GENETICS, BEHAVIOR, AND CHEMICAL ECOLOGY OF INSECT SOCIETIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016993
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720

Performing Department
Insect Biology

Non Technical Summary
Ants are widespread, abundant, and influential members of nearly every terrestrial ecosystem. Their success stems from their sophisticated societies: colonies within which work is efficiently and accurately distributed across thousands or millions of individuals. My research examines the behavioral, genetic, and chemical (pheromonal) mechanisms that are used to coordinate these social activities. The knowledge generated by this research provides a understanding of how societies can be organized, and illuminates methods for potentially controlling ants that are damaging in both natural and human-built ecosystems.

Animal Health Component

35%

Research Effort Categories

Basic

60%

Applied

35%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1060	100%

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

Subject Of Investigation
3110 - Insects;

Field Of Science
1060 - Biology (whole systems);

Keywords

ants

genetics

pheromone

invasive species

genomics

chemical ecology

population genetics

behavior

social insect

Goals / Objectives
In the coming years, we will continue to apply approaches from genetics, genomics, and chemistry to questions regarding the behavior, social structure, and evolution of ants. Morever, we will complement these studies with comparative analysis of the structure and function of colony recognition systems in non-invasive ants. Overall, our objectives are to:(1) Conduct experiments in the laboratory and the field, in several different species, to illuminate the role of cuticular hydrocarbon variation in ant colony recognition.(2) To experimentally test hypotheses for the behavioral basis of colony recognition in Argentine ants and other ant species.(3) Use genomic and functional genetic approaches to reveal the genetic basis of pheromone production and perception systems.(4) Identify pheromones that are used for recognizing different members of the colony(eggs, larvae, queens, etc) and that are used for different purposes (e.g. foraging trails, nestrelocation trails).

Project Methods
Behavioral assay. The level of aggression between ants will be quantified using a standard behavioral assay(Tsutsui, Suarez et al. 2003). For each trial, we will either pair two focal individuals together, or test a single worker with five "tester" individuals (depending on the particular experimental protocol). Each bioassay will be performed in a neutral arena for five minutes and we will score interactions on a scale from 1 to 4: 1 = touch (physical contact, but no aggressive response; includes antennation and trophallaxis), 2 = avoid (after touching, one or both of the ants recoils and retreats), 3 = aggression (attack by one or both of the workers; includes lunges, biting and pulling of legs and antennae), and 4 = fighting (includes prolonged biting and pulling and the use of chemical defenses). Each behavioral assay will be assigned a score based on the highest level of aggression observed during the 5- minute time span. Ten to fifty replicates will be performed (depending on the experiment) and the level of aggression between will be calculated as the mean of these replicates. We will also perform statistical tests on the frequency of aggression (% trials in which aggression occurred).Chemical ecology: To identify and quantify cuticular hydrocarbon pheromones, we will extract samples by immersing them in 5 ml ofn-hexanes for 10 minutes. We will separate non-polar molecules from other compounds by pipetting the extract through 4 cm of activated silica gel (Sigma 70-230 mesh) in Pasteur pipet mini-columns. An additional 5 ml of cleann-hexanes will then be passed through the silica gel. We will evaporate the resulting hydrocarbon extracts under a stream of nitrogen and redissolve them in 60 ul ofn-hexanes for GC/MS analyses.A 3 ul aliquot will be injected into the GC/MS.We will perform the analyses on an Agilent7890AGC equipped with a DB-5, silicacapillary column (30m´0.32mm´0.25µm) coupled with a 5975C Mass Spectrometer (MS) with triple axis detector, operated in splitless mode. We will use helium as the carrier gas at 1ml/min, and a temperature program of 2 min at 80°C, to 270°C at 20°C/min, then to 310°C at 3°C/min(Suarez, Holway et al. 2002).We routinely use these techniques(Suarez, Holway et al. 2002, Torres, Brandt et al. 2007, Brandt, van Wilgenburg et al. 2009, Brandt, Van Wilgenburg et al. 2009).We will useMSD ChemStation (v.E.02.00.493, Agilent Technologies, Santa Clara, CA)to integrate the resulting GC profiles and quantify the percent area under the curve for each peak. We will then test for differences among profiles usingby calculating pairwise Euclidian distances between individuals from the relative peak areas. From the resulting dissimilarity matrix, we will visualize the chemical distances by performed a nonmetric multi-dimensional scaling (NMDS) analysis on the relative peak areas of GC/MS profiles using the packageveganand the functionmetaMDSin the program R(R Core Team 2018).

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

Outputs
Target Audience:Scientists, academics, regional parks districts,professional pest control operators and pest control organizations (state and national), growers, members of the public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the course of this research, we provided training in field and laboratory techniques to four undergraduate students. Five PhD graduate students (Kelsey Scheckel, Brian Whyte, Nina Pak, Amelia Harvey, Rachel Weinberg)were able to continue their research projects on a variety of topics involving the genetics, chemical ecology, and behavior of social insects. In addition, two full-time post-doctoral researchers (Dr. Maria Tonione, Dr. Elizabeth Cash)continued to develop new skills and a professional portfolio as they worked on these research projects. How have the results been disseminated to communities of interest?Although COVID-19 shutdowns led to the cancellation of many planned conferences and symposis, I wasstill able topresentour research in talks at: Genomics Social Hour, California Academy of Sciences. "Using UCEs to understand genetic divergence and speciation inPolyerguskidnapper ants". Online. April 2020. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to complete and publish peer-reviewed papers on: the chemical ecology of desiccation resistance in the invasive Argentine ant (Linepithema humile), the effect of dietary variation on cuticular hydrocarbon pheromones, the adaptation and speciation genetics ofPolyerguskidnapper ants, and the dual function of ant semiochemicals as pheromones and antimicrobials. We will continue to provide training for young scientists at the level of undergraduates, graduate students, and post-doctoral researchers in the laboratory, field, and classroom. We will also continue to disseminate the results of our research at professional conferences, departmental seminars, local symposia, and to the public through a variety of outreach events.

Impacts
What was accomplished under these goals? During this review period, we completed and published several different studies that accomplished some of our goals. In Naughton et al (2020), we examined how the invasive Argentine ant (Linepithema humile) impacts the ant communities of California Channel Island ecosystems. In a series of two papers (Tonione et al 2020 PLOS One, Tonione et al 2020 Ecology and Evolution), we closely examined the physiology and genomics ofthermal adaptation in a cold-specialized ant species, the winter ant (Prenolepis imparis). This research is an important contribution to our general understanding of how insects (and other organisms) are likely to be impacted by global change. In Adams and Tsutsui (2020), we reviewed the central role of chemical pheromones in the divergence and species formation of insects, identifying common processes and general rules. Finally, in Sanchez-Restrepo et al (2020), we advanced our taxonomic understanding ofPseudacteonphorid flies by using a variety of approaches to clearly define species boundaries. Species in this genus are important biological controls of invasive ants, includingSolenopsis?fire ants.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Naughton, I., C. Boser, N. D. Tsutsui, and D. A. Holway. 2020. Direct evidence of native ant displacement by the Argentine ant in island ecosystems. BIOLOGICAL INVASIONS 22:681-691.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Tonione, M. A., S. Cho, G. Richmond, C. Irian, N. D. Tsutsui. 2020. Thermal acclimation ability differs between high- and low- elevation populations of the winter ant, Prenolepis imparis. ECOLOGY & EVOLUTION 8:11385-11398.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Adams, S. A. and N. D. Tsutsui. 2020. The evolution of species recognition labels in insects. PHILOSOPHICAL TRANSACTIONS ROYAL SOCIETY LONDON B. 375: 20190476.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: S�nchez-Restrepo, A. F., L. Chifflet, V. A. Confalonieri, N. D. Tsutsui, M. A. Pesquero, and L. A. Calcaterra. 2020. Species delimitation and molecular phylogenetics of South American fire ant decapitating flies (Diptera: Phoridae: Pseudacteon). PLOS ONE 15(7):e0236086.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Tonione, M. A., K. Bi, and N. D. Tsutsui. 2020. Transcriptomic signatures of cold adaptation and heat stress in the thermally sensitive winter ant (Prenolepis imparis). PLOS ONE 15(10): e0239558 https://doi.org/10.1371/journal.pone.0239558

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

Outputs
Target Audience:Scientists, academics, professional pest control operators and pest control organizations (state and national), growers, members of the public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Maria Tonione - post-doctoral researcher, research on genomics and pheromone evolution. Dr. Elizabeth Cash - post-doctoral researcher, research on genomics and pheromone evolution. Brian Whyte - PhD graduate student. Research on the evolution of self/non-self recognition systems. Kelsey Scheckel - PhD graduate student. Research on the evolution of ant social parasitism. Nina Pak - PhD graduate student. Research on evolution of aquatic/marine life histories of flies. Amelia Harvey - PhD graduate student. Research on the impact of invasive ants on bee pollinators. Rachel Weinberg - PhD graduate student. Research on the evolution of self/non-self recognition systems. Approximately 10 undergraduate researchers have also gain research experience in my lab during the past year, by assisting the above PhD students and post-doctoral researchers with elements of their projects. How have the results been disseminated to communities of interest?We have presented our research in talks at various professional conferences, workshops, and departmental seminars, including: Neil Tsutsui as speaker: Invited seminar speaker. 2019. Kyoto University. "The genetics and chemical ecology of Polyergus kidnapper ants". Kyoto, Japan. June. Invited workshop speaker. Winter Workshop Series for pest control operators, Target Specialty Products. Anaheim, CA. March. Invited symposium speaker. 2018. World Life Science Conference. "Of ants and antennae: How pheromones define the boundaries of both societies and species", Beijing, China. October 2018. Invited seminar speaker. 2018. Chinese Academy of Sciences, Institute of Zoology. "Scents of self: How social context shapes ant colony recognition systems", Beijing, China. October 2018. Other lab members as speaker: Maria Tonione, UC Berkeley, Essig Museum of Entomology Brunch. 3 May 2019. "Genomics and speciation as seen through the kidnapper ant". Jenna Florio, Brian Fisher, Neil Tsutsui - "Kitchens, schools, and backyard swimming pools: Citizen science with the backyard biodiversity project", Entomological Society of America, November 2018, Vancouver, BC. Ali Setayesh, UC Berkeley, Molecular and Cellular Biology Undergrad symposium. May 2019. Ali Setayesh, UC Berkeley, Essig Museum of Entomology Brunch. 10 May 2019. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to write and publish a peer-reviewed review paper on the evolution of insect mating pheromones, complete analysis and writing for two additional papers related to Argentine ant pheromones, publish one more paper on kidnapper ant social and pheromoone evolution, and publish two papers on the evolution of cold-tolerance in Prenolepis winter ants. We will continue to provide training for young scientists at the level of undergraduation, graduate student, and post-doctoral researcher in the laboratory, field, and classroom. We will also continue to disseminate the results of our research at professional conferences, departmental seminars, local symposia, and to the public through a variety of outreach events.

Impacts
What was accomplished under these goals? We have completed several studies on the behavior and chemical ecology of social insects, and published our findings in peer-reviewed journal articles. These findings advance our understanding of how insects communicate, evolve, and interact, which is fundamental to understanding and controlling damaging pest insects, in both agricultural settings and human structures. We have continued to perform research in the lab using genetic, genomic, and chemical tools to explore the evolution, ecology, and behavior of social insects. Specifically, the papers published during this review period address our major objectives: - Buellesbach et al. (2018) presents the results of a large, California-wide study of how dozens of colony recognition pheromones of the invasive Argentine ant vary geographically, and how they are affected by local climate. - Felden et al (2019) examined invasive Argentine ant populations from around the world and showed, using next-generation transcriptome sequencing, that patterns of viral infection and immune gene regulation vary across these regions. - Torres et al (2018) presented the results of a chemical, behavioral, and genetic analysis of kidnapper ants in the genus Polyergus, showing fundamental evolutionary patterns and processes of divergence and speciation.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Buellesbach, J., B. A. Whyte, B., E. Cash, J. D. Gibson, K. J. Scheckel, R. Sandidge, and N. D. Tsutsui. 2018. Desiccation resistance and micro-climate adaptation: Cuticular hydrocarbon signatures of different Argentine ant supercolonies across California. JOURNAL OF CHEMICAL ECOLOGY 44:1101-1114.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Felden, A., C. I. Paris, D. G. Chapple, A. V. Suarez, N. D. Tsutsui, P. J. Lester, and M. A. M. Gruber. 2019. Native and introduced Argentine ant populations are characterised by distinct transcriptomic signatures associated with behaviour and immunity. NEOBIOTA. 49:105-126.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Torres, C. W., M. A. Tonione, S. R. Ram�rez, J. R. Sapp, and N. D. Tsutsui. 2018. Genetic and chemical divergence among host races of a socially parasitic ant. ECOLOGY & EVOLUTION. 8:11385-11398.