Parallel evolution of termite‐egg mimicry by sclerotium‐forming fungi in distant termite groups

Among the great diversity of insect–fungus associations, fungal mimicry of termite eggs is a particularly fascinating consequence of evolution. Along with their eggs, Reticulitermes termites often harbour sclerotia of the fungus Fibularhizoctonia sp., called ‘termite balls’, giving the fungus competitor‐free habitat within termite nests. The fungus has evolved sophisticated morphological and chemical camouflage to mimic termite eggs. To date, this striking insect–fungus association has been found in eight temperate termite species, but is restricted to the lower termite genera Reticulitermes and Coptotermes. Here, we report the discovery of a novel type of termite ball (‘Z‐type’) in the subtropical termite, Nasutitermes takasagoensis. Phylogenetic analysis indicated that the Z‐type termite ball is an undescribed Trechisporoid fungus, Trechispora sp., that is phylogenetically distant from Fibularhizoctonia, indicating two independent origins of termite‐egg mimicry in sclerotium‐forming fungi. Egg protection bioassays using dummy eggs revealed that Reticulitermes speratus and N. takasagoensis differ in egg‐size preference. A comparative study of termite ball size and egg‐size preference of host termites showed that both fungi evolved a termite ball size that optimized the acceptance of termite balls as a unit investment. Termite‐egg mimicry by these fungi offers a model case of parallel evolution. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 531–537.     

Athelia termitophila sp. nov. is the teleomorph of the termite ball fungus Fibularhizoctonia sp.

A new species of Athelia, A. termitophila, from Japan is described and illustrated on the basis of morphological and phylogenetic analyses. Basidiomes of this species are characterized by having hyphae sometimes with clamp connections at the septa, basidia without clamp connections at the basal septa, and ellipsoid to ovoid basidiospores measuring 4.5–6 × 3–4.5 μm. In culture, mycelia produce pale brown, orange-brown to brown, globose sclerotia measuring 0.24–0.41 mm diam. The sclerotia are distinctly different in shape and size from those of other Athelia species, and are occasionally found inside the woody substrate beneath basidiomes. They are identical in shape and size to those of Fibularhizoctonia sp., also known as termite balls. Phylogenetic analysis using internal transcribed spacer (ITS) sequence data revealed that A. termitophila is the teleomorph of Fibularhizoctonia sp.     

Egg care by termite soldiers

The handling and transportation of eggs by soldiers of Prorhinotermes simplex after a nest disturbance is described. The soldiers, apart from performing alarm and defensive activities, tried to handle the eggs on the open surface. They often succeeded in lifting an egg or even a group of eggs, and then searched for a nest entrance. They either entered the nest to come out without the egg(s) within a while, or they passed the egg(s) onto a pseudergate waiting in the opening. As a result, all eggs were rapidly evacuated. Laboratory experimentation showed that pseudergates and nymphs try to deposit the eggs in a dark shelter, whereas the soldiers tend to transfer the eggs to nestmates and drop them after a while if no other termite accepts them. Received 25 February 2005; revised 26 April 2005; accepted 3 May 2005.     

Low temperature enhances the ability of the termite-egg-mimicking fungus Athelia termitophila to compete against wood-decaying fungi

A unique symbiosis exists between subterranean termites and the sclerotium-forming fungus Athelia termitophila, which forms termite-egg-mimicking sclerotia called ‘termite balls’. While the sclerotia gain a competitor-free habitat by being harboured by termite eggs, A. termitophila mycelia have to compete with wood-decay fungi in the life stage without termites. To understand its relationship with termites, the factors that affect the ability of A. termitophila to compete with other wood-decay fungi must be clarified. Here, we show that A. termitophila is competitive against other wood-decay fungi at low temperatures. In Petri dish experiments to evaluate the effects of the physicochemical conditions, that A. termitophila experiences in termite nests, on its competitive ability, A. termitophila overcame surrounding fungi in the winter, when termites are less active. Further studies quantifying the effects of A. termitophila on termites in winter will help us to understand this relationship.     

Reproductives and eggs trigger worker vibration in a subterranean termite

In insect societies, the presence of reproductives or eggs has been shown to shape several biological traits in the colony members. Social interactions are one of these traits that involve modification of the communication system of the entire colony. Many studies described the role of chemical compounds and dominance behaviors in the presence of reproductive but vibratory behaviors received very few investigations. Yet, vibratory behaviors are ideal candidates, particularly for subterranean species like termites, as they could be quickly transmitted through the substrate and could be very diversified (origin, modulation). Here, we investigated whether the presence of reproductives/eggs affects the vibratory behavior (body‐shaking) of workers in the subterranean termite Reticulitermes flavipes. Our results reveal that the presence of reproductives or eggs triggers an increase of workers' body‐shaking, independent of their colony of origin after 24 hr. We hypothesize that vibratory communication could be used to transfer information about the presence of reproductives and eggs to the entire colony, suggesting that vibratory behaviors could serve as an important yet neglected mediator of social regulation.     

Parthenogenetic reproduction in neotenics of the subterranean termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Experiments with artificial colonies consisting of only female workers and those of female nymphs and workers of a subterranean termite, Reticulitermes speratus, showed that neotenics (ergatoids and nymphoids) can reproduce parthenogenetically. The larvae hatched from the eggs produced partheno‐genetically by the neotenics were all female and did not have any aberrant external morphology. They had 42 chromosomes and were diploid. The analysis of genotypes at microsatellite loci revealed that the eggs produced parthenogenetically had only one of the two alleles that the mother neotenics possessed, indicating that the eggs were produced by automixis.     

Seasonal patterns of egg production in field colonies of the termite <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> (Isoptera: Rhinotermitidae)

This is the first report on the annual egg production patterns in mature termite colonies in the field. Data on the seasonal patterns of egg production in field colonies are very important for understanding the annual colony growth schedule, resource allocation, and population dynamics of the termites. However, collecting the eggs from a sufficient number of colonies is extremely difficult in Reticulitermes termites because their multiple-site nesting makes it difficult to find the reproductive center of the colonies. Here, we first show the seasonal pattern of egg production in the subterranean termite Reticulitermes speratus by collecting the reproductive center of ten colonies each month from April through October. We had to destructively examine dozens of nests to find eggs from enough field colonies each month. Mature field colonies began to produce eggs in late May, soon after the swarming season, and the egg production rate (EPR) reached its maximum in early July. The eggs hatched until late October. The EPR was significantly correlated with the average monthly temperature. Additional investigation of the egg distributions in the nests showed that most eggs were kept around the royal cell, which contained the reproductives. The largest colony had 109 supplemental queens and 94,023 eggs, suggesting that each queen produced an average of 24.7 eggs per day, based on the known mean hatching period of an inseminated egg of 34.95±0.12 (SE) days.     

Queen pheromone regulates egg production in a termite

In social insects, resource allocation is a key factor that influences colony survival and growth. Optimal allocation to queens and brood is essential for maximum colony productivity, requiring colony members to have information on the total reproductive power in colonies. However, the mechanisms regulating egg production relative to the current labour force for brood care remain poorly known. Recently, a volatile chemical was identified as a termite queen pheromone that inhibits the differentiation of new neotenic reproductives (secondary reproductives developed from nymphs or workers) in Reticulitermes speratus. The same volatile chemical is also emitted by eggs. This queen pheromone would therefore be expected to act as an honest message of the reproductive power about queens. In this study, we examined how the queen pheromone influences the reproductive rate of queens in R. speratus. We compared the number of eggs produced by each queen between groups with and without exposure to artificial queen pheromone. Exposure to the pheromone resulted in a significant decrease in egg production in both single-queen and multiple-queen groups. This is the first report supporting the role of queen pheromones as a signal regulating colony-level egg production, using synthetically derived compounds in a termite.     

Genetically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colonies

Indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus Shiraki, Isoptera: Rhinotermitidae) were used as shuttle systems to deliver, express and spread foreign genes in termite colonies. The gut bacterium Enterobacter cloacae was transformed with a recombinant plasmid (pEGFP) containing genes encoding ampicillin resistance and green fluorescent protein (GFP). In laboratory experiments, termite workers and soldiers from three colonies were fed with filter paper inoculated with transformed bacteria. Transformed bacteria were detected in termite guts by growing the entire gut flora under selective conditions and checking the cultures visually for fluorescence. We demonstrated that (1) transformed bacteria were ingested within a few hours and the GFP gene was expressed in the termite gut; (2) transformed bacteria established a persistent population in the termite gut for up to 11 weeks; (3) transformed bacteria were efficiently transferred throughout a laboratory colony, even when the donor (termites initially fed with transformed bacteria) to recipient (not fed) ratio was low; (4) transformed E. cloacae were transferred into soil; however, they did not accumulate over time and the GFP plasmid was not transferred to other soil bacteria. In the future, transgenic bacteria may be used to shuttle detrimental genes into termite colonies for improved pest control.     

Worker aggression of ant Lasius japonicus enhanced by termite soldier–specific secretion as an alarm pheromone of Reticulitermes speratus

The subterranean termite Reticulitermes speratus usually nests in rotten wood trunks, which may also be occupied by the Japanese garden ant Lasius japonicus. Few battles were observed between them under ordinary circumstances because they inhabit separate nesting sites. However, once the termite nesting sites were artificially broken, the ant workers invaded and hunted the termites, although the termite soldiers fought against the ants. This study aims to confirm intra‐ and inter‐specific chemical interactions between the termite and ant. Solid phase microextraction–gas chromatograph (SPME‐GC) analyses revealed that R. speratus soldiers secreted caste‐specific sesquiterpene hydrocarbon when they were irritated. Both the hexane extract of the soldiers and its hydrocarbon fraction, as well as the crushed soldier bodies, attracted the soldiers but dispersed the workers when presented on the trails. We also confirmed that the soldier chemicals enhanced aggressiveness of L. japonicus, which rushed around the odor sources and hunted any termites that were present. These findings suggest that: (i) the soldier–specific secretion might serve as an alarm pheromone in termite chemical communication, in which components recruit soldiers and also warn the other colony members away; and (ii) termite communication is eavesdropped on by L. japonicus workers to locate and hunt the termites.     

Irreversible transfer of brood care duties and insights into the burden of caregiving in incipient subterranean termite colonies

1. In incipient termite colonies, biparental brood care rapidly shifts towards alloparental brood care. This transition was suggested to recapitulate the evolutionary trajectory from subsocial wood roach ancestors to eusociality in termites. 2. Incipient colonies of the subterranean termite Coptotermes gestroi (Wasmann) were investigated to determine if this transition was irreversible and if the burden of brood care on the first alloparents could be measured. To do so, the initial size of the work force necessary for an incipient colony to survive once the brood care became alloparental was determined. 3. The results of the study show that within 5 months after foundation, brood care duties were fully transferred to workers and the primary reproductives became irreversibly dependent on these workers for survival, reproduction, and colony growth. 4. Once the brood care became strictly alloparental, the presence of a single worker was enough to maintain the survival of the king and queen, confirming that ‘reversed parental care’ was also achieved. However, major brood loss and suppressed egg‐laying activity from the queen was observed, suggesting that the burden of brood care was too high for a single worker to absorb. Therefore, once brood care has shifted to alloparents, a critical number of workers is necessary to prevent brood loss and initiate colony growth. 5. As the initial cost of brood care is rapidly absorbed with colony growth in termites, the performance in brood care of the first few alloparents in a subsocial wood roach ancestor may have contributed to the emergence of eusociality in this clade.     

Evidence that workers recognize unfertilized queen-laid eggs for male production in stingless bees

The behavioural variation in bee workers raises questions about the reproduction in eusocial bees, as well as possible triggers for ovarian activation and oviposition. However, there are no data on possible differences between queen-laid eggs found alone or together with worker-laid eggs in a brood cell. This study evaluated the ploidy of eggs from queens found together with worker-laid eggs in the brood cells of the stingless bee Scaptotrigona xanthotricha. In the brood cells with two eggs, there was a difference in size between them, with the smaller one being a similar size to those found in brood cells with only one egg. The frequency of diploid eggs in brood cells with one egg was 96.6%, whereas, in those with two or more, it was 39.3%. These results suggest that egg fertilization can be identified by workers, because when there is a worker-laid egg, those from the queen are often haploid, supporting a prediction from Hamilton's theory of kin selection.     

Microsatellite markers in the primitive termite Mastotermes darwiniensis

We developed 10 microsatellite loci in the primitive termite Mastotermes darwiniensis. The number of alleles per locus ranged from four to 15, and the expected heterozygosites spanned from 0.21 to 0.90, in a sample of 40 workers collected from the Northern Territory, Australia. We also determined that only two loci amplified in five other termite species. The low frequency of cross‐amplification probably resulted from the high level of phylogenetic divergence between M. darwiniensis and the other taxa. Thus, although the loci are not widely applicable, they should prove effective in elucidating the genetic structure of M. darwiniensis populations.     

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.     

Queen-specific volatile in a higher termite Nasutitermes takasagoensis (Isoptera: Termitidae)

In social insect colonies, queen-produced pheromones have important functions in social regulation. These substances influence the behavior and physiology of colony members. A queen-produced volatile that inhibits differentiation of new neotenic reproductives was recently identified in the lower termite Reticulitermes speratus. However, there are no known queen-specific volatiles of this type in any other termite species. Here, we report volatile compounds emitted by live queens of the higher termite Nasutitermes takasagoensis. We used headspace gas chromatography mass spectroscopy (HS GC-MS) to analyze volatiles emitted by live primary queens, workers, soldiers, alates, and eggs collected in a Japanese subtropical forest. Among 14 detected compounds, 7 were soldier-specific, 1 was alate-specific, 1 was egg-specific, and 1 was queen-specific. The queen-specific volatile was phenylethanol, which is different than the compound identified in R. speratus. The identification of this queen-specific volatile is the first step in determining its functions in higher termite social regulation. Comparisons of queen pheromone substances regulating caste differentiation among various termite taxa will contribute to a better understanding of the evolution of social systems in termites.     

Egg shape mimicry in parasitic cuckoos

Parasitic cuckoos lay their eggs in nests of host species. Rejection of cuckoo eggs by hosts has led to the evolution of egg mimicry by cuckoos, whereby their eggs mimic the colour and pattern of their host eggs to avoid egg recognition and rejection. There is also evidence of mimicry in egg size in some cuckoo–host systems, but currently it is unknown whether cuckoos can also mimic the egg shape of their hosts. In this study, we test whether there is evidence of mimicry in egg form (shape and size) in three species of Australian cuckoos: the fan‐tailed cuckoo Cacomantis flabelliformis, which exploits dome nesting hosts, the brush cuckoo Cacomantis variolosus, which exploits both dome and cup nesting hosts, and the pallid cuckoo Cuculus pallidus, which exploits cup nesting hosts. We found evidence of size mimicry and, for the first time, evidence of egg shape mimicry in two Australian cuckoo species (pallid cuckoo and brush cuckoo). Moreover, cuckoo–host egg similarity was higher for hosts with open nests than for hosts with closed nests. This finding fits well with theory, as it has been suggested that hosts with closed nests have more difficulty recognizing parasitic eggs than open nests, have lower rejection rates and thus exert lower selection for mimicry in cuckoos. This is the first evidence of mimicry in egg shape in a cuckoo–host system, suggesting that mimicry at different levels (size, shape, colour pattern) is evolving in concert. We also confirm the existence of egg size mimicry in cuckoo–host systems.     

Nutritional implications for sexual cannibalism in a sexually dimorphic orb web spider

Models predicting mechanisms driving sexual cannibalism in spiders with sexual size dimorphism (SSD) often assume that spiders use post‐copulatory food to channel nutrients into eggs and fecundity is altered through changes in clutch size or egg mass. I tested these assumptions using an orb web spider with extreme SSD, Argiope keyserlingi. I fed mated female spiders prey of either high protein‐low energy or low protein‐high energy composition. I measured egg energy density; a measure of the relative volumes of yolk and albumen. I predicted that if A. keyserlingi increase their egg energy density upon feeding on prey of a specific nutrient composition, they could enhance their fecundity by investing in more energy dense eggs. However, if the egg energy densities are dissimilar to their post‐copulatory prey they must be drawing energy from their somatic reserves to invest in eggs. In a further experiment I allowed female spiders to mate with and cannibalize males to determine if cannibalism induces similar effects on egg energy density. Male spider protein energy ratio was measured and found to resemble the high protein‐low energy prey. I found disagreement between the composition of post‐copulatory food and eggs in both experiments. Additionally, spiders fed high protein‐low energy prey lost weight indicating that they draw on their energy reserves to invest in eggs. I thus concluded that spiders that feed on high protein‐low energy prey or on males increase their egg energy density and, possibly, fecundity. However, the nutrient content of the prey or males cannot provide for investment in eggs. The energy invested in eggs is drawn from somatic reserves, probably induced by an as yet undescribed physiological trigger.     

Effects of controlled livestock grazing and annual prescribed fire on epigeal termite mounds in a savannah woodland in Burkina Faso

The diversity and abundance of epigeal termite mounds were investigated in response to controlled livestock grazing and annual prescribed fire in a Sudanian savannah-woodland in Tiogo State Forest. Sampling of termite mounds was carried out in 4×4 subplots of 0.25 ha in a split-plot experimental design during the rainy season in 2002. There were two main plots of which one was fenced to exclude livestock grazing and the second exposed to grazing. Each of the main plots included 4 subplots with annual prescribed fire since 1992 and 4 subplots without fire. Data were collected on the number and characteristics of termite mounds. A mean density of 698 mounds ha⁻¹ was recorded. Mounds built by Trinervitermes spp. were the most abundant followed by Cubitermes spp., Macrotermes subhyalinus and M. bellicosus. The large mound-builders Macrotermes spp. dominated the community interms of basal area (96% of the total) and above-ground volume (99%). The diversity of mound types was notaffected by livestock grazing and annual early prescribed fire (P>0.05). There was no statistical effect of livestock grazing on mound density, whereas a strong depressive effect of annual fire was observed for Trinervitermes spp. mound density (P=0.012). In this ecosystem, annual prescribed fire appeared to be the major determinant for termite mound abundance. Received 2 February 2007; revised 23 October 2007 and 21 January 2008; accepted 22 Feburary 2008.     

Avian egg collections: museum collection bias driven by shape and size

Avian eggs exhibit substantial intra- and interspecific variation in shape, size and colour. Considerable efforts have been made to better understand the evolutionary drivers behind such variation, often using museum egg collections. Usually it is assumed that museum collections accurately represent the variation seen in natural populations, but this may not be the case if there is collection bias. Collection bias may lead to the over-representation of certain egg traits in collections, due to the aesthetic (or other) preferences of collectors. The aim of this study is to begin to look for the occurrence of potential collection bias in museum egg collections by comparing three shape indices (pointedness/asymmetry, elongation and polar asymmetry) and egg volume between subsets of eggs in museum collections with those of recently sampled eggs in the field for three different bird species: common guillemot Uria aalge, razorbill Alca torda and northern fulmar Fulmarus glacialis. We found no evidence of collection bias in our sampled razorbill and northern fulmar museum collection eggs, but some evidence for a bias in sampled museum collection eggs of common guillemots. Since the guillemot's egg differs from most bird eggs in being pyriform, we suggest that collection bias by historic egg collectors may be more prevalent in species with extreme egg traits. Researchers using museum egg collections to examine questions relating to egg shape should be aware of collection bias risks and consider how to minimise the effect of these possible biases on accumulated datasets.