Progress 10/01/19 to 09/30/20
Outputs
Target Audience:As a researcher with a majority research and minor teaching component, my primary target audience included graduate students.The 3-credit graduate course on insect-microbe interactions [ENT-591/791, Special topics in Entomology]offered for the first time in FALL 2019 was received well by course reviewers and students. Among various topics, the course introduced entomology students to the roles of microbial symbionts in wood-feeding insects and the use of entomopathogens to control wood-destroying pests. I have also co-instructed the seminar course ENT 601/801 (Arthropod-microbe interactions, Spring 2019) and guest-lectured on microbial symbioses in BSC 495-083/MB590-002 [Fall 2019], which further complement my research focus on insect-microbe interactions. Although my position lacks a formal extension component, I presented about the use of entomopathogens to control wood-destroying and other urban pests at the Pest Control Training School (Spring 2019) and to major companies in the Research Triangle. Several members of the lab managed a stall at the annual Bugfest outreach festival (Fall 2019) organized by the North Carolina Museum of Natural Sciences and North Carolina State University, where we presented on the biology of termties and other wood-feeding insects. Changes/Problems:The COVID-19 outbreak has substantially impacted and will continue to impact our research program this year. Aside from delays in setting up key long-term experiments in the lab, the travel bans associated with the outbreak have caused us to postpone several sampling plans this year, importantly in March to the Czech Republic and in June/July to Japan for sampling termites with collaborators. My lab is doing the best to adapt to these new conditions, by focusing on bioinformatics-based projects, writing articles and reviews, and preparing proposals. What opportunities for training and professional development has the project provided?The project supported two mentees during this reporting period: a PhD student Melbert Schwarz and an undergraduate researcher Marissa Cole, who joined the lab as a graduate student in Spring 2020. Regular mentoring activities include weekly one-on-one meetings with the mentee for discussing progress updates on projects and career goals. Additionally, the lab holds weekly meetings, where the mentees get an opportunity to present data to the entire lab and get practical feedback and improve science communication skills. Aside from meetings to discuss scientific research, Melbert and Marissa have enrolled as full-time students in the entomology program and conducted coursework relating to insect physiology, insect ecology, insect-microbe interactions. Although our research timelines have been strongly impacted by the pandemic, we have been able to hire a postdoctoral scholar Aaron Ashbrook who joined in June 2020, and will be working on some aspects related to goal 2 (the control of wood-destroying pests.)Additionally, two undergraduate researchers Ezra Bailey and Sophia Copeman have joined the lab to assist with ongoing experiments. How have the results been disseminated to communities of interest?The lab presented at the North Carolina Pest Management Association's PCT school in early 2020, highlighting the importance of studying the gut microbiomes of wood-feeding insects to design targets for pest control. Lab members also educated the public about termites at BugFest, an extremely popular insect-based outreach program held by the North Carolina Museum of Natural Sciences that attracts over 35000 visitors in a single day. The lab has also co-developed educational curricula with Bob Kuhn, a teacher inCentennial High (Atlanta, Georgia) that allow students (grades 4-7) to explore the gut microbiomes of wood-feeding isnects in their labs. What do you plan to do during the next reporting period to accomplish the goals? Explore evolutionary trends between wood-feeding insects and their gut microbiomes Following standardization of the Percoll gradients, we will be commercially sequencing the 16S rRNA genes associated with the fiber-free and fiber fractions from a range of wood- and litter-feeding insects to identify those microbes that are physically associated with wood particles. Phylogenetic trees will be constructed for the fiber-digesting bacterial clades, which will help characterize the ecological and evolutionary factors influencing gut microbiome structure in wood-feeding insects. Functionally interrogate the effect of microbiome perturbation on wood-feeding pests Following the optimization of cohort size and enclosure, we will be feeding termites on different ratios of cellulose and starch to understand the role that dietary recalcitrance might be playing on trophallaxis (the cooperative exchange of gut fluids). The latter component will also require the development of a suitable method to track the exchange of gut fluids. Investigate the role of gut community assembly in determining termite health We will isolate commensal bacteria from termites for use in experiments that will test the role of microbial colonization history in determining termite health.
Impacts
What was accomplished under these goals?
Impact Statement Termites and many other wood-feeding insects obligately depend on microbial symbionts for the digestion of lignocellulose. Many of these insects play critical roles in the turnover of plant material in terrestrial ecosystems, however a small percentage of species are extremely successful structural pests. Understanding the ecological and evolutionary basis of insect-microbe interactions is critical to not only understanding the biology of wood-feeding insects but also to developing sustainable technologies to control wood-destroying pests. Graduate Student Melbert Schwarz has led the development of a method to identify those microbial members that are involved in fiber digestion in several species of termites, cockroaches and beetles, including both pest and non-pest insects. The method relies on separating wood particles in insect guts using their higher densities, enabling the analysis of those microbes that are attached to the particles.Our results showthat all eight species analyzed possesed unique fiber-digesting microbes that were consistently recovered in multiple replicates. The host-specificity of these communities will inform the development of technologies that can target symbiotic digestion only in pest species, without affecting those wood-feeding insects that play critical roles in the environment. Objectives: Explore evolutionary trends between wood-feeding insects and their gut microbiomes Background and experiments conducted: The guts of wood-feeding insects act as tiny ecosystems with hundreds of microbial species, however, only a minority of those microbes directly aid in the symbiotic digestion of wood. Graduate student Melbert Schwarz from the laboratory developed a method to precisely identify symbionts in two species of termites that was optimized to identify symbionts in multiple species of wood-feeding and litter-feeding cockroaches. Although most major wood-destroying pests are termites, there are several species among beetles that have also evolved to be pests; for this reason, we also focused on two species of wood-feeding beetles in the families Passalidae and Micromalthidae that are major model organisms for wood digestion.Themethod of enriching wood particles from insect guts relies on Percoll density gradient centrifugation that relies on the heavier density of wood particles to separate them from the remaining gut content. Results and discussion: The density centrifugation method was optimized to efficiently enrich wood particles from passalid beetle Odontotaenius disjunctus, wood-feeding Cryptocercus punctulatus, and several species of litter-feeding cockroaches. Because the density of the particles varied with differences in the decay status of the plant material consumed by each species, the centrifugation protocol had to be standardized for each species. Among the parameters to be standardized, the most important ones are the concentration of Percoll, whether the gradient is continuous or discontinuous, as well as the temperature and duration of the centrifugation. Importantly, a continuous gradient with 70% equilibriated Percoll solution that worked well with termites did not work for the passalid beetles, and we had to employ a discontinuous gradient instead with a lower layer of 60% and an upper layer of 40% Percoll; the rest of the protocol remained the same (100 uL of luminal fluid; centrifugation at 20,000 g /4 ºC/ 30 minutes). DNA from the the different Percoll fractions was used to characterize the microbiome associated with the wood particles in the termites, cockroaches and beetles. Sequence data was analyzed using mothur and a dedicated reference database developed in the lab [DictDb1]. Key outcomes: a. Our protocols build on previous experiments from our lab that identified key xylan-degrading bacteria in the guts of Japanese non-pest termite Nasutitermes takasagoensis [1], and extend it to two pest species native to the US b. These experiments lay the foundation for a versatile method that can help identify bacteria responsible for fiber degradation in any wood-feeding insect c. This approach will be used to distinguish between the wood-digesting symbionts of pest species and non-pest species, and carefully consider that information when designing targetted pest-control measures. 1Mikaelyan, A., Köhler, T., Lampert, N., Rohland, J., Boga, H., Meuser, K. and Brune, A., 2015. Classifying the bacterial gut microbiota of termites and cockroaches: a curated phylogenetic reference database (DictDb).Systematic and applied microbiology,38(7), pp.472-482. Functionally interrogate the effect of microbiome perturbation on wood-feeding pests Background and experiments conducted:Evolutionarily primitive "lower" termites harbor protozoa in the gut for cellulose digestion. Work from the lab recently confirmed2the ability of starch to "defaunate" or eliminate flagellates from the termite gut, thereby negatively affect their ability to feed on wood. The research ofMasters student Marissa Cole will explore the link between termite diet, (social) behavior and survival. Our exploration of use of bacteriophages had to be temporarily paused due to the impact that COVID-19 had on work schedules, access to equipment, etc. Results and discussion:We are currently running pilot experiments with different combinations of cellulose and starch with both subterranean termiteReticulitermes santonensisand the dampwood termiteZootermopsis nevadensis, in order to standardize the correct cohort size and enclosure. 2Mikaelyan, A., Thompson, C.L., Meuser, K., Zheng, H., Rani, P., Plarre, R. and Brune, A., 2017. High?resolution phylogenetic analysis of Endomicrobia reveals multiple acquisitions of endosymbiotic lineages by termite gut flagellates.Environmental microbiology reports,9(5), pp.477-483. Investigate the role of gut community assembly in determining termite health Nothing to report
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Lampert, N., Mikaelyan, A., & Brune, A. (2019). Diet is not the primary driver of bacterial community structure in the gut of litter-feeding cockroaches. BMC microbiology, 19(1), 1-14.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Critchlow, J., Bordenstein, S., Mikaelyan, A., Zackular, J. and Tate, A.T., 2019, August. The gut microbiome of Tribolium castaneum. In 2019 ESA Annual Meeting (August 11--16). ESA.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Otani, S., Zhukova, M., Kon�, N. G. A., da Costa, R. R., Mikaelyan, A., Sapountzis, P., & Poulsen, M. (2019). Gut microbial compositions mirror caste?specific diets in a major lineage of social insects. Environmental microbiology reports, 11(2), 196-205.
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Progress 04/23/19 to 09/30/19
Outputs
Target Audience:Teaching Since joining NCSU, I developed a 3-credit graduate course on insect-microbe interactions, offered in the fall semester under ENT-591/791 [Special topics in Entomology]. The course covers the entire spectrum of insect-microbe interactions, including mutualism, immunity, diseases, and microbiomes. In addition to lectures, the course includes interactive components, ranging from Socratic lectures, scientific communication, and a card game based on core concepts. For Fall 2019, the course attracted a diverse group of enrollees, includingstudents from the Entomology and Biomath graduate programs, suggesting potential student interest in other departments and graduate programs. I have also co-instructed the seminar course ENT 601/801 (social insects, Spring 2019) and guest-lectured on microbial symbioses in BSC 495-083/MB590-002 [Fall 2018], which further complement my research focus on social insects and insect-microbe interactions. Extension and outreach: My program remains strongly committed to the dissemination of accurate scientific knowledge beyond the academic community, to industry stakeholders and the wider public. Myself and other members of my labhave given talks about structural pests to industrial partners andto the annual pest control technician' school organized by the North Carolina Pest Management Association (NCPMA; 2019). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project supported two mentees during this reporting period:a PhD student Melbert Schwarz and an undergraduate researcher Caroline Elliotte. Regular mentoring activities include weekly one-on-one meetings with the mentee for discussing progress updates on projects and career goals. Additionally, the lab holds weekly meetings, where the mentees get an opportunity to present data to the entire lab and get practical feedback and improve science communication skills. Aside from meetings to discuss scientific research, PhD student Melbert Schwarz was enrolled as a full-time student in the entomology program and conducted coursework relating to insect diversity, insect physiology, and insect-microbe interactions. How have the results been disseminated to communities of interest?The lab presented at the North Carolina Pest Management Association's PCT school in early 2019, highlighting the importance of studying the gut microbiomes of wood-feeding insects to design targets for pest control. Lab members also educated the public about termitesat BugFest, an extremely popular insect-based outreach program held by the North Carolina Museum of Natural Sciences that attracts over 35000 visitors in a single day. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact Statement: Termites and many other wood-feeding insects obligately depend on microbial symbionts for the digestion of lignocellulose. Many of these insects play critical roles in the turnover of plant material in terrestrial ecosystems, however a small percentage of species are extremely successful structural pests.My lab is currently working towards developing strategies to specifically target microbes responsible for symbiotic digestion in structural pests. Our approach involves identifying ecological and evolutionary patterns in the distribution of digestive symbionts accross the various lineages of wood-feeding insects, followed by the generation of technologies to specifically attack the symbionts of wood-destroying pests (while minimizing the impact on other wood-feeding insects).Since the lab became functional in April 2019, we have developed a methodto identifyspecific lignocellulose-digesting symbionts in two species of termites. The objectives of our continuing investigations are to confirm and extend our previous findings to other termite species, whiletestinginnovative approaches for the disruption of symbioses in economically important termite pests. The effectiveness of current methods of chemical control of wood-destroying termites does notnecessitate the discovery new technologies. However, a lot that remains to be discovered in the context of termite control from the perspective of environmental safety and sustainability.We hope our approach will pave the way for using dysbiosis (the disruption of microbiomes) alongside traditional methods to cause mortality in insect pests, while minimizing off-target impacts on ecologically important insects. Objectives: Explore evolutionary trends between wood-feeding insects and their gut microbiomes Background and experiments conducted: The guts of wood-feeding insects act as tiny ecosystems with hundreds of microbial species, however, only a minority of those microbes directly aid in the symbiotic digestion of wood. Graduate student Melbert Schwarz from the laboratory has developed a method to identify precisely those symbionts in any wood-feeding insect that are involved in the digestion of wood. This novel approach relies on the enrichment of wood particles in the insect gut using Percoll density gradient centrifugation that relies on the heavier density of wood particles to separate them from planktonic bacteria in the gut. Results and discussion: We have optimized the method to efficiently enrich wood particles from the pestEastern subterranean termiteReticulitermes flavipes and the dampwood termiteZootermopsis nevadensis.Among the parameters to be standardized, the most important ones are the concentration of Percoll, whether the gradient is continuous or discontinuous, as well as the temperature and duration of the centrifugation. For these two species, we have been able to optimize the method to using a continuous gradient with 70% equilibriated Percoll solution. 100 uL of luminal fluid from the termites is used to separate wood particles at 20,000 g /4 ºC/ 30 minutes. DNA from the the different Percoll fractions will be extracted using a kit from Zymo Research, and used to characterize the microbiome associated with the wood particles. Key outcomes: a.Our protocols build on previous experiments from our lab that identified key xylan-degrading bacteria in the guts of Japanese non-pest termiteNasutitermes takasagoensis [1], and extend it to two pest species native to the US b.These experiments laythe foundation for a versatile method that can help identify bacteria responsible for fiber degradation in any wood-feeding insect c.This approach will be used to distinguish between the wood-digesting symbionts of pest species and non-pest species, and carefully consider that information when designing targetted pest-control measures. Functionally interrogate the effect of microbiome perturbation on wood-feeding pests Background and experiments: All animals depend on their gut microbiomes for nutrition, immunityand development. Viruses, including those infecting bacteria (bacteriophages) occur naturally in animal guts.An imbalance in bacteriophage populations in any gut microbiome can trigger dysbiosis a pathological condition involving a disruption of the gut microbiome. My lab's interest is to understand how dysbiosis can be induced in wood-feeding pests, which rely on the gut microbiome. Our eventual goal is to raise bacteriophages that specifically target symbionts playing a role in digestion (identified in Aim 1). However, as a proof-of-principle, we optimized aprotocol (that we published last year [2]), which allows us to enrich the entire community of bacteriophages from termites using ultracentrifugation (100,000 x g). Results and discussion: A combination of PEG-precipiation and ultracentrifugation was observed to be the best method to enrich the gut viromes of termites and cockroaches. However, feeding the enriched bacteriophage solution to termites did not result insignificant mortality. This suggests that the termite gut microbiome is resilient enough in the face of general perturbation by bacteriophages. Key outcomes: a.We have standardized a method for the enrichment of bacteriophages from termite guts. b.Our experiments to testthe use of a bacteriophage enrichmentfor termite control suggest that the gut microbiome of the termite is resilient and may require the use of individual populations of bacteriophages in stead of using an enriched community. Investigate the role of gut community assembly in determining termite health Nothing to report. References: Tokuda, G., Mikaelyan, A., Fukui, C., Matsuura, Y., Watanabe, H., Fujishima, M. and Brune, A., 2018. Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher termites.Proceedings of the National Academy of Sciences,115(51), pp.E11996-E12004. Leigh, B.A., Bordenstein, S.R., Brooks, A.W., Mikaelyan, A. and Bordenstein, S.R., 2018. Finer-scale phylosymbiosis: insights from insect viromes.Msystems,3(6).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Otani, S., Zhukova, M., Kon�, N.G.A., da Costa, R.R., Mikaelyan, A., Sapountzis, P. and Poulsen, M., 2019. Gut microbial compositions mirror caste?specific diets in a major lineage of social insects. Environmental microbiology reports, 11(2), pp.196-205.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Lampert, N., Mikaelyan, A. and Brune, A., 2019. Diet is not the primary driver of bacterial community structure in the gut of litter-feeding cockroaches. BMC microbiology, 19(1), p.238.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Critchlow, J., Bordenstein, S., Mikaelyan, A., Zackular, J. and Tate, A.T., 2019, August. The gut microbiome of Tribolium castaneum. In 2019 ESA Annual Meeting (August 11--16). ESA.
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