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.

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.

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.

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.

Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: Gene duplication facilitates social evolution.

Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing a transitional taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome, and methylome of the Japanese subterranean termite Reticulitermes speratus Our analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression was prevalent in the R. speratus genome. The duplicated genes comprised diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense), and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but their expression patterns were highly variable, exhibiting caste biases. Some of the assayed duplicated genes were expressed in caste-specific organs, such as the accessory glands of the queen ovary and the frontal glands of soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification, leading to caste-biased expression and subfunctionalization and/or neofunctionalization conferring caste-specialized functions.

The role of epigenetics, particularly DNA methylation, in the evolution of caste in insect societies.

Eusocial insects can be defined as those that live in colonies and have distinct queens and workers. For most species, queens and workers arise from a common genome, and so caste-specific developmental trajectories must arise from epigenetic processes. In this review, we examine the epigenetic mechanisms that may be involved in the regulation of caste dimorphism. Early work on honeybees suggested that DNA methylation plays a causal role in the divergent development of queen and worker castes. This view has now been challenged by studies that did not find consistent associations between methylation and caste in honeybees and other species. Evidence for the involvement of methylation in modulating behaviour of adult workers is also inconsistent. Thus, the functional significance of DNA methylation in social insects remains equivocal. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Silencing of the phosphofructokinase gene impairs glycolysis and causes abnormal locomotion in the subterranean termite Reticulitermes chinensis Snyder.

Phosphofructokinase (PFK) is a rate-limiting enzyme in glycolysis, but its linkage with locomotion in termites is not well understood, despite the demonstrated involvement of this gene in the locomotion of different animals. Here, we investigated the effect of the pfk gene on locomotion in the subterranean termite Reticulitermes chinensis Snyder through RNA interference and the use of an Ethovision XT tracking system. The knockdown of pfk resulted in significantly decreased expression of the pfk gene in different castes of termites. The pfk-silenced workers displayed higher levels of glucose but lower levels of nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) production and decreased activity of the PFK enzyme. Furthermore, abnormal locomotion (decreased distance travelled, velocity and acceleration but increased turn angle, angular velocity and meander) was observed in different castes of pfk-silenced termites. We found caste-specific locomotion among workers, soldiers and dealates. Additionally, soldiers and dealates showed higher velocity in the inner zone than in the wall zone, which is considered an effective behaviour to avoid predation. These findings reveal the close linkage between the pfk gene and locomotion in termites, which helps us to better understand the regulatory mechanism and caste specificity of social behaviours in social insects.

Biochemical, molecular, and morphological variations of flight muscles before and after dispersal flight in a eusocial termite, Reticulitermes chinensis.

Swarming behavior facilitates pair formation, and therefore mating, in many eusocial termites. However, the physiological adjustments and morphological transformations of the flight muscles involved in flying and flightless insect forms are still unclear. Here, we found that the dispersal flight of the eusocial termite Reticulitermes chinensis Snyder led to a gradual decrease in adenosine triphosphate supply from oxidative phosphorylation, as well as a reduction in the activities of critical mitochondrial respiratory enzymes from preflight to dealation. Correspondingly, using three-dimensional reconstruction and transmission electron microscopy (TEM), the flight muscles were found to be gradually deteriorated during this process. In particular, two tergo-pleural muscles (IItpm5 and III-tpm5) necessary to adjust the rotation of wings for wing shedding behavior were present only in flying alates. These findings suggest that flight muscle systems vary in function and morphology to facilitate the swarming flight procedure, which sheds light on the important role of swarming in successful extension and fecundity of eusocial termites.

The relationship between oxidant levels and gut physiology in a litter-feeding termite.

The termite gut is an efficient decomposer of polyphenol-rich diets, such as lignocellulosic biomasses, and it has been proposed that non-enzymatic oxidative mechanisms could be involved with the digestive process in these animals. However, oxidant levels are completely unknown in termites, as well as protective mechanisms against oxidative damage to the termite gut and its microbiota. As the first step in investigating the role oxidants plays in termite gut physiology, this work presents oxidant levels, antioxidant enzymatic defenses, cell renewal and microbiota abundance along the litter-feeding termite Cornitermes cumulans gut compartments (foregut, midgut, mixed segment and hindgut p1, p3, p4, and p5 segments) and salivary glands. The results show variable levels of oxidants along the C. cumulans gut, the production of antioxidant enzymes, gut cell renewal as potential defenses against oxidative injuries and the profile of microbiota distribution (being predominantly inverse to oxidant levels). In this fashion, the oxidative challenges imposed by polyphenol-rich diet seem to be circumvented by the C. cumulans gut, ensuring efficiency of the digestive process together with preservation of tissue homoeostasis and microbiota growth. These results present new insights into the physicochemical properties of the gut in a litter-feeding termite, expanding our view in relation to termites' digestive physiology.

The evolution of sociality in termites from cockroaches: A taxonomic and phylogenetic perspective.

Despite multiple studies and advances, sociality still puzzles evolutionary biologists in numerous ways, which might be partly addressed with the advent of sociogenomics. In insects, the majority of sociogenomic studies deal with Hymenoptera, one of the two groups that evolved eusociality with termites. But, to fully grasp the evolution of sociality, studies must obviously not restrict to eusocial lineages. Multiple kinds of social system transitions have been recorded and they all bring complementary insights. For instance, cockroaches, the closest relatives to termites, display a wide range of social interactions and evolved convergently subsocial behaviors (i.e., brood care). In this context, we emphasize the need for natural history, taxonomic, and phylogenetic studies. Natural history studies provide the foundations on which building hypotheses, whereas taxonomy provides the taxa to sample to test these hypotheses, and phylogenetics brings the historical framework necessary to test evolutionary scenarios of sociality evolution.

Juvenile hormone and hemimetabolan eusociality: a comparison of cockroaches with termites.

Termites are social Dictyoptera that evolved eusociality independently from social Hymenoptera. They are characterized by unique developmental plasticity that is the basis of caste differentiation and social organization. As developmental plasticity is a result of endocrine regulation, in order to understand the evolution of termite sociality it is helpful to compare the endocrine underpinning of development between termites and cockroaches. Nijhout and Wheeler (1982) proposed that varying JH titers determine caste differentiation in termites. Based on current results, we extend this model by adding the importance of social interactions. High JH titers in the presence of soldiers lead to regressive development (decrease in body size, apparent regression in development), while an absence of soldiers induces (pre-)soldier differentiation. On the opposite side, low JH titers in colonies headed by reproductives result in progressive molts toward adults, while an absence of reproductives induces development of replacement reproductives. In cockroaches, transcription factors involved in JH signaling, including the adult specifier E93 (the co-called MEKRE93 pathway) regulate the morphogenetic transition between the nymph and the adult. In termites, we speculate that castes might be determined by social effects playing a modulatory action of JH in the MEKRE93 pathway.

Asexual queen succession mediates an accelerated colony life cycle in the termite Silvestritermes minutus.

Mixed modes of reproduction, combining sexual processes with thelytokous parthenogenesis, occur in all major clades of social insects. In several species of termites, queens maximize their genetic input into nondispersing replacement queens through parthenogenesis, while maintaining genetically diverse sterile offspring and dispersing reproductives via sexual reproduction. This so-called asexual queen succession (AQS) has multiple independent origins and its presumed advantages are diverse as well, ranging from multiplication of colony reproductive potential to extension of its lifespan beyond that of the foundress. However, how AQS shapes colony life cycles under natural conditions remains poorly known. The neotropical termite Silvestritermes minutus inhabits small but conspicuous nests, offering a unique opportunity to investigate the impact of AQS on life history. We report on its breeding system, life cycle and sex allocation using social structure census in 137 nests and genotyping of 12 colonies at 12 microsatellite loci. We show that colonies are established by an outbred pair of primary reproductives. In less than 2 years, the foundress is replaced by multiple neotenic queens, arising mostly through automixis with central fusion. Sterile castes, male and most (93%) female dispersers are produced sexually. Colony reproduction is usually restricted to a single dispersal of alates with unbiased sex ratio, taking place after 3 years. We conclude that S. minutus benefits from AQS to maximize colony growth rate and alate production within a very short life cycle rather than to extend colony lifespan. This highlights the versatile role of AQS in different cases of its polyphyletic origin.

Laboratory Evaluations of Durability of Southern Pine Pressure Treated With Extractives From Durable Wood Species.

Extracts from sawdust of four naturally durable wood species [Alaskan yellow cedar, AYC, Cupressus nootkanansis D. Don 1824; eastern red cedar, ERC, Juniperus virginiana L.; honey mesquite, HM, Prosopis glandulosa Torr.; and black locust, BL, Robinia pseudoacacia L.] were used to treat southern pine, Pt, Pinus taeda L. sapwood blocks. Extractive treated blocks were evaluated for decay resistance in standard soil bottle fungal assays challenged with brown and white rot decay fungi. Results showed that extractives did impart some improvement to decay resistance of Pt blocks. BL- and HM-treated Pt blocks were also used in choice and no-choice assays to determine feeding preference and damage by eastern subterranean termites (Reticulitermes flavipes) Kollar. Minimal feeding on treated blocks was seen in both choice and no-choice assays. In choice assays, there was similar mortality between HM and BL arenas; however, in no-choice assays, complete mortality was recorded for HM-treated Pt and high mortality was seen with BL-treated Pt. Subsequent dose mortality termite assays showed HM to be effective in killing R. flavipes at low concentrations. Both HM and BL show promise as deterrents or termiticidal protectants and will be further evaluated in field studies.

High juvenile hormone titre and abdominal activation of JH signalling may induce reproduction of termite neotenics.

Termite castes are a key example of polyphenism, in which reproductive division of labour is clearly seen in colonies. The reproductive castes in termites include primary and neotenic reproductives; primary reproductives found a new colony whereas neotenics succeed them in the reproductive role when the primary reproductives die or become senescent. Neotenics usually differentiate from nymphs or workers by developing functional gonads while retaining juvenile characteristics; however, the developmental mechanism during neotenic differentiation remains poorly understood. Juvenile hormone (JH) mediates a number of aspects of developmental regulation in caste differentiation in termites. In the present study we quantified JH titres in neotenic reproductives of Reticulitermes speratus, and compared these with other developmental stages. In addition, expression changes in JH signalling gene homologues (Methoprene-tolerant [Met], Krüppel-homolog1, Broad-Complex) in the head, thorax and abdomen were investigated during neotenic differentiation. Finally, we examined the function of Met in reproduction of neotenics by RNA interference (RNAi). Our results showed that the JH titres of neotenics were significantly higher than those of nymphs and workers. JH signalling genes were highly expressed in neotenic abdomens, compared with those in workers and nymphs. Met RNAi resulted in the inhibition of vitellogenin gene expression in newly moulted neotenics. These results suggest that the fertility of neotenics might be controlled by a large increase of JH titres and body-part-specific activation of JH signalling pathways.

Gas exchange pattern transitions in the workers of the harvester termite.

The evolutionary genesis and the current adaptive significance of the use of the discontinuous gas exchange cycle (DGC) for respiration by insects is the subject of intense debate. Years of research have resulted in several leading hypotheses, one of which is the emergent-property hypothesis. This hypothesis states that DGC is an emergent property or consequence of interactions between the O2 and CO2 set points that regulate spiracular function, i.e. opening and closing. Workers of the harvester termite, Hodotermes mossambicus were selected as a model to test this hypothesis. The respiratory patterns of major workers, investigated using flow-through respirometry, were obtained at 100% relative humidity (RH) under varying temperature to evaluate the assumptions of the emergent-property hypothesis. Metabolic rate, measured as VCO2 increased significantly after 15 °C. As VCO2 increased in response to increasing temperature and activity, the gas exchange pattern displayed by workers transitioned to a continuous gas exchange. A true DGC, defined as showing all three phases and a coefficient of variation value close to 2, was not expressed under the experimental conditions. The conclusion drawn from this study of termite workers is that changes in respiratory patterns are most likely an emergent property of the insects' nervous and respiratory system.

Using hidden Markov models to characterize termite traveling behavior in tunnels with different curvatures.

Subterranean termites live underground and build tunnel networks to obtain food and nesting space. After obtaining food, termites return to their nests to transfer it. The efficiency of termite movement through the tunnels is directly connected to their survival. Tunnels should therefore be optimized to ensure highly efficient returns. An optimization factor that strongly affects movement efficiency is tunnel curvature. In the present study, we investigated traveling behavior in tunnels with different curvatures. We then characterized traveling behavior at the level of the individual using hidden Markov models (HMMs) constructed from the experimental data. To observe traveling behavior, we designed 5-cm long artificial tunnels that had different curvatures. The tunnels had widths (W) of 2, 3, or 4mm, and the linear distances between the two ends of the tunnels were (D) 20, 30, 40, or 50mm. High values of D indicate low curvature. We systematically observed the traveling behavior of Coptotermes formosanus shiraki and Reticulitermes speratus kyushuensis and measured the time (τ) required for a termite to pass through the tunnel. Using HMM models, we calculated τ for different tunnels and compared the results with the τ of real termites. We characterized the traveling behavior in terms of transition probability matrices (TPM) and emission probability matrices (EPM) of HMMs. We briefly discussed the construction of a sinusoidal-like tunnels in relation to the energy required for termites to pass through tunnels and provided suggestions for the development of more sophisticated HMMs to better understand termite foraging behavior.

Termites as targets and models for biotechnology.

Termites have many unique evolutionary adaptations associated with their eusocial lifestyles. Recent omics research has created a wealth of new information in numerous areas of termite biology (e.g., caste polyphenism, lignocellulose digestion, and microbial symbiosis) with wide-ranging applications in diverse biotechnological niches. Termite biotechnology falls into two categories: (a) termite-targeted biotechnology for pest management purposes, and (b) termite-modeled biotechnology for use in various industrial applications. The first category includes several candidate termiticidal modes of action such as RNA interference, digestive inhibition, pathogen enhancement, antimicrobials, endocrine disruption, and primer pheromone mimicry. In the second category, termite digestomes are deep resources for host and symbiont lignocellulases and other enzymes with applications in a variety of biomass, industrial, and processing applications. Moving forward, one of the most important approaches for accelerating advances in both termite-targeted and termite-modeled biotechnology will be to consider host and symbiont together as a single functional unit.

Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae).

Beta-glucosidase (BG) is known as a multifunctional enzyme for social maintenance in terms of both cellulose digestion and social communication in termites. However, the expression profiles of each BG gene and their evolutionary history are not well understood. First, we cloned two types of BG homologs (RsBGI and RsBGII) from the termite Reticulitermes speratus (Kolbe). Gene expression analyses showed that RsBGI expression levels of primary queens and kings from 30 to 100 days after colony foundation were high, but those of reproductives dropped after day 400. Extremely low gene expression levels of RsBGI were observed in eggs, whereas workers had significantly higher expression levels than those of soldiers and other colony members. Consequently, RsBGI gene expression levels changed among each developmental stage, and RsBGI was shown to be involved in cellulose digestion. On the other hand, the RsBGII gene was consistently expressed in all castes and developmental stages examined, and notable expression changes were not observed among them, including in eggs. It was indicated that RsBGII is a main component involved in social communication, for example, the egg-recognition pheromone shown in this species previously. Finally, we obtained partial gene homologs from other termite and cockroach species, including the woodroach (genus Cryptocercus), which is the sister group to termites, and performed molecular phylogenetic analyses. The results showed that the origin of the BG gene homologs preceded the divergence of termites and cockroaches, suggesting that the acquisition of multifunctionality of the BG gene also occurred in cockroach lineages.

Cockroaches probably cleaned up after dinosaurs.

Dinosaurs undoubtedly produced huge quantities of excrements. But who cleaned up after them? Dung beetles and flies with rapid development were rare during most of the Mesozoic. Candidates for these duties are extinct cockroaches (Blattulidae), whose temporal range is associated with herbivorous dinosaurs. An opportunity to test this hypothesis arises from coprolites to some extent extruded from an immature cockroach preserved in the amber of Lebanon, studied using synchrotron X-ray microtomography. 1.06% of their volume is filled by particles of wood with smooth edges, in which size distribution directly supports their external pre-digestion. Because fungal pre-processing can be excluded based on the presence of large particles (combined with small total amount of wood) and absence of damages on wood, the likely source of wood are herbivore feces. Smaller particles were broken down biochemically in the cockroach hind gut, which indicates that the recent lignin-decomposing termite and cockroach endosymbionts might have been transferred to the cockroach gut upon feeding on dinosaur feces.