Molecular mechanisms of phenotypic plasticity in social insects.

Polyphenism in insects, whereby a single genome expresses different phenotypes in response to environmental cues, is a fascinating biological phenomenon. Social insects are especially intriguing examples of phenotypic plasticity because division of labor results in the development of extreme morphological phenotypes, such as the queen and worker castes. Although sociality evolved independently in ants, bees, wasps and termites, similar genetic pathways regulate phenotypic plasticity in these different groups of social insects. The insulin/insulin-like growth signaling (IIS) plays a key role in this process. Recent research reveals that IIS interacts with other pathways including target of rapamycin (TOR), epidermal growth factor receptor (Egfr), juvenile hormone (JH) and vitellogenin (Vg) to regulate caste differentiation.

The gut bacterial communities associated with lab-raised and field-collected ants of Camponotus fragilis (Formicidae: Formicinae).

Camponotus is the second largest ant genus and known to harbor the primary endosymbiotic bacteria of the genus Blochmannia. However, little is known about the effect of diet and environment changes on the gut bacterial communities of these ants. We investigated the intestinal bacterial communities in the lab-raised and field-collected ants of Camponotus fragilis which is found in the southwestern United States and northern reaches of Mexico. We determined the difference of gut bacterial composition and distribution among the crop, midgut, and hindgut of the two types of colonies. Number of bacterial species varied with the methods of detection and the source of the ants. Lab-raised ants yielded 12 and 11 species using classical microbial culture methods and small-subunit rRNA genes (16S rRNAs) polymerase chain reaction-restriction fragment-length polymorphism analysis, respectively. Field-collected ants yielded just 4 and 1-3 species using the same methods. Most gut bacterial species from the lab-raised ants were unevenly distributed among the crop, midgut, and hindgut, and each section had its own dominant bacterial species. Acetobacter was the prominent bacteria group in crop, accounting for about 55 % of the crop clone library. Blochmannia was the dominant species in midgut, nearly reaching 90 % of the midgut clone library. Pseudomonas aeruginosa dominated the hindgut, accounting for over 98 % of the hindgut clone library. P. aeruginosa was the only species common to all three sections. A comparison between lab-raised and field-collected ants, and comparison with other species, shows that gut bacterial communities vary with local environment and diet. The bacterial species identified here were most likely commensals with little effect on their hosts or mild pathogens deleterious to colony health.

Mating system evolution and worker caste diversity in Pheidole ants.

The efficiency of social groups is generally optimized by a division of labour, achieved through behavioural or morphological diversity of members. In social insects, colonies may increase the morphological diversity of workers by recruiting standing genetic variance for size and shape via multiply mated queens (polyandry) or multiple-breeding queens (polygyny). However, greater worker diversity in multi-lineage species may also have evolved due to mutual worker policing if there is worker reproduction. Such policing reduces the pressure on workers to maintain reproductive morphologies, allowing the evolution of greater developmental plasticity and the maintenance of more genetic variance for worker size and shape in populations. Pheidole ants vary greatly in the diversity of worker castes. Also, their workers lack ovaries and are thus invariably sterile regardless of the queen mating frequency and numbers of queens per colony. This allowed us to perform an across-species study examining the genetic effects of recruiting more patrilines on the developmental diversity of workers in the absence of confounding effects from worker policing. Using highly variable microsatellite markers, we found that the effective mating frequency of the soldier-polymorphic P. rhea (avg. meN = 2.65) was significantly higher than that of the dimorphic P. spadonia (avg. meN = 1.06), despite a significant paternity skew in P. rhea (avg. B = 0.10). Our findings support the idea that mating strategies of queens may co-evolve with selection to increase the diversity of workers. We also detected patriline bias in the production of different worker sizes, which provides direct evidence for a genetic component to worker polymorphism.

Evolution of novel mosaic castes in ants: modularity, phenotypic plasticity, and colonial buffering.

Many ants have independently evolved castes with novel morphology as well as function, such as soldiers and permanently wingless (ergatoid) queens. We present a conceptual model, based on modularity in morphology and development, in which evolutionary innovation is facilitated by the ancestral ant polyphenism of winged queens and wingless workers. We suggest that novel castes evolved from rare intercastes, anomalous mosaics of winged queens and workers, erratically produced by colonies through environmental or genetic perturbations. The colonial environment is highly accommodating and buffers viable intercastes from individual selection. Their cost is limited because they are diluted by the large number of nestmates, yet some can bring disproportionate benefits to their colonies in the context of defense or reproduction (e.g., wingless intercastes able to mate). Useful intercastes will increase in frequency as their morphology is stabilized through genetic accommodation. We show that both soldiers and ergatoid queens are mosaics of winged queens and workers, and they are strikingly similar to some intercastes. Modularity and developmental plasticity together with winged/wingless polyphenism thus allow for the production of highly variable mosaic intercastes, and colonies incubate the advantageous mosaics.

Highly similar microbial communities are shared among related and trophically similar ant species.

Ants dominate many terrestrial ecosystems, yet we know little about their nutritional physiology and ecology. While traditionally viewed as predators and scavengers, recent isotopic studies revealed that many dominant ant species are functional herbivores. As with other insects with nitrogen-poor diets, it is hypothesized that these ants rely on symbiotic bacteria for nutritional supplementation. In this study, we used cloning and 16S sequencing to further characterize the bacterial flora of several herbivorous ants, while also examining the beta diversity of bacterial communities within and between ant species from different trophic levels. Through estimating phylogenetic overlap between these communities, we tested the hypothesis that ecologically or phylogenetically similar groups of ants harbor similar microbial flora. Our findings reveal: (i) clear differences in bacterial communities harbored by predatory and herbivorous ants; (ii) notable similarities among communities from distantly related herbivorous ants and (iii) similar communities shared by different predatory army ant species. Focusing on one herbivorous ant tribe, the Cephalotini, we detected five major bacterial taxa that likely represent the core microbiota. Metabolic functions of bacterial relatives suggest that these microbes may play roles in fixing, recycling, or upgrading nitrogen. Overall, our findings reveal that similar microbial communities are harbored by ants from similar trophic niches and, to a greater extent, by related ants from the same colonies, species, genera, and tribes. These trends hint at coevolved histories between ants and microbes, suggesting new possibilities for roles of bacteria in the evolution of both herbivores and carnivores from the ant family Formicidae.

Ancestral developmental potential facilitates parallel evolution in ants.

Complex worker caste systems have contributed to the evolutionary success of advanced ant societies; however, little is known about the developmental processes underlying their origin and evolution. We combined hormonal manipulation, gene expression, and phylogenetic analyses with field observations to understand how novel worker subcastes evolve. We uncovered an ancestral developmental potential to produce a "supersoldier" subcaste that has been actualized at least two times independently in the hyperdiverse ant genus Pheidole. This potential has been retained and can be environmentally induced throughout the genus. Therefore, the retention and induction of this potential have facilitated the parallel evolution of supersoldiers through a process known as genetic accommodation. The recurrent induction of ancestral developmental potential may facilitate the adaptive and parallel evolution of phenotypes.

Dynamics of an ant-ant obligate mutualism: colony growth, density dependence and frequency dependence.

In insect societies, worker vs. queen development (reproductive caste) is typically governed by environmental factors, but many Pogonomyrmex seed-harvester ants exhibit strict genetic caste determination, resulting in an obligate mutualism between two reproductively isolated lineages. Same-lineage matings produce fertile queens while alternate-lineage matings produce sterile workers. Because new virgin queens mate randomly with multiple males of each lineage type, and both worker and queen phenotypes are required for colony growth and future reproduction, fitness is influenced by the relative frequency of each lineage involved in the mutualistic breeding system. While models based solely on frequency-dependent selection predict the convergence of lineage frequencies towards equal (0.5/0.5), we surveyed the lineage ratios of 49 systems across the range of the mutualism and found that the global lineage frequency differed significantly from equal. Multiple regression analysis of our system survey data revealed that the density and relative frequency of one lineage decreases at lower elevations, while the frequency of the alternate lineage increases with total colony density. While the production of the first worker cohort is largely frequency dependent, relying on the random acquisition of worker-biased sperm stores, subsequent colony growth is independent of lineage frequency. We provide a simulation model showing that a net ecological advantage held by one lineage can lead to the maintenance of stable but asymmetric lineage frequencies. Collectively, these findings suggest that a combination of frequency-dependent and frequency-independent mechanisms can generate many different localized and independently evolving system equilibria.

The potential for gene flow in a dependent lineage system of a harvester ant: fair meiosis in the F1 generation.

We investigated the potential for gene flow in a dependent lineage (DL) system of the harvester ant Pogonomyrmex. Each DL system is composed of 2 reproductively isolated lineages that are locked in an obligate mutualism. The genetic components that produce the worker phenotype are acquired by hybridizing with the partner lineage. In the mating flight, queens of both lineages mate with multiple males from each lineage. During colony growth and reproduction, eggs fertilized by partner-lineage sperm produce F(1) hybrid workers with interlineage genomes, whereas eggs fertilized by same-lineage sperm result in the development of new queens with intralineage genomes. New males are typically produced from unfertilized eggs laid by the pure-lineage queen but in her absence may be produced by interlineage F(1) workers. We investigated the potential for interlineage gene flow in this system using 2 classes of lineage-specific nuclear markers to identify hybrid genome combinations. We confirmed the production of viable interlineage F(1) reproductive females in field colonies, the occurrence of which is associated with the relative frequencies of each lineage in the population: interlineage F(1) queens occurred only in the rare lineage of the population with dramatically skewed lineage frequencies. In laboratory colonies, we detected fair meiosis in interlineage F(1) workers leading to the production of viable and haploid interlineage F(2) males. We conclude that the genomes of each lineage recombine freely, suggesting that extrinsic postzygotic selection maintains the integrity of each lineage genome. We compare our findings with those of the H1/H2 DL system.

Remaining flexible in old alliances: functional plasticity in constrained mutualisms.

Central to any beneficial interaction is the capacity of partners to detect and respond to significant changes in the other. Recent studies of microbial mutualists show their close integration with host development, immune responses, and acclimation to a dynamic external environment. While the significance of microbial players is broadly appreciated, we are just beginning to understand the genetic, ecological, and physiological mechanisms that generate variation in symbiont functions, broadly termed "symbiont plasticity" here. Some possible mechanisms include shifts in symbiont community composition, genetic changes via DNA acquisition, gene expression fluctuations, and variation in symbiont densities. In this review, we examine mechanisms for plasticity in the exceptionally stable mutualisms between insects and bacterial endosymbionts. Despite the severe ecological and genomic constraints imposed by their specialized lifestyle, these bacteria retain the capacity to modulate functions depending on the particular requirements of the host. Focusing on the mutualism between Blochmannia and ants, we discuss the roles of gene expression fluctuations and shifts in bacterial densities in generating symbiont plasticity. This symbiont variation is best understood by considering ant colony as the host superorganism. In this eusocial host, the bacteria meet the needs of the colony and not necessarily the individual ants that house them.

Evolution of insect metamorphosis: a microarray-based study of larval and adult gene expression in the ant Camponotus festinatus.

Holometabolous insects inhabit almost every terrestrial ecosystem. The evolutionary success of holometabolous insects stems partly from their developmental program, which includes discrete larval and adult stages. To gain an understanding of how development differs among holometabolous insect taxa, we used cDNA microarray technology to examine differences in gene expression between larval and adult Camponotus festinatus ants. We then compared expression patterns obtained from our study to those observed in the fruitfly Drosophila melanogaster. We found that many genes showed distinct patterns of expression between the larval and adult ant life stages, a result that was confirmed through quantitative reverse-transcriptase polymerase chain reaction. Genes involved in protein metabolism and possessing structural activity tended to be more highly expressed in larval than adult ants. In contrast, genes relatively upregulated in adults possessed a greater diversity of functions and activities. We also discovered that patterns of expression observed for homologous genes in D. melanogaster differed substantially from those observed in C. festinatus. Our results suggest that the specific molecular mechanisms involved in metamorphosis will differ substantially between insect taxa. Systematic investigation of gene expression during development of other taxa will provide additional information on how developmental pathways evolve.

Storage protein content as a functional marker for colony-founding strategies: a comparative study within the harvester ant genus Pogonomyrmex.

Claustral colony founding, in which new queens rear their first clutch of workers solely from internal reserves, is common in the higher ant subfamilies and is believed to represent a major innovation in ant life histories. The ability to store large amounts of amino acids contained in storage proteins is an essential physiological trait for claustral colony founding by ant queens. To determine whether there is an association between storage protein content and colony-founding strategy, we identified and quantified two major storage proteins in queens of five harvester ant species in the genus Pogonomyrmex that differ in colony-founding strategy. Queens of the fully claustral nonforaging species Pogonomyrmex rugosus and Pogonomyrmex maricopa contained the greatest amount of these proteins. Facultatively foraging semiclaustral Pogonomyrmex occidentalis queens contained an intermediate amount. Obligately foraging semiclaustral Pogonomyrmex californicus queens from two different populations contained significantly less storage protein than the other independent-founding species. Queens of the dependent-founding social parasite Pogonomyrmex anergismus also contained little storage protein. Our results suggest that storage protein content has evolved in concert with colony-founding strategies in the genus Pogonomyrme and provides a good functional marker for colony-founding strategy.

Presence of a single abundant storage hexamerin in both larvae and adults of the grasshopper, Schistocerca americana.

We identified a single hexameric storage protein in the grasshopper, Schistocerca americana, and monitored its abundance through the last larval instar and up until reproductive competence in adults of both sexes. This storage hexamerin, termed Schistocerca americana Persistent Storage Protein (saPSP) was the most abundant soluble protein in both larvae and adults. In both sexes, saPSP abundance started out low at the onset of the last larval instar and accumulated during feeding, peaking just prior to molting. Adults of both sexes contained significant amounts of saPSP after eclosion. In adult males, saPSP content dropped continuously after eclosion and was lowest once individuals reached reproductive maturity. In contrast, adult females depleted saPSP reserves during the first days of adulthood, but subsequently accumulated significant saPSP stores. In adult females, saPSP stores peaked just prior to the completion of egg provisioning. Given the overall patterns of abundance, saPSP has functions in both larvae and adults. In addition, the observed pattern of storage hexamerin accumulation differs from patterns of accumulation in the other known grasshoppers, Locusta migratoria and Romalea microptera, suggesting that significant functional diversity has evolved in storage hexamerins among the grasshoppers.

A perspective for understanding the modes of juvenile hormone action as a lipid signaling system.

The juvenile hormones of insects regulate an unusually large diversity of processes during postembryonic development and adult reproduction. It is a long-standing puzzle in insect developmental biology and physiology how one hormone can have such diverse effects. The search for molecular mechanisms of juvenile hormone action has been guided by classical models for hormone-receptor interaction. Yet, despite substantial effort, the search for a juvenile hormone receptor has been frustrating and has yielded limited results. We note here that a number of lipid-soluble signaling molecules in vertebrates, invertebrates and plants show curious similarities to the properties of juvenile hormones of insects. Until now, these signaling molecules have been thought of as uniquely evolved mechanisms that perform specialized regulatory functions in the taxon where they were discovered. We show that this array of lipid signaling molecules share interesting properties and suggest that they constitute a large set of signal control and transduction mechanisms that include, but range far beyond, the classical steroid hormone signaling mechanism. Juvenile hormone is the insect representative of this widespread and diverse system of lipid signaling molecules that regulate protein activity in a variety of ways. We propose a synthetic perspective for understanding juvenile hormone action in light of other lipid signaling systems and suggest that lipid activation of proteins has evolved to modulate existing signal activation and transduction mechanisms in animals and plants. Since small lipids can be inserted into many different pathways, lipid-activated proteins have evolved to play a great diversity of roles in physiology and development.

Storage proteins in vespid wasps: characterization, developmental pattern, and occurrence in adults.

Wasps of family Vespidae contain three types of major proteins that have the size, amino acid composition, subunit composition, immunological reactivity, and pattern of occurrence characteristic of storage proteins. The three types of storage protein, which have been identified in other Hymenoptera, are very high density lipoprotein, high glutamine/glutamic acid protein, and hexamerin. The predominant pattern of occurrence for these proteins is as known from most or all Holometabola: synthesis during the last larval instar and utilization as an amino acid source during metamorphosis. Hexamerin also occurred in a large young adult female Monobia quadridens but not a small one, which suggests that carry-over into adult females is a reaction norm response to quantity of larval provisions, because these wasps could not have fed as adults. In two paper wasp species of the genus Polistes, hexamerin was present in large adult females which emerged during the colony cycle phase when reproductive females are typically produced, but not in adult female offspring that emerged earlier in the colony cycle or in adult females that were workers. It cannot be confirmed by these data that the hexamerin in the adult paper wasps represented carry-over from metamorphosis rather than post-emergence feeding, but the pattern of occurrence suggests that presence of storage protein may play a role in caste differentiation in paper wasps. No storage protein was found in any adult Vespula maculifrons, a yellowjacket wasp, suggesting that caste differentiation in vespine wasps does not incorporate storage protein as a component.

Sexual differences in postingestive processing of dietary protein and carbohydrate in caterpillars of two species.

Previous studies indicated that female Heliothis virescens and Estigmene acrea caterpillars use both feeding and postingestive processing as mechanisms to meet nutrient demands. We present utilization and nutrient budget data on the postingestive partitioning of nutrients into pre- and postabsorptive components. Both species utilized carbohydrate efficiently except at very high ingestion rates. Of the carbohydrate utilized, more was unaccounted for as intake increased, and we assume the material unaccounted for served as a respiratory substrate. In contrast, the pattern of nitrogen utilization differed between the species. Larval H. virescens efficiently retained nitrogen except at very high ingestion rates, whereas E. acrea progressively increased nitrogen egestion in response to increased nitrogen ingestion. Overall, females of both species utilized nitrogen more efficiently than did males at most ingestion levels, thus contributing to the greater protein-derived growth we previously reported. In addition, for H. virescens, protein and amino acids accounted for a small proportion of fecal nitrogen so that most of the fecal nitrogen was of a postabsorptive nature. In contrast, nitrogen excreted by E. acrea could be partitioned into both pre- and postabsorptive components. The manner of postingestive processing by these two species reflects differences in their larval diet.

Morphological specializations of the digestive tract of Zacryptocerus rohweri (Hymenoptera: Formicidae).

Light, scanning, and transmission electron microscopy are used to examine the morphology and ultrastructure of the peculiar digestive tract of the turtle ant, Zacryptocerus rohweri. The proventriculus is heavily sclerotized and covered with clusters of small spines. Narrow spine-lined channels converging at the opening to the midgut act as a fine filter of food; particles >12.5 µm are unable to pass through the proventriculus. In the midgut, ultrastructural study reveals bacteria among the microvilli of midgut epithelial cells. The hindgut of Z. rohweri consists of an enlarged, dark-colored pouch filled with masses of bacteria of three major morphotypes. A thick layer of circular muscle and deep infoldings of the epithelium greatly increase surface area for absorption. Newly emerged individuals appear to acquire these microorganisms by soliciting material from the abdomen tip of other older workers in the colony. Whether or not the hindgut bacteria are true symbionts is unknown; their acquisition and presence suggest that they may supplement the ants' limited, liquid diet by supplying essential amino acids and other nutrients. J. Morphol. 234:253-262, 1997. © 1997 Wiley-Liss, Inc.