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RNA interference (RNAi) is an endogenous, sequence-specific gene-silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi-based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double-stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small-interfering RNA/double-stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. © 2018 Society of Chemical Industry.

Animals are thought to use only glucose polymers (glycogen) as energy reserve, whereas both glucose (starch) and fructose polymers (fructans) are used by microbes and plants. Here, it is reported that the gall midge Mayetiola destructor, and likely other herbivorous animal species, gained the ability to utilize dietary fructans directly as storage polysaccharides by a single horizontal gene transfer (HGT) of bacterial levanase/inulinase gene followed by gene expansion and differentiation. Multiple genes encoding levanases/inulinases have their origin in a single HGT event from a bacterium and they show high expression levels and enzymatic activities in different tissues of the gall midge, including nondigestive fat bodies and eggs, both of which contained significant amounts of fructans. This study provides evidence that animals can also use fructans as energy reserve by incorporating bacterial genes in their genomes.

To identify ligands for orphan GPCRs, we searched novel neuropeptide genes in the Drosophila melanogaster genome. Here, we describe CNMa, a novel cyclic neuropeptide that is a highly potent and selective agonist for the orphan GPCR, CG33696 (CNMaR). Phylogenetic analysis revealed that arthropod species have two paralogous CNMaRs, but many taxa retain only one. Drosophila CNMa potently activates CNMaR-2 from Apis mellifera, suggesting both receptors are functional. Although CNMa is conserved in most arthropods, Lepidoptera lack the CNMa gene. However, they retain the CNMaR gene. Bombyx CNMaR showed low sensitivity to Drosophila CNMa, hinting toward the existence of additional CNMaR ligand(s).

Mariner transposons are widespread in eukaryote genomes and have been used as transposon vectors in insect transgenesis. We examined two closely related Helicoverpa species, the cotton bollworm Helicoverpa armigera and corn earworm Helicoverpa zea, for the presence of mariner-like elements (MLEs). Multiple copies of two distinct MLEs, Hamar1 and Hamar2, were isolated in H. armigera, and a MLE showing a high degree of conservation to Hamar1 was detected in H. zea and was named Hzmar1. These MLEs belong to the cecropia subfamily, containing indels in the transposase coding region. Sequence analysis indicated the earlier invasion of Hamar1 and relatively recent activity of Hamar2.

A transgenic line of the pink bollworm, Pectinophora gossypiella, a key lepidopteran cotton pest, was generated previously using the piggyBac transposon IFP2 from Trichoplusia ni. Here we identified an endogenous piggyBac-like element (PLE), designated as PgPLE1, in the pink bollworm. A putatively intact copy of PgPLE1 (PgPLE1.1) presents the canonical features of PLE: inverted terminal repeats with three C/G residues at the extreme ends, inverted subterminal repeats, TTAA target site and an open reading frame encoding transposase with 68% similarity to IFP2. Vectorette PCR revealed large variation in the insertion sites of PgPLE1 amongst worldwide populations, indicating the potential mobility of PgPLE1. The PgPLE1 was undetectable in the genome of Pectinophora endema, implying the recent invasion of PgPLE1 after the divergence of these two closely related species.

Coleopteran insects comprise a highly successful taxon, representing more than 25% of eukaryotic species, many of which are economically important. One of these, the red flour beetle (Tribolium castaneum H.), is a major pest of stored grain and cereal products and is also an excellent genetic model for the Coleoptera. The Tribolium genome sequencing project was recently completed, the first for an agricultural insect pest species. This genome sequence and annotation, in combination with the tractability of Tribolium for genetic dissection and functional genomic analysis, makes it one of the most useful subjects for studies of insect development, genetics and physiology. Tribolium has a number of interesting physiological adaptations, including desiccation tolerance associated with specialized cryptonephridial organs for active rectal absorption of atmospheric water. The study of endocrine regulation of diuresis in this species has led to the identification of a number of osmoregulatory peptides, including the novel arginine-vasopressin-like peptide.

Transposable element (TE) display is a method exhibiting the insertion site variations of TE, serving for multiple dominant markers. We report an improved TE display method evaluated by testing inheritance pattern of the TE insertion sites in two different piggyBac-like elements in a lepidopteran insect tobacco budworm. The specificity of the polymerase chain reaction (PCR)-based method was confirmed by sequencing randomly chosen a total of 15 bands all revealing the junctions between TE-end and the flanking sequence. The improved method removes the steps in the conventional TE display method using radiolabelled primers and running denaturing gel, saving time and costs.

Analysis of transcriptomes from the salivary glands and midgut of Hessian fly larvae Mayetiola destructor (say) identified a set of diverse cDNAs that encode proteins with a relatively high percentage (over 10%) of cysteinyl residues. Structural comparison of these putative proteins with known sequences in GenBank revealed that the positions of the cysteinyl residues in the identified proteins were highly conserved within a family of proteinase inhibitors despite very little overall sequence similarity. Phylogenetic analysis sorted this set of cDNAs into five different groups. To determine if these cDNAs indeed encode proteinase inhibitors, recombinant proteins were generated with two cDNAs from two different groups. Biochemical analysis of the recombinant proteins against commercial and insect gut proteinases demonstrated that the recombinant proteins are strong proteinase inhibitors with different specificities. Northern blot and real-time PCR analysis revealed that the different genes were expressed at different developmental stages and in different tissues. The overall results indicated that M. destructor contains a complex of genes that code for proteinase inhibitors which may regulate proteinase activities in different regulatory pathways. The GenBank accession numbers for the cDNAs in this paper were DQ232690 to DQ232718.

We isolated multiple copies of mariner -like element (MLE) from the pink bollworm (PBW), Pectinophora gossypiella , a key lepidopteran cotton pest. Although all the MLE sequences contain multiple mutations accumulated in their transposase coding region, the consensus sequence revealed a putative ancestral transposase encoding 339 amino acid residues with a D,D(34)D motif, and 36 bp inverted terminal repeats, belonging to the cecropia subfamily, and most similar to the MLE found in Antheraea species. Examining six different pink bollworm populations, we conclude that the MLE in PBW described in this study are ancient and are undergoing the process of accumulating inactivating mutations. This conclusion is supported by the patterns of polymorphisms revealed by genomic Southern hybridization, transposable element displays, and sequences from multiple MLE.