Inter-clonal competition over queen succession imposes a cost of parthenogenesis on termite colonies.

In social insect colonies, selfish behaviour due to intracolonial conflict among members can result in colony-level costs despite close relatedness. In certain termite species, queens use asexual reproduction for within-colony queen succession but rely on sexual reproduction for worker and alate production, resulting in multiple half-clones of a single primary queen competing for personal reproduction. Our study demonstrates that competition over asexual queen succession among different clone types leads to the overproduction of parthenogenetic offspring, resulting in the production of dysfunctional parthenogenetic alates. By genotyping the queens of 23 field colonies of Reticulitermes speratus, we found that clone variation in the queen population reduces as colonies develop. Field sampling of alates and primary reproductives of incipient colonies showed that overproduced parthenogenetic offspring develop into alates that have significantly smaller body sizes and much lower survivorship than sexually produced alates. Our results indicate that while the production of earlier and more parthenogenetic eggs is advantageous for winning the competition for personal reproduction, it comes at a great cost to the colony. Thus, this study highlights the evolutionary interplay between individual-level and colony-level selection on parthenogenesis by queens.

Hybrids of two destructive subterranean termites established in the field, revealing a potential for gene flow between species.

Hybridization between invasive pest species may lead to significant genetic and economic impacts that require close monitoring. The two most invasive and destructive termite species worldwide, Coptotermes formosanus Shiraki and Coptotermes gestroi (Wasmann), have the potential for hybridization in the field. A three-year field survey conducted during the dispersal flight season of Coptotermes in Taiwan identified alates with atypical morphology, which were confirmed as hybrids of the two Coptotermes species using microsatellite and mitochondrial analyses. Out of 27,601 alates collected over three years, 4.4% were confirmed as hybrid alates, and some advanced hybrids (>F1 generations) were identified. The hybrid alates had a dispersal flight season that overlapped with the two parental species 13 out of 15 times. Most of the hybrid alates were females, implying that mating opportunities beyond F1 may primarily be possible through female hybrids. However, the incipient colony growth results from all potential mating combinations suggest that only backcross colonies with hybrid males could sometimes lead to brood development. The observed asymmetrical viability and fertility of hybrid alates may critically reduce the probability of advanced-hybrid colonies being established in the field.

Termites can learn.

It is generally believed that termites can't learn and are not "intelligent". This study aimed to test whether termites could have any form of memory. A Y-shaped test device with one release chamber and two identical test chambers was designed and constructed by 3D printing. A colony of damp wood termites was harvested from the wild. Worker termites were randomly selected for experiment. Repellent odors that could mimic the alarm pheromone for termites were first identified. Among all substances tested, a tea tree oil and lemon juice were found to contain repellent odors for the tested termites, as they significantly reduced the time that termites spent in the chamber treated with these substances. As control, a trail pheromone was found to be attractive. Subsequently, a second cohort of termites were operant conditioned by punishment using both tea tree oil and lemon juice, and then tested for their ability to remember the path that could lead to the repellant odors. The test device was thoroughly cleaned between trials. It was found that conditioned termites displayed a reduced tendency to choose the path that led to expectant punishment as compared with naïve termites. Thus, it is concluded that damp wood termites are capable of learning and forming "fear memory", indicative of "intelligence" in termites. This result challenges established presumption about termites' intelligence.

Termite primary queen - ancestral, but highly specialized eusocial phenotype.

Termite eusociality is accompanied by flagrant caste polyphenism manifested by the presence of several sterile (workers and soldiers) and reproductive (imaginal and neotenic kings and queens) caste phenotypes. Imaginal kings and queens are developmentally equivalent to adults of other hemimetabolous insects but display multiple adaptations inherent to their role of eusocial colony founders, such as long lifespan and high fecundity. Herein, we summarize the recent advances in understanding the biology of imaginal (primary) queens as emblematic examples of termite polyphenism acquired during social evolution. We focus on the control of queen development, on dynamics in physiology and fecundity during the queen's life, on new findings about queen fertility signaling, and on proximate mechanisms underlying queen longevity.

Defensive behavior is linked to altered surface chemistry following infection in a termite society.

The care-kill response determines whether a sick individual will be treated or eliminated from an insect society, but little is known about the physiological underpinnings of this process. We exploited the stepwise infection dynamics of an entomopathogenic fungus in a termite to explore how care-kill transitions occur, and identify the chemical cues behind these shifts. We found collective responses towards pathogen-injected individuals to vary according to severity and timing of pathogen challenge, with elimination, via cannibalism, occurring sooner in response to a severe active infection. However, injection with inactivated fungal blastospores also resulted in increased albeit delayed cannibalism, even though it did not universally cause host death. This indicates that the decision to eliminate an individual is triggered before pathogen viability or terminal disease status has been established. We then compared the surface chemistry of differently challenged individuals, finding increased amounts of long-chained methyl-branched alkanes with similar branching patterns in individuals injected with both dead and viable fungal blastospores, with the latter showing the largest increase. This coincided with the highest amounts of observed cannibalism as well as signs of severe moribundity. Our study provides new mechanistic insight into the emergent collective behaviors involved in the disease defense of a termite society.

Secretory oviducts contribute to the high egg-laying rate of physogastric termite queens (Isoptera: Termitidae).

Physogastric termite queens are characterized by a notorious enlargement of the abdomen triggered by an equal development of the ovaries. Other physogastry-related modifications have been reported on the fat body, cuticle, midgut, tracheal system, and hemolymph. Surprisingly, modifications on the lateral oviducts of these females, important sites for ovulation and egg transport, have received little attention. Here we took advantage of the high fecundity of physogastric queens in three termitid species to evaluate ovary development and also to compare the morphophysiological features of the lateral oviducts between early-mated and physogastric queens of Cornitermes cumulans. Older queens show well-developed ovaries, with numerous ovarioles connected to the lateral oviducts through pedicels. At these sites, several corpora lutea were observed, residual follicle cells from previous ovulation events. Such features were absent among early-mated queens and reflect then the maturity and ageing of the queens. Histological and histochemical analyses indicated that secretory activity of the lateral oviducts was also restricted to physogastric queens, in which proteins, but not polysaccharides, are secreted into the oviduct lumen. The likely function of these proteins, based on previous studies, is to lubricate the lateral oviducts and stimulate muscular contractions to the egg transport. The physogastry of termite queens is a notorious feature, characterized by several body modifications, especially concerning the ovaries. Our results shed light on the physogastry-related changes in the lateral oviducts of termite queens, as their increasing secretory activity is in agreement with the high number of eggs produced and transporting through these structures. Thus, such changes correspond to an important step allowing the high egg-laying rate shown by physogastric termite queens.

α-Terpineol affects social immunity, increasing the pathogenicity of entomopathogenic nematodes to subterranean termites (Isoptera).

Biocontrol of subterranean termites is largely impeded by their social immune responses. Studies on biocontrol agents combined with natural insecticides and their possible effects on the immune defense mechanisms of termites are limited. In this study, we investigated the effects of a combined biocontrol strategy using a plant-derived insect ATPase inhibitor, α-terpineol, with the entomopathogenic nematodes (EPNs) Steinernema carpocapsae against the subterranean termite Coptotermes formosanus Shiraki. Survival assays showed that even a low lethal concentration of α-terpineol significantly increased the EPNs-induced virulence in C. formosanus. α-terpineol treatment majorly inhibited the activity of Na+- K+- ATPase, which disturbed the EPNs-induced enhancement of locomotor activity and grooming behavior in termites treated with the combined strategy. Furthermore, the combination treatment had a synergistic inhibitory effect on innate immune responses in C. formosanus, which were measured as changes in the expression of immune-related genes and activities of immune system enzymes. In conclusion, α-terpineol can weaken the immune defense of termites against EPNs at low lethal concentrations, and is a suitable non-synthetic insecticide to prove the biocontrol efficiency of EPNs on C. formosanus. This study provides a theoretical basis and technical reference for a novel biocontrol strategy that promises to overcome the problems of host immune defense in termites.

Exploring the effects of high curvature section distribution and density in termite tunnels on food transport efficiency: A simulation study.

This study explores the food transport efficiency of termite using an individual-based model. Termites are believed to have evolved tunneling patterns that optimize food search and transport efficiency. However, few studies have investigated transport efficiency due to the difficulty of field observations. The model is characterized by four control variables: the number of simulated termites participating in transport (k1), the distribution of high curvature sections of the termite tunnel (k2), a quantity related to the density of the tunnel sections (k3), and the duration of traffic jams (k4). As k3 increases, the total length of the high curvature section decreases. Our simulation results show that the E(k1, k2) maps for k3 and k4 contain two modes: Mode A shows that E decreases with increasing k1 due to an increase in traffic jams, while Mode B shows E increasing with increasing k1 due to a decrease in the density of curved sections and an increase in jamming resolution time. The partial rank correlation coefficient analysis reveals that k1 and k2 have a negative effect on E, while k3 and k4 have a positive effect, with k1 having the greatest influence on E, followed by k3, k4, and k2. The ecological implications of the simulation results are briefly described, and the limitations of the model are discussed.

Desiccation tolerance of Termitidae termites in relation to their nest type.

The family Termitidae is renowned for its diverse nesting behaviors, with the evolution of epigeal and arboreal nests hypothesized to increase desiccation stress due to greater exposure to air. However, these nests may also alleviate desiccation stress through humidity regulation. To explore the implications of acquiring epigeal and arboreal nests, we investigated desiccation tolerance traits in 16 Termitidae termite species with varying nest types and analyzed trait correlations. Principal component analysis revealed that termites constructing epigeal and arboreal nests exhibited reduced water loss rates and enhanced survival under desiccated conditions. Furthermore, termites building arboreal nests displayed a notably higher water content. Redundancy analysis demonstrated that nest types accounted for a substantial portion (57.2%) of the observed variation in desiccation tolerance. These findings support the hypothesis that epigeal and arboreal nests in termites are associated with increased desiccation stress and increased desiccation tolerance. These findings highlight the role of nest type in influencing desiccation tolerance mechanisms and water regulation strategies in termites.

Earthen mounds (heuweltjies) of South Africa and their termite occupants: applicability of concepts of the extended phenotype, ecosystem engineering and niche construction.

Heuweltjies are earthen mounds found throughout the Succulent Karoo of South Africa and are inhabited by the termite Microhodotermes viator. Many have assumed that heuweltjies are constructed by the occupying termites. Consequently, heuweltjies have been used as an example of several important concepts in ecology and evolution: the extended phenotype, ecosystem engineering and niche construction. However, recent findings demonstrate that M. viator does not directly construct heuweltjies. Rather, termite colonies enrich the soil around their nests with plant nutrients, which promotes development of widely separated patches of denser vegetation. Eventual formation of heuweltjies represents a response of the physical environment to the windbreak effect of the denser vegetation patches (localized reduction of wind velocity and resultant deposition and accumulation of airborne sediment). Other structures constructed by the termites are justifiably regarded as extended phenotypes. Identification and investigation of a complex cascade of processes are required to more precisely assess the manner in which this termite species functions as an ecosystem engineer or niche constructor, thereby significantly influencing the availability of resources within local ecosystems. Environmental alterations that are either directly or indirectly generated by social animals that construct large, communal nests represent ecological processes that contribute significantly to local biodiversity. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

Effects of soil moisture on tunneling, survivorship, and food consumption of the Formosan and eastern subterranean termites (Blattodea: Rhinotermitidae).

Soil moisture is a critical environmental factor for the survival and behavior of subterranean termites (family Rhinotermitidae). The invasive Formosan subterranean termite, Coptotermes formosanus Shiraki, and the native eastern subterranean termite, Reticulitermes flavipes (Kollar), co-occur in the southeastern United States, while R. flavipes is distributed in a wider geoclimatic range. Previous studies showed that subterranean termites preferred higher soil moisture levels for tunneling and feeding; however, the impacts of constant moisture remained to be characterized to understand their moisture tolerance. In this study, we hypothesized that different soil moisture regimes can alter termite foraging and survival, and that the effects differ between the two species. The tunneling activity, survivorship, and food consumption of termites were documented for 28 days with different sand moisture conditions ranging from no moisture to full saturation (0%, 1%, 5%, 15%, 25%, and 30%). We found that there were no significant differences in the responses between C. formosanus and R. flavipes. In both species, termites did not survive or tunnel with 0% moisture. Termites performed tunneling with only 1% sand moisture, although they did not survive for 28 days. A minimal of 5% sand moisture was required for survival, and there were no significant differences in survivorship, tunneling activity, or food consumption among moisture contents of 5-30%. The results suggest that subterranean termites are resilient to moisture extremes. Colonies can tolerate low moisture conditions in their foraging environment for extended times, which may allow them to tunnel and find new moisture sources for colony survival.

Screening of reference genes for microRNA analysis in the study of solider caste differentiation of Formosan subterranean termite Coptotermes formosanus Shiraki.

The soldier caste differentiation is a complex process that is governed by the transcriptional regulation and post-transcriptional regulation. microRNAs (miRNAs) are noncoding RNAs that control a wide range of activities. However, their roles in solider caste differentiation are barely studied. RT-qPCR is a powerful tool to study the function of genes. A reference gene is required for normalization for the the relative quantification method. However, no reference gene is available for miRNA quantification in the study of solider caste differentiation of Coptotermes formosanus Shiraki. In this research, in order to screen the suitable reference genes for the study of the roles of miRNAs in solider caste differentiation, the expression levels of 8 candidate miRNA genes were quantified in the head and thorax + abdomen during soldier differentiation. The qPCR data were analyzed using geNorm, NormFinder, BestKeeper, ΔCt method and RefFinder. The normalization effect of the reference genes was evaluated using the let-7-3p. Our study showed that novel-m0649-3p was the most stable reference gene, while U6 was the least stable reference gene. Our study has selected the most stable reference gene, and has paved the way for functional analysis of miRNAs in solider caste differentiation.

Plastic brain structure changes associated with the division of labor and aging in termites.

Division of labor is a prominent feature of social insect societies, where different castes engage in different specialized tasks. As brain differences are associated with behavioral differences, brain anatomy may be linked to caste polymorphism. Here, we show that termite brain morphology changes markedly with caste differentiation and age in the termite, Reticulitermes speratus. Brain morphology was shown to be associated with reproductive division of labor, with reproductive individuals (alates and neotenic reproductives) having larger brains than nonreproductives (workers and soldiers). Micro-computed tomography (CT) imaging and dissection observations showed that the king's brain morphology changed markedly with shrinkage of the optic lobes during their long life in the dark. Behavioral experiments showed that mature primary kings lose visual function as a result of optic lobe shrinkage. These results suggested that termites restructure their nervous systems to perform necessary tasks as they undergo caste differentiation, and that they also show flexible changes in brain morphology even after the final molt. This study showed that brain morphology in social insects is linked to caste and aging, and that the evolution of the division of labor is underpinned by the development of diverse neural systems for specialized tasks.

King- and queen-specific degradation of uric acid contributes to reproduction in termites.

Caste-based reproductive division of labour in social insects is built on asymmetries in resource allocation within colonies. Kings and queens dominantly consume limited resources for reproduction, while non-reproductive castes such as workers and soldiers help reproductive castes. Studying the regulation of such asymmetries in resource allocation is crucial for understanding the maintenance of sociality in insects, although the molecular background is poorly understood. We focused on uric acid, which is reserved and used as a valuable nitrogen source in wood-eating termites. We found that king- and queen-specific degradation of uric acid contributes to reproduction in the subterranean termite Reticulitermes speratus. The urate oxidase gene (RsUAOX), which catalyses the first step of nitrogen recycling from stored uric acid, was highly expressed in mature kings and queens, and upregulated with differentiation into neotenic kings/queens. Suppression of uric acid degradation decreased the number of eggs laid per queen. Uric acid was shown to be provided by workers to reproductive castes. Our results suggest that the capacity to use nitrogen, which is essential for the protein synthesis required for reproduction, maintains colony cohesion expressed as the reproductive monopoly held by kings and queens.

The effect of ergosterol on the allogrooming behavior of termites in response to the entomopathogenic fungus Metarhizium anisopliae.

Termites have physiological and behavioral immunities that make them highly resistant to pathogen infections, which complicates biocontrol efforts. However, the stimuli that trigger the pathogen-avoidance behaviors of termites are still unclear. Our study shows that workers of Coptotermes formosanus exposed to the conidia of Metarhizium anisopliae exhibited a significantly higher frequency and longer duration of allogrooming behaviors compared with untreated termites. Volatile compounds in the cuticle of control termites and termites previously exposed to a suspension of M. anisopliae conidia were analyzed and compared using a gas chromatography-mass spectrometer (GC-MS). Our results showed that the amount of ergosterol differed between the fungus-exposed and control termites. Choice tests showed that termites significantly preferred to stay on filter paper treated with ergosterol (0.05, 0.1, or 1.0 mg/mL) compared with control filter paper. In addition, termites exposed to ergosterol followed by M. anisopliae conidia were allogroomed at a significantly higher frequency and for a longer duration than termites exposed to alcohol (the solvent used with the ergosterol in the ergosterol trials) alone followed by M. anisopliae conidia. These results showed that ergosterol may enhance the allogrooming behavior of termites in the presence of entomopathogenic fungi.

Toxicological, Behavioral, and Horizontal Transfer Effects of Cycloxaprid Against Formosan Subterranean Termites (Blattodea: Rhinotermitidae).

Cycloxaprid, 9-((6-chloropyrid-3-yl)methyl)-4-nitro-8-oxa-10,11-dihydroimidazo-[2,3-a]-bicyclo-[3,2,1]-oct-3-ene, is a cis-configuration neonicotinoid insecticide. In the present study, the lethal and sublethal effect of cycloxaprid against Formosan subterranean termites, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae), was evaluated and compared with fipronil. Toxicity bioassays showed that cycloxaprid had slightly lower toxicity than fipronil. The minimum cycloxaprid concentration in sand and soil that causes 100% termite mortality was 100 ppm. Similar to fipronil, cycloxaprid significantly reduced wood consumption and tunneling activities of termites. In the tunneling-choice tests, termite tunneling activity measured in both length and area was significantly lower in sand treated with cycloxaprid (10 or 100 ppm) than that in untreated sand. In the aggregation-choice tests, cycloxaprid exhibited inhibition to termite aggregation starting from 100 ppm. In addition, cycloxaprid exhibited significant horizontal transfer effect at 10 ppm. In conclusion, our study showed that cycloxaprid is slightly less toxic than fipronil and more repellent to C. formosanus than fipronil. Future studies are needed to evaluate the effectiveness of cycloxaprid against subterranean termites in the field.

Thermal tolerance of an invasive drywood termite, Cryptotermes brevis (Blattodea: Kalotermitidae).

West Indian drywood termites (Cryptotermes brevis, Blattodea: Kalotermitidae) are an important invasive termite in many countries including Australia where they are spreading across two eastern states. Fumigation is often used to eliminate infestations, but it is costly, has negative environmental effects and does not prevent reinfestation. Heat treatment has been suggested as an alternative. Many insect pest mitigation strategies recommend 30 min exposure at 56 °C, but this may be difficult to achieve in structural applications. The potential for heating at lower temperatures was explored to determine the effect on termite survival and gut fauna. Exposure to 40 °C up to an hour did not kill the termites; however, 1-h exposure at 45 °C was lethal. Exposure for little as 3 min at 50 °C or 2 min at 55 °C was lethal. Protozoa levels were lower in termites that survived shorter exposures, but there appeared to be some recovery over time. The results suggest that short term exposures to 50 or 55 °C could be used to eliminate infestations, creating an opportunity for localized spot heating as a mitigation measure.

Termite sociogenomics: evolution and regulation of caste-specific expressed genes.

Termite genomes have been sequenced in at least five species from four different families. Genome-based transcriptome analyses have identified large numbers of protein-coding genes with caste-specific expression patterns. These genes include those involved in caste-specific morphologies and roles, for example high fecundity and longevity in reproductives. Some caste-specific expressed genes belong to multi-gene families, and their genetic architecture and expression profiles indicate they have evolved via tandem gene duplication. Candidate regulatory mechanisms of caste-specific expression include epigenetic regulation (e.g. histone modification and non-coding RNA) and diversification of transcription factors and cis-regulatory elements. We review current knowledge in the area of termite sociogenomics, focussing on the evolution and regulation of caste-specific expressed genes, and discuss future research directions.

Termites have wider thermal limits to cope with environmental conditions in savannas.

The most diverse and abundant family of termites, the Termitidae, evolved in African tropical forests. They have since colonised grassy biomes such as savannas. These open environments have more extreme conditions than tropical forests, notably wider extremes of temperature and lower precipitation levels and greater temporal fluctuations (of both annual and diurnal variation). These conditions are challenging for soft-bodied ectotherms, such as termites, to survive in, let alone become as ecologically dominant as termites have. Here, we quantified termite thermal limits to test the hypothesis that these physiological limits are wider in savanna termite species to facilitate their existence in savanna environments. We sampled termites directly from mound structures, across an environmental gradient in Ghana, ranging from wet tropical forest through to savanna. At each location, we quantified both the Critical Thermal Maxima (CTmax ) and the Critical Thermal Minima (CTmin ) of all the most abundant mound-building Termitidae species in the study areas. We modelled the thermal limits in two separate mixed-effects models against canopy cover at the mound, temperature and rainfall, as fixed effects, with sampling location as a random intercept. For both CTmax and CTmin , savanna species had significantly more extreme thermal limits than forest species. Between and within environments, areas with higher amounts of canopy cover were significantly associated with lower CTmax values of the termite colonies. CTmin was significantly positively correlated with rainfall. Temperature was retained in both models; however, it did not have a significant relationship in either. Sampling location explained a large proportion of the residual variation, suggesting there are other environmental factors that could influence termite thermal limits. Our results suggest that savanna termite species have wider thermal limits than forest species. These physiological differences, in conjunction with other behavioural adaptations, are likely to have enabled termites to cope with the more extreme environmental conditions found in savanna environments and facilitated their expansion into open tropical environments.

Lifespan prolonging mechanisms and insulin upregulation without fat accumulation in long-lived reproductives of a higher termite.

Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it.