Biodegradation is currently the most eco-friendly approach to breaking down complex plastic into less harmful products. Luckily, a number of insects and microorganisms have the capability to digest plastic polymers, and several studies have shown that insect guts can biodegrade plastics faster than environmental microbes. To tackle the global—and mounting—plastic waste problem, researchers look to these critters in hopes of adapting their enzymatic capabilities into efficient systems that can degrade plastic waste at scale.

In a recent study published in the June issue of G3: Genes, Genomes, Genetics, Young et al. report an improved reference genome for the greater wax moth Galleria mellonella as a tool to identify enzymatic pathways with plastic biodegradation properties.

Well-known as a honeybee pest, greater wax moth larvae feed on beeswax, which contains long-chain hydrocarbons. Since long-chain hydrocarbons are also the major constituent in polyethylene (PE), researchers are quite interested in the enzymes responsible for beeswax degradation; in fact, the hexamerin and arylphorin proteins, found in larval saliva, have demonstrated PE-degrading abilities. Evidence suggesting wax moth larvae can degrade other plastics like polystyrene and polypropylene makes them attractive for plastic biodegradation research. The extent to which moth larvae possess plastic catabolizing enzymes is unclear; however, since both the larvae themselves and their gut microbiota have been implicated in PE biodegradation.

Since the existing reference genome for G. mellonella was fragmented, Young et al. combined short- and long-read sequencing approaches to generate a new assembly with improved continuity, identifying an additional 3,000 mRNA sequences. This new reference genome also supported phylogenetic comparisons with other Lepidoptera members such as moths, butterflies, and silkworms, allowing the authors to begin constructing an understanding of the evolutionary history of PE-degrading enzymes in winged insects.

Secreted proteins have a much better chance of playing a role in long-chain hydrocarbon degradation than intracellular proteins, so the authors investigated 3,865 proteins identified as secreted in their assembly, finding numerous hydrolases, transferases, oxidoreductases, ligases, lyases, and isomerases. They propose that these secretory enzymes, which may have evolved to catabolize a variety of exogenous and insoluble polymers, must also be capable of processing long-chain polymers like polyethylene. Several of the identified hydrolases and oxidoreductases are members of enzyme classes known to degrade plastic. They also found 135 hydrolases and 10 oxidoreductases that are predicted to act on ester bonds and peroxide, which may make them capable of breaking polyethylene. This genome is one of many sequenced by the Applied Genomics Initiative at the Commonwealth Scientific and Industrial Research Organisation in Australia. The initiative aims to sequence the genomes of a variety of organisms of interest to enable translational research in areas such as conservation, biosecurity, and health. The improved reference genome for the greater wax moth will continue to aid researchers in uncovering the molecular mechanisms behind its ability to degrade long-chain hydrocarbons; hopefully, these larvae can become a powerhouse for developing industrial and bioremediation applications in reducing plastic waste.

We’re taking time to get to know the members of the GSA’s Early Career Scientist Committees. Join us to learn more about our early career scientist advocates.

Rupinder Kaur
Career Development Subcommittee
Pennsylvania State University

Research Interest

I am a cell and molecular biologist interested in exploring host-symbiont interactions with relevance to human health outcomes. Mosquito-borne diseases, especially dengue, have become an emerging global threat to mankind. The existing vector control strategies—such as diminishing mosquito breeding sites, insecticide use, chemical spraying, and personal protective measures—have been found ineffective and do not confer long-term protection. Moreover, risks surrounding climate change have created an urgency for alternative vector control strategies. The prospect of using symbiotic microorganisms to save millions of lives with positive human health outcomes is highly promising. The bacterium Wolbachia is a prime example, which is human- and environment-friendly and can play a significant role in controlling dengue and other mosquito-borne viruses on the ground. Wolbachia expresses two key traits in these control strategies: virus-blocking, in which Wolbachia reduces virus replication in the salivary glands of virus-transmitting mosquito females, and reproductive manipulation called cytoplasmic incompatibility (CI), during which embryos die when Wolbachia-infected males mate with uninfected females, thus crashing the mosquito population.

In my research, I’m digging deeper into the mechanism of CI to better grasp how Wolbachia bacteria influence the genes and pathways governing insect reproduction. Using Drosophila melanogaster and Aedes aegypti carrying Wolbachia, I identified that CI-causing genes disrupt an evolutionary-conserved process of histone-to-protamine transition during sperm development. This transition is crucial for maintaining male fertility. When embryos are fertilized by these abnormally developed sperm, their nuclei fail to divide properly and embryos ultimately die. I am further keen on understanding the intricacies of the flip side of CI, known as “rescue,” where female insects infected with Wolbachia can prevent embryonic death. My goal is to enhance methods utilizing these bacteria to control mosquito populations, thereby making them even more effective and sustainable in the fight against diseases.

As a PhD-trained scientist, you have many career options. What interests you the most?

As someone who loves diving into the unknown to uncover new things, I find being a scientist incredibly rewarding. I enjoy brainstorming new ideas, formulating hypotheses, and troubleshooting experiments to bring them to life. Even though science can be tough and challenging at times, those moments when everything clicks and years of hard work culminate in a breakthrough are truly amazing. Each discovery feels like finding a missing piece of a puzzle. At that point, more than just a career option, it becomes a passion that keeps me curious and eager to share what I learn with others in the scientific community.

Moreover, I recently explored the intricacies of grant writing, a crucial skill for securing essential research funding. I learned that grant writing is not just about acquiring resources; it’s about articulating the potential impact of my work on the scientific community and society at large. I acquired the skill of translating my scientific vision into actionable proposals, ensuring that the future research direction is not only intellectually stimulating but also socially relevant. It bridges the gap between innovative ideas and transformative research outcomes, reinforcing my commitment to making a meaningful difference in the world of science.

In addition to your research, how do you want to advance the scientific enterprise?

Science advances significantly when diverse fields intersect, sparking new and creative ideas. In addition to my research pursuits, my vision for advancing the scientific enterprise is firmly grounded in the principles of collaboration, outreach, and mentorship. I work towards creating an environment where scientists from different backgrounds can come together to create ideas that address scientific challenges. I have shared my research through seminar presentations with several universities, companies, and scientific organizations in the United States. By facilitating dialogue and knowledge exchange, I assisted them in developing specific assays tailored to their research programs.

I am actively engaged in initiatives that expand the horizons of STEM education and promote inclusivity within the scientific community. For instance, as a judge in the ENVISION research competition, I play a pivotal role in evaluating the innovative project proposals generated by women and genderqueer high school students. I provide valuable feedback and recognition, foster their passion for scientific inquiry, and encourage them to pursue careers in STEM fields. Furthermore, I participate in mentoring initiatives aimed at bridging the opportunity gap for students from disadvantaged backgrounds. Volunteering my time and expertise, I create research opportunities for these aspiring scientists by guiding them through the research process, helping them understand scientific articles, and assisting with formulating hypotheses for scientific experiments. I not only provide essential scientific guidance but also instill confidence and inspire a greater sense of possibility. By empowering young minds, recognizing and nurturing their talent, dismantling barriers, and fostering inclusivity, I am dedicated to creating a scientific community that reflects the diversity and potential of our world.

As a leader within the Genetics Society of America, what do you hope to accomplish?

As a member of GSA’s Early Career Leadership Program, I am committed to advancing the career growth of fellow GSA members. One of my primary objectives within this role is to establish a robust mentorship network. I aim to provide guidance, insight, and support by connecting early-career scientists with experienced mentors in their respective fields. By organizing symposiums, networking events, panel discussions, and virtual forums at conferences, I aim to facilitate interdisciplinary collaborations and encourage sharing of ideas and expertise to open doors to new opportunities. This collaborative environment will not only enrich the scientific discourse within GSA but also expose early-career scientists to diverse research areas, promoting a spirit of curiosity and innovation.

Further, I intend to organize targeted professional development workshops and training sessions. These sessions will cover a wide array of topics, including grant writing, science communication, leadership skills, and work-life balance. By providing access to these resources, I hope to equip early-career scientists with the skills and knowledge necessary for a successful and fulfilling career in genetics. Last, in line with my commitment to diversity and inclusivity, I will advocate for programs that specifically support underrepresented individuals within the GSA community. I aim to level the playing field and ensure that everyone, regardless of their background, has equal access to resources and opportunities for career growth. Through these initiatives, I hope to empower the next generation of geneticists, leaving a lasting legacy of mentorship, support, and inclusivity.

Previous leadership experience

1. Editorial Board Member, mSystems, American Society for Microbiology (2024-2027)
2. Early-career editorial board member, mBio, American Society for Microbiology (ASM) (2024-present)
3. Panelist in the Science Communication panel, How to have an accessible conference experience, The Allied Genetics Conference (2024)
4. Judge, Poster session at the One Health Microbiome Symposium, Penn State University, PA (2024)
5. Judge, Poster session at the Undergraduate Exhibition, Penn State University, PA (2024)
6. Elected member in ASM’s Future Leaders Mentoring Fellowship program (2023-present)
7. Member, Early Career Leadership Program, Genetics Society of America (2023-present)
8. Judge, ENVISION research competition for high school girls and genderqueer students (2022-present)
9. Mentor, Summer research program by Talaria Summer Institute, founded by the nonprofit organization ATHENA (2022-present)
10. Organized and moderated the virtual Career Exploration panel, the 64th Annual Drosophila Research Conference (2023)
11. Mentor to undergraduate and graduate students, technicians, and research staff in the lab
12. Active volunteer for national/international virtual and in-person science outreach programs

You can contact Rupinder via email at r.kaur at psu.edu, on Twitter, and on LinkedIn.

When female Anopheles mosquitoes take a blood meal from someone with malaria, a tiny Plasmodium parasite enters the mosquito’s digestive tract. That parasite can invade the mosquito’s salivary tissues, so when the insect takes another blood meal, the intruder can slip into the next human host and start a new malaria infection. Malaria is a life-threatening condition that infected 241 million people in 2020 and disproportionately affects vulnerable populations.

To combat the disease, researchers from the University of California, Irvine are developing genetically modified African malaria mosquitoes (Anopheles gambiae) that can’t transmit human malaria, alongside a gene-drive system that can quickly spread those genes and block the spread of the malaria parasite through the population. While this system usually operates with nearly 100 percent efficiency, a small number of mosquitoes will still wind up with mutant alleles that resist the gene drive. Could these mutant alleles sabotage the whole approach? In a paper published in GENETICS, Carballar-Lejarazú et al. looked at this phenomenon and found these mutations didn’t hamper the gene drive in their system.

Malaria-Resistant Mosquitoes

Contributing author Anthony James began exploring genetic methods for controlling vector-borne disease in the mid-1980s. Eventually he exploited mosquito genes that are only turned on in female mosquitoes after a blood meal and linked them with mouse antibodies that protect mice from human malaria parasites.

When these malaria-busting synthetic genes are inserted into mosquitoes, they can’t transmit malaria. And if it mates with a regular mosquito, the beneficial gene will be inherited like any other gene, gradually building up in the mosquito population. But what if that process could happen faster?

That’s when CRISPR gene editing technology hit the scene. “It seems like overnight when you work 10 or 15 years on something to make it work and then something new comes along and—in less than a year—you have it working,” says James.

Gene editing operates on the germline—the cells that will eventually become sperm or eggs—by snipping the normal chromosome and inserting the new sequence, in this case, the malaria fighting gene. In male mosquitoes, this works so well that each male passes on the new gene to nearly 100% of its offspring.

It’s a bit more complicated in female mosquitoes because egg cells are massive compared with sperm. When the system snips the normal chromosome and inserts the synthetic sequence, the second chromosome may be too far away to trigger the repair mechanism that sews the cut chromosome back up while including the system. That means there’s a chance the snipped chromosome will just stick itself back together—called nonhomologous end joining—possibly resulting in a mutant allele that resists the gene drive.

Exploring Gene Drive Mutations

To figure out if those mutant alleles could pose a problem for the gene drive system, the researchers linked the system to a somatic gene for eye color and marked it with a fluorescent protein. Then, the team performed various crosses to see how the genes passed on to future generations. Non-mutant progeny had black eyes (before adulthood) while those with the mutant allele had pink eyes. And all the progeny carrying the gene drive had eyes that fluoresced blue under light.

To make things a bit more complicated, some progeny were mosaics, with a mix of alleles and more complex eyes, but since the eye color gene isn’t part of the germline—it won’t pass on to the next generation—most of those mosaic mosquitoes still passed on the gene drive.

In the lab, about 25 percent of the first generation of progeny received the gene drive. By the fourth generation, the entire population had fluorescent blue eyes—meaning none of those mosquitoes could transmit malaria.

“Four generations is sufficient,” says James.  “That’s well short of one transmission season.”

James is quick to point out that this isn’t a magic bullet for malaria and there is more research to be done. There are also many thorny issues and debates for scientists and the broader community to work through before everyone is comfortable deploying gene drive mosquitoes in the wild. But James is hopeful the project to which he’s dedicated so many years may one day help ease the malaria crisis.

“There was a famous scientist who said a new idea doesn’t take hold because you change people’s minds; it takes hold because there’s a whole new generation of people that have grown up hearing about it,” says James. “I wish it was a little faster, but we’ll do our part, and hopefully people will take it up. It may not be me, but we have something to hand off.”

CITATION:

Cas9-mediated maternal effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae

Rebeca Carballar-Lejarazú, Taylor Tushar, Thai Binh Pham, Anthony A James

GENETICS

2022: iyac055

https://doi.org/10.1093/genetics/iyac055

Melissa Mayer is a freelance science writer based in Portland, Oregon.

Researchers used a forward genetic approach to identify genes that affect a social behavior in honey bees.

For more than 30 years, honey bee geneticist Robert E. Page, Jr. and his colleagues have sought the genes that influence a colony trait that only emerges from interactions between thousands of individual bees — a social phenotype.

Such traits are notoriously difficult to study. As hard as it is to disentangle a gene’s effect on behavior from environmental influences, these challenges are greatly multiplied by the complex interactions between genetically different individuals forming and altering their own social environments. As a result, mapping quantitative trait loci (QTLs) for social behavior long seemed out of reach. A recent paper published in GENETICS surveys the three decades of work that culminated in identifying genes that affect a complex, socially-regulated foraging behavior.

“Everything flowed from our observations of the behaviors that were associated with foraging behavior and division of labor and the resulting impact on the amount of pollen stored in the comb,” says Page, a researcher at Arizona State University. “Over time, we adapted new technologies to ask questions about those phenomena in different ways. By bringing together a host of different toolkits, we were able to build a story of the underlying genetic basis of a very complex social trait.”

Pollen hoarders

In a honey bee colony, some of the bees specialize in collecting pollen and hoarding this protein-rich food in wax cells near the “nursery” where eggs and larvae develop. Some of the bees eat the pollen and produce glandular secretions to feed the larvae; in turn, the larvae produce pheromones that stimulate foragers to collect pollen.

Despite the complexity of factors involved — thousands of individual bees, larvae, their interactions, and their environment — the total amount of pollen stored in the colony is a regulated trait. In a previous study, ASU reseacher Professor Jennifer established this by adding and removing pollen from honey bee colonies and observing the changes in foraging behavior. Each colony had a set level of stored pollen that the bees collectively sought to maintain; when researchers added pollen, foraging decreased until excess pollen had been consumed, and when researchers removed pollen, foraging increased until pollen again reached the colony’s standard level.

Using selective breeding, Page’s team generated strains of honey bees that substantially differ in amount of pollen stored within just three generations. “People have tried for decades to breed bees that store more honey, but honey storage is a very sloppily regulated trait,” says Page. “As long as nectar is available, bees will bring it back and stick it anywhere they can find space in the hive. In contrast, pollen storage offers excellent consistency of measurement.”

Phenotypic and genotypic analysis

Using a wide range of methods, Page and his colleagues studied the phenotypes and genotypes of these strains for 42 generations of selection. Phenotypic mapping revealed that bees in colonies that store more pollen are likely to have more ovarioles (the egg-producing structures in the insect ovary) and to be more sensitive to sugar than those from the colonies with less pollen. One research collaborator, Ying Wang, went so far as to painstakingly graft ovarioles from worker bees into recipients from a colony that produced less, which in turn affected the recipients’ behavior.

When they began to map genetic trait determination, the researchers expected that individual behavior would be more selectable than complex social traits playing out across large groups. Instead, they were surprised to find that genotype explained roughly 41 percent of colony variance in stored pollen but only two percent of individual variance in pollen collection.

Digging down to the genetic level, Page and his colleagues performed QTL mapping for the social phenotype of pollen hoarding along with individual foraging behavior, physiology, and anatomical traits. From the gene lists for each QTL they identified candidate genes of interest based on the phenotypic architecture and assessed them using expression assays and gene knockdown. Ultimately, they identified three genes of special interest, all of which have some association with ovary size. Future studies will involve examining the effects of these genes in developing larvae more closely.

A vertical approach

Page attributes his success in part to his tenacity in pursuing a “vertical approach”—interrogating a single question at every level of influence, starting with social interaction and working down to examine individual behavior, morphological differences, physiology, developmental processes, and genetic variation.

Pursuing this vertical approach over so many years involved the work of countless expert apiculturists, laboratory technicians, students, and fellow researchers alongside Page. “Our success came from combining the right people with the right tools as they became available,” he says. “Everyone who came through my lab made it better and brought something new.”

CITATION:
Societies to genes: can we get there from here?
Robert E Page, Jr.
GENETICS 2021; iyab104
https://doi.org/10.1093/genetics/iyab104

Termites may have evolved sterile castes through hormone signaling changes in their cockroach ancestors.

Eusocial insects, like ants, bees, and termites, have rigid caste systems: some individuals reproduce, while others are temporarily sterile workers or permanently sterile soldiers. This division of labor allows colonies to thrive, but the question remains:  how did these species evolve such drastically different developmental possibilities? In a report in GENETICS, Masuoka et al. looked into the genetics underlying caste differentiation by comparing termites with their close evolutionary cousins—cockroaches.

Both termites and wood-eating Cryptocercus cockroaches evolved from cockroach-like ancestors. However, while termites differentiate into castes, roaches don’t. By comparing how the two insects develop, the authors were able to glean insights into what drives specific caste differentiation in termites.

They found that both the termite species Zootermopsis nevadensis and its woodroach relative  Cryptocercus punctulatus are induced to molt by juvenile hormone (JH) or its analogs, suggesting that both termites and Cryptocercus share a JH-dependent molting system. In termites, JH specifically induces development toward the soldier caste, which is thought to be the earlier of the two sterile termite castes to evolve.

The authors then used RNA-mediated inhibition to knock down expression of the JH receptor or genes downstream of it. Knocking down the JH receptor inhibited molting in both species, and in termites, it prevented differentiation into the soldier caste. JH receptor knockdown in both species also repressed genes involved in the synthesis and signaling of 20-hydroxyecdysone (20E), a hormone crucial for insect molting.

JH knockdown affected different genes in the two species. HR39, a gene that is involved in 20E signaling, was dysregulated following JH receptor inhibition in termites but was unaffected by the same treatment in cockroaches. Knocking down HR39 in termite nymphs prevented differentiation into the soldier caste, suggesting that this gene is critical for soldier differentiation and that tweaks to 20E signaling may have been key in the evolution of complex termite societies from their cockroach forebears.

CITATION:

A crucial caste regulation gene detected by comparing termites and sister group cockroaches

Yudai Masuoka, Kouhei Toga, Christine A. Nalepa, Kiyoto Maekawa

A Crucial Caste Regulation Gene Detected by Comparing Termites and Sister Group Cockroaches

Photosensitive alternative splicing of a malt fly circadian clock gene varies between northern and southern populations.

Over the course of a day, most organisms undergo profound changes. Over the course of a season, the changes can be even more dramatic. For example, insects’ responses to the brisk nights and cooler days of fall and winter often involve transformations of both physiology and behavior, including reproduction, activity level, and metabolism. Whether in insects or humans, these daily and seasonal transitions are in part controlled by the internal circadian clock, with the expression of circadian genes responding to rhythmic environmental fluctuations.

Tapanainen et al. wondered how alternative splicing—a common gene regulatory mechanism—of the core circadian gene timeless might be linked to light and temperature in the fly Drosophila montana, a much more cold-tolerant relative of the familiar lab model D. melanogaster. They discovered that in D. montana, timeless splicing is regulated only by the amount of daily light exposure, not by temperature—in contrast to the thermal regulation seen in D. melanogaster. There was also no evidence that northern or southern D. montana had a particular timeless splice variant found in many cold-adapted D. melanogaster populations.

The group also made a peculiar observation: the way timeless splicing was regulated in D. montana differed depending on where the flies originated. For any given number of hours of light per day, if a splice variant was more abundant in flies from northern populations in North America and Europe, it was less abundant in flies from southern populations of the same continents, and vice versa. That the regulation of critical genes can be completely reversed even in two populations of the same species is a reminder of the importance of studying individuals from different populations and regions—perhaps especially in research on something as complex as the circadian clock.

CITATION:
Photosensitive Alternative Splicing of the Circadian Clock Gene timeless Is Population Specific in a Cold-Adapted Fly, Drosophila montana
Riikka Tapanainen, Darren J. Parker, Maaria Kankare
G3: Genes|Genomes|Genetics 2018 8: 1291-1297; https://doi.org/10.1534/g3.118.200050
http://www.g3journal.org/content/8/4/1291

Beer, doughnuts, and genetics textbooks have one thing in common: they were all made possible by collaborations between humans and yeast. Our fungal ally Saccharomyces cerevisiae resides not only in breweries, bakeries, and laboratories, but also sometimes in our own bodies—where, on rare occasions, it betrays us.

S. cerevisiae is increasingly being reported as an opportunistic human pathogen, sometimes even in non-immunocompromised people. Infections remain rare overall, but studying them presents an exceptional opportunity to understand how benign fungi can turn destructive. Such lethal transformations are more common in some other fungi, but those species are often difficult to grow and manipulate in the lab. In contrast, the long history of S. cerevisiae use in laboratory research means there are many powerful tools for working with the species. If baker’s yeast and other fungi become pathogenic through similar mechanisms, understanding S. cerevisiae infections could have broad implications.

With this in mind, Phadke et al. searched for traits that allow S. cerevisiae to invade a model host: the larval form of the greater wax moth Galleria mellonella. This moth is often used in studies of innate immunity, and because it doesn’t naturally cohabitate with S. cerevisiae, the yeast shouldn’t have any preexisting adaptations that would allow it to infect the moth. This means investigating how yeast infect it can inform researchers about fungal pathogenesis in general.

Comparing yeast strains derived from clinical samples to ones from environmental samples revealed that strains from both groups were equally likely to be pathogenic in the moths. Although this conflicts with findings in mice that clinically isolated strains are more likely to be pathogenic, the studies do concur that pathogenic strains are more likely to be able to form pseudohyphae—elongated strands of cells that remain joined after cell division.

The researchers used a library of yeast deletion mutants to screen for strains that grow better or worse in the moth larvae than in standard conditions. They noticed that one strain that struggled to infect the moths lacked the gene slt2, which encodes a protein that helps maintain part of the yeast’s cell wall. This part of the cell wall normally shields fungal molecules that the host could recognize. If these molecules were noticed by the moth immune system, it could cause a defensive response against the yeast, providing a possible explanation for the strain’s inability to infect the moths. This finding aligns with previous observations that cell wall integrity genes are important for other opportunistic fungal pathogens to infect hosts.

Several genes encoding mitochondrial proteins were also more important for yeast growing in the moth larvae than they were for cells growing in vitro, but since mitochondria have so many roles in the cell, it’s difficult to say how exactly these genes affect pathogenesis. The screen also turned up genes involved in metabolism of some aromatic compounds. In most cases, strains with one of these genes deleted grew better in the moths than they did in vitro, indicating that the genes aren’t important for pathogenesis—but two strains suffered dramatic fitness hits. Both strains were mutant for genes required for the synthesis of two aromatic amino acids, tyrosine and phenylalanine. In mice, S. cerevisiae infection is greatly attenuated if the yeast cannot synthesize aromatic amino acids, and infection in the moths may be similar in this regard.

This work asserts that some of the genetic underpinnings of pathogenesis are shared among S. cerevisiae and other fungal opportunistic pathogens and fuels hope for development of a broad-spectrum treatment targeting numerous fungal infections. It also demonstrates the power of our alliances with laboratory models—even when they go on the attack.

CITATION:

Phadke, S.; Maclean, C.; Zhao, S.; Mueller, E.; Michelotti, L.; Norman, K.; Kumar, A.; James, T. Genome-Wide Screen for Saccharomyces cerevisiae Genes Contributing to Opportunistic Pathogenicity in an Invertebrate Model Host.
G3, 8(1), 63-78.
DOI: 10.1534/g3.117.300245
http://www.g3journal.org/content/8/1/63

Susan A. Gerbi

The Genetics Society of America’s George W. Beadle Award honors individuals who have made outstanding contributions to the community of genetics researchers and who exemplify the qualities of its namesake. The 2017 recipient is Susan A. Gerbi, who has been a prominent leader and advocate for the scientific community.

In the course of her research on DNA replication, Gerbi helped develop the method of Replication Initiation Point (RIP) mapping to map replication origins to the nucleotide level, improving resolution by two orders of magnitude. RIP mapping also provides the basis for the now popular use of λ-exonuclease to enrich nascent DNA to map replication origins genome-wide. Gerbi’s second area of research on ribosomal RNA revealed a conserved core secondary structure, as well as conserved nucleotide elements (CNEs). Some CNEs are universally conserved, while other CNEs are conserved in all eukaryotes but not in archaea or bacteria, suggesting a eukaryotic function. Intriguingly the majority of the eukaryotic-specific CNEs line the tunnel of the large ribosomal subunit through which the nascent polypeptide exits.

Gerbi has promoted the fly Sciara coprophila as a model organism ever since she used its enormous polytene chromosomes to help develop the method of in situ hybridization during her PhD research in Joe Gall’s lab. The Gerbi lab maintains the Sciara International Stock Center and manages its future, actively spreading Sciara stocks to other labs. Gerbi has also served in many leadership roles, working on issues of science policy, women in science, scientific training, and career preparation.

An abridged version of this interview was published in the December 2017 issue of GENETICS.

How did you get involved with the March for Science?

As scientists, we can enjoy doing science because those of us fortunate enough to have research grants from the NIH and NSF receive them through tax dollars. So, we have an obligation to share with the public what our science is about. It’s important for scientists to learn how to speak to the public because the worst thing we can do is speak in such technical terms that their eyes will glaze over and they say, “this is why I didn’t want to study science in the first place!” Of course, this has always been true, but it seems especially true in the current era. There seems to be a disregard for science as a methodology. I was really spurred on by [GSA President] Lynn Cooley at the fly meeting, where she challenged me when she was presenting me with the Beadle Award. She mentioned that I had played a role in public policy through the American Society for Cell Biology [ASCB] and through FASEB [Federation of American Societies for Experimental Biology], as well as through the AAMC [American Association of Medical Colleges]. And then she said, “we need you now!”

I went home and I thought: yes, the field needs people to be actively involved in public policy at this particular time in history. So, with some difficulty, I found the local leaders for the March for Science in Rhode Island and then played an active role in mobilizing the Brown community. The underpinning of the March for Science was applauding the importance of science and the scientific approach. I enjoyed that experience and I really thank Lynn for her challenge to me, as well as her inspirational writings about how the community of geneticists really needs to be vocal.

Even though the March for Science itself was amazingly successful, it must go on beyond that. We need to speak to our congressional representatives, we need to speak to the general public, and to our neighbors about what we do, why it’s exciting, and why it’s important for advances in our society.

What inspired you to become a scientist?

Years ago, there was a study reported at the ASCB [American Society for Cell Biology] that found that many prominent women scientists have their fathers as their role model—that was certainly true for me. My father was a physician scientist. He grew up in Italy and went to medical school there, but he also was involved in renal hypertension research. During World War II he came to this country and was ultimately affiliated with Columbia University College of Physicians and Surgeons. When I was a youngster he would bring me to lectures at the New York Academy of Sciences, which was terribly exciting. I would be learning about things in high school biology and then would get to hear talks by the people making the discoveries. Holley spoke about the structure of tRNA, for example, and Palade about ribosomes, and Nirenberg about cracking the genetic code.

What drew you to studying chromosomes?

I became interested in chromosomes in high school after reading a Scientific American article by J. Herbert Taylor who had discovered that replication of chromosomes was semiconservative, which temporally paralleled the discovery by Matt Meselson at the DNA level. Then when I went to Barnard College I had the opportunity to take a molecular genetics course with Herb Taylor, and that confirmed my interest in chromosomes and replication. I knew I wanted to do a PhD on chromosomes, and one of the emerging leaders in the field at the time was Joe Gall. I was planning to apply to the University of Minnesota, where he was at the time, but I learned he was going to join the faculty at Yale, so I applied to Yale. The rest is history, as they say!

It was a fortuitous time to be in his lab because the method of molecular hybridization had just emerged from the work of Spiegelman, where radiative probes are hybridized to DNA captured on nitrocellulose filters. It was a no-brainer to try to expand that to the chromosome level. Joe Gall went to a meeting in South America where several scientists brainstormed about how they might best apply this method. They all went home to their labs and got hung up on the controls. But Gall, being a fabulous biologist, said he was going to use a system where he knew what the biological answer should be and then he would work things out from there.

He and my fellow grad student Mary-Lou Pardue worked out the initial method of in situ hybridization. They used the stage of meiosis in Xenopus oocytes where you find thousands of nucleoli, and everything in the field pointed to the fact they contained amplified genes for ribosomal RNA, and indeed that turned out to be the case. The next step was to apply the method to chromosomes themselves rather than amplified nucleoli, and I was part of that effort. We did the first in situ hybridization to chromosomes using the gigantic polytene chromosomes from the salivary glands of the lower dipteran Sciara, as well as Drosophila. Sciara polytene chromosomes are a bit larger than those of Drosophila because they have a few more rounds of endoduplication.

How did your interest in ribosomes begin?

The probe we used in the in situ hybridizations was ribosomal RNA labeled with tritiated uridine, and we used Xenopus rRNA because it was available from tissue culture cells. I wondered how Xenopus RNA could hybridize to fly chromosomes. I thought there must be some sequences that have been retained during evolution, and that started me on the long path of studying eukaryotic ribosomal RNA using evolution as a guide.

So, we reasoned that sequences with strong functional consequences should be evolutionarily conserved. We started with Xenopus rRNA because it was the first eukaryotic gene ever cloned. By hybridization we found there were regions of conservation even between bacteria and eukaryotes. Then we produced the first rRNA sequence from a metazoan. We modeled the secondary structure of rRNA using principles of compensatory base changes—where base-pairing in hairpin stem regions would be retained even if the sequence changes—and we found that there was a core structure that was conserved between Xenopus, yeast, and E. coli.

Our subsequent studies found that eukaryotic rRNA is larger because of insertions that were highly variable in sequence length and nature. We called them expansion segments, and initially people thought they were a remnant of evolution and didn’t have any function, but current studies by John Woolford making mutations in yeast and by other groups doing X-ray crystallography and cryo-electron microscopy are starting to zero in on whether they may indeed be playing functional roles.

There are now an enormous number of rRNA sequences that have become available across the three domains of life. We did a bioinformatic study and confirmed that there were some sequences that were universally conserved, and in addition we discovered a new category: sequences that are fully conserved within one domain of life, such as eukaryotes, but not present in that sequence composition in the other two domains. That points to the possibility that they carry out a domain-specific function. Intriguingly the majority of them line the tunnel of the large ribosomal subunit through which the nascent polypeptide exits. Whether it plays a regulatory role feeding back to the nearby peptidyl transferase center is something worthy of future study.

What can we learn from understanding Sciara re-replication?

DNA re-replication leading to gene amplification is a hallmark of many cancers, but the underlying mechanism isn’t fully understood. Whether re-replication is an alternate or a primary mechanism that subsequently leads to breakage and rejoining and recombination hasn’t been studied. One cannot induce amplification in cells in a way that allows you to study the initiating events; you only see the final outcomes of amplification. So, it became very desirable to look for model systems where this is a natural part of development.

There are two known cases of developmentally programmed locus-specific re-replication: Drosophila follicle cells, and salivary gland polytene chromosomes from the end of Sciara larval life. We want to understand how these origins of replication bypass normal cellular controls. Once we figure that out, this may serve as a paradigm to understand whether the same thing is happening in cancer cells.

What is the function of developmentally programmed re-replication?

The areas that undergo re-replication in the Sciara polytene chromosomes are called DNA puffs (to contrast them from Drosophila RNA puffs). The DNA puffs have undergone extra rounds of replication, and are templates for a massive amount of transcription that is translated into the proteins needed to make the pupal case in the next stage of development.

In both Sciara late larvae and in Drosophila follicles there’s a very short window in which a massive amount of protein is needed. In Drosophila it’s for the chorion that forms the egg shell, and in Sciara it’s for the pupal coat. And so the strategy in both systems is gene amplification. You might ask why other cell types don’t use the same strategy. The problem is that once you’ve undergone re-replication you now have nested replication forks and a structure called an onion-skin that is potentially very unstable when the cell tries to divide. But in both Sciara polytene chromosomes and the polyploid cells of Drosophila follicle cells there is no mitosis, so the onion-skin structure is not damaging. In addition, both tissues are destined to be destroyed soon after the re-replication event, so they wouldn’t have to live with the consequences anyway. If such onion-skin structures occur in dividing cells—such as in the cells that become cancerous—this might lead to breakage and recombination and eventually lead to amplification.

What have you learned about re-replication?

The first thing we had to do is to understand what an origin of replication looks like at the sequence level. This has been a very elusive target for the replication community because no specific origin of replication sequence has emerged for any organism except for budding yeast. Other organisms seem to have initiation zones rather than point origins, and no specific sequence. We developed a method that we called Replication Initiation Point (RIP) mapping. This was done with Anja Bielinsky, who was a postdoc in my lab. We needed an enriched population of newly replicated DNA to start with, and for this we popularized the use of the enzyme λ-exonuclease. This will digest DNA from its 5′ end in an exonucleolytic fashion, but not if there’s an RNA primer at the end, such as there is after re-replication. We first piloted the method using SV40 and then using yeast ARS1 [an origin of replication]. The ARS1 structure was very well established, but it wasn’t known whether there was a specific nucleotide where initiation starts, or whether it involves a larger area. We were able to show that indeed DNA replication begins at a unique start site. We were then able to identify where the Sciara DNA puff re-replication starts at the nucleotide level. There too we saw a unique start site for synthesis, even though there’s an apparently larger initiation zone seen by 2-D gels. Maybe at each end of the initiation zone there are preferred sites to start DNA synthesis, and that gives rise to the appearance of a zone.

Once we established where DNA synthesis starts in re-replication, we could look at the surrounding sequence and see if anything jumped out at us that might be a regulatory element. Directly adjacent to the start site, where the origin of replication complex binds, we found a potential binding site for an ecdysone receptor. This is the master regulator of insect development, and it was the first transcription factor ever discovered. We’re trying to test whether it is also acting as a replication factor. If so, the question is whether —in hormonally sensitive cancers such as breast cancer—the estrogen receptor might also serve as an amplification factor.

What role do you think the ecdysone receptor might play in re-replication?

We don’t have direct evidence it is a replication factor, only smoking gun evidence. But there is some precedence for transcription factors also acting as replication factors in certain animal viruses. In the case of Sciara we imagine a couple of scenarios. One possibility is that the ecdysone receptor is interacting with some of the replication machinery that’s sitting adjacent to it on the chromosome, keeping it in an “on” state. Another possibility of course is that it’s acting only as a transcription factor and triggering a cascade of events that lead to re-replication. The difficulty is you would expect this to impact all origins in the genome, not specific subsets. In Sciara there are 18 DNA puffs; what distinguishes them is still a mystery, but to me it suggests there’s something in the local environment—either at the sequence level or the chromatin level or in neighboring proteins such as the ecdysone receptors.

You are a great advocate for Sciara. What’s so compelling about this species?

Sciara is an amazing model organism with many unique biological features. Geneticists usually figure out how things work by making mutations. But, if you will, the unique features in Sciara are like God-given mutations; they are variations of canonical processes that can shed light on the underlying mechanism.

Around 1914 Charles Metz decided to study Sciara for his PhD thesis at Columbia. He captured it in the pigeon house at Cold Spring Harbor Laboratory on the suggestion of a friend. It took him quite a number of years to figure out the chromosome mechanics, but he ultimately succeeded and ended up dedicating his career to studying Sciara.

In the 1930s geneticists had a meeting at Cold Spring Harbor and realized that they would make more progress if they all worked on the same organism. They discussed which to choose, and the two finalists were Sciara and Drosophila. We all know who won! The reason Drosophila was chosen was because geneticists of the 1930s relied on making mutations by X-irradiation, and Sciara turns out to be extremely resistant to X-irradiation. This is another of its unique biological features, but it was not good at the time. Sciara surfaced again in 1970–71 when Sydney Brenner spent two years in the library trying to figure out a good model system for developmental biology. Sciara made his final shortlist of six organisms, but the winner of that competition was the nematode worm C. elegans.

Fast forward to the current time, and of course now we don’t have to rely on X-irradiation for mutation. Thanks to genome sequencing and other methods there’s been an explosion of emerging model systems where one can now reap the benefits of studying unique aspects of biology. In our lab, a senior staff member, Yutaka Yamamoto, has succeeded in developing a germline transformation method for Sciara. Moreover, former graduate student John Urban has sequenced and assembled the Sciara genome. Thus, we’ve established the toolbox of a genome sequence and a methodology to transform Sciara, so the time is now ripe for the scientific community to study all the unique features of Sciara. I’ve been trying to encourage other lab groups to start to work with Sciara. I’m thrilled that several labs have already started and others are on the horizon. We give a 1–2 day workshop in my lab for anyone who wants to learn how to work with Sciara.

What are some of the unique features of Sciara?

One is sex determination. There’s no Y chromosome, and sex is determined by the mother. There are two types of females: those with two copies of the X have only sons; those with one X and one X’ (an X with a long paracentric inversion) have only daughters. How that works is if the haploid egg came from an X/X’ mother, then the egg (when fertilized) will become a daughter. Whereas if it’s an egg from a mother that was X/X, the fertilized egg will become a son. Something, possibly in the cytoplasm, is conditioned by the mother at an early stage prior to meiosis when the X’ and X separate.

Spermatogenesis is also unique. In the first meiotic division in males there’s a monopolar spindle. Monopolar spindles have been studied in cases where they’re induced, but in Sciara it’s a normal occurrence. The chromosomes move from what looks like prophase, skipping metaphase, directly into an anaphase-like configuration, and ultimately telophase. What’s remarkable is in the anaphase-like configuration all the paternally-derived homologs move towards the nonpolar end of the spindle. That’s instead of it being random whether a maternally derived homologue will go to one pole end of the spindle or the other. This was the first example of imprinting, where the cell can recognize the paternal or maternal origin of chromosomes. It was noticed by Helen Crouse, who worked with Metz for a few years, and she coined the term imprinting in her 1960 GENETICS paper. It was later studied much more in-depth in mammalian systems, but it is not yet clear whether in Sciara it occurs by modification systems, such as methylation, as it does in mammals.

So, all of the paternally derived homologs move away from the single pole and are then discarded in a little bud of cytoplasm. In a way, this is a system en route to parthenogenesis because—at least in sperm—it’s not using the paternally-derived chromosomes of the previous generations. The chromosomes that move towards the single pole are maternally derived, and of course, how chromosomes move to this pole is a fascinating subject that is worthy of study in itself.

Then in meiosis II a bipolar spindle is established, though there’s only a single centrosome at one end, the one that came from the previous monopolar spindle. So now the chromosomes do align on a metaphase plate and then segregate, with the exception of the X. The X instead stays locked into the single centrosome, and the result is two products of meiosis II: one is nullo-X and the other has two copies of X (the X dyad). The nullo-X product is also encapsulated in a small bud of cytoplasm and degenerates. So, the only product of spermatogenesis is a single cell that has two copies of an X and is haploid for the autosomes. At fertilization, you have one X from the egg and two from the sperm, and the zygote ends up with three copies. But, of course, you can’t keep doing this every generation! You would accumulate more and more X chromosomes. So, in an early cleavage division some of the X chromosomes are eliminated.

And this is where sex determination comes into play: if the offspring is going to be male, it eliminates one of the three Xs; if the offspring is going to be female, it eliminates two of the three Xs. The final chromosomal complement in the soma of males is a single X, and females are either X/X or X/X’. Now imprinting comes into play. The eliminated Xs are always paternally derived. The X chromosomes that will be eliminated line up on the metaphase plate and start to separate—in that their centromeres disjoin and start to be pulled to one pole or the other—but the arms of the Xs fail to separate. So, it’s as if there’s a chromosome-specific failure of the cohesins to dissolve.

It turns out there’s a region that was genetically identified by Crouse that she called the controlling element (CE). It governs the X dyad nondisjunction in meiosis II, as well as the X chromosome elimination in embryogenesis. You can move the CE locus to any of the three autosomes by reciprocal translocations, and now you’ve fooled the cell into treating the autosomes as if they were the X: The translocation autosome will undergo non-disjunction in meiosis II and chromosome elimination in early embryogenesis, and the X that now lacks the controlling element no longer undergoes those unique behaviors.

What is the controlling element and how does it regulate these processes? We’d like to know more. The controlling element is located within the tandem array of 50 copies of ribosomal RNA genes—it’s right in the middle of the array and is flanked by translocation breakpoints. So, we would like to be able to zero in on it with long read sequencing and terrific genome assemblies. We know already know there is some non-rDNA sequence within the tandem array which in itself is interesting – then the question is what part of that is functional and how does it function. Is it, for example, like the XIST locus, which creates an RNA that coats the entire chromosome? That’s one hypothesis because the controlling element acts on the chromosome on which it’s sitting.

In addition to the sex determination mechanism and the unusual behaviors imparted by the controlling element, Sciara also has germline-limited chromosomes called the L chromosomes, whose roles are totally unknown. And, in addition, Sciara has locus-specific re-replication in DNA puffs of polytene chromosomes and other unique features.

Who have been your most important mentors?

Joe Gall without a doubt, and I’m still in very close touch with him. He’s most important cell biologists of our generation. He was always fascinated by the biology uniquely offered by particular eukaryotic species, including less well-studied organism, and that was one reason I went to study with him. He wasn’t wedded to one biological system, but being such a well-rounded biologist he would ask, what is the best biological system to study the question at hand? Rather than the other way around, which is, I have this biological system, now what questions can I ask with it? I think that’s what makes him quite remarkable and unique. A sequel lesson learned from that is it’s safest if we do experiments in a biological context—rather than try to dissect everything through test-tube biology or even cells in culture. I’ve always tried to do experiments in the biological system itself because then you’re less likely to have changes in unknown parameters that will give you the wrong answer.

What’s the best advice you ever received?

My colleague at Brown Art Landy once gave me some advice when we were worried about being scooped. He said if you know the answer to the experiment you are doing, you can jump ahead to where that was going to take you and plan the next experiment. If others arrive at a conclusion that you trust, then you can simply fast forward to the next logical question.

What advice would you give to younger scientists?

Treasure your exceptions. Sciara is an exception to the way things normally happen, but it can give you an enormous amount of insight into the basic canonical mechanisms that are shared by most other organisms. If you get a result in the lab that is unexpected, don’t throw up your hands in despair and say, things aren’t working, and I must have done something wrong, and this is not what the field would have predicted. You may in fact have opened up a whole new line of pursuit! After you repeat it and do the appropriate controls, it could change the mindset of the field and let people know that the current hypothesis or model might need some tweaking.

A green, iridescent bee perches on a pink flower, extending its proboscis to reach the sweet nectar inside. He’s not just after a meal—he’s also collecting fragrant substances to store inside his hollow rear legs. Later, he’ll buzz his wings to release the aroma with the hope of attracting a mate. The cover of the September issue of G3 features a photograph of this eye-catching insect: a type of orchid bee called Euglossa dilemma. Orchid bees inhabit the neotropical realm, a region encompassing most of South America, some of Central America, and a tiny fraction of southern North America. There, these bees are some of the most important pollinators of flowering plants.

In the same issue of G3, Brand et al. report a draft assembly of the nuclear and mitochondrial genome of E. dilemma, the first draft genome of any species in the genus Euglossa. The genome revealed several interesting facts about the bees; for example, they have one of the largest genomes of any insect, loaded with repetitive sequences. Their assembly will also be a boon to bee researchers, from those seeking to know more about how to conserve these essential pollinators to those studying bee evolution.

Of particular interest is the evolution of one of many bees’ most fascinating traits: the ability to form intricate social structures. E. dilemma, unlike its close relatives the honeybee, stingless bee, and bumble bee, doesn’t actually live in communal hives. If the male orchid bee succeeds in seducing a female with his foraged scents, she’ll lay their eggs in a small nest of up to twenty cells, where she’ll feed the larvae nectar and pollen. Orchid bees may build their nests near each other, giving the impression of a loosely connected society, but groups of nests don’t form communities like hives. However, daughter bees sometimes stay in their mothers’ nests to help her raise a new generation—a type of social interaction that may have been one of the evolutionary stepping stones toward hives in insects like honeybees. Using the new genome as a starting point, researchers might be able to learn more about the evolution of this complex behavior, increasing our knowledge of many more types of these industrious insects.

CITATION:
Brand, P.; Saleh, N.; Pan, H.; Li, C.; Kapheim, K.; Ramírez, S. The Nuclear and Mitochondrial Genomes of the Facultatively Eusocial Orchid Bee Euglossa dilemma.
G3: Genes|Genomes|Genetics, 7(9), 2891-2898.
DOI: 10.1534/g3.117.043687
http://www.g3journal.org/content/7/9/2891

Genes involved in male reproduction tend to evolve rapidly. This has been observed in many different species and is thought to be due to sexual selection as males compete over mating opportunities. But in the August issue of GENETICS, Whittle and Extavour present results that flip this paradigm upside down. They find that in the yellow-fever mosquito, female-biased genes expressed in the ovaries evolve faster than their male counterparts. This fascinating break from the trend could be due to increased competition between females for mates, adaptive evolution during egg-sperm attraction, and/or limited sperm competition in this species.

First, the authors identified genes more highly expressed in either male or female gonads using whole transcriptome data. They found that ovarian-biased expression was typically due to elevated expression in females, not just reduced expression in males as has been observed in other species. They then identified nucleotide changes that altered the protein composition in these genes to compare the rates of protein evolution. Although a small subset of testis-biased genes were evolving rapidly, on average transcripts with ovary-biased expression showed a significantly higher protein evolution rate than those with testis-biased expression. Genes expressed only in the ovaries had the fastest protein evolution rate of all. They determined that the rapid evolution of some of these genes is most likely due to positive selection using a phylogenetic analysis including two other mosquito species.   

Interestingly, members of this set of rapidly-evolving, ovary-specific genes have functions preferentially related to the mosquito’s olfactory system, including odor molecule binding and smell receptor activity. Olfactory signaling appears to be important for mosquito mating; groups of males will gather together and swarm females, who are lured over by their scent. These types of chemical cues may also be important for guiding the sperm to the egg or directing females to store sperm after mating. Like some other insects, female mosquitoes have special storage organs that allow them to keep enough sperm from a single mating to fertilize all their eggs throughout their entire lives. There may be strong selective pressure on proteins that drive evolution of these critical reproductive functions.

The yellow-fever mosquito’s mating system is likely behind its unusual rapid evolution of ovary-biased genes. There might be competition between females to attract males or male mate choice, which could result in strong sexual selection on ovary-expressed genes involved in chemical sensing. These females usually mate once, and the male deposits a “copulation plug” in the female’s reproductive tract. This physical and chemical barrier prevents the sperm of another male from passing through, ensuring the first male will father all her offspring. The plug cuts off nearly any chance of competition between the sperm of multiple males, in contrast to many other organisms where the rapid evolution of testis-biased genes could be due to the pressure of this sperm competition arms race.

A combination of these diverse factors likely influences the rapid protein evolution of ovary-biased genes in yellow-fever mosquitoes. These results offer a fascinating glimpse into how ecology and reproductive lifestyle can affect genome evolution and illustrate how there are notable exceptions for every trend observed in nature.

CITATION:

Rapid Evolution of Ovarian-Biased Genes in the Yellow Fever Mosquito (Aedes aegypti)

Carrie A. Whittle and Cassandra G. Extavour

Genetics August 2017 206: 2119-2137

https://doi.org/10.1534/genetics.117.201343

http://www.genetics.org/content/206/4/2119