A long non-coding RNA regulates cadherin transcription and susceptibility to Bt toxin Cry1Ac in pink bollworm, Pectinophora gossypiella.

Extensive planting of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has spurred increasingly rapid evolution of resistance in pests. In the pink bollworm, Pectinophora gossypiella, a devastating global pest, resistance to Bt toxin Cry1Ac produced by transgenic cotton is linked with mutations in a gene (PgCad1) encoding a cadherin protein that binds Cry1Ac in the larval midgut. We previously reported a long non-coding RNA (lncRNA) in intron 20 of cadherin alleles associated with both resistance and susceptibility to Cry1Ac. Here we tested the hypothesis that reducing expression of this lncRNA decreases transcription of PgCad1 and susceptibility to Cry1Ac. Quantitative RT-PCR showed that feeding susceptible neonates small interfering RNAs (siRNAs) targeting this lncRNA but not PgCad1 decreased the abundance of transcripts of both the lncRNA and PgCad1. Moreover, neonates fed the siRNAs had lower susceptibility to Cry1Ac. The results imply that the lncRNA increases transcription of PgCad1 and susceptibility of pink bollworm to Cry1Ac. The results suggest that disruption of lncRNA expression could be a novel mechanism of pest resistance to Bt toxins.

Induction of stress- and immune-associated genes in the Indian meal moth Plodia interpunctella against envenomation by the ectoparasitoid Bracon hebetor.

Envenomation is an important process in parasitism by parasitic wasps; it suppresses the immune and development of host insects. However, the molecular mechanisms of host responses to envenomation are not yet clear. This study aimed to determine the transcription-level responses of the Indian meal moth Plodia interpunctella against envenomation of the ectoparasitoid Bracon hebetor. Quantitative real-time reverse-transcription PCR was used to determine the transcriptional changes of 13 selected genes, which are associated with development, metabolism, stress, or immunity, in the feeding and wandering fifth instar larvae over a 4-day period after envenomation. The effects of envenomation on the feeding-stage larvae were compared with those of starvation in the transcriptional levels of the 13 genes. Most selected genes were altered in their expression by either envenomation or starvation. In particular, a heat shock protein, hsp70, was highly upregulated in envenomated larvae in both the feeding and wandering stages as well as in starved larvae. Further, some genes were upregulated by envenomation in a stage-specific manner. For example, hsp25 was upregulated after envenomation in the feeding larvae, but hsp90 and an immune-associated gene, hemolin, were upregulated in the wandering larvae. However, both envenomation and starvation resulted in the downregulation of genes associated with development and metabolism. Taken together, P. interpunctella upregulated stress- and immune-responsive genes, but downregulated genes associated with development and metabolism after envenomation. This study provides important information for understanding the molecular mechanisms of host responses to parasitism.

De novo sequencing and transcriptome analysis of venom glands of endoparasitoid Aenasius arizonensis (Girault) (=Aenasius bambawalei Hayat) (Hymenoptera, Encyrtidae).

Aenasius bambawalei Hayat (Encyrtidae: Hymenoptera) has been synonymized with Aenasius arizonensis (Girault) is a small, newly discovered endoparasitoid of the cotton mealybug Phenacoccuss solenopsis Tinsley (Pseudococcidae: Hemiptera), which completes its life cycle inside the body of its host and it is a potential insect control tool. Despite the acquired knowledge regarding host-parasitoid interaction, little information is available on the factors of parasitoid origin able to modulate mealybug physiology. The components of A. arizonensis venom have not been well studied but venom from other parasitoids and wasps contain biologically active proteins that have potential applications in pest management or may be of medicinal importance. To provide an insight into the transcripts expressed in the venom gland of A. arizonensis, a transcriptomic database was developed utilizing high throughput RNA sequencing approaches to analyze the genes expressed in venom glands of this endoparasitic wasp. The resulting A. arizonensis RNA sequences were assembled de-novo with contigs then blasted against the NCBI non-redundant sequence database. Contigs which matched database sequences were mostly homologous to genes from hymenopteran parasitoids such as Nasonia vitripennis, Copidosoma floridanum, Fopius arsenus and Pteromalas puparium. Further analysis of the A. arizonensis database was then performed which focused on selected genes encoding proteins potentially involved in host developmental arrest, disrupting the host immune system, host paralysis, and transcripts that support these functions. Sequenced mRNAS predicted to encode full length ORFs of Calreticulin, Serine Protease Precursor and Arginine kinase proteins were identified and the tissue specific expression of these putative venom genes was analyzed by RT-PCR. In addition, results also demonstrate that de novo transcriptome assembly allows useful venom gene expression analysis in a species lacking a genome sequence database and may provide useful information for devising control tools for insect pests and other applications.

De novo sequencing and transcriptome analysis of female venom glands of ectoparasitoid Bracon hebetor (Say.) (Hymenoptera: Braconidae).

Venom is a key-factor in the regulation of host physiology by parasitic Hymenoptera and a potentially rich source of novel bioactive substances for biotechnological applications. The limited study of venom from the ectoparasitoid Bracon hebetor, a tiny wasp that attacks larval pest insects of field and stored products and is thus a potential insect control agent, has not described the full complement and composition of these biomolecules. To have a comprehensive picture of genes expressed in the venom glands of B. hebetor, a venom gland transcriptome was assembled by using next generation sequencing technologies followed by de novo assemblies of the 10.81 M sequence reads yielded 22,425 contigs, of which 10,581 had significant BLASTx hits to know genes. The majority of hits were to Diachasma alloeum, an ectoparasitoid from same taxonomic family, as well as other wasps. Gene ontology grouped the sequences into molecular functions in which catalytic activity with 42.2% was maximum, cellular components in which cells with 33.8% and biological processes among which metabolic process with 30% had the most representatives. In this study, we highlight the most abundant sequences, and those that are likely to be functional components of the venom for parasitization. Full length ORFs of Calreticulin, Venom Acid Phosphatase Acph-1 like protein and arginine kinase proteins were isolated and their tissue specific expression was studied by RT-PCR. Our report is the first to characterize components of the B. hebetor venom glands that may be useful for developing control tools for insect pests and other applications.