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.     

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.     

Cloning and characterization of hexamerin in Spodoptera exigua and the expression response to insecticide exposure

Hexamerins are hemolymph-proteins, which are mainly considered as storage proteins for non-feeding stages, and also undertake other roles during insect development and growth, however the characterization of hexamerin proteins in Spodoptera exigua is less understood. In this study five new hexamerin genes were identified and a total seven hexamerin genes were reported in S. exigua. These hexamerins contain the typical domains of hemocyanin at the N-terminal, C-terminal and in the middle of their protein sequences. These genes are mainly expressed in fat body, and the signal peptide sequences at their N-terminal of protein sequences can drive the expressed protein to excrete into hemolymph after synthesis. The phylogenetic analysis and amine acid composition revealed S. exigua express five different types of hexamerins: 1) Storage protein rich in methionine residue (MRSP), 2) Storage protein moderately rich in methionine (MMRSP), 3) Hexamerin with high composition of aromatic amino acids (Arylphorin), 4) Arylphorin-like hexamerin, and 5) Riboflavin-binding hexamerin (RbH). The phylogenetic pattern combined with the comparison of conserved histidine residues in copper binding sites of hexamerins revealed basal position of RbH and the evolutionary pathway in lepidopteran hexamerins. Finally, the induction expression of hexamerins by insecticide, lambda-cyhalothrin, were analyzed, results showed that lambda-cyhalothrin exposure may down-regulate their expression. This study increased the gene number of hexamerin to seven, and reported their expression and structural characterizations, the finding will facilitate the understand of hexamerin in other insects.     

Decrease in Methoprene tolerant and Taiman expression reduces juvenile hormone effects and enhances the levels of juvenile hormone circulating in males of the linden bug Pyrrhocoris apterus

Juvenile hormone (JH) produced by the corpus allatum (CA) stimulates vitellogenesis and reduces the synthesis of hexamerin proteins in adult females of Pyrrhocoris apterus. At present it is unknown whether the signaling pathway involving the JH receptor gene Methoprene tolerant (Met) and its binding partner Taiman (Tai), regulates the synthesis of accessory gland proteins (ACPs) and hexamerin proteins or effects male survival. Knockdown of genes by injecting Met dsRNA or Tai dsRNA, reduced the amount of ACPs whilst enhancing the amount of hexamerin mRNA in the fat body and the release of hexamerin proteins into haemolymph, as occurs after the ablation of CA. Lifespan was enhanced by injecting Met but not Tai dsRNA. Diapause associated with the natural absence of JH had a stronger effect on all these parameters than the ablation of CA or the knockdown of genes. This indicates there is an additional regulating agent. Both Met and Tai dsRNA induced a several fold increase in JH (JH III skiped bisepoxide) but a concurrent loss of Met or Tai disabled its function. This supports the view that the Met/Tai complex functions as a JH receptor in the regulation of ACPs and hexamerins.     

Expression and evolution of hexamerins from the tobacco hornworm,Manduca sexta,and other Lepidoptera

Hexamerins are large hemolymph-proteins that accumulate during the late larval stages of insects. Hexamerins have emerged from hemocyanin, but have lost the ability to bind oxygen. Hexamerins are mainly considered as storage proteins for non-feeding stages, but may also have other functions, e.g. in cuticle formation, transport and immune response. The genome of the hornworm Manduca sexta harbors six hexamerin genes. Two of them code for arylphorins (Msex2.01690, Msex2.15504) and two genes correspond to a methionine-rich hexamerin (Msex2.10735) and a moderately methionine-rich hexamerin (Msex2.01694), respectively. Two other genes do not correspond to any known hexamerin and distantly resemble the arylphorins (Msex2.01691, Msex2.01693). Five of the six hexamerin genes are clustered within ∼45 kb on scaffold 00023, which shows conserved synteny in various lepidopteran genomes. The methionine-rich hexamerin gene is located at a distinct site. M. sexta and other Lepidoptera have lost the riboflavin-binding hexamerin. With the exception of Msex2.01691, which displays low mRNA levels throughout the life cycle, all hexamerins are most highly expressed during pre-wandering phase of the 5th larval instar of M. sexta, supporting their role as storage proteins. Notably, Msex2.01691 is most highly expressed in the brain, suggesting a divergent function. Phylogenetic analyses showed that hexamerin evolution basically follows insect systematics. Lepidoptera display an unparalleled diversity of hexamerins, which exceeds that of other hexapod orders. In contrast to previous analyses, the lepidopteran hexamerins were found monophyletic. Five distinct types of hexamerins have been identified in this order, which differ in terms of amino acid composition and evolutionary history: i. the arylphorins, which are rich in aromatic amino acids (∼20% phenylalanine and tyrosine), ii. the distantly related arylphorin-like hexamerins, iii. the methionine-rich hexamerins, iv. the moderately methionine rich hexamerins, and v. the riboflavin-binding hexamerins.     

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.     

The Evolution of Hexamerins and the Phylogeny of Insects

The evolutionary relationships among arthropod hemocyanins and insect hexamerins were investigated. A multiple sequence alignment of 12 hemocyanin and 31 hexamerin subunits was constructed and used for studying sequence conservation and protein phylogeny. Although hexamerins and hemocyanins belong to a highly divergent protein superfamily and only 18 amino acid positions are identical in all the sequences, the core structures of the three protein domains are well conserved. Under the assumption of maximum parsimony, a phylogenetic tree was obtained that matches perfectly the assumed phylogeny of the insect orders. An interesting common clade of the hymenopteran and coleopteran hexamerins was observed. In most insect orders, several paralogous hexamerin subclasses were identified that diversified after the splitting of the major insect orders. The dipteran arylphorin/LSP-1-like hexamerins were subject to closer examination, demonstrating hexamerin gene amplification and gene loss in the brachyceran Diptera. The hexamerin receptors, which belong to the hexamerin/hemocyanin superfamily, diverged early in insect evolution, before the radiation of the winged insects. After the elimination of some rapidly or slowly evolving sequences, a linearized phylogenetic tree of the hexamerins was constructed under the assumption of a molecular clock. The inferred time scale of hexamerin evolution, which dates back to the Carboniferous, agrees with the available paleontological data and reveals some previously unknown divergence times among and within the insect orders. Received: 4 August 1997 / Accepted: 29 October 1997     

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.     

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.     

Soldier-specific modification of the mandibular motor neurons in termites

Social insects exhibit a variety of caste-specific behavioral tendencies that constitute the basis of division of labor within the colony. In termites, the soldier caste display distinctive defense behaviors, such as aggressively attacking enemies with well-developed mandibles, while the other castes retreat into the colony without exhibiting any aggressive response. It is thus likely that some form of soldier-specific neuronal modification exists in termites. In this study, the authors compared the brain (cerebral ganglion) and the suboesophageal ganglion (SOG) of soldiers and pseudergates (workers) in the damp-wood termite, Hodotermopsis sjostedti. The size of the SOG was significantly larger in soldiers than in pseudergates, but no difference in brain size was apparent between castes. Furthermore, mandibular nerves were thicker in soldiers than in pseudergates. Retrograde staining revealed that the somata sizes of the mandibular motor neurons (MdMNs) in soldiers were more than twice as large as those of pseudergates. The enlargement of MdMNs was also observed in individuals treated with a juvenile hormone analogue (JHA), indicating that MdMNs become enlarged in response to juvenile hormone (JH) action during soldier differentiation. This enlargement is likely to have two functions: a behavioral function in which soldier termites will be able to defend more effectively through relatively faster and stronger mandibular movements, and a developmental function that associates with the development of soldier-specific mandibular muscle morphogenesis in termite head. The soldier-specific enlargement of mandibular motor neurons was observed in all examined species in five termite families that have different mechanisms of defense, suggesting that such neuronal modification was already present in the common ancestor of termites and is significant for soldier function.     

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.     

Juvenile hormone regulates vitellogenin gene expression through insulin-like peptide signaling pathway in the red flour beetle, Tribolium castaneum

Our recent studies identified juvenile hormone (JH) and nutrition as the two key signals that regulate vitellogenin (Vg) gene expression in the red flour beetle, Tribolium castaneum. Juvenile hormone regulation of Vg synthesis has been known for a long time in several insects, but the mechanism of JH action is not known. Experiments were conducted to determine the mechanism of action of these two signals in regulation of Vg gene expression. Injection of bovine insulin or FOXO double-stranded RNA into the previtellogenic, starved, or JH-deficient female adults increased Vg mRNA and protein levels, thereby implicating the pivotal role for insulin-like peptide signaling in the regulation of Vg gene expression and possible cross-talk between JH and insulin-like peptide signaling pathways. Reduction in JH synthesis or its action by RNAi-mediated silencing of genes coding for acid methyltransferase or methoprene-tolerant decreased expression of genes coding for insulin-like peptides (ILPs) and influenced FOXO subcellular localization, resulting in the down-regulation of Vg gene expression. Furthermore, JH application to previtellogenic female beetles induced the expression of genes coding for ILP2 and ILP3, and induced Vg gene expression. FOXO protein expressed in baculovirus system binds to FOXO response element present in the Vg gene promoter. These data suggest that JH functions through insulin-like peptide signaling pathway to regulate Vg gene expression.     

Diversity of stonefly hexamerins and implication for the evolution of insect storage proteins

Hexamerins are large storage proteins of insects in the 500 kDa range that evolved from the copper-containing hemocyanins. Hexamerins have been found at high concentration in the hemolymph of many insect taxa, but have remained unstudied in relatively basal taxa. To obtain more detailed insight about early hexamerin evolution, we have studied hexamerins in stoneflies (Plecoptera). Stoneflies are also the only insects for which a functional hemocyanin is known to co-occur with hexamerins in the hemolymph. Here, we identified hexamerins in five plecopteran species and obtained partial cDNA sequences from Perla marginata (Perlidae), Nemoura sp. (Nemouridae), Taeniopteryx burksi (Taeniopterygidae), Allocapnia vivipara (Capniidae), and Diamphipnopsis samali (Diamphipnoidae). At least four distinct hexamerins are present in P. marginata. The full-length cDNA of one hexamerin subunit was obtained (PmaHex1) that measures 2475 bp and translates into a native polypeptide of 702 amino acids. Phylogenetic analyses showed that the plecopteran hexamerins are monophyletic and positioned at the base of the insect hexamerin tree, probably diverging about 360 million years ago. Within the Plecoptera, distinct hexamerin types evolved before the divergence of the families. Mapping amino acid compositions onto the phylogenetic tree shows that the accumulation of aromatic amino acids (and thus the evolution of "arylphorins") commenced soon after the hexamerins diverged from hemocyanins, but also indicates that hexamerins with distinct amino acid compositions reflect secondary losses of aromatic amino acids.