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Peptidoglycan recognition proteins (PGRPs) are one of the receptors in insects' immune pathways, essential for insects to recognize the exogenous pathogens in order to activate the Toll and immune deficiency (IMD) pathway. In the silkworm Bombyx mori, previous studies focused on the short PGRPs and less is known about the long PGRPs. In this study, a long PGRP in silkworm BmPGRP‐L4 was cloned and its expression and function were analysed. The results showed that BmPGRP‐L4 contains a transmembrane region, a conserved PGRP domain, and an amidase‐2 domain. The expression profile demonstrated that BmPGRP‐L4 existed in diverse tissues including epidermis, fat body, midgut, and silk glands, with remarkably high expression in the midgut in the 5th instar. Oral infection with Escherichia coli and Staphylococcus aureus significantly induced BmPGRP‐L4 in the midgut and epidermis, as well as in the fat body and silk glands. Peptidoglycan also induced the expression of BmPGRP‐L4 in midgut tissue ex vivo and BmN4 cells in vitro. RNAi of BmPGRP‐L4 was effective in the midgut and epidermis, while the efficiency in the fat body was transient. RNAi‐mediated knock‐down of BmPGRP‐L4 reduced the weight and growth of the silkworm, possibly due to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae. [ABSTRACT FROM AUTHOR]

Arboviruses are capable to establish long-term persistent infections in mosquitoes that do not affect significantly the physiology of the insect vectors. Arbovirus infections are controlled by the RNAi machinery via the production of viral siRNAs and the formation of RISC complexes targeting viral genomes and mRNAs. Engineered arboviruses that contain cellular gene sequences can therefore be transformed to "viral silencing vectors" for studies of gene function in reverse genetics approaches. More specifically, "ideal" viral silencing vectors must be competent to induce robust RNAi effects while other interactions with the host immune system should be kept at a minimum to reduce non-specific effects. Because of their inconspicuous nature, arboviruses may approach the "ideal" viral silencing vectors in insects and it is therefore worthwhile to study the mechanisms by which the interactions with the RNAi machinery occur. In this review, an analysis is presented of the antiviral RNAi response in mosquito vectors with respect to the major types of arboviruses (alphaviruses, flaviviruses, bunyaviruses, and others). With respect to antiviral defense, the exo-RNAi pathway constitutes the major mechanism while the contribution of both miRNAs and viral piRNAs remains a contentious issue. However, additional mechanisms exist in mosquitoes that are capable to enhance or restrict the efficiency of viral silencing vectors such as the amplification of RNAi effects by DNA forms, the existence of incorporated viral elements in the genome and the induction of a non-specific systemic response by Dicer-2. Of significance is the observation that no major "viral suppressors of RNAi" (VSRs) seem to be encoded by arboviral genomes, indicating that relatively tight control of the activity of the RNA-dependent RNA polymerase (RdRp) may be sufficient to maintain the persistent character of arbovirus infections. Major strategies for improvement of viral silencing vectors therefore are proposed to involve engineering of VSRs and modifying of the properties of the RdRp. Because of safety issues (pathogen status), however, arbovirus-based silencing vectors are not well suited for practical applications, such as RNAi-based mosquito control. In that case, related mosquito-specific viruses that also establish persistent infections and may cause similar RNAi responses may represent a valuable alternative solution. [ABSTRACT FROM AUTHOR]

Highlights: [•] Generation of Bm5 transformed cell line that expresses BmToll9-1. [•] Higher transcriptional response of Dcr2 by dsRNA in BmToll9-1 expressing cells. [•] Lower transcriptional response of innate immune pathway and AMP genes to LPS in BmToll9-1 expressing cells. [•] No direct modulation of RNAi process by BmToll9-1. [ABSTRACT FROM AUTHOR]

Abstract: A DNA/RNA non-specific alkaline nuclease (BmdsRNase) was isolated from the digestive juice of Bombyx mori. While originally reported to be produced by the midgut only, in this project it was found that the mRNA of this enzyme was also expressed in the epidermis, fat body, gut, thoracic muscles, Malpighian tubules, brain, and silk glands of 5th instar larvae, indicating additional functions to its reported role in nucleic acid digestion in the midgut. In order to study the functional properties of BmdsRNase, three pEA-BmdsRNase expression constructs were generated, characterized by presence or absence of a signal peptide and a propeptide, and used for expression in lepidopteran Hi5 tissue culture cells. Western blot indicated that these different forms of BmdsRNase protein were not secreted into the growth medium, while they were detected in the pellets and supernatants of Hi5 cell extracts. Nucleic acids cleavage experiments indicated that full-length BmdsRNase could digest dsRNA and that the processed form (absence of signal peptide and propeptide) of BmdsRNase could degrade both DNA and dsRNA in Hi5 cell culture. Using a reporter assay targeted by transfected homologous dsRNA, it was shown that the digestive property of the processed form could interfere with the RNAi response. Immunostaining of processed BmdsRNase protein showed asymmetric localization in the cellular cytoplasm and co-localization with Flag-tagged Dicer-2 was also observed. In conclusion, our in vitro studies indicated that intracellular protein isoforms of BmdsRNase can be functional and involved in the regulation of nucleic acid metabolism in the cytoplasm. In particular, because of its propensity to degrade dsRNA, the enzyme might be involved in the innate immune response against invading nucleic acids such as RNA viruses. [Copyright &y& Elsevier]

RNAi is considered a major antiviral defense mechanism in insects, but its relative importance as compared to other antiviral pathways has not been evaluated comprehensively. Here, it is attempted to give an overview of the antiviral defense mechanisms in Drosophila that involve both RNAi and non-RNAi. While RNAi is considered important in most viral infections, many other pathways can exist that confer antiviral resistance. It is noted that very few direct recognition mechanisms of virus infections have been identified in Drosophila and that the activation of immune pathways may be accomplished indirectly through cell damage incurred by viral replication. In several cases, protection against viral infection can be obtained in RNAi mutants by non-RNAi mechanisms, confirming the variability of the RNAi defense mechanism according to the type of infection and the physiological status of the host. This analysis is aimed at more systematically investigating the relative contribution of RNAi in the antiviral response and more specifically, to ask whether RNAi efficiency is affected when other defense mechanisms predominate. While Drosophila can function as a useful model, this issue may be more critical for economically important insects that are either controlled (agricultural pests and vectors of diseases) or protected from parasite infection (beneficial insects as bees) by RNAi products. [ABSTRACT FROM AUTHOR]