Soldier Caste Differentiation Research Articles

Regulation of soldier caste differentiation by microRNAs in Formosan subterranean termite (Coptotermes formosanus Shiraki).

The soldier caste is one of the most distinguished castes inside the termite colony. The mechanism of soldier caste differentiation has mainly been studied at the transcriptional level, but the function of microRNAs (miRNAs) in soldier caste differentiation is seldom studied. In this study, the workers of Coptotermes formosanus Shiraki were treated with methoprene, a juvenile hormone analog which can induce workers to transform into soldiers. The miRNomes of the methoprene-treated workers and the controls were sequenced. Then, the differentially expressed miRNAs (DEmiRs) were corrected with the differentially expressed genes DEGs to construct the DEmiR-DEG regulatory network. Afterwards, the DEmiR-regulated DEGs were subjected to GO enrichment and KEGG enrichment analysis. A total of 1,324 miRNAs were identified, among which 116 miRNAs were screened as DEmiRs between the methoprene-treated group and the control group. A total of 4,433 DEmiR-DEG pairs were obtained. No GO term was recognized as significant in the cellular component, molecular function, or biological process categories. The KEGG enrichment analysis of the DEmiR-regulated DEGs showed that the ribosome biogenesis in eukaryotes and circadian rhythm-fly pathways were enriched. This study demonstrates that DEmiRs and DEGs form a complex network regulating soldier caste differentiation in termites.

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.

Regulation of host phenotypic plasticity by gut symbiont communities in the eastern subterranean termite (Reticulitermes flavipes).

Termites are eusocial insects that host a range of prokaryotic and eukaryotic gut symbionts and can differentiate into a range of caste phenotypes. Soldier caste differentiation from termite workers follows two successive molts (worker-presoldier-soldier) that are driven at the endocrine level by juvenile hormone (JH). Although physiological and eusocial mechanisms tied to JH signaling have been studied, the role of gut symbionts in the caste differentiation process is poorly understood. Here, we used the JH analog methoprene in combination with the antibiotic kanamycin to manipulate caste differentiation and gut bacterial loads in Reticulitermes flavipes termites via four bioassay treatments: kanamycin, methoprene, kanamycin+methoprene, and an untreated (negative) control. Bioassay results demonstrated a significantly higher number of presoldiers in the methoprene treatment, highest mortality in kanamycin+methoprene treatment, and significantly reduced protist numbers in all treatments except the untreated control. Bacterial 16S rRNA gene sequencing provided alpha and beta diversity results that mirrored bioassay findings. From ANCOM analysis, we found that several bacterial genera were differentially abundant among treatments. Finally, follow-up experiments showed that if methoprene and kanamycin or untreated termites are placed together, zero or rescued presoldier initiation, respectively, occurs. These findings reveal that endogenous JH selects for symbiont compositions required to successfully complete presoldier differentiation. However, if the gut is voided before the influx of JH, it cannot select for the necessary symbionts that are crucial for molting. Based on these results, we are able to provide a novel example of linkages between gut microbial communities and host phenotypic plasticity.

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

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a serious pest worldwide. Juvenile hormone analog (and its analogs such as methoprene) can induce the transformation of the worker caste into soldier caste in C. formosanus. However, several factors, such as feeding substrate and colony origin, influence the proportion of solider formation. The molecular mechanism of worker to soldier transformation of C. formosanus is still not clear. RT-qPCR is a powerful tool for molecular studies. Accurate gene quantification by the relative quantification method requires a stable expressed gene as the reference gene. However, no reference genes were available for this species in the methoprene bioassay. To study the problem of gene response to methoprene by RT-qPCR we have to first screen reference genes in C. formosanus. Workers were fed with methoprene. Termites were collected during the methoprene bioassay and separated into head and thorax+abdomen. Expression profiles of 10 candidate reference genes in the two body part types were investigated using RT-qPCR. The results were analyzed by a set of established methods (geNorm, NormFinder, BestKeeper, and RefFinder) as well as comparative ΔCt method. Our results suggest that RPS18 is the most stably-expressed gene both in the head and thorax+abdomen for expression analysis in the methoprene bioassay of C. formosanus. The screening of suitable reference genes in C. formosanus establishes the foundation for the molecular study of soldier caste differentiation in this species.

Differential gene expression in response to juvenile hormone analog treatment in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Archotermopsidae)

Termite societies are characterized by a highly organized division of labor among conspicuous castes, groups of individuals with various morphological specializations. Termite caste differentiation is under control of juvenile hormone (JH), but the molecular mechanism underlying the response to JH and early events triggering caste differentiation are still poorly understood. In order to profile candidate gene expression during early soldier caste differentiation of the damp-wood termite, Hodotermopsis sjostedti, we treated pseudergates (workers) with a juvenile hormone analog (JHA) to induce soldier caste differentiation. We then used Suppressive Subtractive Hybridization to create two cDNA libraries enriched for transcripts that were either up- or downregulated at 24h after treatment. Finally, we used quantitative PCR to confirm temporal expression patterns. Hexamerins represent a large proportion of the genes upregulated following JHA treatment and have an expression pattern that shows roughly an inverse correlation to intrinsic JH titers. This data is consistent with the role of a JH “sink”, which was demonstrated for hexamerins in another termite, Reticulitermes flavipes. A putative nuclear protein was also upregulated a few hours after JHA treatment, which suggests a role in the early response to JH and subsequent regulation of transcriptional events associated with soldier caste differentiation. Some digestive enzymes, such as endogenous beta-endoglucanase and chymotrypsin, as well as a protein associated to digestion were identified among genes downregulated after JHA treatment. This suggests that JH may directly influence the pseudergate-specific digestive system.

Cyp15F1: A NOVEL CYTOCHROME P450 GENE LINKED TO JUVENILE HORMONE‐DEPENDENT CASTE DIFFERENTION IN THE TERMITE Reticulitermes flavipes

Termites are eusocial insects that jointly utilize juvenile hormone (JH), pheromones, and other semiochemicals to regulate caste differentiation and achieve caste homeostasis. Prior EST sequencing from the symbiont-free gut transcriptome of Reticulitermes flavipes unexpectedly revealed a number of unique cytochrome P450 (Cyp) transcripts, including fragments of a Cyp15 family gene (Cyp15F1) with homology to other insect Cyp15s that participate in JH biosynthesis. The present study investigated the role of Cyp15F1 in termite caste polyphenism and specifically tested the hypothesis that it plays a role in JH-dependent caste differentiation. After assembling the full-length Cyp15F1 cDNA sequence, we (i) determined its mRNA tissue expression profile, (ii) investigated mRNA expression changes in response to JH and the caste-regulatory primer pheromones γ-cadinene (CAD) and γ-cadinenal (ALD), and (iii) used RNA interference (RNAi) in combination with caste differentiation bioassays to investigate gene function at the phenotype level. Cyp15F1 has ubiquitous whole-body expression (including gut tissue); is rapidly and sustainably induced from 3 h to 48 h by JH, CAD, and ALD; and functions at least in part by facilitating JH-dependent soldier caste differentiation. These findings provide the second example of a termite caste regulatory gene identified through the use of RNAi, and significantly build upon our understanding of termite caste homeostatic mechanisms. These results also reinforce the concept of environmental caste determination in termites by revealing how primer pheromones, as socioenvironmental factors, can directly influence Cyp15 expression and caste differentiation.

Group size effect on worker juvenile hormone titers and soldier differentiation in Formosan subterranean termite

In a finite environment, population growth can lead to crowding, increased densities and stress. Termites live in highly organized societies and densities can increase astronomically as colonies grow. However, little is known about juvenile hormone (JH) changes in these insects as numbers increase, despite the fact that JH is a critical caste regulator in this insect. Using Formosan subterranean termites as a model, we evaluated minimum group size requirements for soldier differentiation and the effect of density (50, 100, 500, and 1000 individuals/experimental unit) on worker JH titers either with or without initial soldiers being present. The minimum group size investigation indicated that groups initiated with 5 workers were insufficient for soldier differentiation. Soldiers were produced in groups initiated with 10 or more workers. As density increased from 50 to 1000 individuals per experimental unit, worker JH levels were elevated. Presence of soldiers lessened the effect of density on rising worker JH titers, indicating that soldiers have the ability to down-regulate worker JH. The study provides direct evidence of a population density effect on JH in eusocial insects and sheds light on understanding of the regulatory mechanisms associated with termite soldier caste differentiation.

Effects of Soldier-Derived Terpenes on Soldier Caste Differentiation in the Termite Reticulitermes flavipes

Primer pheromones play key roles in regulating division of labor, which is a fundamental and defining aspect of insect sociality. Primer pheromones are chemical messengers that transmit hormone-like messages among colony members; in recipients, these messages can either induce or suppress phenotypic caste differentiation. Here, we investigated soldier caste-derived chemicals as possible primer pheromones in the lower termite Reticulitermes flavipes, a species for which no primer pheromones have yet been identified. We determined that soldier head extracts (SHE), when provided to totipotent workers along with the insect morphogenetic juvenile hormone (JH), significantly enhanced soldier caste differentiation. When applied alone, however, SHE had no impacts on caste differentiation, survivorship, or any other aspect of worker biology. These findings support a function of soldier chemicals as primer pheromones that enhance the action of the endogenous JH. In accord with previous studies, gamma-cadinene and the corresponding aldehyde, gamma-cadinenal, were identified by gas chromatography-mass spectrometry and nuclear magnetic resonance analyses as the two most abundant components of R. flavipes SHE. Validative bioassays with commercially available cadinene confirmed activity. Several other terpenes, previously identified in R. flavipes soldiers, also were found to be active. These findings reveal a novel primer pheromone-like function for soldier-derived terpenes in termites and further suggest convergent evolution of terpene functions in enhancing JH-dependent soldier caste differentiation.

Histology of the hormone-producing glands in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Termopsidae): A focus on soldier differentiation

Caste differentiation in termites depends on complex hormonal changes during postembryonic development. Juvenile hormone (JH) is a central player in this process. The present study examined histological changes in the main hormone-producing endocrine glands, the corpora allata and molt glands, in the Japanese dampwood termite Hodotermopsis sjostedti. We focused on the soldier caste differentiation pathway, which can be induced artificially using an analogue of JH. The corpora allata exhibited volumetric changes during soldier induction, reflecting variations in the quantity of cytoplasm. Corpora allata from alates and neotenics clearly showed differentiation accompanied by cell proliferation, preparing for the high-level JH production necessary for reproduction. However, the volume increase of corpora allata was not always correlated to high JH titers. In contrast, molt glands degenerated in the reproductive castes. The JH analogue induced hypertrophy of the molt glands, along with the formation of lacunae, possibly related to ecdysteroid production. The JH analogue effect, inducing soldier differentiation, was suggested to require both mimic of high JH titers and stimulation of the molt glands.

Juvenile hormone titers and caste differentiation in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Termopsidae)

Termites are social insects, presenting morphologically distinct castes, performing specific tasks in the colony. The developmental processes underlying caste differentiation are mainly controlled by juvenile hormone (JH). Although many fragmentary data support this fact, there was no comparative work on JH titers during the caste differentiation processes. In this study, JH titer variation was investigated using a liquid chromatography-mass spectrometry (LC-MS) quantification method in all castes of the Japanese damp-wood termite Hodotermopsis sjostedti, especially focusing on the soldier caste differentiation pathway, which was induced by treatment with a JH analog. Hemolymph JH titers fluctuated between 20 and 720 pg/μl. A peak of JH was observed during molting events for the pseudergate stationary molt and presoldier differentiation, but this peak was absent prior to the imaginal molt. Soldier caste differentiation was generally associated with high JH titers and nymph to alate differentiation with low JH titers. However, JH titer rose in females during alate maturation, probably in relation to vitellogenesis. In comparison, JH titer was surprisingly low in neotenics. On the basis of these results in both natural and artificial conditions, the current model for JH action on termite caste differentiation is discussed and re-appraised.

Regulation of polyphenic caste differentiation in the termiteReticulitermes flavipesby interaction of intrinsic and extrinsic factors

Polyphenism is a key strategy used by solitary insects to adapt to changing environmental conditions and by eusocial insects for existing collaboratively in a social environment. In social insects, the morphogenetic juvenile hormone (JH) is often involved in directing the differentiation of polyphenic behavioral castes. The present study examines the effects of JH, environment and feeding on caste polyphenism in a eusocial insect, the termite Reticulitermes flavipes (Kollar). Our approach included a combination of model JH bioassays, SDS-PAGE and western blotting. Our findings revealed significant temperature-dependent effects on (1) JH-induced soldier caste differentiation, (2) abundance of soldier-inhibitory hexamerin proteins, and (3) JH-sequestration by hexamerin proteins. Additionally, though it appears to be dependent on a complex interaction of factors, feeding apparently plays a significant upstream role in enhancing hexamerin accumulation under normal colony conditions. These findings offer important new information on termite eusocial polyphenism by providing the first mechanistic evidence linking an intrinsic caste regulatory factor (hexamerin proteins) to an upstream extrinsic factor (environment) and a downstream response (caste differentiation). These observations are consistent with the hypothesis that the hexamerins serve as an environmentally and nutritionally responsive switching mechanism that regulates termite caste polyphenism.

Hexamerin-based regulation of juvenile hormone-dependent gene expression underlies phenotypic plasticity in a social insect

Worker termites of the genus Reticulitermes are temporally-arrested juvenile forms that can terminally differentiate into adultsoldier- or reproductive-caste phenotypes. Soldier-caste differentiation is a developmental transition that is induced by high juvenile hormone (JH) titers. Recently, a status quo hexamerin mechanism was identified, which reduces JH efficacy and maximizes colony fitness via the maintenance of high worker-caste proportions. Our goal in these studies was to investigate more thoroughly the influences of the hexamerins on JH-dependent gene expression in termite workers. Our approach involved RNA interference (RNAi), bioassays and quantification of gene expression. We first investigated the expression of 17 morphogenesis-associated genes in response to RNAi-based hexamerin silencing. Hexamerin silencing resulted in significant downstream impacts on 15 out of the 17 genes, suggesting that these genes are members of a JH-responsive genomic network. Next, we compared gene-expression profiles in workers after RNAi-based hexamerin silencing to that of (i) untreated workers that were held away from the colony; and (ii) workers that were also held away from the colony, but with ectopic JH. Here, although there was no correlation between hexamerin silencing and colony-release effects, we observed a significant correlation between hexamerin silencing and JH-treatment effects. These findings provide further evidence supporting the hypothesis that the hexamerins modulate JH availability, thus limiting the impacts of JH on termite caste polyphenism. Results are discussed in a context relative to outstanding questions on termite developmental biology, particularly on regulatory gene networks that respond to JH-, colony- and environmental-cues.

Juvenile hormone and colony conditions differentially influence cytochrome P450 gene expression in the termite Reticulitermes flavipes*

In lower termites, the worker caste is a totipotent immature stage that is capable of differentiating into other adult caste phenotypes. We investigated the diversity of family 4 cytochrome P450 (CYP4) genes in Reticulitermes flavipes workers, with the specific goal of identifying P450s potentially involved in regulating caste differentiation. Seven novel CYP4 genes were identified. Quantitative real-time PCR revealed the tissue distribution of expression for the seven CYP4s, as well as temporal expression changes in workers in association with a release from colony influences and during juvenile hormone (JH)-induced soldier caste differentiation. Several fat-body-related CYP4 genes were differentially expressed after JH treatment. Still other genes changed expression in association with removal from colony influences, suggesting that primer pheromones and/or other colony influences impact their expression. These findings add to a growing database of candidate termite caste-regulatory genes, and provide explicit evidence that colony factors influence termite gene expression.

Juvenile hormone III, hydroprene and a juvenogen as soldier caste differentiation regulators in three Reticulitermes species: potential of juvenile hormone analogues in termite control

The efficacy of juvenile hormone III (JH III) and two JH-mimicking compounds was compared in laboratory experiments with Reticulitermes lucifugus (Rossi), R. santonensis Feytaud and R. virginicus (Banks). Induction of presoldier, soldier and/or soldier-worker intercaste differentiation was taken as positive response to the treatment. The novel juvenogen, a fatty acid ester of a juvenoid alcohol, induced greatest soldier differentiation in all species tested, followed by hydroprene. JH III was less effective. Representatives of three Reticulitermes species showed similar trends in soldier induction rates. Differences in mortality in treatments of termites of the same species from different colonies with the same compound were observed and evidently were caused by differences in the conditions of respective colonies.

Effects of juvenile hormone III on Reticulitermes flavipes: changes in hemolymph protein composition and gene expression

Termites express polyphenism during caste differentiation that is mostly undefined at the molecular level. Using the eastern subterranean termite, Reticulitermes flavipes Kollar, we wanted (1) to test juvenile hormone (JH) model assays for their ability to induce detectable molecular changes in worker termites and (2) to investigate hemolymph proteins and their corresponding genes during JH-induced soldier caste differentiation. Our results illustrate pronounced changes in two hemolymph proteins after JH treatment, as well as differences among several caste phenotypes. Significant increases in the expression of four genes encoding hemolymph proteins, including two vitellogenins and two hexamerins, were observed after JH exposure. These findings are the first to demonstrate such protein and gene expression changes during termite caste differentiation. These results also validate the utility of JH model assays for inducing detectable molecular changes in worker termites that have begun presoldier differentiation.

Gene expression profiles among immature and adult reproductive castes of the termite Reticulitermes flavipes

Array-based genomic studies were conducted with the goal of identifying immature (i.e. nymph) and adult reproductive caste-biased gene expression in the termite Reticulitermes flavipes. Using cDNA macro-arrays, we identified thirty-four nymph-biased genes falling into eight ontogenic categories. Based on gene expression profiles among diverse castes and developmental stages (determined by quantitative PCR), several important trends emerged. These findings highlight the importance of several developmental and survival-based factors among immature and adult termite reproductives, including: vitellogenesis, nutrient storage, juvenile hormone sequestration, ribosomal translational and filtering mechanisms, fatty acid biosynthesis, apoptosis inhibition, and both endogenous and symbiont cellulase-assisted nutrition. These findings are highly significant as they are the first to elucidate the molecular biology underlying termite reproductive caste differentiation and reproductive caste-specific biology. Other gene expression results are in agreement with previous findings that suggest roles for vitellogenin-like haemolymph proteins in soldier caste differentiation.

Morphogenesis and gene expression in the soldier-caste differentiation of termites

Since almost all termite species possess a soldier caste, there must be ubiquitous mechanisms of soldier differentiation throughout isopteran species. In order to reveal the caste differentiation mechanisms, observations during the soldier morphogenesis and identification of soldier specific gene expression are thought to be important. In this article, I summarize research approaches for analyzing caste differentiation in termites, and introduce two of our studies in Hospitalitermes medioflavus (Termitidae) and Hodotermopsis japonica (Termopsidae).¶Colonies of the nasute termite H. medioflavus have soldiers with a frontal projection (nasus) on the head, from which defensive substances are secreted. During soldier differentiation from male minor worker to presoldier, the most dynamic morphogenesis occurs. In the presumptive nasus epithelium of minor workers, a disc-like structure termed "soldier-nasus disc" rapidly develops to form the nasus of presoldiers. This rapid growth is associated with two folding layers of cuticle and epithelium.¶To identify genes specifically expressed in soldiers of the damp wood termite Hodotermopsis japonica, a differential display using RT-PCR was tried, comparing mRNA from the heads of soldiers and pseudergates. An identified gene candidate termed SOL1 was expressed specifically in terminally differentiated mature soldiers, and the product of the gene was suggested to encode a novel protein with a putative signal peptide at the N-terminus. This gene was shown to be expressed in the mandibular glands which actually develop during the soldier differentiation. Thus, these molecular techniques are applicable to reveal the proximate mechanisms of caste determination in termites and other social insects.