Chad C. Smith

Independent effects of male and female density on sexual harassment, female fitness, and male competition for mates in the western mosquitofish Gambusia affinis

Operational sex ratio (the ratio of sexually active males to fertilizable females) has a major influence on male competition for mates and male–female interactions. The contributions of male and female density per se to mating system dynamics, however, are rarely examined, and the fitness consequences are often inferred rather than quantified. Male mosquitofish (Gambusia affinis) compete aggressively and frequently harass females for copulations, a behavior thought to reduce female fitness. Female fitness can also be reduced by increases in female density, which may affect food availability, cannibalism rates, and chemical interactions between females. I manipulated male and female densities of G. affinis to measure their effects on male–male aggression, male harassment toward females, and female fitness. I found that males chased rivals more often and attempted fewer copulations when female density decreased, but surprisingly male density had no significant effect on the frequency of these male behaviors. In contrast, males’ agonistic displays toward other males increased with male density, but display behavior was unaffected by female density. These results suggest that male and female density do not always contribute equally or at all to the patterns of behavior we observe. Female fitness declined as female density increased, the opposite pattern expected if male harassment is costly to females. This suggests that a strong, negative effect of female density overwhelmed any potential costs of male harassment. Sources of female density dependence and the consequences of changes in male and female density to patterns of male behavior are discussed.

Tactic-dependent plasticity in ejaculate traits in the swordtail Xiphophorus nigrensis

In species with alternative reproductive tactics, males that sneak copulations often have larger, higher quality ejaculates relative to males that defend females or nest sites. Ejaculate traits can, however, exhibit substantial phenotypic plasticity depending on a males mating role in sperm competition, which may depend on the tactic of his competitor. We tested whether exposure to males of different tactics affected sperm number and quality in the swordtail Xipophorus nigrensis, a species with small males that sneak copulations and large males that court females. Sperm swimming speed was higher when the perceived competitor was small than when the competitor was large. Plasticity, however, was only exhibited by small males. Sperm number and viability were invariant between social environments. Our results suggest sperm quality is role-dependent and that plastic responses to the social environment can differ between male reproductive tactics.

Spatial Structure of the Mormon Cricket Gut Microbiome and Its Predicted Contribution to Nutrition and Immune Function

The gut microbiome of insects plays an important role in their ecology and evolution, participating in nutrient acquisition, immunity, and behavior. Microbial community structure within the gut is heavily influenced by differences among gut regions in morphology and physiology, which determine the niches available for microbes to colonize. We present a high-resolution analysis of the structure of the gut microbiome in the Mormon cricket Anabrus simplex, an insect known for its periodic outbreaks in the western United States and nutrition-dependent mating system. The Mormon cricket microbiome was dominated by 11 taxa from the Lactobacillaceae, Enterobacteriaceae, and Streptococcaceae. While most of these were represented in all gut regions, there were marked differences in their relative abundance, with lactic-acid bacteria (Lactobacillaceae) more common in the foregut and midgut and enteric (Enterobacteriaceae) bacteria more common in the hindgut. Differences in community structure were driven by variation in the relative prevalence of three groups: a Lactobacillus in the foregut, Pediococcus lactic-acid bacteria in the midgut, and Pantoea agglomerans, an enteric bacterium, in the hindgut. These taxa have been shown to have beneficial effects on their hosts in insects and other animals by improving nutrition, increasing resistance to pathogens, and modulating social behavior. Using PICRUSt to predict gene content from our 16S rRNA sequences, we found enzymes that participate in carbohydrate metabolism and pathogen defense in other orthopterans. These were predominately represented in the hindgut and midgut, the most important sites for nutrition and pathogen defense. Phylogenetic analysis of 16S rRNA sequences from cultured isolates indicated low levels of divergence from sequences derived from plants and other insects, suggesting that these bacteria are likely to be exchanged between Mormon crickets and the environment. Our study shows strong spatial variation in microbiome community structure, which influences predicted gene content and thus the potential of the microbiome to influence host function.

Biogeography of Mutualistic Fungi Cultivated by Leafcutter Ants

Leafcutter ants propagate co‐evolving fungi for food. The nearly 50 species of leafcutter ants (Atta, Acromyrmex) range from Argentina to the United States, with the greatest species diversity in southern South America. We elucidate the biogeography of fungi cultivated by leafcutter ants using DNA sequence and microsatellite‐marker analyses of 474 cultivars collected across the leafcutter range. Fungal cultivars belong to two clades (Clade‐A and Clade‐B). The dominant and widespread Clade‐A cultivars form three genotype clusters, with their relative prevalence corresponding to southern South America, northern South America, Central and North America. Admixture between Clade‐A populations supports genetic exchange within a single species, Leucocoprinus gongylophorus. Some leafcutter species that cut grass as fungicultural substrate are specialized to cultivate Clade‐B fungi, whereas leafcutters preferring dicot plants appear specialized on Clade‐A fungi. Cultivar sharing between sympatric leafcutter species occurs frequently such that cultivars of Atta are not distinct from those of Acromyrmex. Leafcutters specialized on Clade‐B fungi occur only in South America. Diversity of Clade‐A fungi is greatest in South America, but minimal in Central and North America. Maximum cultivar diversity in South America is predicted by the Kusnezov–Fowler hypothesis that leafcutter ants originated in subtropical South America and only dicot‐specialized leafcutter ants migrated out of South America, but the cultivar diversity becomes also compatible with a recently proposed hypothesis of a Central American origin by postulating that leafcutter ants acquired novel cultivars many times from other nonleafcutter fungus‐growing ants during their migrations from Central America across South America. We evaluate these biogeographic hypotheses in the light of estimated dates for the origins of leafcutter ants and their cultivars.

Sexual transmission of beneficial microbes

Beneficial sexually transmitted infections (STIs) are an understudied phenomenon with important implications for the evolution of cooperation and host reproductive behavior. Challenging the prevailing expectation that sexual transmission leads to pathogenesis, these symbionts provide new opportunities to examine how STIs might influence sexual selection and the evolution of promiscuity.

Polyandry and paternity in a wild population of the swordtail Xiphophorus nigrensis

Genetic analyses of parentage provide crucial information about the prevalence of polyandry and the potential for sexual selection to operate in wild populations. In the swordtail Xiphophorus nigrensis, large males are thought to have a competitive advantage due to their superiority in male–male contests and attractiveness to females, who are presumed to mate multiply. I examined the distribution of paternity within broods, the relationship between male body size and paternity and the effect of sire number on fecundity from females collected in the field. Sixty-one percent of females produced offspring from two to four males, with 70% of the offspring typically sired by one of the males represented in the brood. Male body size did not affect paternity share or whether females were multiply mated, as predicted if precopulatory sexual selection has a strong effect on the outcome of postcopulatory sexual selection. Female fecundity increased with the number of sires; however, this relationship was not observed when the smallest broods, where multiple mating is more difficult to detect, were excluded from the analysis. The high levels of multiple paternity and reproductive skew suggest that postcopulatory sexual selection has important evolutionary consequences in X. nigrensis. Traits important in precopulatory sexual selection, such as male body size, however, are more likely to affect sexual selection by increasing the number of mates obtained rather than paternity share within broods.

Partitioning the effects of mating and nuptial feeding on the microbiome in gift-giving insects

Mating is a ubiquitous social interaction with the potential to influence the microbiome by facilitating transmission, modifying host physiology, and in species where males donate nuptial gifts to females, altering diet. We manipulated mating and nuptial gift consumption in two insects that differ in nuptial gift size, the Mormon cricket Anabrus simplex and the decorated cricket Gryllodes sigillatus, with the expectation that larger gifts are more likely to affect the gut microbiome. Surprisingly, mating, but not nuptial gift consumption, affected the structure of bacterial communities in the gut, and only in Mormon crickets. The change in structure was due to a precipitous drop in the abundance of lactic-acid bacteria in unmated females, a taxon known for their beneficial effects on nutrition and immunity. Mating did not affect phenoloxidase or lysozyme-like antibacterial activity in either species, suggesting that any physiological response to mating on host-microbe interactions is decoupled from systemic immunity. Protein supplementation also did not affect the gut microbiome in decorated crickets, suggesting that insensitivity of gut microbes to dietary protein could contribute to the lack of an effect of nuptial gift consumption. Our study provides experimental evidence that sexual interactions can affect the microbiome and suggests mating can promote beneficial gut bacteria.

Artificial Microbiome-Selection to Engineer Microbiomes That Confer Salt-Tolerance to Plants

We develop a method to artificially select for rhizosphere microbiomes that confer salt-tolerance to the model grass Brachypodium distachyon. We differentially propagate microbiomes within the background of a non-evolving, highly-inbred plant population, and therefore only microbiomes evolve in our experiment, but not the plants. To optimize methods, we conceptualize artificial microbiome-selection as a special case of indirect selection: We do not measure microbiome properties directly, but we use host performance (e.g., biomass; seed set) as an indicator to infer association with rhizosphere microbiomes that confer salt-tolerance to a plant. We previously called this indirect-selection scheme host-mediated indirect selection on microbiomes (Mueller & Sachs 2015). Our methods aim to maximize evolutionary changes due to differential microbiome-propagation, while minimizing some (but not all) ecological processes affecting microbiome composition. Specifically, our methods aim to maximize microbiome perpetuation between selection-cycles and maximize response to artificial microbiome-selection by (a) controlling microbiome assembly when inoculating seeds at the beginning of each selection cycle; (b) using low-carbon soil to enhance host-control mediated by carbon secretions of plants during initial microbiome assembly and subsequent microbiome persistence; (c) fractionating microbiomes before transfer between plants to perpetuate and select only on bacterial and viral (but not fungal) microbiome components; and (d) ramping of salt-stress between selection-cycles to minimize the chance of over-stressing plants. Our selection protocol generates microbiomes that enhance plant fitness after only 1-3 rounds of artificial selection on rhizosphere microbiomes. Relative to fallow-soil control treatments, artificially-selected microbiomes increase plant fitness by 75% under sodium-sulfate stress, and by 38% under aluminum-sulfate stress. Relative to null control treatments, artificially-selected microbiomes increase plant fitness by 13% under sodium-sulfate stress, and by 12% under aluminum-sulfate stress. When testing microbiomes after nine rounds of differential microbiome propagation, the effect of bacterial microbiomes selected to confer tolerance to sodium-sulfate stress appears specific (these microbiomes do not confer tolerance to aluminum-sulfate stress), but the effect of microbiomes selected to confer tolerance to aluminum-sulfate stress appears non-specific (selected microbiomes ameliorate both sodium- and aluminum-sulfate stresses). Complementary metagenomic analyses of the artificially selected microbiomes will help elucidate metabolic properties of microbiomes that confer specific versus non-specific salt-tolerance to plants.

Structure and culture of the gut microbiome of the Mormon cricket Anabrus simplex

The gut microbiome of insects plays an important role in their ecology and evolution, participating in nutrient acquisition, immunity, and behavior. Microbial community structure within the gut is thought to be heavily influenced by differences among gut regions in morphology and physiology, which determine the niches available for microbes to colonize. We present a high-resolution analysis of the structure of the gut microbiome in the Mormon cricket Anabrus simplex, an insect known for its periodic outbreaks in the Western United States and nutrition-dependent mating system. We found the Mormon cricket microbiome was dominated by eleven bacterial phylotypes from the Lactobacillaceae, Enterobacteriaceae, and Streptococcaeae. While most of these were represented in all gut regions, there were marked differences in their relative abundance, with lactic-acid bacteria (Lactobacillaceae) more common in the foregut and midgut and enteric (Enterobacteriaceae) bacteria more common in the hindgut. Differences in community structure were driven by variation in the relative prevalence of three groups: a Lactobacillus phylotype in the foregut, Pediococcus lactic-acid bacteria in the midgut, and Pantoea agglomerans, an enteric bacterium, in the hindgut. These taxa have been shown to have beneficial effects on their hosts in insects or other animals by improving nutrition, increasing resistance to pathogens, and modulating social behavior. Phylogenetic analysis of 16s rRNA sequences from cultured isolates indicated low levels of divergence from sequences derived from plants and other insects, suggesting that these bacteria are likely to be frequently exchanged between Mormon crickets and the environment. Our study provides the foundation for future work on an economically important insect and emerging model for the study of how social interaction influence host-microbe symbiosis.The gut microbiome of insects plays an important role in their ecology and evolution, participating in nutrient acquisition, immunity, and behavior. Microbial community structure within the gut is heavily influenced by differences among gut regions in morphology and physiology, which determine the niches available for microbes to colonize. We present a high-resolution analysis of the structure of the gut microbiome in the Mormon cricket Anabrus simplex, an insect known for its periodic outbreaks in the western United States and nutrition-dependent mating system. The Mormon cricket microbiome was dominated by eleven taxa from the Lactobacillaceae, Enterobacteriaceae, and Streptococcaeae. While most of these were represented in all gut regions, there were marked differences in their relative abundance, with lactic-acid bacteria (Lactobacillaceae) more common in the foregut and midgut and enteric (Enterobacteriaceae) bacteria more common in the hindgut. Differences in community structure were driven by variation in the relative prevalence of three groups: a Lactobacillus in the foregut, Pediococcus lactic-acid bacteria in the midgut, and Pantoea agglomerans, an enteric bacterium, in the hindgut. These taxa have been shown to have beneficial effects on their hosts in insects and other animals by improving nutrition, increasing resistance to pathogens, and modulating social behavior. Using PICRUSt to predict gene content from our 16S rRNA sequences, we found enzymes that participate in carbohydrate metabolism and pathogen defense in other orthopterans. These were predominately represented in the hindgut and midgut, the most important sites for nutrition and pathogen defense. Phylogenetic analysis of 16S rRNA sequences from cultured isolates indicated low levels of divergence from sequences derived from plants and other insects, suggesting that these bacteria are likely to be exchanged between Mormon crickets and the environment. Our study shows strong spatial variation in microbiome community structure, which influences predicted gene content and thus the potential of the microbiome to influence host function.

Mating promotes lactic-acid gut bacteria in a gift-giving insect

Mating is a ubiquitous social interaction with the potential to influence the microbiome by facilitating transmission, modifying host physiology, and in species where males donate nuptial gifts to females, altering diet. We manipulated mating and nuptial gift consumption in two insects that differ in nuptial gift size, the Mormon cricket Anabrus simplex and the decorated cricket Gryllodes sigillatus, with the expectation that larger gifts are more likely to affect the gut microbiome. Surprisingly, mating, but not nuptial gift consumption, affected bacterial community structure, and only in Mormon crickets. The change in structure was due to a precipitous drop in the abundance of lactic-acid bacteria in unmated females, a taxon known for their beneficial effects on nutrition and immunity. Mating did not affect phenoloxidase or lysozyme-like antibacterial activity in either species, suggesting that any physiological response to mating on host-microbe interactions is decoupled from the systemic immunity. Protein supplementation also did not affect the gut microbiome in decorated crickets, suggesting that insensitivity of gut microbes to dietary protein could contribute to the lack of an effect of nuptial gift consumption. Our study provides experimental evidence that sexual interactions can affect the microbiome and suggests mating can promote beneficial gut bacteria.