Soldier caste influences on candidate primer pheromone levels and juvenile hormone-dependent caste differentiation in workers of the termite Reticulitermes flavipes

Caste systems and the division of labor they make possible are common underlying features of all social insects. Multiple extrinsic factors have been shown to impact caste composition in social insect colonies. Primer pheromones are one type of extrinsic caste-regulatory factor; they are chemical signaling molecules produced by certain colony members to impact developmental physiology of recipient nestmates. However, only limited evidence exists regarding primer pheromones and their actions in eusocial termites. In previous research we identified two soldier-produced terpenes, γ-cadinene (CAD) and γ-cadinenal (ALD), as candidate primer pheromones of the lower termite Reticulitermes flavipes. In the present study we tested hypotheses related to CAD and ALD action in recipient individuals. We examined the influences of terminally developed soldier termites on (1) CAD and ALD levels and (2) caste differentiation in developmentally totipotent workers. Our findings show CAD and ALD (respectively) are caste stimulatory and inhibitory components of chemical blends present in soldier heads, ALD levels increase significantly (10.9×) in workers only in the presence of soldiers, and soldiers can reduce developmental-hormone response thresholds of workers, presumably via ALD action. These findings provide novel evidence supporting that CAD and ALD are authentic caste-regulatory primer pheromones in Reticulitermes.     

A novel gene from the takeout family involved in termite trail-following behavior

This study investigated physiological and behavioral functions of a novel gene identified from the termite Reticulitermes flavipes. The gene, named deviate, encodes an apparent ligand binding protein from the takeout-homologous family. Initial studies were conducted to investigate deviate mRNA expression among termite castes and body regions, and changes in response to light-dark conditions, starvation, temperature, and juvenile hormone (JH). Deviate has ubiquitous caste and tissue expression, including antennal expression. Consistent with characteristics of other takeout family members, deviate expression is responsive to photophase conditions (p<0.1), and feeding, temperature, and JH (p<0.05). Using RNA-interference (RNAi) techniques, short-interfering RNAs (siRNAs) homologous to the deviate gene were synthesized and injected into worker termites, which were then subjected to bioassays designed to (1) induce caste differentiation or (2) measure various behavioral aspects of foraging and trail following. No impacts on JH-dependent caste differentiation were observable. However, trail following accuracy was significantly reduced in termites that received deviate siRNA injections, and this pattern generally mirrored deviate mRNA attenuation and recovery after RNAi. In a subsequent distance foraging bioassay, deviate-silenced termites exhibited equal feeding levels to controls, suggesting the deviate gene is not linked to general vigor or the ability/motivation of termites to move and forage. These findings are among the first linking the expression of a termite gene with eusocial behavior; they illustrate the connection between deviate expression and trailing behavior, which is a key evolutionary adaptation vital to subterranean social insects such as termites and ants.     

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.     

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.     

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 720pg/microl. 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.     

Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when α‐naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub‐cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)‐derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional‐genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut. © 2009 Wiley Periodicals, Inc.     

Soldier presence suppresses presoldier differentiation through a rapid decrease of JH in the termite Reticulitermes speratus

The regulation of caste differentiation is essential to insect eusociality. Termite soldiers are sterile and cannot eat by themselves because they have specialized mouth morphology. Almost all termite species have a soldier caste, and the soldier ratio per colony is maintained at a low level, probably by elaborate regulatory mechanisms. Although the soldier presence is considered to negatively affect soldier differentiation in all examined species, the detailed mechanism remains unclear. Presoldier differentiation can be induced artificially by juvenile hormone (JH) application to workers, showing that JH is a key factor underlying the regulation of soldier differentiation. In this study, to elucidate physiological changes in workers because of the soldier presence during the molt into presoldiers, JH III applications and JH titer quantifications were carried out in the rhinotermitid termite Reticulitermes speratus. Firstly, the effects of soldier presence before the molt into presoldiers induced by JH III application to workers were investigated. The rates of presoldier molt induced by the treatments with soldiers were significantly lower than those without soldiers. Secondly, worker JH titers in the presence or absence of soldiers were quantified by LC-MS on day 0, 5, 10, and 15 after JH application. Results indicated that the worker JH titers (endogenous + applied JH III) in the presence of soldiers were significantly lower than those without soldiers on day 5 after the JH treatment. On days 10 and 15, such soldier effects were not observed. Finally, the effective duration of soldier presence after the JH application was elucidated. A 4 day period of co-existence with soldiers suppressed presoldier differentiation, suggesting that the soldier presence rapidly decreased the JH titer in other colony members (i.e., workers), resulting in the inhibition of presoldier production.     

白蚁的分子生物学研究进展

白蚁是危险性社会昆虫,建立安全有效的白蚁防治方法有赖多学科的参与,分子生物学已经成为白蚁研究的重要工具。目前,DNA序列分析方法已应用于白蚁鉴定和分类,其中线粒体基因是最通用的分子标记;基因工程技术成功构建了可用于白蚁防治的白蚁肠道工程菌;Hexamerin、COX Ⅲ、纤维素酶等白蚁功能基因以及白蚁品级分化相关的若干蛋白相继得到了分离和鉴定。文章从白蚁分类、防治、功能基因、品级分化4个方面综述白蚁分子生物学的研究进展,为白蚁的防治提供新的方法和思路。     

Histology of the hormone-producing glands in the damp-wood termite <Emphasis Type="Italic">Hodotermopsis sjostedti</Emphasis> (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. Received 12 November 2007; revised 2 June 2008; accepted 14 July 2008.     

On the origin of termite workers: weighing up the phylogenetic evidence

Resolving the phylogenetic history of a 'true' worker caste in termites is essential to our understanding of termite eusocial evolution. Whether this caste is ancient and monophyletic or derived and polyphyletic will have a tremendous impact on our interpretation of termite eusocial history and remains an outstanding question in termite biology. Recent work has begun to re-examine this question in light of new phylogenetic information, but new questions have now arisen about how best to model character state changes in termite caste systems. In the present paper, we compare the models of Grandcolas and D'Haese [J. Evol. Biol. 15 (2002) 885] and Thompson et al. [J. Evol Biol. 13 (2000) 8691 and attempt to make explicit how these proposals differ with respect to the number of, and homology between, character states. We highlight the support each model has for the two principal, but competing, evolutionary hypotheses outlined above.     

Expression of methyl farnesoate epoxidase (mfe) and juvenile hormone esterase (jhe) genes and their relation to social organization in the stingless bee Melipona interrupta (Hymenoptera: Apidae)

Social organization in highly eusocial bees relies upon two important processes: caste differentiation in female larvae, and age polyethism in adult workers. Juvenile Hormone (JH) is a key regulator of both processes. Here we investigated the expression of two genes involved in JH metabolism - mfe (biosynthesis) and jhe (degradation) - in the context of social organization in the stingless bee Melipona interrupta. We found evidence that the expression of mfe and jhe genes is related to changes in JH levels during late larval development, where caste determination occurs. Also, both mfe and jhe were upregulated when workers engage in intranidal tasks, but only jhe expression was downregulated at the transition from nursing to foraging activities. This relation is different than expected, considering recent reports of lower JH levels in foragers than nurses in the closely related species Melipona scutellaris. Our findings suggest that highly eusocial bees have different mechanisms to regulate JH and, thus, to maintain their level of social organization.     

蜜蜂级型分化相关生理因子研究进展

蜜蜂Apis melliferaL.是典型的社会性昆虫,蜂群内蜂王和工蜂的级型分化现象吸引了众多研究者的高度关注。蜜蜂级型分化是一个极为复杂的生长发育调节过程,许多重要生理因子参与其中,包括保幼激素、蜕皮激素、胰岛素/胰岛素类似物信号通路等。本文概述了蜜蜂级型分化相关生理因子研究进展:保幼激素决定着蜜蜂级型发育的轨迹并能阻止卵巢发育过程中细胞的程序性死亡;蜕皮激素协同保幼激素发挥调节作用;蜜蜂蜂王与工蜂的躯体大小及相关器官大小的级型特异性差异与胰岛素/胰岛素类似物信号通路密切相关。此外,本文还介绍了蜜蜂级型分化模型,该模型系统描述了上述各生理因子对级型分化的综合作用。     

Rates of juvenile hormone synthesis control caste differentiation in the stingless bee Scaptotrigona postica depilis

Summary In social insects the expression of caste-specific characters is controlled by juvenile hormone (JH) during definite sensitive periods in preimaginal development. For a number of stingless bee species the existence of such a JH-sensitive period has already been demonstrated. Queen development can be induced by topical JH applications during the cocoon spinning phase of the last larval instar. Neither JH titers nor rates of JH synthesis were known so far for this subfamily of eusocial bees distinguished by a pronounced caste dimorphism. As the pantropically distributed stingless bees with approximately 400 recent species are the largest group of social bees, JH synthesis was studied in one of the species that can be kept under laboratory conditions. An in vitro radiochemical assay was used to measure stage- and caste-specific activities of the corpora allata (CA). For the first time in a eusocial hymenopteran species it was demonstrated how the endocrine system is reacting to trophogenic stimuli capable to induce caste differentiation during larval development. Generally JH synthesis in queen CA was found to be 30–80% higher than in workers during the penultimate and last larval instar, but a strong and distinct caste-specific modulation of JH synthesis was only observed right before the onset of a JH-sensitive period in the cocoon spinning phase of the fifth instar.     

Knockdown of the juvenile hormone receptor gene inhibits soldier-specific morphogenesis in the damp-wood termite Zootermopsis nevadensis (Isoptera: Archotermopsidae)

The Methoprene-tolerant (Met) protein has been established as a juvenile hormone (JH) receptor. Knockdown of the Met gene caused precocious metamorphosis and suppression of ovarian development. However, the function of Met in caste development of social insects is unclear. In termites, JH acts as a central factor for caste development, especially for soldier differentiation, which involves two molts from workers via a presoldier stage. Increased JH titer in workers is needed for the presoldier molt, and the high JH titer is maintained throughout the presoldier period. Although presoldiers have the fundamental morphological features of soldiers, the nature of the cuticle is completely different from that of soldiers. We expected that JH signals via Met are involved in soldier-specific morphogenesis of the head and mandibles during soldier differentiation, especially in the presoldier period, in natural conditions. To test this hypothesis, we focused on soldier differentiation in an incipient colony of the damp-wood termite Zootermopsis nevadensis. Met homolog (ZnMet) expression in heads increased just after the presoldier molt. This high expression was reduced by ZnMet double stranded (dsRNA) injection before the presoldier molt. Although this treatment did not cause any morphological changes in presoldiers, it caused strong effects on soldiers, their mandibles being significantly shorter and head capsules smaller than those of control soldiers. Injection of ZnMet dsRNA throughout the presoldier stage did not affect the formation of soldier morphology, including cuticle formation. These results suggested that the rapid increase in ZnMet expression and subsequent activation of JH signaling just after the presoldier molt are needed for the formation of soldier-specific weapons. Therefore, besides its established role in insect metamorphosis, the JH receptor signaling also underlies soldier development in termites.     

RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA

RNA interference (RNAi) represents a breakthrough technology for conducting functional genomics research in non-model organisms and for the highly targeted control of insect pests. This study investigated RNAi via voluntary feeding in the economically important pest termite, Reticulitermes flavipes. We used a high-dose double-stranded (ds) RNA feeding approach to silence two termite genes: one encoding an endogenous digestive cellulase enzyme and the other a caste-regulatory hexamerin storage protein. Contrary to results from previous low-dose studies that examined injection-based RNAi, high-dose silencing of either gene through dsRNA feeding led to significantly reduced group fitness and mortality. Hexamerin silencing in combination with ectopic juvenile hormone treatments additionally led to lethal molting impacts and increased differentiation of presoldier caste phenotypes (a phenotype that is not capable of feeding). These results provide the first examples of insecticidal effects from dsRNA feeding in a termite. Additionally, these results validate a high-throughput bioassay approach for use in (i) termite functional genomics research, and (ii) characterizing target sites of conventional and novel RNAi-based termiticides.     

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.