Edward L. Vargo

Colony Size as a Buffer Against Seasonality: Bergmann's Rule in Social Insects

In eusocial species, the size of the superorganism is the summed sizes of its component individuals. Bergmanns rule, the cline of decreasing size with decreasing latitude, applies to colony size in ants. Using data from the literature and our own collections, we show that colony sizes of tropical ant species are about one-tenth the average size of temperate species. This pattern holds when species or genera are sample units. Further, this trend is shown in 17 of 19 genera and five of six subfamilies. Bergmanns rule may arise if seasonal famine favors larger organisms, given their increased energy reserves. We constructed three colony sizes of the ant Solenopsis invicta. We deprived these colonies of food, or food and water. Queens, when surrounded by 102 workers or 104 workers, survived longer than solitary queens. When deprived only of food, days of queen survival had an allometry of M0.21 (where M is mass), not significantly different from the predicted M0.25 for unitary organisms. We propose that shorter growing seasons in the temperate latitudes cull small-colony species through overwintering starvation, which contributes to Bergmanns rule in social insects.

Molecular traces of alternative social organization in a termite genome

Although eusociality evolved independently within several orders of insects, research into the molecular underpinnings of the transition towards social complexity has been confined primarily to Hymenoptera (for example, ants and bees). Here we sequence the genome and stage-specific transcriptomes of the dampwood termite Zootermopsis nevadensis (Blattodea) and compare them with similar data for eusocial Hymenoptera, to better identify commonalities and differences in achieving this significant transition. We show an expansion of genes related to male fertility, with upregulated gene expression in male reproductive individuals reflecting the profound differences in mating biology relative to the Hymenoptera. For several chemoreceptor families, we show divergent numbers of genes, which may correspond to the more claustral lifestyle of these termites. We also show similarities in the number and expression of genes related to caste determination mechanisms. Finally, patterns of DNA methylation and alternative splicing support a hypothesized epigenetic regulation of caste differentiation.

Biology of Subterranean Termites : Insights from Molecular Studies of Reticulitermes and Coptotermes

Molecular genetic techniques have made contributions to studies on subterranean termites at all levels of biological organization. Most of this work has focused on Reticulitermes and Coptotermes, two ecologically and economically important genera. DNA sequence data have significantly improved our understanding of the systematics and taxonomy of these genera. Techniques of molecular biology have provided important new insights into the process of caste differentiation. Population genetic markers, primarily microsatellites, have furthered our understanding of the life history, population biology, community ecology, and invasion biology of subterranean termites. Recent results on the behavioral ecology of subterranean termites reveal a picture different from long-held views, especially those concerning colony breeding structures and foraging ranges. As additional molecular tools and genomic resources become available, and as more subterranean termite researchers incorporate molecular techniques into their approaches, we can expect accelerating advances in all aspects of the biology of this group.

BIOCHEMICAL PHENOTYPIC AND GENETIC STUDIES OF TWO INTRODUCED FIRE ANTS AND THEIR HYBRID (HYMENOPTERA: FORMICIDAE)

Two introduced fire ants, Solenopsis invicta and S. richteri, and their hybrid were studied using phenotypic markers from gas chromatographic analysis of hydrocarbons and venom alkaloids, as well as genetic markers from enzyme electrophoresis. Both methods show that extensive gene introgression is occurring over a distance of at least 120 km at the contact zone between the two forms in eastern Mississippi. Genetic analyses suggest that the hybrid population does not depart dramatically from panmixia. Also, recombinant genotypes predominate in the hybrid zone, indicating that F1 hybrids are viable. Allele frequency clines through the hybrid zone are apparent for four polymorphic loci. Data sets generated by the chromatographic and electrophoretic methods are highly concordant in that they differentiate completely between the two forms and agree in designating colonies from the contact zone as hybrid or parental in a high proportion (90%) of cases. The two methods can serve as complementary tools for studying closely related but genetically distinct populations in this, and perhaps other, groups of insects.

Identification of a pheromone regulating caste differentiation in termites

The hallmark of social insects is their caste system: reproduction is primarily monopolized by queens, whereas workers specialize in the other tasks required for colony growth and survival. Pheromones produced by reining queens have long been believed to be the prime factor inhibiting the differentiation of new reproductive individuals. However, there has been very little progress in the chemical identification of such inhibitory pheromones. Here we report the identification of a volatile inhibitory pheromone produced by female neotenics (secondary queens) that acts directly on target individuals to suppress the differentiation of new female neotenics and identify n-butyl-n-butyrate and 2-methyl-1-butanol as the active components of the inhibitory pheromone. An artificial pheromone blend consisting of these two compounds had a strong inhibitory effect similar to live neotenics. Surprisingly, the same two volatiles are also emitted by eggs, playing a role both as an attractant to workers and an inhibitor of reproductive differentiation. This dual production of an inhibitory pheromone by female reproductives and eggs probably reflects the recruitment of an attractant pheromone as an inhibitory pheromone and may provide a mechanism ensuring honest signaling of reproductive status with a tight coupling between fertility and inhibitory power. Identification of a volatile pheromone regulating caste differentiation in a termite provides insights into the functioning of social insect colonies and opens important avenues for elucidating the developmental pathways leading to reproductive and nonreproductive castes.

Queen Succession Through Asexual Reproduction in Termites

The evolution and maintenance of sexual reproduction may involve important tradeoffs because asexual reproduction can double an individuals contribution to the gene pool but reduces diversity. Moreover, in social insects the maintenance of genetic diversity among workers may be important for colony growth and survival. We identified a previously unknown termite breeding system in which both parthenogenesis and sexual reproduction are conditionally used. Queens produce their replacements asexually but use normal sexual reproduction to produce other colony members. These findings show how eusociality can lead to extraordinary reproductive systems and provide important insights into the advantages and disadvantages of sex.

Wolbachia infections in native and introduced populations of fire ants (Solenopsis spp.)

Wolbachia are cytoplasmically inherited bacteria that induce a variety of effects with fitness consequences on host arthropods, including cytoplasmic incompatibility, parthenogenesis, male‐killing and feminization. We report here the presence of Wolbachia in native South American populations of the fire ant Solenopsisinvicta, but the apparent absence of the bacteria in introduced populations of this pest species in the USA. The Wolbachia strains in native S. invicta are of two divergent types (A and B), and the frequency of infection varies dramatically between geographical regions and social forms of this host. Survey data reveal that Wolbachia also are found in other native fire ant species within the Solenopsis saevissima species complex from South America, including S. richteri. This latter species also has been introduced in the USA, where it lacks Wolbachia. Sequence data reveal complete phylogenetic concordance between mtDNA haplotype in S. invicta and Wolbachia infection type (A or B). In addition, the mtDNA and associated group A Wolbachia strain in S. invicta are more closely related to the mtDNA and Wolbachia strain found in S. richteri than they are to the mtDNA and associated group B Wolbachia in S. invicta. These data are consistent with historical introgression of S. richteri cytoplasmic elements into S. invicta populations, resulting in enhanced infection and mtDNA polymorphisms in S. invicta. Wolbachia may have significant fitness effects on these hosts (either directly or by cytoplasmic incompatibility) and therefore these microbes potentially could be used in biological control programmes to suppress introduced fire ant populations.

Polymorphism at trinucleotide microsatellite loci in the subterranean termite Reticulitermes flavipes.

The subterranean termite Reticulitermes flavipes (Rhinotermitidae) is widespread throughout the eastern U.S.A., where it is important both as a decomposer of wood and as an economic pest. The social organization and modes of colony foundation in Reticulitermes spp. are highly flexible (reviewed by Thorne 1998). However, the cryptic nesting and foraging habits of these subterranean species has made it difficult to conduct extensive studies of their social and spatial organization. Consequently, we have little information on the relative frequencies of the alternate forms of colony organization and modes of reproduction, or how these vary in response to ecological conditions. Genetic markers have great potential for elucidating colony organization and population structure, but there have only been a few studies on Reticulitermes spp. using either allozymes To provide a sensitive tool for investigating colony and population structure, I developed microsatellite markers for R. flavipes. Termites were collected from infested logs of pine and hardwood trees at various locations in North Carolina, USA. Heads of five workers from each of five colonies were used to construct the genomic library. DNA from pooled tissue was extracted using the Wizard Genomic DNA Purification Kit (Promega) after grinding in liquid nitrogen. Library construction and screening was performed largely following the protocol of Glenn (1996). The genomic DNA was digested with Sau 3AI, and fragments 300 –700 bp were selected for cloning into pZErO-2 plasmids (Invitrogen). I transformed TOP10 F ′ cells to obtain a 20 000-clone library. Plated colonies were lifted onto nylon membranes which were probed with synthetic oligonucleotides consisting of 8–10 repeats of a trinucleotide motif rich in AT (AAT, AAC, ATC, AAG and ACT). Thirty-six positive clones were sequenced. Southern blots of plasmid DNA confirmed 36 positives, for which I obtained 23 sequences containing five or more repeats. From these sequences, 20 primer pairs were designed. Microsatellite analysis was performed largely according to the methods of Oetting et al. (1995) in which the first 19 bp of the M13 forward sequencing primer (CACGACGTTGTAAAA-CGAC) is added to the 5 ′ end of one of the specific primers in each pair. The tail was attached to the left primer in each pair, except in the case of Rf 5 –10, in which it was added to the right primer. A fluorescent labelled M13 (M13F-29/IRD 800, Li-Cor) primer was included in the polymerase chain reaction (PCR), yielding labelled product which was detected in a Li-Cor …

HIERARCHICAL ANALYSIS OF COLONY AND POPULATION GENETIC STRUCTURE OF THE EASTERN SUBTERRANEAN TERMITE, RETICULITERMES FLAVIPES, USING TWO CLASSES OF MOLECULAR MARKERS

Abstract Termites (Isoptera) comprise a large and important group of eusocial insects, yet, in contrast to the eusocial Hymenoptera (ants, bees, wasps), the breeding systems of termites remain poorly understood. In this study, I inferred the breeding system of the subterranean termite Reticulitermes flavipes based on colony and population genetic structure as determined from microsatellite and mitochondrial DNA markers. Termites were sampled from natural wood debris from three undisturbed, forested sites in central North Carolina. In each site, two transects separated by 1 km were sampled at approximately 15‐m intervals. A total of 1272 workers collected from 57 collection points were genotyped at six microsatellite loci, and mitochondrial DNA haplotype was determined for a subset of these individuals using either restriction fragment length polymorphism or sequence variation in the AT‐rich region. Colonies appeared to be localized: workers from the 57 collection points represented 56 genetically distinct colonies with only a single colony occupying two collection points located 15 m apart. Genetic analysis of family structure and comparisons of estimates of F‐statistics (FIT, FIC, FCT) and coefficients of relatedness (r) among nestmate workers with results of computer simulations of potential breeding systems suggested that 77% of all colonies were simple families headed by outbred monogamous pairs, whereas the remaining colonies were extended (inbred) families headed by low numbers of neotenics (about two females and one male) who were the direct offspring of the colony founders. There was no detectable isolation by distance among colonies along transects, suggesting that colony reproduction by budding is not common and that dispersal of reproductives during mating flights is not limited over this distance. Higher‐level analysis of the microsatellite loci indicated weak but significant differentiation among sites (FST= 0.06), a distance of 16–38 km, and between transects within sites (FST ‐0.06), a distance of 1 km. No significant differentiation at either the transect or site level was detected in the mitochondrial DNA sequence data. These results indicate that the study populations of R. flavipes have a breeding system characterized by monogamous pairs of outbred reproductives and relatively low levels of inbreeding because most colonies do not live long enough to produce neotenics, and those colonies that do generate neotenics contain an effectively small number of them.

Colony genetic organization and colony fusion in the termite Reticulitermes flavipes as revealed by foraging patterns over time and space

Temporal and spatial analyses are seldom utilized in the study of colony genetic structure, but they are potentially powerful methods which can yield novel insights into the mechanisms underlying variation in breeding systems. Here we present the results of a study which incorporated both of these dimensions in an examination of genetic structure of subterranean termites in the genus Reticulitermes (primarily R. flavipes). Most colonies of this species (70%) were simple families apparently headed by outbred primary reproductives, while most of the remaining (27% of the total) colonies contained low effective numbers of moderately inbred reproductives. Mapping the spatial distribution of colony foraging sites over time revealed that despite the high colony density, the absolute foraging boundaries of most R. flavipes colonies were persistent and exclusive of other conspecific colonies, which suggests that this species is more territorial than has been implied by laboratory studies of intraspecific aggression. Nevertheless, we found a single colony (3% of all colonies) which contained the offspring of more than two unrelated reproductives. Although other studies have also described subterranean termite colonies with a similarly complex genetic composition, we demonstrate here that such colonies can form under natural conditions via the fusion of whole colonies. This study underscores how repeated sampling from individual colonies over time and space can yield information about colony spatial and genetic structure that cannot be obtained from conventional analyses or sampling methods.