INTEGRATIVE RESEARCH ON THE OVERWINTERING BIOLOGY OF INSECTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010996
• Project Start Date: Oct 29, 2016
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Entomology

Non Technical Summary
Climate change is a major threat to both natural and agricultural ecosystems. While much research has focused on responses to climate change during the growing season, climate change is also having a significant impact on winter conditions. As cold-blooded animals, insects are particularly sensitive to winter temperatures and make a good model for understanding biological responses to climate change. Here, we seek to enhance understanding of the overwintering biology of insects. Successful overwintering is a key determinant of an insect's potential range, and overwintering biology has important implications for pest control and human health, which are detailed below.In this project, we investigage three aspects of insect overwintering biology: 1) Physiological adaptations to rapid change in temperature, 2) Mechanisms of freeze tolerance in insects, and 3) Extreme adaptations of Antarctic arthropods. Our research approach integrates physiology, genetics, genomics, and cell biology to understand the mechanisms used by insects to survive harsh winter conditions. Below is a summary of our three goalsand their potential benefit to science and society.Goal 1: Adaptations to rapid change in temperature.In recent years, many groups have investigated seasonal responses to winter in insects. These studies have revealed the molecular and physiological mechanisms that allow an insect to transition from an actively feeding and reproducing phase to a dormant overwintering phase. However, insects are also capable of responding to low temperatures on a much shorter time scale. In a process called rapid cold hardening, brief exposure to mild chilling (i.e., minutes to hours) significantly enhances survival at extreme low temperatures. This process allows insects to cope with sudden cold snaps and diurnal changes in temperature. Rapid cold hardening offers protection against cold stress at times of year such asspring and fall when temperature can be unpredictable. With climate change leading to increased thermal variability, adaptations to sudden temperature change, like rapid cold hardening, will be critical to the continued success of insects.While the ecological importance of rapid cold hardening is well established, we don't have a good understanding of how it works at the physiological and molecular levels. Because it occurs so quickly, rapid cold hardening seems to be mostly regulated by cell signaling pathways. Previous work by the PI demonstrated that chilling causes calcium to enter the cell, and this calcium entry triggers processes that protect insects against cold stress. Work by others demonstrated that chilling also activates a protein called p38 MAP kinase, although the exact function of this protein during rapid cold hardeninghas not been established.Thus, in this objective, we will test whether calcium signaling and p38 MAP kinase are part of the same signaling pathway that activates rapid cold hardening. These experiments will lead to a significant increase in our understanding of this critical ecological process. Knowledge of the mechanism of rapid cold hardening will facilitate predictions into which insect species will be able to cope with increased thermal variability, and these mechanisms may also have implications for pest control.Goal 2: Understand the genetic mechanisms of freeze tolerance.Among the insects, most survive sub-freezing conditions in a "supercooled" state. This means that the body fluids remain in a liquid state even when temperatures are below the freezing point of water, and these insects die if internal ice formation occurs. However, a minority of insects can survive internal freezing, and these species are referred to as "freeze-tolerant." While some of the physiological and biochemical mechanisms of freeze tolerance have been described, we have no understanding of the genetic mechanisms that allow some insects to survive freezing. None of the classic insect models (e.g., fruit flies, mosquitoes, silk moths, flour beetles, etc.) are capable of surviving freezing, so new models are needed to study this phenotype.A promising model for freeze tolerance research is the drosophilid fly Chymomyza costata.This fly can survive immersion in liquid nitrogen, making it the most complexmulticellular organism capable of surviving liquid nitrogen temperatures. Thus, we propose to use this species as a model to understand the genetic mechanisms of freeze tolerance. First, we will use genomic methods to identify genes and pathways that are activated by freezing. Second, we will develop transgenic methods for this species, so that we can manipulate gene expression and function to identify the critical genes that underscore the capacity for freeze tolerance. Our goal is to fully describe the genetic mechanisms that allow flies likeC. costatato survive complete freezing.This work may have significant implications for human health. Currently, a bottleneck for human organ transplantation is the availability of viable organs. The ability to freeze organs for long periods of time would significantly increase the shelf-life of organs and lead to better transplant outcomes. Understanding the secrets of freeze tolerance ininsects likeC. costatamay lead to improved strategies for human cryopreservation.Goal 3: Genomic mechanisms of extreme adaptation in insects.Insects are the most abundant land animals on the planet, both in terms of numbers and biomass. However, in Antarctica, the cold dry climates are inhospitable for insects and other arhthropods. As a result, arhtropod diversity and abundance is extremely low in Antarctica compared to the rest of the planet. There is only one insect species, the midgeBelgica antarctica,that is native to Antarctica, and this species is the world's southernmost insect. This insect is extremely tolerant of environmental stress,surviving 9 months a year of freezing conditions and capable of severe dehydration. In previous work, we sequenced the genome for this species to identify genetic mechanisms associated with extreme adaptation. However, a lack of genomes from closely related species made these analyses difficult. Here, we will sequence the genomes of two closely related species, the midgesBelgica albipesandEretemoptera murphyi,neither of which are native to Antarcitca. Genome information from these species will allow us to make formal comparisons with the Antarctic midge genome to describe genome-level changes that contribute to adaptation to extreme environments.This information will help us understand the nature of extreme adaptation. These insights will inform our work in temperate climates, as often biological processes are most apparent by studying extreme cases (for example, many of the mechanisms of human oxygen transport were better understood by studying populations in the Himilayas). Also, climate warming is happening faster in Antarctica than anywhere else, so it is critical that we study these Antarctic species to predict how they might be impacted by climate warming.Societal benefits.The primary societal benefit from this work will be a significant gain in knowledge of our understanding of insect overwintering biology. This information is critical to understand how insects thrive in nearly every environment on work, and it will help us make predictions on how insects will respond to climate change. In addition, there are some potential practical applications of this work, including human cryoprservation and pest control, that are detailed above. Finally, this work facilitates the training of the next generation of agricultural scientists. Our research is cutting edge and uses a diversity of techniques and approaches, so students trained on these projects will be well-situated to lead the future of agricultural research.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1020	50%
211	3110	1030	20%
211	3110	1040	15%
211	3110	1080	15%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1080 - Genetics; 1020 - Physiology;

Keywords

insect physiology

climate change

cold tolerance

freeze tolerance

antarctica

extreme biology

Goals / Objectives
Goal #1:Understand the physiological and molecular mechanisms that regulated rapid cold hardening in insects. Objective 1: Test the hypothesis that calcium signaling pathways activated by low temperatures regulate rapid cold hardening. Objective 2: Test the hypothesis that rapid activation of p38 MAP kinase by chilling is critical for rapid cold hardening. Objective 3: Test the hypothesis that p38 MAP kinase is activated by calcium signaling during rapid cold hardening.Goal #2: Identify genetic mechanisms associated with freeze tolerance. Objective 1: Use RNA-seq to identify candidate mechanisms of freeze tolerance in the drosophilid fly,Chymomyza costata,which is capable of surviving at liquid nitrogen temperatures. Objective 2: Develop genetic transformation methods forC. costatato validate candidate genes for freeze tolerance.Goal #3: Understand the evolutionary genetic mechanisms of adaptation to extreme Antarctic environments. Objective 1: Sequence the genomes ofBelgica aplbipesandEretmoptera murphyi. These species are closely related to the Antarctic midge,B. antarctica,which is the only native insect in Antarctica and the world's southernmost insect. We have previously sequenced the genome of this species. Objective 2: Use comparative genomics with these three species (B. antarctica, B. albipes, and E. murphyi) to identfiy genomic signatures of extreme adaptation.

Project Methods
Goal 1, Objective 1:We will primarily use pharmacological methods to test the hypothesis that calcium signaling is a key regulator of rapid cold hardening inDrosophila melanogaster.Various tissues will be dissected from adult flies (e.g., midgut, Malpighian tubules, fat body, brain) and exposed to conditions that elicit rapid cold hardening. Tissues will be treated both under normal calcium conditions and in the presence of drugs that manipulatecalcium signaling. The intracellular calcium chelator, BAPTA-AM, will be used to remove intracellular calcium. Additional samples will be treated with lanthanum chloride (a general calcium channel blocker) andW-7 (an inhibitor of the calcium-sensing protein calmodulin). After treatment, cell viabilty will be measured with both a dye exclusion assay and the TUNEL assay, which specifically measures apoptotic cell death. We expect that blocking calcium signaling will prevent rapid cold hardening in tissues, and thus cell survival will be lower in drug-treated tissues exposed to rapid cold hardening conditions. Calcium signaling will also be manipulated genetically, using transgenic RNAi to knockdown expression of key calcium signaling genes such as calmodulin.Goal 1, Objective 2. To assess the role of p38 MAP kinase during rapid cold hardening we will measure the activiation and function of p38. We will first measure activation of p38 MAP kinase using Western blot, and these results will indicate the time course and magnitude of p38 MAP kinase during rapid cold hardening. To test whether this activiation is critical for rapid cold hardening, we will use transgenic lines harboring RNAi for p38 to knockdown expression. If p38 is critical for rapid cold hardening, we expect that knocking down expression will reduce the capacity for rapid cold hardening. Survival will be measured at both the whole animal and cellular level.Goal 1, Objective 3. In this experiment, we will test whether p38 activiation during rapid cold hardening is calcium dependenent, and thus whether these two pathways are operating in concert. Calcium signaling will be manipulated using either pharmacology or RNAi, and we will test whether blocking calcium signaling prevents activation of p38 during rapid cold hardening. We will also test the hypothesis that cross-talk between calcium signaling and p38 is mediated by apoptosis signal-regulating kinase (ASK1). In mammals, this protein mediates calcium-dependent activiation of p38, but its role in insects has not been explored. ASK1 activation will be measured with Western blot, and the functional role of ASK1 will be tested with RNAi.Goal 2, Objective 1: We will use RNA-seq to profile the genes and pathways activated by freezing inChymomyza costata.Samples for gene expression profiling will be collected from the following groups: 1) Warm acclimated larvae, 2) Cold acclimated larvae, 3) Larvae sampled immediately after a freezing event, 4) Larvae sampled after being chilled (but not frozen), 5) Larvae sampled after 24 h of freezing, 6) Larvae sampled after 24 h of chilling, and 7) Larvae after recovery from freezing. After sampling, RNA will be extractracted, converted to cDNA Illumina sequencing libraries, and sequenced with Illumina sequencing technologies. Genome-wide expression patterns for each group will be calculated and compared using widely available bioinformatic tools.Goal 2, Objective 2:For this objective, we will develop methods to make transgenicChymomyza costata,which will facilitate future functional genomics work in this system. Methods for germline transformation will be adapted from closely relatedDrosophila melanogaster.Our first approach will use the piggyBac transformation system to insert transgenes into the genome.In brief, embyros will be collected within 1 h of egg lay, chemically dechorionated, and injectedwith a plasmid mix containing a piggyBac plasmid with the gene of interest and a helper plasmid to provide a source of transposase enzyme. Injected flies that survive to adulthood will be backcrossed to wild type. Our transgene will contain a fluorescent marker so that transformants can be easily identified. Once we establish methods for germline transformation, we will begin screening canddiate genes for freeze tolerance. For example, proline synthesis has been demonstrated to contribute to freeze tolerance, so we will design constructs to either overexpress or knockdown expression of key proline synthesis genes, and determine the impacts on freeze tolerance. Candidate genes identified in Goal 2, Objective 1, will also be tested using transgenics to determine which genes are critical for the development of freeze tolerance.Goal 3, Objective 1:Specimens ofBelgica albipesandEretmoptera murphyiwill be obtained from collaborators as part of the Functional Biogeogrpahy Project of Antarctica. This group will be participating in the Antarctic Circumnavigation Expedition, a research cruise that will extensively sample arthropods at sub-Antarctic Islands around the continent. I will receive specimens from this cruise for genome sequencing. Genomic DNA will be extracted from larvae ofB. albipesandE. murphyi,converted to Illumina sequencing libraries, and sequenced on an Illumina HiSeq 2500 at the University of Kentucky. The software Velvet will be used forde novoassembly, and the draft genomes will be annotated with the MAKER pipeline. This work will result in a completely sequenced, assembled, and annotated genome for these species, which will complement our existing genomic work on the Antarctic midge,Belgica antarctica.Goal 3, Objective 2:With the genome sequences from Objective 1, we will use comparative genomics to identify genomic signatures of extreme adaptation. First, we will analyze synteny and gene content between the three species, to determine whether there are major changes in genomic architecture bewteen the species. Gene Ontology (GO) enrichment analysis will be used to determine whether the functional composition of the genome differs between species. This analysis will indicate whether any classes of genes have been duplicated or deleted inB. antarcticaas a result of Antarctic adaptation. We will also determine orthologs between species and test the Ka/Ks ratio of each orthologue to identify genes that are under positive selection. Taken together, these analyses will indicate which genes have been altered via natural selection to support adaptation to extreme Antarctic environments.Evaluation Plan:Project progress will be evaluated weekly through regularly schedule lab meetings. Annual presentations at national meetings will also be used to ensure that projects are progressing. Conference presentations will be used to present preliminary data and solicit feedback from experts. Student progress will also be monitored using the AAS Individual Development Plan tool (myidp.sciencecareers.org) to ensure that students are meeting project and training milestones.

Progress 10/29/16 to 09/30/21

Outputs
Target Audience:Our target audience for this project has primarily been scientists working in insect physiology and genetics. Our work has been disseminated in high quality journals with international scope (see Products), and we have presented this research at several scientific conferences, including the Entomological Scoeity of America and Society for Integrative and Comparative Biology annual meetings. We have also conducted a number of outreach events primarily geared towards K-12 students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This Hatch project has provided multiple opportunities for professional development, primarily through the training of scientists at the undergraduate, graduate, and postdoctoral level. The research detailed in this report and reflected in the Products section has been contributed to by five different PhD students, two postdoctoral scholars, and >10 undergraduate researchers. These trainees receive extensive instruction in all aspects of research, including experimental design, technical research skills (i.e., insect physiology, molecular biology, field work), data analysis (statistical analyses and bioinformatics), and scientific presentation (written and oral). The graduate students and postdocs also have opportunities to develop their teaching skills through guest lecturing in courses, serving as teaching assistants, and participating in teaching workshops at the university. Finally, all trainees receive extensive opportunities for professional development at scientific meetings. Many of the conference presentations in the Products section were given by students and postdocs, and our work has been presented at several national and international forums. How have the results been disseminated to communities of interest?Results have primarily been disseminated to communities of interest through peer-reviewed journal articles. All of the publications in the Products section are in reputable peer-reviewed journals with international scope. We also disseminate research through conference presentations, including regular presentations at the Entomological Society of America meeting and the Society for Integrative and Comparative Biology meeting. We have also presented work at several international forums, including the Scientific Council for Antarctic Research (SCAR) and the International Symposium on the Environmental Physiology of Ectotherms and Plants. Work from our lab is also disseminated through public outreach events, primarily at the K-12 level. We have led hands-on insect-themed activities at several schools in the Fayette County Public Schools district, and we have led community events through the Living Arts and Science Center in Lexington, KY. Finally, we designed an implemented a week-long "Genetics Bootcamp" for local high school students in which they learned skills in insect collection and molecular barcoding. 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: An insect's cold tolerance, or its ability to survive at low temperatures, is a critical determinant of species distribution. Understanding insect cold tolerance is critical for understanding population dynamics for pest species and for predicting insect responses to climate change. In our project, we investigate several aspects of insect overwintering biology, including how insects deal with short-term fluctuations in temperature, the genetic architecture of thermal tolerance in insects, and the overwintering biology of important pest and beneficial species. We are also investigating the mechanisms of extreme freeze tolerance in insects, using a combination of genomics and physiology. This work is important for understanding how insects evolve to cope with extreme climates, and there may be practical applications of this work for improving cryopreservation efforts. Some of the most impactful outcomes of our project include: 1) We showed that cultured cells are capable of rapidly responding to low temperature, indicating that cold acclimation occurs (at least in part) at the cellular level without input from the nervous or endocrine system. 2) Distinct cold tolerance traits are not correlated to one another across genotypes, indicating that these traits can evolve independently, and that multiple measures of cold tolerance must be accounted for when characterizing the overwintering biology of a particular insect. 3) Spotted wing drosophila has genetic variation in both cold tolerance and insecticide resistance. 4) While the Antarctic midge is highly freeze-tolerant, it is susceptible to winter warming, so this species may be vulnerable to climate change. Goal 1: Cold tolerance physiology in insects. Objectives 1-3: Mechanisms of rapid cold hardening. Throughout this project period we have increased our understanding of rapid cold hardening (RCH), an important adaptation in insects for coping with sudden changes in temperature. First, we showed that cultured Drosophila S2 cells are capable of undergoing RCH, extending upon our earlier work that isolated tissues are capable of RCH. In the Antarctic midge, B. antarctica (also a focal species for Goal 3), nonlethal freezing reduces locomotor ability and metabolic rate, causes damage to tissues, leads to protein denaturation, and leads to energetic deficits. However, all of these types of injury are at least partially attenuated by RCH, indicating that RCH protects insects from cold injury at several levels of organization. We are also conducting similar experiments in D. melanogaster, and these experiments suggest that RCH can also protect against cold stress-mediated declines in reproductive output. Overall this work in is important in showing that RCH plays an essentially role in preserving insect function under ecologically relevant temperature conditions. Objective 4: Genetic correlation of cold tolerance traits. We tested the hypothesis that cold tolerance traits independently vary across distinct genotypes of D. melanogaster. Specifically, for 12 lines of flies, we measured critical thermal minimum (CTmin), cold shock tolerance, chill coma recovery time, and changes in locomotor behavior after cold stress. These results indicated that these different metrics of cold tolerance are not correlated across lines and thus can evolve independently. Furthermore, neither baseline expression nor inducibility of two cold-stress associated genes (hsp70 and frost) were associated with cold tolerance, indicating that stress gene expression patterns are not predictive of cold tolerance. Objective 5: Local adaptation spotted wing drosophila. Spotted wing drosophila (SWD), D. suzukii, is major invasive fruit pest across the US. We established lines of SWD from across the US to test for variation in cold tolerance in insecticide resistance. Lines show considerable differences in both cold tolerance and insecticide resistance, indicating there is segregating for both traits within these populations. Using RNA-seq, we are currently investigating the gene expression mechanisms that underly variation in cold tolerance and insecticide resistance. Objective 6: Cold tolerance of winter active wolf spiders. Wolf spiders are important predators in both natural and agricultural ecosystems, but their overwintering biology has been largely uncharacterized. We demonstrated that these spiders are winter-active, which allows them to actively hunt in the winter, and as a result juvenile spiders grow during the winter. We also characterized cryoprotectant profiles in overwintering spiders and showed that they accumulate modest levels of several polyols and amino acids that likely protect against cold stress in the winter months. In a second study, we conducted a laboratory assessment of cold tolerance in wolf spiders. We showed that spiders have a lot CTmin relative to other temperate species, which likely facilitates their ability to remain active in the winter. We also showed that CTmin is plastic and responds to environmental temperature, such that winter warming could make spiders more susceptible to sudden cold snaps. Goal 2: Genomics of extreme freeze-tolerance. As detailed in previous progress reports, most of this work is now being conducted by collaborators. However, we are leading a genome sequencing project for the fly Chymomyza costata, which is the only insect capable of surviving exposure to liquid nitrogen. In collaboration with geneticists at Stanford, we have obtained a high-quality genome assembly for C. costata, and we are currently in the process of annotating this genome and testing hypotheses regarding the genomic mechanisms of extreme freeze tolerance. We have also recently collected RNA samples from each life stage, and these samples were sent away for transcriptome sequencing, so that we can improve our gene annotations. Goal 3: Evolutionary genetics and physiology of extreme adaptation in Antarctic insects. As detailed in previous progress reports, this aim has been delayed by the pandemic, as we were relying on international collaborators that were more disrupted by shutdowns. However, our collaborators have obtained genome sequences for Eretmoptera murphyi, and our lab will be collecting samples for transcriptomic analyses of freeze-tolerance in this species, and these sequences will also be used to improve genome annotation. While progress on genomics studies has been delayed, we have completed several studies on the physiology and ecology of Antarctic insects. We demonstrated that when larvae of B. antarctica are exposed to cold for long-periods of time (up to two weeks), it is more energetically costly to be frozen than it is to remain supercooled. Using laboratory simulations of ecologically relevant overwintering conditions, we showed that warm winters are detrimental for B. antarctica. Specifically, larvae experiencing a warm winter had lower survival and reduced energy stores relative to larvae in cold winter environments. In ongoing work, we are using RNA-seq to identify genes involved in recovery from freezing stress. At the ecological level, we identified environmental factors that govern the extremely patchy distribution of this species. Specifically, we showed that larvae tend to have larger populations when terrestrial algae is present, and that biotic factors play in important role in determining this species' distribution, in addition to the well-established role of abiotic conditions. Finally, we showed that there is considerable variation in microhabitat conditions over small spatial scales (i.e., hundreds of meters), and that this microclimate variation influences the metabolism, energetics, and phenology of midges living there. This study indicates that it is critical to consider fine-scale variation in temperature when making predictions about insect responses to climate change.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Teets, N.M., Meuti, M.E. 2021. Hello darkness, my old friend: A tutorial of Nanda-Hamner protocols. Journal of Biological Rhythms 36, 221-225. doi.org/10.1177/0748730421998469
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Littler, A.S., Garcia, M.J., Teets, N.M. 2021. Laboratory diet influences cold tolerance in a genotype-dependent manner in Drosophila melanogaster. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 257, 110948. doi.org/10.1016/j.cbpa.2021.110948
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Teets, N.M., Hayward, S.A.L. 2021. Editorial on combatting the cold: Comparative physiology of low temperature and related stressors in arthropods. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 257, 111037. doi.org/10.1016/j.cbpa.2021.111037
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Spacht, D.E., Gantz, J.D., Devlin, J.J., McCabe, E.A., Lee, R.E., Denlinger, D.L. Teets, N.M. 2021. Fine-scale variation in microhabitat conditions influences physiology and metabolism in an Antarctic insect. Oecologia, 197, 373-385. doi.org/10.1007/s00442-021-05035-1
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Dias, V.S., Caceres, A.G., Parker, A.G., Pereira, R., Demirbas-Uzel, G., Abd-Alla, A.M.M., Teets, N.M., Schetelig, M.F., Handler, A.M., Hahn, D.A. 2021. Mitochondrial superoxide dismutase overexpression and low oxygen conditioning hormesis improve the performance of irradiated sterile males. Scientific Reports 11, 1-15. doi.org/10.1038/s41598-021-99594-1
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Teets, N.M., Harner, D.H. Enhancing local environmental education programming through higher education research partnerships. Kentucky Association for Environmental Education webinar series, May 20, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Awde, D.N., Teets, N.M. Genetic variation in phenotypic plasticity of thermal limits in Drosophila melanogaster. Society for Integrative and Comparative Biology, January 2021. Presented virtually.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Unfried, L.M., Teets, N.M. Ability of RCH to protect against physiological damage from sublethal chilling in Drosophila melanogaster. Society for Integrative and Comparative Biology, January 2021. Presented virtually.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Perez-Galvez, F.R., Awde, D., McCabe, E.A., Teets, N.M. Computer assisted analysis to improve throughput and precision of knockdown time assays. Society for Integrative and Comparative Biology, January 2021. Presented virtually.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Teets, N.M., Spacht, D.E., Potts, L.J., Gantz, J.D., Lee, R.E., Denlinger, D.L. Microhabitat diversity influences physiology and phenology in an Antarctic insect. Society for Integrative and Comparative Biology, January 2021. Presented virtually.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Teets, N.M., Lecheta, M.C. Molecular regulation of diapause in two corn rootworm species (Diabrotica spp.). Entomological Society of America, Orlando, FL. November 19, 2020.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Our primary target audience for this reporting period was scientists working in the fields of insect physiology and egenetics. We published articles in several internationally recognized peer-reviewed journals, and research was disseminated in numerous conference presentations. These products are detailed in the Products section. We also reached the general public through various outreach and education presentations, particularly presentatiosn geared towards K-12 students. Changes/Problems:We are having issues receiving our phosphoproteomics data for Goal 1. Despite several attempts to contact the company, I can't get a response from them. We are still hopeful they will come through, but if not, we won't be able to complete Goal 1, Objective 1 as planned. This problem has delayed progress on other parts of this goal, but we have switched personnel from another project to make sure we make progress on this Goal in the coming year. What opportunities for training and professional development has the project provided?The project has provided multiple opportunities for professional development. A lab technician is supported directly by Hatch funds, and she is also enrolled part-time as a PhD student. She is receiving extensive training in insect husbandry, field work, laboratory methods, etc., and she is taking courses through the entomology PhD program at University of Kentucky. In addition, several graduate students and postdocs contribute to parts of the research accomplishments presented in this report. In total, two PhD students and two postdocs are working directly on the work presented here. These trainees are gaining experience in experimental design, physiology, molecular biology, data analysis, and scientific communication, among other skills. Finally, our lab also includes undergraduate researchers in our projects, and several undergraduate students from the College of Agriculture have contributed to this work. Students participating in these projects were also provided opportunities to present their work at national meetings (for example, the Entomological Society of America and the Society for Integrative and Comparative Biology) and publish papers in reputable journals. See Products for details. How have the results been disseminated to communities of interest?Results have primarily been disseminated through scientific publications and presentations. Results were published in reputable journals such as Journal of Experimental Biology, Journal of Insect Physiology, Evolution, and others. Presentations were giving at the Entomological Society of America annual meeting, the Society for Integrative and Comparative Biology annual meeting, and the Saltiel Life Sciences Symposium. We also disseminated information to the general public through several outreach presentations, primarily to K-12 students at local schools. What do you plan to do during the next reporting period to accomplish the goals?Goal 1, Objective 1: This objective is mostly complete. We are still working with the phosphoproteomics company to get our results, which will allow us to complete the objective. Goal 1, Objectives 2-3: In the coming year, we will also begin new experiments on the role of p38 MAPK in rapid cold hardening. Specifically, we will use p38 mutants and RNAi lines to test the hypothesis that reduced p38 expression prevents rapid cold hardening in flies. Goal 1, Objective 4: This Objective is complete Goal 1, Objective 5: The experiments for these objectives are complete. In the coming year, we will conclude the data analysis for the RNA-seq portions of these studies and submit these studies for publication. Goal 1, Objective 6: This Objective is complete and we are in the final stages of submitting this work for publication. Goal 2, Objective 1: This work is no longer part of our project and is being completed by collaborators. Goal 2, Objective 2: For this Objective, we will complete genome assembly and annotation for Chymomyza costata. The genome will be annotated with the MAKER pipeline, which uses several lines of evidence to delineate gene models in a genome. We will also use Gene Ontology enrichment analysis to test for evidence of gene expansion/retraction in this species relative to other drosophilids. Finally, we will use Ka/Ks analysis to identify classes of genes that are under selection and may contribute to the extreme freeze tolerance of this species. Goal 3, Objective 1: As discussed above, this work was delayed due to the covid pandemic. In the coming year, assuming a postdoc can be hired, the sequencing data for E. murphyi and B. albipes will be assembled using the de novo assembler Velvet, and we will use the MAKER pipeline to annotate these genomes. Goal 3, Objective 2: Once the genomes are assembled and annotated, will use GO enrichment analysis, Ka/Ks analysis, and synteny analysis to identify genomic-level changes between various species of cold-adapted midges.

Impacts
What was accomplished under these goals? Impact: The ability to survive cold and other stressors is a critical determinant of insect distributions. We seek to understand the genetic and physiological basis of cold tolerance in insects so that we can 1) predict species distributions in response to changing environments, and 2) use this information to inform applications like cryopreservation. During this reporting period, we have made important progress in both of these areas. For #1, we published two studies on the genetic basis of thermal tolerance in flies. In one, we showed that distinct cold tolerance traits are not correlated, indicating these physiological responses to cold can evolve independently. Thus, when using information on cold tolerance to predict species distributions, it is important to consider several distinct cold tolerance traits simultaneously, rather than relying on a single measure. Also, we completed a large study of the genetic basis of cold and heat tolerance in flies. In brief, we showed that there is significant overlap between alleles that predict variation in thermal tolerance and genes that are differentially expressed in direct response to thermal stress. In addition, this work allowed us to identify several important candidate genes that we can potentially use to disrupt or augment thermal tolerance. For #2, we have been making progress on understanding the fundamental processes involved in freeze tolerance. We have shown that ice formation in insects is energetically costly, which is a potential barrier to successful cryopreservation of other organisms. In ongoing work, we have identified key changes in gene expression that accompany freezing (but not cold per se), which will allow us to identify the key genes that involved in surviving ice formation. Finally, during this reporting period, we completed an experiment on long-term freezing survival, to identify mechanisms that allow Antarctic insects to survive the long, cold winters. Previous work in this area has only used short-term freezing bouts, so while these long-term freezing experiments are challenging and time-consuming, they are an important step towards achieving perpetual cryopreservation. Goal 1: Objective 1: This objective is mostly complete. Our work on rapid cold hardening in Drosophila cells was published in Journal of Experimental Biology. Objectives 2-3: To set the stage for these objectives, we have been conducting experiments to demonstrate that rapid cold hardening protects against sublethal cold injury. In short, when flies are directly cold shocked at a nonlethal temperature, there is a damage to tissues and a reduction in fecundity, but rapid cold hardening prior to cold shock prevents or reduces these damages. These experiments will provide conditions for mechanistic work, because it is easier to study physiological mechanisms in live flies. The mechanistic experiments with p38 and calcium signaling have been delayed due to issue with the company that ran our phosphoproteomics analyses; see "Changes/Problems." These experiments will be restarted in the coming year with a postdoc who was recently switched from a different project, so we hope to make significant progress on these objectives in the coming year. Objective 4: Genetic correlation of different cold tolerance traits. This Objective is complete. Objective 5: Local adaptation in spotted wing drosophila. In the previous reporting period, we generated isogenic lines of spotted wing drosophila and showed that these lines have considerable variation in cold tolerance. Using RNA-seq, we measured gene expression differences between the most susceptible and hardy lines. The susceptible lines show bigger changes in gene expression when challenged by cold, and we are currently working through the specific functions of those genes. In addition, we measured the susceptibility of these lines to a commonly used insecticide, Mustang Maxx, and there was considerably variation in the ability of these lines to survive an insecticide challenge. We also used RNA-seq to measure gene expression between lines with the highest and lowest susceptibility to insecticides, and we see that the resistant lines have higher expression of detoxification genes, such as cytochrome P450 genes. We are currently finishing these analyses and preparing these manuscripts for publication. Objective 6: Cold tolerance of winter active wolf spiders. This Objective is complete, and we are preparing the final manuscript for publication. Goal 2: Objective 1: As discussed in the previous annual report, we are no longer conducting these experiments; the responsibilities have shifted to our collaborators. Objective 2: We have identified some collaborators at Stanford who are helping with this Objective. They have provided Nanopore long-read DNA sequences and a preliminary genome assembly. Our lab is using available RNA-seq data and in silico annotation software to generate gene models for this species. Goal 3: Objective 1-2: Progress on these Objectives was delayed due to covid-19. Our collaborators in the UK were in the process of hiring a postdoctoral associate to conduct these analyses, but hiring was shut down due to the pandemic. Nonetheless, we have data in hand for this Objective, and we expect to make swift progress when the pandemic restrictions are lifted. In addition to the stated goals and objective, our new NSF funding has allowed us to expand on this work. Specifically, during the current reporting period, we made the following accomplishments: 1) We collected samples of B. antarctica from ~20 islands around the Antarctic Peninsula for a large-scale population genomics study. This sample set is the largest population genetics collection for an Antarctic arthropod and will greatly expand our understanding of local adaption, gene flow, and biogeography. 2) We conducted a study on multiple stress tolerances in B. antarctica. We defined limits of tolerance to cold, heat, desiccation, and osmotic stress, and we will use RNA-seq to compare gene expression responses to all stresses. These experiments will allow us to determine the extent to which conserved and stress-specific molecular pathways contribute to the ability of B. antarctica to survive a variety of environmental extremes. 3) We conducted experiments on responses to long-term freezing and long-term recovery in B. antarctica. Early results suggest that mild winters are detrimental to larvae, as survival was considerably lower when larvae are subjected to a warm simulated Antarctic winter.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Garcia, M.J., Littler, A.S., Sriram, A., Teets, N.M. 2020. Distinct cold hardiness traits independently vary across genotypes in Drosophila melanogaster. Evolution 74, 1437-1450. doi.org/10.1111/evo.14025
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Awde, D.N., Fowler, T.E., Galvez-Perez, F., Garcia, M.J., Teets, N.M. 2020. High-throughput assays of critical thermal limits in insects. Journal of Visualized Experiments, e61186. doi:10.3791/61186
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Potts, L.J., Garcia, M.J., Teets, N.M. 2020. Chilling in the cold: Using thermal acclimation to demonstrate phenotypic plasticity in animals. CourseSource. doi.org/10.24918/cs.2020.21
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Teets, N.M., Gantz, J.D., Kawarasaki, Y. 2020. Rapid cold hardening: Ecological relevance, physiological mechanisms and new perspectives. Journal of Experimental Biology 223, jeb203448. doi:10.1242/jeb.203448
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Nadeau, E.A.W., Teets, N.M. 2020. Evidence for a rapid cold hardening response in cultured Drosophila S2 cells. Journal of Experimental Biology 223, jeb212613. doi:10.1242/jeb.212613
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Teets, N.M., Dalrymple, E.G., Hillis, M.H., Gantz, J.D., Spacht, D.E., Lee, R.E. Jr., Denlinger, D.L. 2020. Changes in energy reserves and gene expression elicited by freezing and supercooling in the Antarctic midge, Belgica antarctica. Insects 11, 18. doi.org/10.3390/insects11010018
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Mercer, N.H., Teets, N.M., Bessin, R.T., Obrycki, J.J. 2020. Supplemental foods affect energetic reserves, survival, and spring reproduction in overwintering adult Hippodamia convergens (Coleoptera: Coccinellidae). Environmental Entomology 49, 1-9. doi.org/10.1093/ee/nvz137
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Potts, L.J., Koat�l, V., Simek, P., Teets, N.M. 2020. Energy balance and metabolic changes in an overwintering wolf spider, Schizocosa stridulans. Journal of Insect Physiology 126, 104112. doi.org/10.1016/j.jinsphys.2020.104112
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Potts, L.J., Gantz, J.D., Kawarasaki, Y., Philip, B.N., Gonthier, D.J., Law, A.D., Moe, K., Unrine, J.M., McCulley, R.L., Lee, R.E. Jr., Denlinger, D.L., Teets, N.M. 2020. Environmental factors influencing fine-scale distribution of Antarcticas only endemic insect. Oecologia 194, 529-539. doi.org/10.1007/s00442-020-04714-9
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lecheta, M.C., Awde, D.N., OLeary, T.S., Unfried, L.N., Jacobs, N.A., Whitlock, M.H., McCabe, E., Powers, B., Bora, K., Waters, J.S., Axen, H.J., Frietze, S., Lockwood, B.L., Teets, N.M., Cahan, S.H. 2020. Integrating GWAS and transcriptomics to identify the molecular underpinnings of thermal stress responses in Drosophila melanogaster. Frontiers in Genetics 11, 658. doi.org/10.3389/fgene.2020.00658
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Teets, N.M. Cryopreservation of multicellular animals: Lessons from extreme insects. University of Michigan Life Sciences Symposium, Ann Arbor, MI. September 30, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bredlau, J.P.!, Perez, F.P., Teets, N.M. Potential for resistance to conditionally lethal transgenes used for Sterile Insect Technique. Entomological Society of America, St. Louis, MO, November 17, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Teets, N.M., Dalrymple, E.G., Hillis, M.H., Lee, R.E. Jr., Denlinger, D.L. To freeze or not to freeze: Cold tolerance strategies in an Antarctic midge. Society for Integrative and Comparative Biology, Austin, TX, January 7, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Littler, A., Garcia, M.J., Teets, N.M. Does a well-balanced diet keep you going when the going gets cold? Society for Integrative and Comparative Biology, Austin, TX, January 6, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Awde, D.N., Lecheta, M.C., Unfried, L.N., Jacobs, N.A., Powers, B., Bora, K., Waters, J.S., Axen, H.J., Frietze, S.E., Lockwood, B.L., Cahan, S.H., Teets, N.M. Genetic mechanisms of basal thermal tolerance in Drosophila melanogaster. Society for Integrative and Comparative Biology, Austin, TX, January 4, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Garcia, M.J., Teets, N.M. Genetic variation and molecular regulation of cold hardiness in spotted wing drosophila. Society for Integrative and Comparative Biology, Austin, TX, January 4, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Teets, N.M., Lecheta, M., Awde, D. Genetic architecture of thermal tolerance in Drosophila melanogaster. Entomological Society of America, St. Louis, MO, November 19, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lecheta, M., Teets, N.M. Transcriptional mechanisms of diapause in the corn rootworm complex. Entomological Society of America, St. Louis, MO, November 19, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Garcia, M.J., Teets, N.M. Genetic variation and molecular regulation of cold hardiness in spotted-wing drosophila. Entomological Society of America, St. Louis, MO, November 19, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Littler, A., Teets, N.M., Garcia, M.J. Does a well-balanced diet keep you going when the going gets cold? Entomological Society of America, St. Louis, MO, November 18, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Potts, L.J., Teets, N.M. Overwintering spiders: Biochemical and metabolic responses to the winter season. Entomological Society of America, St. Louis, MO, November 18, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Nadeau, E.A.W., Obrycki, J., Teets, N.M. Transcriptional regulation of reproductive diapause in the convergent lady beetle. Entomological Society of America, St. Louis, MO November 18, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Unfried, L.N., Teets, N.M. Benefits of rapid cold hardening at sublethal temperatures in Drosophila melanogaster. Society for Integrative and Comparative Biology, Austin, TX, January 5, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Helms Cahan, S., Frietze, S.E., Gerrard, D.L., Bora, K., Kaplan, I., Perez, M., Lockwood, B.L., Teets, N.M., Waters, J.S., Axen, H.J. Developmental temperature alters brain gene expression in adult Drosophila melanogaster. Society for Integrative and Comparative Biology, Austin, TX, January 4, 2020.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Our primary target audience for this reporting period was scientists working in the fields of insect physiology and egenetics. We published articles in several internationally recognized peer-reviewed journals, and research was disseminated in numerous conference presentations. These products are detailed in the Products section. We also reached the general public through various outreach and education presentations, particularly presentatiosn geared towards K-12 students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided several opportunities for professional development. Several PhD students have worked on the objectives of this project, one of which received her own USDA fellowship to continue her work. This project has also provided opportunities for postdoctoral scholars and a full-time research technician. Several of the publications and presentations in this report include student authors. This project has also provided opportunities for undergraduate and high school researchers. One of these students has presented research at a national meeting and will be coauthor on two publications. All students, both at the graduate and undergraduate level, have received essential training in insect physiology, genetics, molecular biology, and statistical analyses. Students and postdocs are also involved in writing and presenting results from the project. How have the results been disseminated to communities of interest?Results have been disseminated through scientific publications and presentations. Results were published in several respected and well-read journals, including Journal of Experimental Biology and Proceedings of the Royal Society B. Presentations were given at several venues, including the Entomological Society of America annual meeting and the Society for Integrative and Comparative Biology annual meeting. Information was also presented to stakeholders through K-12 outreach initiatives and an invited presentation to the Household Commercial Products Association annual meeting. What do you plan to do during the next reporting period to accomplish the goals?For Goal 1, we should obtain our phosphoproteomics results from the proteomics company. These results will then be synthesized and published in a scientific journal. These results will also generate specific hypotheses that can be used to complete Objectives 2 and 3. For the rapid cold hardening objectives, we are also testing the ability of rapid cold hardening to protect against sublethal cold in fruit flies. This work will increase the ecological relevance of this work and provide new approaches for testing mechanistic hypotheses. Objective 4 of Goal 1 is complete. Objective 5 is nearly complete; we have tanscriptomics data in hand and will analyze these results and publish them along with the phenotypic data. These results will be the first attempt to identify genetic factors that explain variation in cold tolerance in different lineages of spotted wing drosophila and will provide clues into how this species has been able to invade regions much colder than its native range. The experiments for Objective 6 on spider overwintering biology are complete, and this year we will publish these results in two papers. For Goal 2, we are continuing to work on the genome of Chymomyza costata. Progress has been slow, but the student in charge of this objective has completed her training and should be able to make substantial progress this year. We are also going to receive some long-read sequence data from a collaborator, which should allow us to improve our initial assembly, which was highly fragmented. This year, we will complete de novo assembly with all sequence reads and begin annotating using with in silico methods. We will also use publicly available RNA-seq data for this species to improve assembly and annotation. For Goal 3, we will assemble and annotate the genomes of E. murphyi and B. albipes. We will also obtain sequence data for a third species, Halirytus magellanicus. Raw Illumina reads will be assembled with de novo assembly methods, and gene models will be annotated with in silico methods. We will map orthologs between these three species and our focal species, the midge Belgica antarctica, and these midge genomes will then be compared for differences in gene content and evolution of protein sequences.

Impacts
What was accomplished under these goals? Impact: Cold tolerance is a significant determinant of the current and future distributions of insects, including both beneficial and pest species.Furthermore, some insects are capable of extreme freeze tolerance, and knowledge gained from these insects can inform cryopreservation strategies. This project deals with several aspects of the overwintering biology of insects. We investigate the mechanisms by which insects cope with sudden changes in temperature, including the cell biology of this process. We also investigate the evolutionary genetics of cold tolerance in model insects, which will help our ability to predict evolutionary responses to changes in environmental temperature. Our project also includes investigations of cold tolerance in economically important species, like invasive fruit pests and spiders that function as biological control agents. Our second two objectives deal with the capacity for extreme freeze tolerance in certain insects. We investigate the genetic and physiological basis of freeze tolerance in several species of cold-adapted flies, with the hopes that this information can be used to store other animals (for example important insect strains or even human organs) in the cold. During this reporting period, we made several important discoveries in these areas. First, we showed that insect cells cultured outside of the body are capable of sensing and physiologically responding to cold, which opens new doors to investigate the cell biology of cold tolerance. Second, we showed that different cold tolerance traits in flies are not phenotypically correlated, suggesting that the evolution of cold tolerance is more complicated than we predicted. Third, we showed that the cold tolerance of an invasive fruit fly is strongly dependent on its genetic makeup, and we are using this information to identify the genetic processes that determine cold tolerance in this economically important pest. Fourth, we provided the first comprehensive characterization of overwintering biology for wolf spiders in North America, which will help understand how these ecologically important spiders cope with winter conditions and respond to climate change. Finally, for our work on freeze tolerance, we have identified a way that insects can rapidly enhance their freeze tolerance, and we are working on sequencing the genomes of several freeze-tolerant species to allow for an evolutionary genomics approach to understanding the biological basis of freeze-tolerance. Together, the results of this project have significantly enhanced our understanding of the physiology and evolutionary genetics of cold tolerance, which is an important physiological trait for determining the geographic range of insect species. Goal 1: Objective 1:To support this objective, we recently completed our study showing that cultured Drosophila cells can undergo rapid cold hardening. This work was submitted for publication to the Journal of Experimental Biology, and it will be published pending minor revisions. These same conditions were used to generate samples for phosphoproteomics, so that we can determine the extent to which calcium-dependent signaling mechanisms are activated by cold in Drosophila cells. We expected to have these results, but the company we are working with has been delayed in finished our analyses. Nonetheless, the finding that Drosophila S2 cells are capable of RCH will create many new directions of research for this field. Objectives 2-3: The phosphorpteomics results we are still waiting for are critical for continuing this objective. Objective 4: Genetic correlation of different cold tolerance traits. This Objective is completed and is in revision for publication. Objective 5: Local adaptation in spotted wing drosophila. We created 14 isogenic lines and tested the cold tolerance of these lines. Cold tolerance significantly varies across these lines, indicating that cold tolerance has a strong genetic component in this species. For the lines with the highest and lowest cold tolerance, we conducted RNA-seq and flies to quantify gene expression before and after a cold stress. There were many differences in gene expression between the cold hardy and susceptible lines, and we are working through these results to identify the gene functions that contribute to variation in thermal tolerance in this economically important invasive species. Objective 6: Cold tolerance of winter-active wolf spiders. We have completed two studies on the winter biology of wolf spiders. In the first, we assessed biochemical changes in field-collected overwintering wolf spiders, Schizocosa stridulans. Spiders were collected each month from October to March, and we assessed growth rates, energy reserves, and cryorprotectant profiles. The spiders grew considerably over the winter, providing support for the hypothesis that winter activity allows for somatic growth and a jumpstart on spring reproduction. However, lipid energy stores decreased over time, suggesting spiders either have difficulty maintaining energy stores over the winter, or there are phenological changes in lipid content as spiders grow and mature. We also identified a suite of cryoprotectants used by these spiders to cope with winter conditions. In a second study, we simulated three overwintering environments in the lab, a normal winter, a warmer winter, and a warmer and more variable winter. The warmer winter environments led to plastic changes in the spiders' ability cope with extreme low temperatures, and winter warming led to a significant decrease in carbohydrate energy stores. Thus, while warmer winters may extend the growing season for spiders, they may leave them more susceptible to cold snaps and cause reductions in certain energetic stores, depending on prey availability. Outside of these stated objectives, we have pursued other questions related to rapid cold hardening that are relevant to this project. First, while rapid cold hardening has a well-defined role in preventing mortality from cold, its ability to protect against nonlethal cold stress has received less attention. To test the hypothesis that rapid cold hardening protects against sublethal cold injury, we exposed larvae of the Antarctic midge, Belgica antarctica, to three conditions: Control, direct freezing, and rapid cold hardening before freezing. Compared to larvae that were directly frozen, larvae that were treated with rapid cold hardening prior to freezing recovered more quickly, had higher metabolic rates, reduced tissue damage, reduced protein damage, and improved energy balance. In addition, we showed that multiple environmental cues can trigger rapid cold hardening. In addition to cold, rapid cold hardening can be triggered by desiccation, heat, osmotic stress, UV radiation, and changes in pH. This rapid cross-tolerance to distinct stressors is important because environmental stressors are rarely experienced in isolation. Goal 2: Objective 1: As discussed in the previous annual report, we are no longer conducting these experiments; the responsibilities have shifted to our collaborators. Objective 2: We are continuing to work on assembling and annotating the genome to support this objective. In addition to sequence data we have previously obtained, we are working with some collaborators to generate long-read sequences to improve our assembly. Goal 3: Objective 1: We have obtained NSF funding to travel to Antarctica and support this project. In this new project, we will work with collaborators in the UK to sequence these genomes. The genome for E. murphyi has been sequenced and is currently being analyzed. The specimens for B. albipes were collected by a collaborator in France and will be sequenced shortly. Objective 2: This Objective will begin in earnest this year, as it is a component of our NSF-funded project.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Teets, N.M., Kawarasaki, Y., Potts, L.J., Philip, B.N., Gantz, J.D., Denlinger, D.L., Lee, R.E. 2019. Rapid cold hardening protects against sublethal freezing injury in an Antarctic insect. Journal of Experimental Biology 222, jeb206011.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Teets, N.M., Dias, V.S., Pierce, B.K., Schetelig, M.F., Handler, A.M., and Hahn, D.A. 2019. Overexpression of an antioxidant enzyme improves male mating performance after stress in a lek-mating fruit fly. Proceedings of the Royal Society B 286, 20190531.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kawarasaki, Y., Teets, N.M., Philip, B.N., Potts, L.J., Gantz, J.D., Denlinger, D.L., Lee, R.E. 2019. Characterization of drought-induced rapid cold-hardening in the Antarctic midge, Belgica antarctica. Polar Biology 42, 1147-1156.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Teets, N.M. Winter climate change and insects: Is warmer always better? Household Commercial Products Association Impact2019 Conference, Washington, DC, May 2, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Teets, N.M., Garcia, M.J., and Nadeau, E.AW. From cells to populations: Towards an integrative understanding of how insects cope with low temperature stress. P-IE Section Symposium: Stressors Across Space and Time: Energy Sources, Enemies, and Environmental Influences. Entomological Society of America annual meeting, Vancouver, BC, November 14, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Teets, N.M. Nonlethal freezing injury in the Antarctic midge Belgica antarctica. 8th International Symposium on the Environmental Physiology of Ectotherms and Plants, Buenos Aires, Argentina, August 1, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Teets, N.M., Kawarasaki, Y., Potts, L.J., Gantz, J.D., Philip, B.P., Denlinger, D.L., Lee, R.E. Rapid cold hardening provides sublethal benefits in an Antarctic extremophilic insect. Society for Integrative and Comparative Biology Annual Meeting, Tampa, FL, January 6, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Teets, N.M., Dias, V., Schetelig, M.F., Handler, A.M., Hahn, D.A. Making macho males by transgenic overexpression of a mitochondrial antioxidant enzyme. Society for Integrative and Comparative Biology Annual Meeting, Tampa, FL, January 6, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Garcia, M.J., Srirarm, A., Littler, A., Teets, N.M. Genetic variance in cold tolerance and its molecular underpinnings. Society for Integrative and Comparative Biology Annual Meeting, Tampa, FL, January 6, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Perez-Galvez, F.R., Teets, N.M. Genetic and environmental factors influencing the efficacy of transgenic Sterile Insect Technique. Society for Integrative and Comparative Biology Annual Meeting, Tampa, FL, January 5, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kawarasaki, Y., Teets, N.M., Philip, B.N., Potts, L.J., Gantz, J.D., Denlinger, D.L., Lee, R.E. Characterization of drought-induced rapid cold-hardening in the Antarctic midge, Belgica antarctica. Society for Integrative and Comparative Biology Annual Meeting, Tampa, FL, January 5, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Littler, A.S., Srirarm, A., Garcia, M.J., Teets, N.M. Out in the cold: Genetic correlation of cold tolerance traits in Drosophila melanogaster. Society for Integrative and Comparative Biology Annual Meeting, Tampa, FL, January 5, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Potts, L.J., Teets, N.M. Overwintering spiders: Physiological responses to the winter season. Society for Integrative and Comparative Biology Annual Meeting, Tampa, FL, January 4, 2019.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Garcia, M. and Teets, N. (2019). Cold stress results in sustained locomotor and behavioral deficits in Drosophila melanogaster. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology. 331. 10.1002/jez.2253.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Teets, N.M. Mechanisms of environmental stress tolerance in Antarcticas only endemic insect. Arthropod Genomics Symposium, Manhattan, KS, June 13, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Teets, N.M., Kawarasaki, Y., Potts, L.J., Gantz, J.D., Philip, B.N., Denlinger, D.L., Lee, RE. Sublethal benefits of rapid cold hardening in Antarcticas only endemic insect. Entomological Society of America Annual Meeting, Vancouver, BC, November 14, 2018.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Our primary target audience for this reporting period was other scientists working the fields of insect physiology, ecology, and genetics. Our primary route of dissemination for this reporting period was scientific conferences, as work supported by this Hatch project was presented at several regional and nationalvenues. The PI and graduate student also reached the general public through various outreach presentations. Changes/Problems:Goal #2 is a collaborative effort with a lab in the Czech Republic, and the division of labor for this collaborationhas changed slightly. Our lab will no longer be doing gene expression analysis in C. costata, the species of interest for this Goal. Our efforts will continue to focus on assembling and annotating a genome so that we can eventually apply genome editing techniques to this species. As such. Goal #2, Objective #1 is no longer being undertaken in our lab, with the exception of building a genome that will benefit the RNA-seq experiments being conducted in the Czech Republic. What opportunities for training and professional development has the project provided?The project has provided several opportunities for professional development. Two PhD students and a postdoctoral scholar contributed to this project in the past reporting period. All three have received training in laboratory methods (e.g., physiology, genetics, bioinformatics), data analysis, and scientific presentation (written and oral). All three trainees have also attended national conferences to present their work. Our laboratory has also been actively involved in undergraduate research. Several students have contributed to this work, including two who conducted their senior research projects in our lab. We are also involved in a research program for local high school students, and we have mentored two high school students as part of these efforts. These undergraduate and high school students have received invaluable research training and the opportunity to interact with graduate students, postdocs, and PIs involved in the project. How have the results been disseminated to communities of interest?The results have been primarily disseminated through scientific publications and presentations. Work was published in international peer-reviewed journals and at several scientific venues, including the Entomological Society of America annual meeting. Our group also dissemintated information through community outreach activities. In particular, members of our group designed and implemented lessons at the Living Arts and Science Center's Science Explorers program, an after school STEM program for underpriveleged schools. Results from Goal #3 in particular were highlighted as part of this program. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Objective 1:We have submitted samples for phosphoproteomic profiling to identify key signaling processes that are activated by rapid cold hardening. We expect those data soon, and we will conduct bioinformatic analyses to characterize the key proteins and biological processes that are activated by rapid cold hardening. Candidate genes will be testedin vivousing reverse genetic techniques in flies. Objective 2:We will use RNAi and null mutatnts to test the hypothesis that p38 MAP Kinase is an essential signaling mechanism for rapid cold hardening. We will also manipulate p38 signaling pharmacologically in isolated tissues and cell culture to test these hypotheses at the cellular level. Objective 3:We are still waiting on results from Objectives 1 and 2 from this Goal to begin this Objective. Objective 4:This objective is nearly complete. We will write up our results from this Objective (summarized above) and submit for publication. Objective 5:We are currently measuring thermal tolerance and insecticide resistance in the 13 isogenic spotted wing drosophila lines our lab has generated. We will use these resutls to select lines and conditions for RNA-seq analysis. Speficially, we will select the two most and two least cold tolerant lines and measure gene expression before and after cold stress. These experiments will identify the key expression differences that are associated with phenotypic variation in cold tolerance. Similar experiments will be conducted for insecticide resistance by measuring gene expression before and after insecticide exposure in lines with high resistance to insecticides and lines with low resistance to insecticides. Taken together, these experiments will identify key genetic process that drive variation in thermal tolerance and insecticide resistance, two field-relevant traits that contribute to the success of this invasive pest. Objective 6:We will conduct metabolomics analyses of spiders collected over the winter to identify cryoprotective compounds present in these species. We will also repeat the witner warming experiments to test the hypothesis that warmer winters impair the cold tolerance, growth, and energy balance of winter-active wolf spiders. Goal 2: Objective 1:As explained above, these experiments are being conducted by our collaborators in the Czech Republic. We have identified these changes in the "Changes/Problems" section. Objective 2:We are cotinuing to build a genome for C. costata. Once a high quality genome is in hand, we will identify potential genes to mutate with CRISPR/Cas9 to develop gene editing capabilities in this species. Goal 3: Objective 1:Genome sequencing for both species is underway. Also forB. antarcitcawe have collected RNA-seq data of larvae that were both frozen and supercooled at the same temperature. We will be working through these data to gain a comprehensive understanding of the molecular processes that are specifically activated by freezing. These results will indicate the unique adaptations that are required for freezing tolerance and provide clues into the mechanisms that have allowedB. antarcticaadapt to Antarctica's harsh terrestrial environments. Objective 2:We are waiting for genome sequence data from collaborators to begin these analyses. Once we receive data, we will use dn/dS analysis to identify genes that are under selection. We will also use Gene Ontology enrichment analyses to identify classes of genes that have undergone expansion or reduction in the Antarctic species.

Impacts
What was accomplished under these goals? Impact: Our project has three specific goals, and the impacts associated with each of those goals areas follows: 1) Goal 1: Adaptations for rapid change in temperature. We demonstrated that cultured insect cells are capable of rapid cold hardening, a physiological response to sudden drops in temperature. This result is exciting because it indicates that rapid cold hardening occurs at the level of individual cells and doesn't require input from the nervous systems or hormones. This knowledge will allow us to generate identify the underliying cellular mechanisms of rapid cold hardening. For this Goal, we also showed that molecular responses to cold stress show significant genetic variation, indicating that different genotypes may use different mechanisms to cope with cold stress.2) Goal 2: Understand the genetic mechanisms of freeze tolerance. We are working through a genome assembly for Chymomyza costata, the most freeze-tolerant insect known to scientists. When this objective is completed by the end of this Hatch project, it will allow us to test in-depth hypotheses regarding the genetic nature of freeze tolerance. 3) Goal 3: Genomic mechanisms of extreme adaptation in insects. We showed that rapid cold hardening (also a topic of Goal 1) protects midges from sublethal freezing injury. We have also collected RNA-seq data from midges that are both frozen and supercooled at the same temperature, so that we can identify the specific molecular processes that are activated by freezing. Preliminary analyses indicate that distinct transcriptional events occur when midges are frozen, but that most of the molecular activity is occuring during recovery from freezing (rather than during the freezing event). Below are details regarding the specific accomplishments for each Goal and Objective. We have included some additional Objectvies that are not listed above but were added in last year's progress report. Goal 1: Objective 1: Test the hypothesis that calcium signaling pathways activated by low temperatures regulate rapid cold hardening. For this Objective, we have been working to establish conditions for eliciting rapid cold hardening inDrosohpila S2 cells. We demonstrated that when cells are exposed to a 2 h cold shock, there is a progressive reduction in viability as temperature decreases, and minimum viability is reached at -8ºC. However, when cells are first exposed to 4ºC for 2 h and then dropped to -8ºC for 2 h, viability returns to control levels. Thus, much like whole insects, cultured cells are able to detect and quickly respond to changes in temperature. We have recently submitted samples for phosphoproteomics analysis to identify and quantify the cell signaling processes that are activted during rapid cold hardening. In the upcoming reporting period, we will test the function of these identified proteinsin vivousing reverse genetic techniques. These results are also critical for Objectives 2-3 of this Goal. Objective 2:Results from Objective 1 also support the upcoming activities for Objective 2. Objective 3:Results from Objective 1 also support the upcoming activities for Objective 3. Objective 4: Genetic correlation of different cold tolerance traits.One of the challenges of overwintering biology is selectingan appropriate measure of cold tolerance that is robust and repeatable yet ecologically relevant. As such, several metrics of cold tolerance have been developed that attempt to simulate different aspects of cold stress. Here, using distinct isogenic lineages of flies, we show that there is little phenotypic correlation betweenthese different traits, suggesting they have disctinct underlying mechanisms and may have the capacity to independently evolve. For this Objective we also assessed the extent to which well-characterized molecular responses to cold stress are associated with phenotypic variation in cold tolerance. Specifically, we measured mRNA expression of 70-kDa heat shock protein (hsp70) and Frost before and after cold stress in 12 distinct fly lines. Both of these genes have a well-defined role in cold stress responses. However, expression of these genes showed little correlation with cold tolerance across these lines. We did observe that induction of both genes was negatively associated with critical thermal minimum, which indicates that genotypes that are more resistant to cold stress have a weaker molecular response to an equivalent stressor. Overall, these results show that molecular and physiological responses to cold show significant variation across genotypes, which is an important consideration when modeling the effects of cold stress on insect populations. Objective 5: Local adaptation in spotted wing drosophila.For this objective, we have finished generating the isogenic lines needed for our project. A total of 13 isogenic, isofemale lines have been established that were originally collected from several locations in Kentucky, Florida, and Wisconsin. We are currently measuring thermal tolerance and insecticide resistance in these lines, and in the upcoming reporting period we will use an RNA-seq candidate gene approach to identify the key molecular processes that drive variation in thermal tolerance and insecticide resistance in this economically important, globally invasive insect pest. Objective 6: Cold tolerance of winter-active wolf spiders. We have continued our work on the overwintering biology of these important arthropod predators. The spiders are able to maintain activity until around -3ºC, which is impressive for arthropods at KY latitudes. This adaptation allows them to maintain activity in the winter and take advantage of feeding opportunites when most arthropods are dormant. We simulated three different winter conditions in the laboratory, and thus far we haven't observed any evidence that winter warming is detrimental to spider physiology or development. We are repeating these winter warming experiments this coming year to increase sample sizes. Goal 2: Objective 1:Use RNA-seq to identify candidate mechanisms of freeze tolerance in the drosophilid fly,Chymomyza costata,which is capable of surviving at liquid nitrogen temperatures.Our collaborators int he Czech Republic are coordinating this objective, and thus it is no longer a part of this Hatch project. See Changes/Problems for more information. Our lab is now doing similar experiments in a different species, the midgeBelgica antarctica,but these experiments are more relevant to include withGoal #3. Objective 2: Develop genetic transformation methods for C. costata to validate genes for freeze tolerance.We are continuing to work on sequencing the genome ofC. costata insupport of this objective. We have obtained genomic sequencing reads from two different labs working on this species, and a PhD student in our group is assembling and annotating the genome as part of her dissertation. Goal 3: Objective 1: Sequence the genomes ofBelgica albipes and Eretmoptera murphyi.Our collaborators in France have isolated a specimen ofB. albipesneeded to to complete this objective. At the same time, collaborators in the UK are coordinating the sequencing of E. murphyiand will make the data available to us when it is available. We have also sought additional funding from NSF to support these efforts, which require travel to remote field sites. Objective 2: Use comparative genomics with these three species (B. antarctica, B. albipes,andE. murphyi)to identify genomic signatures of extreme adaptation.There is nothing to report for this objective, as it is dependend on the results of the previous objective.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Spacht DE, Teets NM, Denlinger DL. 2018. Two isoforms of Pepck in Sarcophaga bullata and their distinct expression profiles through development, diapause, and in response to stresses of cold and starvation. Journal of Insect Physiology 111, 41-46.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Garcia, M. J., Teets, N. M., Society for Integrative and Comparative Biology Annual Meeting, "Neuromuscular Performance as a Measure of Thermal Tolerance," Society for Integrative and Comparative Biology, Accepted, National, San Francisco, CA, United States. (January 4, 2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Teets, N. M., Garcia, M. J., Entomological Society of America Annual Meeting, "Drosophila suzukii Population Collection (DSPC): A tool for studying local adaptation in an invasive pest.," Entomological Society of America, Accepted, National, Denver, CO, United States. (November 6, 2017).
• Type: Other Status: Other Year Published: 2018 Citation: Teets, N. M., Palmer Station Science Talks, "GMO 101: The future of agriculture, or are Mark and KC slowly killing us?," Palmer Station, Invited, National. (January 30, 2018).
• Type: Other Status: Other Year Published: 2017 Citation: Teets, N. M., Ohio State University Department of Entomology Seminar Series, "Genetic approaches for improving the management of invasive fruit flies.," Ohio State University Department of Entomology, Invited, University, Columbus, OH, United States. (November 15, 2017).

Progress 10/29/16 to 09/30/17

Outputs
Target Audience:Our primary target audience for this reporting period was other scientists working the fields of insect physiology, ecology, and genetics. Our primary route of dissemination for this reporting period was scientific conferences, as work supported by this Hatch project was presented at regional, national, and international venues. The PI and graduate student also reached the general public through various outreach presentations. Changes/Problems:The primary changes to this project is that we have added Objectives toGoal 1. Based on new collaborations and personnel, we are working on some projects that fall under the purview ofGoal 1and are partially supported by this Hatch project. Below is a brief description of these new objectives, and See "Accomplishments" section for an overview of the results obtained for these projects. Goal 1, Objective 4:Genetic and physiological correlation of cold tolerance traits in fly populations.Our work has identified key physiological mechanisms of cold tolerance, while work of others has demonstrated that there is heritable genetic variation in cold tolerance traits. However, few studies have attempted to link genetic variation with the underlying physiological processes that explain this variation. There are several commonly used metrics of cold that assess how well insects survive cold, at what temperatures they remain active, how fast they recover from non-lethal cold stress, and what are the long-term consequences of cold stress. However, the degree to which these various cold tolerance traits are genetically linked is unknown. Here, we test for correlation of various physiological measures of cold tolerance across a panel of fly lines with natural variation in cold tolerance. Then, we test whether genetic variation in key physiological processes thought to be important for cold tolerance (e.g., osmotic regulation, stress protein expression, cell membrane composition) predicts variation in cold tolerance. Ultimately, these experiments will shed light into the evolutionary physiology of cold tolerance in insects. Goal 1, Objective 5: Local adaptation in cold tolerance in spotted wingDrosophila.Spotted wingDrosophila (Drosophila suzukii)is an invasive fruit pest that has rapidly spread to all 50 states and is causing significant economic damage to fruit growers. Despite originating from warm climates in southeast Asia, this fly is capable of surviving in cold climates in North America. We are testing whether there is evidence of rapid evolution of cold tolerance in spotted wingDrosophilaacross its geographic range. We have collected fly populations from Wisconsin, Kentucky, and Florida, and we are currently in the process of generating isogenic fly lineages from these populations. These lines will be used to test the hypothesis that local adaptation to environmental conditions partly explains the rapid spread of spotted wingDrosophila.These lines will also be used to assess the evolutionary potential for thermal tolerance within individual populations. Overall, this information will improve our ability to predict population dynamics and establish management recommendations for this pest. Goal 1, Objective 6: Overwintering biology of winter-active wolf spiders.Spiders are voracious predators of other arthropods and as such provide valuable top-down regulation of herbivores in both natural and agricultural ecosystems. A handful of spider species are capable of remaining active and hunting during winter, but the overwintering physiology of these spiders (or any spiders for that matter) is poorly understood. In this Objective, we will 1) assess basic cold tolerance parameters of winter active wolf spiders, 2) characterize biochemical adaptations of overwintering wolf spiders, 3) Assess the impacts of winter warming on the overwintering physiology and performance of wolf spiders, and 4) Assess the effect of prey availability on the overwintering physiology of winter active spiders. These experiments will identify key adaptations that contribute to the success of these key biological control agents. What opportunities for training and professional development has the project provided?The project has provided several opportunities for training. A postdoc was partially supported by these funds, and he is contributing to Goal 1. The postdoc is working on cold tolerance of Drosophila melanogasterand spotted wing Drosophila,and A PhD student in my lab also contributed to results under Goal 1, specifically the spider research. The project also provided research experiences for undergraduates. Several undergraduates participated in these projects, including an Honors biology student who is largely responsible of the results obtained in Goal 3 thus far. How have the results been disseminated to communities of interest?These results have been primarily disseminated at scientific conferences, including presentations at Scientific Council on Antarctic Research Biology Symposium in Belgium, and the American Arachnological Society Annual Meeting in Mexico. The PI Teets also presented work supported by this project at invited seminars in Kentucky and Canada. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Objective 1: We will continue our work using cell culture to identify signaling mechanisms that are activated by cold stress. We will use proteomics to identify calcium-dependent signaling mechanisms activated by cold. Objective 2: Using mutants obtained from collaborators, we will test whether mutations in p38 MAP kinase affects flies' ability to undergo rapid cold hardening. Objective 3: This objective is dependent on the results of Objectives 1 and 2, and thus we are unlikely to make progress on this objective during the next reporting period. Objective 4: In the coming year, we will compare variousmeasures of cold tolerance across genetically distinct lines that naturally vary in their ability to survive cold. Specifically, we will compare cold shock tolerance, critical thermal minimum, lethal time at 0"C, chill coma recovery time, and climbingperformance after cold across multiple fly lines that differ in cold tolerance. We will determine which cold tolerance phenotypes are genetically correlated, and we will also determine the underlying physiological mechanisms that predict this variation in cold tolerance. Objective 5: The spotted wing drosophila lines will be fully generated by the spring. We will measure cold tolerance in all lines across five populations. Objective 6: We are using metabolomics to identify cryoprotective molecules in winter active wolf spiders, and we are simulating winter climate warming in the lab to determine its impacts on winter-active wolf spiders. Goal 2: Objective 1: We will continue working towards assembling and annotating the genome for C. costata. In brief, raw Illumina reads will be assembled into scaffolds with Velvet, and gene models will be predicted using the MAKER pipeline. The output of these analysis will be a complete collection of gene models for C. costata that can be used in downstream comparative genomics analysis, transcriptomics, and functional genomics. Objective 2: Objective 2 is dependent on the completion of Objective 1 and will likely not be started during this reporting period. Goal 3: Objective 1: We will obtain frozen samples of E. murphyiandB albipesin the coming months and will submit these for sequencing. Objective 2: Our comparative work will begin withE. murphyiin the coming months, as we will generate preliminary gene expression and population genetic data for this species.

Impacts
What was accomplished under these goals? Impact: Our project has three specific goals, and the impacts associated with each of those goals is as follows: 1) Goal 1:Adaptations for rapid change in temperature.We have shown that sub-lethal effects of cold stresshave long-lasting impacts on insect performance and fitness. We have also demonstrated that winter active spiders, which provide important pest control services, are able to take advantage of winter activity to actively feed and grow throughout the winter. Finally, we have preliminary data that geographically isolated populations ofspotted wingDrosophila,an invasive fruit pest, show differences in cold tolerance, even over a short geographic distance. This result suggests that this pest species is capable of rapid local adaptation, which means that differences between populations must be accounted for when making management recommendations. 2)Goal 2: Understand the genetic mechanisms of freeze tolerance.We have obtained genome sequence data for a highly freeze-tolerant fly,Chymomyza costataand are in the process of assembling and annotating these data. These data will provide critical insights into the nature of extreme freeze tolerance, and this information may provide information toimprove cryopreservation strategies that would impact human health. 3) Goal 3:Genomic mechanisms of extreme adaptation in insects.We have showed that Antarctic insects seasonally enhance their ability to survive freezing conditions, and we are in the process of identifying molecular mechanisms that underliethis freeze tolerance. This information will allow us to better understand the nature of cold adaptation, which is important not only for understanding Antarctic insects, but will also provide insights into how temperate, economically important species tolerate extreme environments. Specific Accomplishments for Each Goal: Goal #1: Objective 1: We have begun developing protocols to use insect cell culture to investigate cellular mechanisms of rapid cold hardening.We do not have any results to report yet, but using a simplified cell culture system will make it easier to investigate the complex networkof cellular events that occur when insects experience cold. Other objectives for Goal 1not provided in project initiation: Due to opportunities for collaboration and the interests of key personnel, there are some additional objectives that were completed under the purview of Goal 1. These new objectives are also highlighted in the "Changes" section. Objective 4: Genetic correlation of different cold tolerance traits. We have generated a new cold tolerance metric, based on a negative geotaxis climbing assay, that will allow us to compare both short- and long-term consequences of cold stress in insects.We collected data on flies' ability to recover climbing performance after a cold stress event, the effect of duration of cold stress on climbing performance, and the ability of cold acclimation to preserve climbing performance after cold stress. Climbing assays are widely used to measure neuromuscular performance in flies over time, but these experiments have not been applied to the study of cold tolerance. Our experiments show that flies exposed to cold stress have deficits in climbing performance as long as 72 h after cold stress, suggesting that the effects of sub-lethal cold stress last throughout a fly's life. We also demonstrated that climbing deficits are highly dependent on the duration of cold stress, but that rearing flies at a cooler temperature (i.e., cold acclimation) preserves climbing performance after cold stress.The key outcome is that we have demonstrated that mild cold stress has long-lasting impacts on neuromuscular performance. Objective 5: Local adaptation in spotted wingDrosophila We have collected populations of spotted wing drosophila from Wisconsin, Kentucky, and Florida, andwe generted preliminary data on cold tolerance in the Kentucky populations. The northern Lexington population was significantly more cold tolerant, even though these populations are only separated by about 150 km. These data suggest that spotted wing drosophila are capable of rapid local adaptation, so assessing population differences will be critical for forecasting the spread of this species, and for establishing local management recommendations.We are establishing a panel of lines that can be used to investigate several aspects of the population biology of spotted wing drosophila. Objective 6: Cold tolerance of winter-active wolf spiders. We have measured basic parameters of cold tolerance in a winter-active wolf spider,Schizocosa stridulans,and we have assessed biochemical characteristics of spiders collected throughout the winter.We collected data on basic parameters of cold tolerance, and we measured body size and energy reserves in spiders collected throughout the winter.Spiders are able to remain active at temperatures around 0°C, which allows them to actively hunt throughout the winter. The spiders also grow during the winter, which is rare among arthropods, and they are able to increase levels of certain energy reserves. This winter activity and growth throughout the winter likely gives these spiders a selective advantage by giving them a jumpstart on reproduction in the spring.It has been long-known that some spiders remain active in the winter, and we have provided a physiological basis for this winter activity, and we have showed that this winter activity likely affords these spiders an advantage over competitors by allowing them to grow and obtain energy throughout the winter. Goal 2: Objective 1: We have obtained genomic DNA sequence data for Chymomyzacostata, and these data will allow us to build a genome that will serve as the foundation for this entire goal. Data analysis is still underway. The flyChymomyza costatais the most cold tolerant insect that has been described, and this genome sequence data will yield critical insights into its biology in the coming years. Goal 3: Objective 1: We have established collaborations with scientists in France and theUK who will be providing us with difficult-to-obtain samples ofB. albipesandE. murphyiin the coming months.These international collaborationswill set the stage for some powerful experiments to identify the nature of extreme adaptation in Antarctic insects. Objective 2: We have investigated the freeze tolerance ofBelgica antarctica,which will be used to establish conditions for future comparative genomics/transcriptomics studies of freeze tolerance across these three Antarctic species. Specifically, we have measured the ability ofB. antarcticato survive long periods of freezing stress, and we are in the process of characterizing gene expression changes that accompany this freeze tolerance.The midges survive in a frozen state at least two weeks with no apparent mortality. Ongoing experiments will determine if there are any sublethal consequences to this freezing stress.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Halbritter, D.A., Teets, N.M., Williams, C.M., and Daniels, J.C. Differences in winter cold hardiness support the geographic range disjunction of Neophasia menapia and Neophasia terlooii (Lepidoptera: Pieridae). In revision at Journal of Insect Physiology.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Teets, N.M., Hahn, D.A. Genetic variation in the shape of cold survival curves in a single fly population suggests potential for selection from climate variability. In revision at Journal of Evolutionary Biology.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mercer, M., Teets, N.M., Bessin, R.T., Obrycki, J.J. Impact of winter feeding on overwintering Hippodamia convergens (Coccinellidae) survival and spring reproduction. Entomological Society of America North Central Branch Meeting, June 5, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Teets, N.M. A tiny genome for a tiny midge: physiology and genomics of the world's southernmost insect. Scientific Council on Antarctic Research Biology Symposium, Leuven, Belgium, July 11, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Potts, L., and Teets, N.M. Biochemical adaptations of overwintering spiders. American Arachnological Society Annual Meeting, Queretaro, Mexico, July 25, 2017.