Common and Distinct Roles of Juvenile Hormone Signaling Genes in Metamorphosis of Holometabolous and Hemimetabolous Insects

Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis

Murine and Bovine γδ T Cells Enhance Innate Immunity against Brucella abortus Infections

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα−/−, and GMCSF−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections

Search for Limiting Factors in the RNAi Pathway in Silkmoth Tissues and the Bm5 Cell Line: The RNA-Binding Proteins R2D2 and Translin

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed

Unravelling the evolution of the Allatostatin-Type A, KISS and Galanin Peptide-Receptor gene families in Bilaterians: insights from Anopheles Mosquitoes

Allatostatin type A receptors (AST-ARs) are a group of G-protein coupled receptors activated by members of the FGL-amide (AST-A) peptide family that inhibit food intake and development in arthropods. Despite their physiological importance the evolution of the AST-A system is poorly described and relatively few receptors have been isolated and functionally characterised in insects. The present study provides a comprehensive analysis of the origin and comparative evolution of the AST-A system. To determine how evolution and feeding modified the function of AST-AR the duplicate receptors in Anopheles mosquitoes, were characterised. Phylogeny and gene synteny suggested that invertebrate AST-A receptors and peptide genes shared a common evolutionary origin with KISS/GAL receptors and ligands. AST-ARs and KISSR emerged from a common gene ancestor after the divergence of GALRs in the bilaterian genome. In arthropods, the AST-A system evolved through lineage-specific events and the maintenance of two receptors in the flies and mosquitoes (Diptera) was the result of a gene duplication event. Speciation of Anophelesmosquitoes affected receptor gene organisation and characterisation of AST-AR duplicates (GPRALS1 and 2) revealed that in common with other insects, the mosquito receptors were activated by insect AST-A peptides and the iCa(2+)-signalling pathway was stimulated. GPRALS1 and 2 were expressed mainly in mosquito midgut and ovaries and transcript abundance of both receptors was modified by feeding. A blood meal strongly up-regulated expression of both GPRALS in the midgut (p < 0.05) compared to glucose fed females. Based on the results we hypothesise that the AST-A system in insects shared a common origin with the vertebrate KISS system and may also share a common function as an integrator of metabolism and reproduction. Highlights: AST-A and KISS/GAL receptors and ligands shared common ancestry prior to the protostome-deuterostome divergence. Phylogeny and gene synteny revealed that AST-AR and KISSR emerged after GALR gene divergence. AST-AR genes were present in the hemichordates but were lost from the chordates. In protostomes, AST-ARs persisted and evolved through lineage-specific events and duplicated in the arthropod radiation. Diptera acquired and maintained functionally divergent duplicate AST-AR genes.Foundation for Science and Technology, Portugal (FCT) [PTDC/BIA-BCM/114395/2009]; European Regional Development Fund (ERDF) COMPETE - Operational Competitiveness Programme; Portuguese funds through FCT Foundation for Science and Technology [PEst-C/MAR/LA0015/2013, UID/Multi/04326/2013, PEst-OE/SAU/LA0018/2013]; FCT [SFRH/BPD/89811/2012, SFRH/BPD/80447/2011, SFRH/BPD/66742/2009]; auxiliary research contract FCT Pluriannual funds [PEst-C/MAR/LA0015/2013, UID/Multi/04326/2013]info:eu-repo/semantics/publishedVersio

Second-Generation Sequencing Supply an Effective Way to Screen RNAi Targets in Large Scale for Potential Application in Pest Insect Control

The key of RNAi approach success for potential insect pest control is mainly dependent on careful target selection and a convenient delivery system. We adopted second-generation sequencing technology to screen RNAi targets. Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies were used to screen optimal RNAi targets from Ostrinia furnalalis. Total 14690 stage specific genes were obtained which can be considered as potential targets, and 47 were confirmed by qRT-PCR. Ten larval stage specific expression genes were selected for RNAi test. When 50 ng/µl dsRNAs of the genes DS10 and DS28 were directly sprayed on the newly hatched larvae which placed on the filter paper, the larval mortalities were around 40∼50%, while the dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities reached 73% to 100% at 5 d after treatment. The qRT-PCR analysis verified the correlation between larval mortality and the down-regulation of the target gene expression. Topically applied fluorescent dsRNA confirmed that dsRNA did penetrate the body wall and circulate in the body cavity. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost less and an easy way to select the candidate target genes for RNAi. More importantly, it demonstrated that dsRNAs are able to penetrate the integument and cause larval developmental stunt and/or death in a lepidopteron insect. This finding largely broadens the target selection for RNAi from just gut-specific genes to the targets in whole insects and may lead to new strategies for designing RNAi-based technology against insect damage

Hmgcr in the Corpus Allatum Controls Sexual Dimorphism of Locomotor Activity and Body Size via the Insulin Pathway in Drosophila

The insulin signaling pathway has been implicated in several physiological and developmental processes. In mammals, it controls expression of 3-Hydroxy-3-Methylglutaryl CoA Reductase (HMGCR), a key enzyme in cholesterol biosynthesis. In insects, which can not synthesize cholesterol de novo, the HMGCR is implicated in the biosynthesis of juvenile hormone (JH). However, the link between the insulin pathway and JH has not been established. In Drosophila, mutations in the insulin receptor (InR) decrease the rate of JH synthesis. It is also known that both the insulin pathway and JH play a role in the control of sexual dimorphism in locomotor activity. In studies here, to demonstrate that the insulin pathway and HMGCR are functionally linked in Drosophila, we first show that hmgcr mutation also disrupts the sexual dimorphism. Similarly to the InR, HMGCR is expressed in the corpus allatum (ca), which is the gland where JH biosynthesis occurs. Two p[hmgcr-GAL4] lines were therefore generated where RNAi was targeted specifically against the HMGCR or the InR in the ca. We found that RNAi-HMGCR blocked HMGCR expression, while the RNAi-InR blocked both InR and HMGCR expression. Each RNAi caused disruption of sexual dimorphism and produced dwarf flies at specific rearing temperatures. These results provide evidence: (i) that HMGCR expression is controlled by the InR and (ii) that InR and HMGCR specifically in the ca, are involved in the control of body size and sexual dimorphism of locomotor activity

R\ue9vision du genre Xylodes Waterhouse (Coleoptera, Ptinidae)

Volume: 95Start Page: 401End Page: 42

Le genre Trymolophus nov. (Coleoptera, Ptinidae) des \ueeles Mascareignes

Volume: 97Start Page: 55End Page: 7

Lapidoniptus nov. gen. pour Piarus hispanicus Pic, 1953 (Coleoptera, Ptinidae)

Volume: 4Start Page: 69End Page: 7