have certain misconceptions about animals and animal diversity. So the idea from this project came out of trying to better understand how college students think about animals because there’s a lot of literature on how younger students, like K-12 populations think about animals, but not so much about how college students or biology majors think about animals.

Laci Gerhart: I’m Laci Gerhart. I’m an Assistant Professor of teaching in Evolution & Ecology. So Hannah and I got together on this project because she was proposing doing this as part of her dissertation. So we talked about various ideas and then I was brought in on her committee to advise on this project specifically. So there are a couple of prongs to this project. The one that’s furthest along is looking at some of the instructional materials and how biased they are, kind of as a guide to inform what might be driving some of the biases. Then the biggest dataset is the survey project where we send surveys out to all of the students in the Introductory Biology series (BIS 002) and a couple of more specific elective courses to get a sense of students’ perceptions on this as they enter a class. And then if and how different types of classes shift the needle and how that happens across a series of courses. One of the cool pieces of this was because we did it through one whole academic year, there were a small number of students that we caught across the entire Bis 2 series. So we could look at how their perspective has progressed long-term across the entire introductory series.

AP: What initially got you into this research? What has the research shown about younger students’ perspectives on animal diversity and how can that impact more work down the line?

HH: I think a lot of my interest in this research came out of recognizing my own biases and how those have changed over time. So I definitely grew up thinking that most animals were furry and four-legged, especially coming from the Midwest. Like I didn’t encounter marine organisms unless on vacation. So to me, that was an animal and it wasn’t until I took invertebrate zoology when I was in college that suddenly I realized, the vast majority of animals are these spineless, weird, slimy things, not the furry four-legged creatures that I saw everywhere on TV and in books. And so for me, it came out of going through that transition myself and wondering how often students here also go through that transition and what causes them to come to a more expert level of thinking about animals. 

LG: I was interested in this from a really different perspective. I mostly teach BIS 2B and that class is so huge that I don’t feel like I get to have a lot of one-on-one conversations with students about their perspectives and how they engage with the material. I do a little bit during office hours, but that’s a really small number of students. So throughout the time that I’ve been teaching here, I’ve been involved in a variety of survey projects that get at student perspectives of different things. And I find those really valuable as an instructor because it gives me a much better window into the diversity of perspectives in the class. Is there one perspective that falls out, or are we seeing a lot of variety? And it gives me a way to get to know the student’s perspectives a little bit better. So that’s part of what I was excited about with this, was getting an idea about how my students are thinking about this when they come into the class and is my instruction of this course changing their thinking at all. Is the introductory series broadly changing it at all? The other classes we included are later in their degree or for non-majors so are we able to see any sort of shifting in these perspectives or not? 

AP: Can you talk a little bit more about how you designed each part of the study and what went into that process? 

HH: A lot of iteration. We had a pilot survey, where we wrote a first draft and then tried it out in a class. And while those are not results we are ever going to publish or share publicly, they were informative in terms of how long does this survey take? Are we getting the responses we expect or not? That helped us develop the final survey. And then since we’re studying human populations, there’s a lot of special permissions and processes you have to go through. Still, it is much different than working with invertebrates, which is all my previous work. 

LG: In basically any sort of research where humans are the subject, every university and some non-university organizations will have to review your protocol and make sure that it’s not going to harm anyone. There’s a lot of review on making sure that we’re doing that in an ethical, appropriate way. And so there were early steps on getting the survey approved through the IRB (Institutional Review Board). And what sorts of things can we ask the students and what does the consent language need to look like to make sure we’re being transparent with the study design. And so that process is very iterative as well. You submit something to the IRB office, they review it, they let you know if things need to be changed. And then once you get through all of those, then you have the approval to do the project. 

AP: Could you talk about how the survey is structured? 

HH: We paid attention to a lot to previous studies and what kinds of questions they were asking. Because ideally, you don’t want your study to be in isolation. You want to be able to compare your results to previous work. So we did try to ask the same types of questions, if not the same questions, that previous work had done so that we could compare. The survey had three main parts. The first part was asking students and experts to just name five animals. And we sorted those animals into their taxonomic groups. So like if you said dog, that’s a chordate. If you said jellyfish, that’s a cnidarian. So we could figure out, do all five things they’re naming belong to a single phylum? Or is it a diverse representation spanning the tree of life? And that kind of question has been asked on previous studies. The second part of the survey was trying to get at how students think about animals in terms of their characteristics and how often animals belong to certain groups. So this section was a series of sliders. So we asked, what percentage of described animal species do you think have hair or fur? And the real answer is quite small. We could compare with all these questions how far the survey participant was from what we think of as the real or correct answer. Then we also asked specifically, what percentage of described animals do you think are insects or mammals? We spent a lot of time over designing those sliders and how they should be worded. And part of the reason we asked questions such as “How common do you think this trait is?” and “How common do you think this taxonomic group is?” is so that we could tease apart if we were getting responses that were overestimating those. Is it because the person thinks that most species are mammals? Or is it that they think this trait is true of more groups than just mammals? Asking the same question in slightly different ways allows us to tease apart how the person who’s responding to the survey is thinking about these things. 

AP: What have you been seeing in the results of the survey and other parts of your research so far? 

HH: Biases and misconceptions about animals are really prevalent. Not just in student, and college student populations, but even in the experts, we saw many of the same biases where they would basically consistently overestimate the abundance of mammals and underestimate how many invertebrate species are out there. I think one of the most surprising results was the third part of the survey, which was sorting pictures into two categories: animals or non-animals. And we found that after taking biology classes, students definitely broadened their idea of what animals were. But often they broadened it a little bit too far and started including things like bacteria or carnivorous plants, even though they’re not animals. Overall, we had over 2,000 undergraduate UC Davis students take the survey across three quarters (Fall ’21-Spring ’22) and 246 professional biologists (faculty, postdoctoral scholars, graduate students, and staff who research biology at UC Davis).

LG: Another aspect that was interesting was in a couple of places, we included a question on the survey that asked how confident the person was in their answer. We could get a sense of if you’re just guessing but maybe after you have taken a class, you’re a little more certain. And we actually saw that how well someone did on the survey in terms of getting close to the correct answers, was actually negatively correlated with their confidence. And I don’t know if you’re familiar with the Dunning-Kruger effect. Basically, it’s like a psychological process by which when you learn a little bit about something you think you know way more than you do. But then as you keep learning, you realize how little you know about that thing, and your confidence shifts accordingly. We interpret that reduction in confidence as these classes are helping students realize how much more diverse the animal kingdom is than they thought when they first came in. So even though they’re doing better on the survey, they now know how much they don’t know. Their confidence is actually going down a little bit, which sounds a little bit sad as a result. But actually, a really important part of the learning process is realizing the scope of what you do and don’t know. And so that was a really exciting result, even though it sounds a little sad. 

AP: What are the potential impacts of this bias of which organisms are considered “animals?” How could this bias affect the field of biology as a whole and the ways that we interact with animals in our lives, such as through conservation? 

LG: Well, one of the parts that Hannah did a lot of research on when we were first doing this was how these sorts of biases can manifest in other things like conservation. How this assumption that most organisms are one group then drives funding for research or interest in imperiled species, right? There is formal literature on how non-charismatic groups don’t get as much attention for conservation, or beyond the animal community, fungi and bacteria are hard to get support for because they’re not things that we generally identify with. While our work in this project is focused mostly on just the magnitude of this misconception, there are a lot of downstream effects about how biases like this can play out in terms of decision-making that is really impactful.

AP: Could you explain how you analyze the survey results? 

HH: The survey was conducted in Qualtrics, a website for distributing surveys. We did most of the analysis using R, which is a statistical programming language. Now, we did have some conversations about how to score the survey in terms of correct and incorrect. For some of those, that was pretty straightforward, right? How far off the estimate of the number of species was is easy to quantify. And then we did some scoring around the number of correct or incorrect sorting in the photos. How many of the photos did you sort correctly or not? And then the opening question we scored based on how many different phyla you were doing. And so collating all those together in a way that then ranked the three parts of the survey roughly equally and scored. We had some conversations around how to try to do that to get an idea of if students were improving when they took the survey multiple times. That part got a little bit interesting in terms of how to quantify each of those steps. 

AP: Is there anything else that you want to talk about or that you think the wider student population should know, especially those who might not have taken the survey?

LG: I’m curious how students feel about being studied in their classes by their faculty. I don’t know what their perspective is on that. I hope that they find it an interesting way to reflect on their own perspectives and a way to have a dialogue with faculty on such a large campus and in a large class setting. I could also see students not really liking the feeling that they’re being studied. And so I don’t actually have a good sense of how students feel about these sorts of projects being done in the classes. And I’d be curious about that perspective. Something I would want to add is that the study is really meant to be sort of a baseline. So the next step would be testing different interventions to see which one is the most effective. We got some responses actually from some of the experts who e-mailed us after taking the survey saying they felt ashamed that they didn’t know the answers. And definitely, that was not our intention! And I think it’s really easy to feel like everyone should know this, but we cannot emphasize enough that these kinds of biases are so pervasive, not just among students but among experts. And you can look everywhere from zoo collections to textbooks to what research gets published in journals. There’s a strong mammal and vertebrae bias everywhere. So it’s not a matter of anyone being prepared or not prepared. We’re kind of equally in the same boat. 

The Impact of COVID-19 Lockdowns on the Progression of Macular Degeneration

By Jessie Lei, Neurobiology, Physiology, & Behavior and minor in Human Development, ’24

Author’s Note: Every person and every facet of life was uniquely impacted by the effects of the COVID-19 lockdowns, yet how deep this influence runs can be unclear. Through the eyes of my grandfather, I witnessed first-hand just how detrimental the pandemic was on the progression of his chronic retinal eye disease. In an effort to learn more about current findings on the collateral damage from lockdowns on macular degeneration patients, this review seeks to synthesize relevant research on this growing topic. The hope is that readers come to recognize the real-time irreversible effects that are occurring in patients because of a temporary event and make connections with this information in any way to bring overall awareness.

ABSTRACT 

Purpose of Review: Neovascular age-related macular degeneration (nAMD) is an unrelenting disease that leads to complete loss of vision in the global elderly population. Though there have been recent advances in treatment, namely anti-VEGF (vascular endothelial growth factor) injections that slow the progression, its success is contingent on repeated administration. These injections slow the secretion of VEGF by retinal cells to prevent the growth of abnormal blood vessels in the retina. When COVID-19 lockdowns were enforced in 2020, nAMD patients were forced to stop their essential ophthalmic visits. The purpose of this review is to synthesize the results from 5 retrospective studies, which explored the long-term effects nAMD patients were subjected to due to the termination of their eye care during the COVID pandemic.

Main Findings: Severe declines in visual acuity were observed from 2020 to 2021, implying that the visual ability for nAMD patients was declining. This was observed even after treatment visits were resumed after lockdown. An additional set of parameters was used to measure the effects on the structural anatomy of the eye, but results were inconclusive due to contradicting data presented from different studies. For example, the damage inflicted on the macula from abnormal fluid accumulation was reversible after anti-VEGF injections resumed, while in other studies, it was determined that these structural parameters only continued to worsen over time. 

Keywords: macular degeneration, neovascular, injections, COVID-19, delay of care 

INTRODUCTION 

Globally, one of the primary causes of irreversible blindness in older generations is age-related macular degeneration (AMD) [1].  This progressive disease is characterized by a gradual loss of one’s central vision due to the degradation of the macula, an area located in the lining of the back of the eye. It is categorized into two clinical forms, dry and wet, with the latter being responsible for severe central vision loss in 90% of AMD cases [2]. Although the pathogenesis of neovascular, or wet, AMD (nAMD) is still unknown, it is characterized by the proliferation of abnormal blood vessels in the retina. These blood vessels are recruited due to the secretion of the vascular endothelial growth factor (VEGF) by retinal cells. Consequently, the atypical vessels will leak fluid and damage the dense population of color photoreceptors within the macula, causing distorted vision. To track the progression of nAMD, current research supports utilizing best-corrected visual acuity (BCVA) measurements and the analysis of optical coherence tomography (OCT) images [3]. BCVA helps assess the functional health of one’s vision and is measured by having patients read letters of decreasing size from a set distance. OCT scans are best simplified as cross-sectional images of the layers in the retina that help visualize a number of parameters indicative of the eye’s structural integrity. 

Treatment Options 

Despite nAMD’s prevalence in the aging population, only recently has an effective treatment been developed that helps stabilize vision by preventing damage from blood vessel growth [4].  Prior to this discovery, photodynamic, or light therapy was the main modality of care where light-activated medicine was stimulated by lasers to seal off abnormal blood vessels. But, it had many complications and only focused on reducing the likelihood of further vision loss. Currently, anti-VEGF drugs are the treatment of choice by ophthalmologists because the intravitreal injections into the eye have fewer adverse effects and outperform previous treatments by partially restoring vision [2]. The mechanism of action is through the inhibition of the VEGF pathway expression, which produces the main glycoprotein that causes leaky blood vessels [4]. However, the caveat is that the drug must be continuously administered every few weeks for a variable amount of time (usually 12 weeks) to take effect. This is the only treatment regimen that has seen significant improvements in patients’ control over nAMD progression [5]. 

COVID-19 and Macular Degeneration 

When the COVID-19 pandemic brought global lockdowns in March of 2020, this treatment plan grew difficult to follow because hospitals were placed under strict public health measures. nAMD patients experienced an unplanned discontinuation of injections since virtual ophthalmic visits were unfeasible for this type of care. Adherence to regular treatment visits dropped to as low as 46% during lockdowns [6],  and a recent systematic review by Im et al. illustrates how compared to other common retinal eye disorders, nAMD patients were the most susceptible to deterioration of the eye after treatment suspension [7]. Therefore, this literature review seeks to understand how the delay of injection care impacted long-term disease progression after COVID-19 lockdowns in 2020 for patients diagnosed with nAMD that were already receiving treatment. 

RESULTS 

Decline of Visual Acuity (VA) 

​​As one of the key determinants in nAMD patients’ overall functional capabilities, BCVA is heavily prioritized by researchers. It provides an accurate representation of how sharp their vision is and tracks changes that occur over time. The study conducted by Stattin et al. assessed how VA was compromised in 98 nAMD patients one year after a 9-week treatment deferral due to COVID-19 restrictions [8]. All patients had ophthalmic exams and injections every few weeks after restrictions were lifted where additional data on BCVA changes was collected. By using 95% confidence intervals with p-values of <0.0001 and linear regression models, the analysis revealed a loss of 4.1 ± 8.1 letters in 1 year after lockdown, which was compared against a pre-lockdown average of 2.9 ± 12.7 letters loss in 3 years [8]. Given the context of using small p-values in the analysis, the results yield large ranges but the significant difference in letter loss still illustrates a profound decline in visual acuity. In essence, the typical BCVA decline that would have been observed in nAMD patients over the course of 5 years was witnessed within 1 year due to unintended treatment breaks. Furthermore, because the delay in care expedites the rate of neovascular AMD in patients, anti-VEGF injections offer limited help in slowing that progression down. 

Along a similar line, Rego Lorca et al. also demonstrated that 12 months of routine visits were unable to mitigate the immediate VA consequences that stemmed from suspension of treatment in 242 nAMD patients from Spain [9]. Additionally, a statistical model was developed for the sample population to demonstrate what visual loss as part of natural disease progression looked like. This serves as a point of comparison to help identify what percentage of the results could be attributed to injection delay. It was found that an average of 7.2 letters were lost in patients’ BCVA over the duration of COVID-19 lockdowns and the 12 months of resumed follow-up care [9]. Based on the approximated natural rate of vision loss calculation, a 2.5 letter deficit can be credited, thus leaving an average of 4.7 letters lost over the aforementioned time period [9]. This means that capacity wise, nAMD patients experienced a functional decrease in visual clarity by 4.7 letters that can not be explained as part of inevitable nAMD progression. Another way that Rego Lorca et al. presented their findings is that pre-pandemic, 1.6 letters lost/year was expected; but post-pandemic, even with treatments restarted, an average of 3.1 letters lost/year was observed [9]. All of these figures point to a continuing theme that sudden drops in BCVA scores is the direct product of treatment discontinuation from the pandemic. Sevik et al. further supported this pattern in a paper that directly compared the outcomes of two different groups – those that delayed their appointments by an average of 3 months during the pandemic (group 1) and those that remained consistent (group 2) [10]. Each group consisted of 30 to 50 nAMD patients from Istanbul and were evaluated periodically for 6 months after lockdowns. It was discovered that with normal injections and follow-up post-lockdown, BCVA remained stable in group 1 while group 2 continued to suffer from statistically significant decreases that were not regained within 6 months [10]. With other possible confounding variables accounted for during the inclusion and exclusion of patient selection in this retrospective study, the only variable that can be attributed for the difference in functional outcomes is the postponement in treatment care. 

Variable Impacts on Structural Eye Health 

In contrast to the prior parametric analysis, there is not one single measurement that can appropriately represent the complexities of the eye as a whole and determine its structural health. Instead, the majority of researchers will examine a variety of anatomical characteristics that can be seen on OCT images. For Kim et al., they chose to study the maximum subretinal fluid (SRF) height, which conveys how much abnormal fluid has accumulated under the retina, as the main indicator of disease activity in 57 Korean nAMD patients [11]. They found that SRF height initially improved but by the 6-month check-up, had significantly worsened compared to baseline. At the end of the study, SRF height increased by 37 μm, which was about 18.9% higher than the recorded baseline value prior to COVID-19 lockdowns [11]. Because of the unique methodology employed where follow-ups occurred in 2-month intervals, researchers were able to catch nuanced fluctuations in progress results, like the temporary morphological improvements in SRF height following the return to treatment regimen. But that quickly deteriorated by the 6-month mark, emphasizing the rapid and unpredictable effects that delayed injections could have on disease activity. 

Interestingly, the article by Rego Lorca et al. completely contradicts previous conclusions from Kim et al. [11] by exhibiting that their sample population was able to anatomically recover to pre-pandemic state with 12 months of continuous treatment [9]. Active disease was defined as showing evidence of SRF and macular neovascularization (MNV), which is the proliferation of atypical blood vessels on the retina, on OCT images. Prior to the lockdown, SRF was present in 25.7% of the sample population, which increased to 28.2% during lockdowns, and decreased to 14.7% after 12 months of routine check-ups. Similarly, the percentage of MNV within nAMD participants before lockdown was 65.3%, increased to 79.6% during lockdowns, and decreased to 51% after [9]. Thus, the structural deterioration of the eye was demonstrated to be fully reversed and improved across two separate parameters in this specific paper. 

Rozon et al. brings in a plausible explanation for the previous discrepancies in their research which assesses the influence that delayed follow-ups had on 351 nAMD patients in Canada [12]. SRF and central foveal thickness (CFT) parameters on OCT images were used to define the structural integrity of the eye. CFT refers to how far off the thickness of the retinal center is from the normal range, because the thicker it is, the higher the likelihood of unwanted fluid accumulation within the retina. After 6 months of rigorous injections, the delay in follow-ups was linked to significant worsening of SRF parameters, while CFT measurements were normalized within the same time frame. SRF increased from 12.4% to 19.1% by 6 months; CFT levels began at 256.1 μm and fluctuated throughout the 6 months, but eventually ended up at 255.7 μm [12]. This provides further clarification on anatomical impacts because multiple different characteristics on OCT images were considered. It illustrated how some features may

improve over the 6-month period while others remain at a deficit, therefore depending on the parameter chosen by researchers, it will yield different data. 

DISCUSSION 

Analysis of Findings 

Amongst these reviewed articles, the combination of statistics highlight the persistent decline in visual loss that appears to be irreversible since pre-pandemic functional capabilities were not recovered in any nAMD patients [8-10]. Even after undergoing intense care following the interruption of visits, visual acuity continues to decline at alarming rates that current treatments cannot seem to dampen. On the other hand, no definite structural outcome could be confidently concluded because the data of each paper do not fully corroborate with one another [9, 11-12].  Some depict the possibility of restoring any anatomical damage sustained during the delay [10], while others express the worsening of structural parameters in the long-term [11-12]. Therefore, it is evident that additional research which explores a multitude of OCT features is needed before a conclusion on the reversibility of damage on the structural integrity of the eye can be made. However, a contradicting paradigm starts to form as there is a high possibility that the functional and structural impacts are complete opposites, which may imply a more complex issue. 

Strengths and Limitations 

Limitations of this review and its sources may help explain why this contradicting paradigm emerged from the findings. One of the biggest weaknesses is that the majority of the studies employed similar experimental designs that were retrospective in nature. Since it would be unethical to create a scenario in which treatment is purposefully delayed, this limitation is inevitable but should still be addressed because retrospective papers tend to supply a degree of selective bias during patient selection. Furthermore, in the article by Rozon et al.,  additional focus on risk factors and minimal acknowledgement of the COVID-19 pandemic’s contribution to the delayed follow-ups act as notable limitations to the relevancy of this source [12]. However, it still provided invaluable insight into why inconsistencies associated with using OCT measurements as a dependent variable occur that other researchers did not identify. Despite these disadvantages, there are still many notable strengths that this review has to offer. Researchers were able to eliminate possible confounding variables and establish a cause-effect relationship by including control groups [10], and statistical models of natural nAMD history [8-9]. Additionally, there is a wide diversity of patients from different countries represented, ensuring that the results of this review can be extrapolated and applied to nAMD patients on a global scale. 

Application of Findings 

As for implications, it is clear that nAMD injection treatments should be given priority in the event of another lockdown because consistency is necessary for positive effects. Patients and health professionals should maintain strict care regimens to ensure that further regression is not seen. It is recommended that research still be conducted in this topic, especially because there is potential to examine what effects from this lockdown period remain 5 or 10 years into the future and provide more evidence on anatomical impacts.

CONCLUSION 

When thoroughly considering all five core sources, there is a distinguishable response to the primary research question about the long-term deleterious effects on functional and structural eye health that were felt by nAMD patients after COVID-19 lockdowns caused unexpected gaps in treatment. Specific themes about the profound negative impacts had on visual acuity and the uncertainty regarding ramifications on the morphological health of the eye were uncovered. To limit the collateral effect of this pandemic on patients’ ophthalmic health, healthcare providers should proactively check in on nAMD patients. They should also place emphasis on performing additional research to better understand the long-term effects of lockdowns and if morphological damage sustained in the eye can be fully restored. 

The author declares no conflict of interest.

REFERENCES (FULL)

  1.   Learn about age-related macular degeneration. Centers for Disease Control and Prevention. Published November 23, 2020. Accessed March 14, 2023. https://www.cdc.gov/visionhealth/resources/features/macular-degeneration.html
  2. Flores R, Carneiro Â, Vieira M, Tenreiro S, Seabra MC. Age-Related Macular Degeneration: Pathophysiology, Management, and Future Perspectives. Ophthalmologica. 2021;244(6):495-511. doi:10.1159/000517520
  3. Ho AC, Albini TA, Brown DM, Boyer DS, Regillo CD, Heier JS. The Potential Importance of Detection of Neovascular Age-Related Macular Degeneration When Visual Acuity Is Relatively Good. JAMA Ophthalmol. 2017;135(3):268–273. doi:10.1001/jamaophthalmol.2016.5314
  4. Song D, Liu P, Shang K, Ma YB. Application and mechanism of anti-VEGF drugs in age-related macular degeneration. Front Bioeng Biotechnol. 2022;10. doi:10.3389/fbioe.2022.943915
  5. Augsburger M, Sarra G-M, Imesch P. Treat and extend versus pro re nata regimens of ranibizumab and aflibercept in neovascular age-related macular degeneration: A comparative study. Graefes Arch ClinExp Ophthalmol. 2019;257(9):1889-1895. doi:10.1007/s00417-019-04404-0
  6. Rothaus K, Kintzinger K, Heimes-Bussmann B, Faatz H, Lommatzsch AP. Impact of the COVID 19 Pandemic on Treatment of nAMD via a Portal-Based Collaboration. Klin Monbl Augenheilkd. 2022;10.1055/a-1806-2474. doi:10.1055/a-1806-2474
  7. Im JHB, Jin Y-P, Chow R, Dharia RS, Yan P. Delayed anti-VEGF injections during the COVID-19 pandemic and changes in visual acuity in patients with three common retinal diseases: A systematic review and meta-analysis. Surv Ophthalmol. 2022;67(6):1593-1602. doi:10.1016/j.survophthal.2022.08.002
  8. Stattin, M., Ahmed, D., Graf, A. et al. The Effect of Treatment Discontinuation During the COVID-19 Pandemic on Visual Acuity in Exudative Neovascular Age-Related Macular Degeneration: 1-Year Results. Ophthalmol Ther. 2021;10, 935–945. doi:10.1007/s40123-021-00381-y
  9. Rego-Lorca D, Valverde-Megías A, Fernández-Vigo JI, et al. Long-term consequences of covid-19 lockdown in neovascular AMD patients in Spain: Structural and functional outcomes after 1 year of standard follow-up and treatment. J Clin Med. 2022;11(17):5063. doi:10.3390/jcm11175063
  10. Sevik, M.O., Aykut, A., Özkan, G. et al. The effect of COVID-19 pandemic restrictions on neovascular AMD patients treated with treat-and-extend protocol. Int Ophthalmol. 2021;41, 2951–296. doi:10.1007/s10792-021-01854-6
  11. Kim J-G, Kim YC, Kang KT. Impact of delayed intravitreal anti-vascular endothelial growth factor (VEGF) therapy due to the coronavirus disease pandemic on the prognosis of patients with neovascular age-related macular degeneration. J Clin Med. 2022;11(9):2321. doi:10.3390/jcm1109232
  12. Rozon J, Hébert M, Laverdière C, et al.. Delayed Follow-Up in Patients with Neovascular Age-Related Macular Degeneration Treated Under Universal Health Coverage. Retina. 2022; 42 (9): 1693-1701. doi:10.1097/IAE.0000000000003512

Olive Oil Harvesting

By J Capone, Agriculture and Environmental Education, ’24

It was a warm Saturday morning in November when Sam rounded the corner and asked if I wanted to join the harvest. He looked like a laureate in the light, with a crown of olive branches placed upon his wide-brimmed field hat. We were taking Intro to Sustainable Agriculture together, and I had heard how he wanted to do a community olive harvest this weekend at the Student Farm, a 23 acre agriculture program at UC Davis. I was next door when I came across him and his friends talking around a field bin, strategizing the day forthcoming. Later, I would join him in the orchard, amazed at the crowd of people collected, the energy exuding from them as joyous as a traditional field harvest. 

“It’s crazy,” he told me “that they were just going to let the olives rot on the tree instead of doing something with them”. 

He had learned through a friend that the olive grove at the Student Farm would not be harvested this season. There was simply not enough manpower. Within a week, he had organized a team effort and convinced the head of the Student Farm to let him and his friends hand-pick as many olives as they could and bring them to a community milling project outside of Livermore, CA. One had a truck, another brought music and blankets, and yet another brought more friends until this manual labor event seemed more like a block party, with clean, empty, dark wine bottles collected to later fill up with the products of their harvest. 

Sam quickly gave me the safety rundown – and Tim gave me the waiver to sign, pinky promising to not sue if my head gets chopped off in the process. The ground under the grove was littered with ankle-splintering holes, dug by ground squirrels to nest close to a fallen food source. I carefully stepped my way to a tree, where a nice girl introduced herself and showed me the strategy for harvesting the olives – taking a big stick, usually with a rake attached to the end of it, and smacking the hell out of the branches, pushing and pulling to entice the tree to release some of her swollen fruit. Together we took turns bashing, raking, holding, and scrubbing until the tarp under us was littered with ripe, firm, green olives. We each grabbed two ends and brought our bounty over to the sorting groups, who cheered as we deposited our load into piles. 

I sat down in one of the circles, and a neighbor showed me exactly how they picked which olives were good and could be pressed and milled, and which were bad and would spoil the batch. Crouched on my feet, I scooped up a handful and admired the pale farina clouding the surface of the fruit. Slowly, I turned it around in my hand, looking for pits or worms or the dreaded Olive Fruit Fly, before tossing it in the “Good” basket. Around me, the fellow harvesters who had been doing this for hours already would scoop up a handful while chatting, roll it around in their palms as they scanned, and quickly made their selection of good and bad fruits, all while singing, chatting, and joking around while the music played. Clearly, there was a rhythm I had to learn. 

In case you were ever interested in the selection process for these olives, here’s what you looked for: a uniform color on a solid, firm skin, no holes or lesions, and especially no scales or larvae. Olives naturally had small pits and divots, but the key difference was the indication of something burrowing into or out of the flesh and skin of the fruit.  The Olive Fruit Fly, an invasive pest that destroys the olive crop in this way, would spoil the batch if too many were detected. Their life cycle depends on laying their eggs in forgotten, fallen crops, and we had to be careful to properly dispose of rejected fruits lest we exacerbate the problem in next year’s harvest.

If we hadn’t collected these olives – even though we left many on the trees – the ground would be littered with rotting olives, a useful food source for pesky pests and other critters on the student farm. It would also be a habitat for insects, like the Olive Fruit Fly, and could bolster their population the following year, even spreading to and worsening the situation on neighboring farms. We made sure all of the soft, scarred, and unsavory olives made their way into the compost pile, so they could be repurposed as agricultural waste into food for next season’s crops. 

As I got into the groove of things, another neighbor made a joke about working under the warm sun. “I can feel my Greek ancestors frowning at me, going ‘We left for America so you wouldn’t be doing farmwork!’” The group laughed at his joke, but it had me thinking about my own connections to the olives, how this group of harvesters made me feel more connected to my ancestors than any other part of modern life. 

Instead, olive tree growers in the south Mediterranean have their own pest problems to worry about. A new virus, called xyzella fastidiosa, has infected thousands of olive trees in Italy and has ravaged orchards [3]. With no natural defense against the introduced pathogen, farmers are having to pull every trick out of their toolbox to protect their trees before it totally destroys the industry, or spreads to any neighboring countries. An already parched basin, Italy and other olive-producing countries have faced drought and intense heat as climate change spreads, altering the global weather patterns. Coupled with these pre-existing climate troubles with the desertification of the Mediterranean, the olive industry, which has flourished for thousands of years in the fertile valleys of southern Europe, is on the ropes. 

As the day stretched on, we all grew hungry and tired under the warm sun, standing up to stretch and daring each other to eat the tempting fruit we were sorting under our fingers. Unfortunately, uncured olives like the ones we were handling taste extremely bitter, and the offensive taste tends to linger in one’s mouth. By the time the sun started setting, we had almost filled the entirety of the truck bed up with gorgeous, colorful olives, ranging from candy apple green to a wine-dark skin. We had only harvested from 6 of the 28 trees in the grove, with not enough people or time to attack the entire orchard in the limited sunlight. Sam and Tim rounded up the last buckets, as everyone helped fold up tarps, put away supplies, and swapped photos taken of the harvest. The next day, Sam and Tim would drive down to the Olivina, an olive orchard and mill in Livermore, that participated in a once-yearly Community Milling Day, where we could get our olives milled for free. In order to make the freshest extra virgin olive oil, we had to get these picked olives milled as quickly as possible, within 24 hours. Promises were made to meet back up in a week, where a taste-testing party with bread would be provided at the Domes. 

At the end of the day, I brought my hands up to my face and inhaled deeply at the fresh, earthy smell of olives still dusting my hands, the farina settled and coating the crevices of my hands and nail beds. Within a week, I was meeting up with Sam and Tim again, and finally getting my own bottle of this liquid gold. A total of 470 lbs of olives were harvested, resulting in 10 gallons of oil to distribute. Gathered around the tasting table, I nabbed some bread and took a dunk in the oil. Instead of a mellow flavor most grocery store oils held, this tasted like a kick in the teeth, the peppery notes coating my mouth and tingling on the way down. Paired with bread and balsamic, or added into a focaccia recipe, this hand-harvested olive oil tasted like satisfaction and a hard day’s sweat under the sun, with the benefit of caring for the land and next year’s crop.