Genetic caste determination in termites: out of the shade but not from Mars

Several ant species are known with genetic effects on caste determination but, for termites, the role of environment has been assumed to be omnipotent. Now Hayashi et al. report that commitment to the nymph and worker pathways in Reticulitermes speratus follows a simple model involving two alleles at a sex-linked locus. The spread of this system of genetic caste determination seems best explained by selection at the colony level. This remarkable system may be widely applicable throughout termites, although it cannot be universal, and may provide a window into causal aspects of the molecular biology of caste determination.     

Identification of two subterranean termite species (Isoptera: Rhinotermitidae) using cellulase genes

To identify subterranean termite species, we designed a pair of common primers that amplified 381-bp fragments from cDNAs encoding the endo-beta-1,4-glucanases (EGases) of Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe). cDNAs from C. formosanus and R. speratus, and genomic DNA from R. speratus, were amplified by polymerase chain reaction (PCR) by using this primer pair and then cloned and sequenced. Sequences amplified from C.formosanus cDNA displayed 97-99% identity to cDNA encoding the EGase of C. formosanus (CfEG), and 92-94% identity to cDNA encoding the EGase of R. speratus (RsEG). By contrast, cDNA from R. speratus displayed 99-100% identity to RsEG cDNA and 93-94% identity to CfEG cDNA. CfEG and RsEG cDNAs can therefore be used as markers for the identification of these termite species. This is the first report of the successful identification of termite species by using cDNA and genomic DNA sequences of termite origin.     

Origin of a new Reticulitermes termite (Isoptera, Rhinotermitidae) inferred from mitochondrial and nuclear DNA data

The Holoarctic termite genus Reticulitermes is widely distributed in Europe. A new Reticulitermes species, R. sp. nov, was recently found in France and Italy. Its phylogenetic position was investigated using a 743-bp fragment of mitochondrial 16S rRNA-ND1 genes and 382-bp of the nuclear ITS2 region. Phylogenies for these sequences were estimated by neighbor-joining, maximum-parsimony and maximum-likelihood analysis. The results strongly supported a relationship between R. sp. nov. and the termite species from the eastern Mediterranean area including Reticulitermes balkanensis from the Balkans, Reticulitermes lucifugus from Turkey and Reticulitermes clypeatus from Israel. The hypothesis of a relationship between R. sp. nov. and the Japanese Reticulitermes speratus was rejected by parametric bootstrap. The current distribution of R. sp. nov. could be linked to postglacial colonization routes between Balkan refuge and northern regions.     

Compound eye development during caste differentiation in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

We morphologically examined postembryonic compound eye development in Reticulitermes speratus (Kolbe) to understand developmental regulation during caste differentiation. The eye primordia were shown to exist from the larval stage. The number of ommatidia and compound eye size greatly increased over the course of imaginal development. Nymphoids (second-form reproductives) possessed a developed compound eye structure on the surface of the cuticle and thick optic nerves, but individual ommatidia were not clearly discriminated. However, in the line of apterous workers and soldiers, although the outer rim of the eye was observed from second-stage workers, there were few morphological differences among instars, including ergatoids (third-form reproductives). Both nymphoids and ergatoids are slightly physogastric and have highly developed reproductive organs. These results suggest that eye development in the apterous line could be strongly arrested and that there is a weak developmental correlation between the eyes and reproductive organs in R. speratus.     

Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination

Background

DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events.

Methodology/Principal Findings

Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucleotides in P. barbatus. We also found methylation of non-CpG sequences. This validated two bioinformatics methods for predicting gene methylation, the bias in observed to expected ratio of CpG dinucleotides and the density of CpG/TpG single nucleotide polymorphisms (SNP). Frequencies of genomic DNA methylation were determined for different developmental stages and castes using ms-AFLP assays. The genetic caste determination system (GCD) is probably the product of an ancestral hybridization event between P. barbatus and P. rugosus. Two lineages obligately co-occur within a GCD population, and queens are derived from intra-lineage matings whereas workers are produced from inter-lineage matings. Relative DNA methylation levels of queens and workers from GCD lineages (contemporary hybrids) were not significantly different until adulthood. Virgin queens had significantly higher relative levels of DNA methylation compared to workers. Worker DNA methylation did not vary among developmental stages within each lineage, but was significantly different between the currently hybridizing lineages. Finally, workers of the two genetic caste determination lineages had half as many methylated cytosines as workers from the putative parental species, which have environmental caste determination.

Conclusions/Significance

These results suggest that DNA methylation may be a conserved regulatory mechanism moderating division of labor in both bees and ants. Current and historic hybridization appear to have altered genomic methylation levels suggesting a possible link between changes in overall DNA methylation and the origin and regulation of genetic caste determination in P. barbatus.     

Intraspecific molecular phylogeny, genetic variation and phylogeography of Reticulitermes speratus (Isoptera: Rhinotermitidae)

Population structure was investigated in Reticulitermes speratus populations in the Korean Peninsula and the Japanese Archipelago. All trees derived from analyses of the combined sequence dataset of two mitochondrial genes, COII and COIII, showed that R. speratus populations cluster into two major clades comprising the Korean/southern Japanese populations and the north-ern Japanese populations. Analysis of population ge-netic structure showed strong genetic partitioning between populations of the two clades. To understand historical migration routes and current distributions, the phylogeographic history of R. speratus was inferred from intra-/interspecific phylogeny and diver-gence times estimated between the clades of the phylogenetic tree. The estimated migration route and divergence time of ancestral R. speratus are congruent with recent paleogeographic hypotheses involving land-bridge connections between the Asian continent and the Japanese Archipelago. We suggest that ancestral R. speratus separated into northern and southern Japanese populations after its migration into the Japanese main islands from East China during the early Pleistocene via the East China Sea basin, which may have been exposed during that period. The Korean populations seem to have diverged recently from southern Japanese populations; this may explain the current distribution of R. speratus in the Japanese Arachipelago, and account for why it is restricted to northern areas of the Tokara Strait.     

The conundrum of the yellow crazy ant (Anoplolepis gracilipes) reproductive mode: no evidence for dependent lineage genetic caste determination

Asexual reproduction and hybridisation are often found among highly invasive plants and marine invertebrates. Recently, it has been suggested that clonality may enhance the success of invasive ants. In contrast, obligate hybridisation (dependent lineage genetic caste determination or DL GCD in ants) may decrease the chances of population persistence if one lineage is less prevalent than the other (asymmetry in lineage ratio). Genetic data available for the invasive yellow crazy ant (Anoplolepis gracilipes) suggest that it has an unconventional mode of reproduction that may involve asexual reproduction by workers or queens, or a form of genetic caste determination. Here, we investigated whether A. gracilipes reproduction involved DL GCD. The potential for worker reproduction was also assessed. We used microsatellite markers to assess the population structure of A. gracilipes workers, males, queens and sperm in queen spermathecae, from field collections in Arnhem Land. We found that a single queen lineage is present in Arnhem Land. The presence of a single lineage of queens discounts the possibility of DL GCD. Population structure separated queens and workers into different lineages, suggesting that these castes are determined genetically in A. gracilipes, or the mode of reproduction differs between workers and queens. Evidence for worker reproduction was weak. We conclude that the reproductive mode of A. gracilipes does not involve DL GCD. The resolution of the reproductive mode of A. gracilipes is complicated by a high prevalence of diploid males. The determination of the A. gracilipes reproductive mode remains a fascinating research question, and its resolution will improve our understanding of the contribution of the reproductive system to invasion success.     

The potential and limits of termites (Isoptera) as decomposers of waste paper products

Termites (Isoptera) have often been proposed as decomposers oflignocellulosic waste, such as paper products, while termite biomass could be harvested for food supplements. Groups of Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe) were kept for 4 and 8 wk, respectively, in the laboratory and given up to 10 different types of paper as their food source. Paper consumption, survival, caste composition, and lipid content were recorded. Corrugated cardboard was by far the most consumed paper product, although survival on it was not necessarily favorable. In R. speratus, lipid reserves and neotenic numbers were quite high, but no breeding occurred. Cardboard may be the "junk food" equivalent for termites. Within the tested period, termites did not perform well on paper products that form the bulk of waste paper--corrugated cardboard, newsprint, and pamphlets and magazines. On all paper products (except recycled office paper), neotenic reproductives were formed, but larvae were observed only on kraft pulp and tissue paper. That all waste paper products contain lignocellulosic fibers does not automatically make them suitable for decomposition by termites. Each paper product has to be assessed on its own merit to see whether termites can reproduce on this diet, if it were to be a candidate for sustainable "termidegradation" and termite biomass production.     

Asexual queen succession in the subterranean termite Reticulitermes virginicus

Termite colonies are founded by a pair of primary reproductives. In many species, including subterranean termites (family Rhinotermitidae), the primary king and queen can be succeeded by neotenic reproductives that are produced from workers or nymphs within the colony. It is generally believed that these neotenics inbreed within the colony, sometimes for many generations. Here, we show that primary queens of the North American subterranean termite, Reticulitermes virginicus, are replaced by numerous parthenogenetically produced female neotenics. We collected functional female neotenics from five colonies of R. virginicus in North Carolina and Texas, USA. Genetic analysis at eight microsatellite loci showed that 91-100% of the neotenics present within a colony were homozygous at all loci, indicating that they were produced through automictic parthenogenesis with terminal fusion. In contrast, workers, soldiers and alates were almost exclusively sexually produced by mating between the female neotenics and a single king. This is the second termite species shown to undergo asexual queen succession, a system first described in the Japanese species, Reticulitermes speratus. Thus, the conditional use of sexual and asexual reproduction to produce members of different castes may be widespread within Reticulitermes and possibly other subterranean termites.     

Identification of Reticulitermes spp. (Isoptera: Reticulitermatidae) from south central United States by PCR-RFLP

Because of morphological ambiguity, traditional identification of Reticulitermes Holmgren termites has always been difficult and unreliable. A molecular diagnostic method is presented for differentiating Reticulitermes species occurring in the south central United States, which are economically important urban pests. A 379-bp region of the mtDNA COII gene and a 415-bp region of the mtDNA 16S rRNA gene were amplified using polymerase chain reaction (PCR) and sequenced from Reticulitermes flavipes (Kollar), Reticulitermes virginicus (Banks), Reticulitermes hageni Banks, and Reticulitermes tibialis (Banks). Applying DNA sequence data, the PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of two restriction enzymes each for the COII amplicon and the 16S amplicon, were diagnostic for all of the Reticulitermes species analyzed. Based on putative mutation rates, >87% and 97% of the samples should be successfully identified to species with PCR-RFLP of COII and 16S, respectively. To verify the accuracy of our predictions, we examined unclassified Reticultermes populations from Arkansas, Louisiana, Missouri, Oklahoma, Texas, and Virginia using PCR-RFLP. Applying PCR-RFLP, 97 samples were correctly classified to species. This technique allows the use of field-collected specimens preserved in alcohol and can identify termite specimens regardless of caste. PCR-RFLP, resolved with agarose or polyacrylamide gel electrophoresis, provided an efficient method for identification of Reticulitermes species from the south central United States for diagnostic purposes.     

Nestmate recognition mediated by intestinal bacteria in a termite, Reticulitermes speratus

Discrimination and aggressive responses toward non-nestmates have been observed in a number of termite species, but the mechanism is poorly understood. Here I present the novel hypothesis that differential intestinal bacteria composition leads to production of colony-specific chemical cues that enable nestmate recognition.
The intestinal microflora of a lower termite, Reticulitermes speratus , consisted of many bacteria species. The composition of the intestinal bacteria was exclusively colony-specific. Termites that had adsorbed an unfamiliar odor of bacteria sampled from another colony were fiercely attacked by nestmates. Experimental manipulation of the composition of bacteria by antibiotics successfully changed the recognition behavior toward nestmates. These results indicate that intestinal bacteria play an important role in nestmate recognition.     

Phylogeography of Pogonomyrmex barbatus and P. rugosus harvester ants with genetic and environmental caste determination

We present a phylogeographic study of at least six reproductively isolated lineages of new world harvester ants within the Pogonomyrmex barbatus and P. rugosus species group. The genetic and geographic relationships within this clade are complex: Four of the identified lineages show genetic caste determination (GCD) and are divided into two pairs. Each pair has evolved under a mutualistic system that necessitates sympatry. These paired lineages are dependent upon one another because their GCD requires interlineage matings for the production of F1 hybrid workers, and intralineage matings are required to produce queens. This GCD system maintains genetic isolation among these interdependent lineages, while simultaneously requiring co-expansion and emigration as their distributions have changed over time. It has also been demonstrated that three of these four GCD lineages have undergone historical hybridization, but the narrower sampling range of previous studies has left questions on the hybrid parentage, breadth, and age of these groups. Thus, reconstructing the phylogenetic and geographic history of this group allows us to evaluate past insights and hypotheses and to plan future inquiries in a more complete historical biogeographic context. Using mitochondrial DNA sequences sampled across most of the morphospecies’ ranges in the U.S.A. and Mexico, we conducted a detailed phylogeographic study. Remarkably, our results indicate that one of the GCD lineage pairs has experienced a dramatic range expansion, despite the genetic load and fitness costs of the GCD system. Our analyses also reveal a complex pattern of vicariance and dispersal in Pogonomyrmex harvester ants that is largely concordant with models of late Miocene, Pliocene, and Pleistocene range shifts among various arid-adapted taxa in North America.     

A microsatellite-based test of the <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> genetic caste determination model in <Emphasis Type="Italic">Coptotermes lacteus</Emphasis>

Coptotermes lacteus, a termite found in eastern Australia, is a central-site nester that constructs mounds up to 2 m in height. Mature colonies typically contain the primary king and queen, and up to a million or more worker and soldier offspring. Once a year, male and female nymphs are produced in approximately equal numbers and develop into new alates. Experimental removal of the queen in the field and subsequent mating of the king with a replacement queen, results in an unusual phenomenon whereby the production of female nymphs completely ceases, while the production of male nymphs, and workers of both sexes, continues. The proximate cause of this nymph male-bias is yet to be discovered. The production of male nymphs and workers of both sexes in de-queened C. lacteus colonies is equivalent to offspring production patterns in laboratory crosses of male nymphoid (nymph-derived) with female ergatoid (worker-derived) replacement reproductives in the related species Reticulitermes speratus. An X-linked genetically influenced caste determination (GCD) mechanism has been proposed to account for such offspring patterns in R. speratus. We examined microsatellite genotypes in C. lacteus to test a prediction arising from the R. speratus GCD model: that nymphs should result from reproduction by neotenics, rather than the primary pair. In five of six colonies examined, genotypes indicated that all workers and nymphs were derived from a single reproductive pair. In three of these cases, the primary queen was also located and examined; her genotype matched that inferred from worker and nymph genotypes. These results suggest that the GCD model proposed for R. speratus does not apply to C. lacteus, at least under field conditions. The male-nymph bias following queen removal therefore remains an unresolved issue in C. lacteus.     

Soldier‐biased gene expression in a subterranean termite implies functional specialization of the defensive caste

In a termite colony, reproduction is typically monopolized by a small number of sexuals that are supported by reproductively altruistic soldiers and workers. We expect caste differentiation to be associated with clear‐cut differences in gene expression, and for these differences to reflect caste function and development. Here, we use RNA‐Sequencing to compare the gene expression profiles of sexual nymphs and two non‐reproductive helper castes (i.e., workers and soldiers) of the Eastern subterranean termite Reticulitermes flavipes. We found that of n = 93 genes that are strictly expressed as a function of caste, a majority (78%) show a soldier‐specific pattern. This conspicuous soldier‐bias in genome‐wide expression suggests that this defensively specialized caste is functionally well‐differentiated from both the reproductive and the other non‐reproductive caste of this species, despite a shared developmental program with workers. Gene ontology analysis supports the notion of functional specialization by soldiers, as soldier‐biased gene sets are enriched for novel biological processes. Whether this pattern reflects ancient or more recent bouts of selection for caste novelty at the gene‐regulatory level is not known, but because soldiers are sterile and thus have no direct fitness, any selection for novelty must have been mediated indirectly, through reproducing relatives.     

Characterization and distribution of Pogonomyrmex harvester ant lineages with genetic caste determination

Genetic caste determination has been described in two populations of Pogonomyrmex harvester ants, each comprising a pair of interbreeding lineages. Queens mate with males of their own and of the alternate lineage and produce two types of diploid offspring, those fertilized by males of the queens' lineage which develop into queens and those fertilized by males of the other lineage which develop into workers. Each of the lineages has been shown to be itself of hybrid origin between the species Pogonomyrmex barbatus and Pogonomyrmex rugosus, which both have typical, environmentally determined caste differentiation. In a large scale genetic survey across 35 sites in Arizona, New Mexico and Texas, we found that genetic caste determination associated with pairs of interbreeding lineages occurred frequently (in 26 out of the 35 sites). Overall, we identified eight lineages with genetic caste determination that always co-occurred in the same complementary lineage pairs. Three of the four lineage pairs appear to have a common origin while their relationship with the fourth remains unclear. The level of genetic differentiation among these eight lineages was significantly higher than the differentiation between P. rugosus and P. barbatus, which questions the appropriate taxonomic status of these genetic lineages. In addition to being genetically isolated from one another, all lineages with genetic caste determination were genetically distinct from P. rugosus and P. barbatus, even when colonies of interbreeding lineages co-occurred with colonies of either putative parent at the same site. Such nearly complete reproductive isolation between the lineages and the species with environmental caste determination might prevent the genetic caste determination system to be swept away by gene flow.     

Termite-egg mimicry by a sclerotium-forming fungus

Mimicry has evolved in a wide range of organisms and encompasses diverse tactics for defence, foraging, pollination and social parasitism. Here, I report an extraordinary case of egg mimicry by a fungus, whereby the fungus gains competitor-free habitat in termite nests. Brown fungal balls, called 'termite balls', are frequently found in egg piles of Reticulitermes termites. Phylogenetic analysis illustrated that termite-ball fungi isolated from different hosts (Reticulitermes speratus, Reticulitermes flavipes and Reticulitermes virginicus) were all very similar, with no significant molecular differences among host species or geographical locations. I found no significant effect of termite balls on egg survivorship. The termite-ball fungus rarely kills termite eggs in natural colonies. Even a termite species (Reticulitermes okinawanus) with no natural association with the fungus tended termite balls along with its eggs when it was experimentally provided with termite balls. Dummy-egg bioassays using glass beads showed that both morphological and chemical camouflage were necessary to induce tending by termites. Termites almost exclusively tended termite balls with diameters that exactly matched their egg size. Moreover, scanning electron microscopic observations revealed sophisticated mimicry of the smooth surface texture of eggs. These results provide clear evidence that this interaction is beneficial only for the fungus, i.e. termite balls parasitically mimic termite eggs.     

Diversity of Nitrogen Fixation Genes in the Symbiotic Intestinal Microflora of the Termite Reticulitermes speratus

The diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of a lower termite, Reticulitermes speratus, was investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut and were clonally isolated. The phylogenetic analysis of the nifH product amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. A large number of the termite nifH sequences were most closely related to those of a firmicute, Clostridium pasteurianum, with a few being most closely related to either the (gamma) subclass of the proteobacteria or a sequence of Desulfovibrio gigas. Some of the others were distantly related to those of the bacteria and were seemingly derived from the domain Archaea. The phylogenetic positions of these nifH sequences corresponded to those of genera found during a previous determination of rRNA-based phylogeny of the termite intestinal microbial community, of which a majority consisted of new, yet-uncultivated species. The results revealed that we have little knowledge of the organisms responsible for nitrogen fixation in termites.     

引诱白蚁的食用菌腐朽物的筛选

作者在实验室内用6种白蚁:栖北网(尉虫)Reticulitermes speratus(Kolbe)、黄胸网(尉虫)R.flaviceps(Osh-ima)、圆唇网(尉虫)R.labralis Hsia et Fan、尖唇网(尉虫)R.aculabialis Tsai et Huang、普见乳(尉虫)Coptoter-mes communis Hsia et He和囟土(尉虫)Odontotermes fontanellus Kemner对5种真菌的腐朽木屑进行了单纯选择和复合选择的生测试验。这5种真菌,一种是木腐菌,即密粘褶菌Gloeophyllum trabeum(Pers.ex Fr.)Murr.,另外4种均为腐木性粮用真菌:银耳Tremella fueiformis、黑木耳Auricularia auricula、猴头菌Hericium erinaceus和香菇Lentinus edodes。生测结果表明上述4种木腐性食用真菌中除猴头菌对白蚁引诱力极微外,其余3种和密粘褶菌对供试的多种白蚊均具有引诱力。其中以银耳腐木屑的引诱力最强而持久,黑木耳次之,密粘褶菌、香菇又次之。有关银耳和黑木耳腐朽木屑中对白蚁具引诱力的活性物质的化学成份和结构尚需进一步研究。