Gene silencing through RNA interference (RNAi) has revolutionized the study of gene 98 function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) 99 RNAi has many times proven to be difficult to achieve. Most of the negative results have been 100 anecdotal and the positive experiments have not been collected in such a way that they are 101 possible to analyze. In this review, we have collected detailed data from more than 150 102 experiments including all to date published and many unpublished experiments. Despite a 103 large variation in the data, trends that are found are that RNAi is particularly successful in the 104 family Saturniidae and in genes involved in immunity. On the contrary, gene expression in 105 epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding 106 dsRNA requires high concentrations for success. Possible causes for the variability of success 107 in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further 108 investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the 109 innate immune response. Our general understanding of RNAi in Lepidoptera will be further 110 aided in the future as our public database at http://insectacentral.org/RNAi will continue to 111 gather information on RNAi experiments.

Photorhabdus is a virulent pathogen that kills its insect host by overcoming immune responses. The bacterium also secretes a range of antibiotics to suppress the growth of other invading microorganisms. Here we show that Photorhabdus produces a small-molecule antibiotic (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene (ST) that also acts as an inhibitor of phenoloxidase (PO) in the insect host Manduca sexta. The Photorhabdus gene stlA encodes an enzyme that produces cinnamic acid, a key precursor for production of ST, and a mutation in stlA results in loss of ST production and PO inhibitory activity, which are both restored by genetic complementation of the mutant and also by supplying cinnamic acid. ST is produced both in vitro and in vivo in sufficient quantities to account for PO inhibition and is the only detectable solvent-extractable inhibitor. A Photorhabdus stlA ؊ mutant is significantly less virulent, proliferates slower within the host, and provokes the formation of significantly more melanotic nodules than wild-type bacteria. Virulence of the stlA ؊ mutant is also rescued by supplying cinnamic acid. The proximate cause of the virulence effect, however, is the inhibition of PO, because the effect of the stlA ؊ mutation on virulence is abolished in insects in which PO has been knocked down by RNA interference (RNAi). Thus, ST has a dual function both as a PO inhibitor to counter host immune reactions and also as an antibiotic to exclude microbial competitors from the insect cadaver.Photorhabdus luminescens ͉ RNA interference ͉ stilbene ͉ virulence

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In response to microbial infection, insects mount several defense reactions including the induction of proteolytic cascades that lead to localized melanization and coagulation. Melanization requires the activation of prophenoloxidase (proPO) to its active form phenoloxidase (PO), a key enzyme that leads to the formation of melanin at wound sites and around intruding microorganisms in the hemolymph. Clotting is critical in limiting hemolymph loss and initiating wound healing following injury; it quickly acts to form a solid barrier against infection by immobilizing microorganisms and promoting their killing. Recent advances in Drosophila and other insects imply a possible link between PO and the coagulation system, although the exact molecular mechanisms controlling this interaction appear to be complex and are still not well defined. The development of hemolymph experimental techniques in Drosophila larvae together with proteomic analysis have further led to the identification of proPO as a cross-linking component that is involved in the hardening and melanization of clots. However, clot PO activity varies between insect species and life stages, depending on physiological and ecological conditions. Here we review our current knowledge of the association between PO and coagulation and discuss the implications of the previous findings on insect innate immunity and hemostasis.

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Insects have evolved obligate, mutualistic interactions with bacteria without further transmission to other eukaryotic organisms. Such long-term obligate partnerships between insects and bacteria have a profound effect on various physiological functions of the host. Here we provide an overview of the effects of endosymbiotic bacteria on the insect immune system as well as on the immune response of insects to pathogenic infections. Potential mechanisms through which endosymbionts can affect the ability of their host to resist an infection are discussed in the light of recent findings. We finally point out unresolved questions for future research and speculate how the current knowledge can be employed to design and implement measures for the effective control of agricultural insect pests and vectors of diseases.

The immune response of a host to a pathogen is typically described as either innate or adaptive. The innate form of the immune response is conserved across all organisms, including insects. Previous and recent research has focused on the nature of the insect immune system and the results imply that the innate immune response of insects is more robust and specific than previously thought. Priming of the insect innate immune system involves the exposure of insects to dead or a sublethal dose of microbes in order to elicit an initial response. Comparing subsequent infections in primed insects to non-primed individuals indicates that the insect innate immune response may possess some of the qualities of an adaptive immune system. Although some studies demonstrate that the protective effects of priming are due to a “loitering” innate immune response, others have presented more convincing elements of adaptivity. While an immune mechanism capable of producing the same degree of recognition specificity as seen in vertebrates has yet to be discovered in insects, a few interesting cases have been identified and discussed.

In response to bacterial and fungal infections in insects and mammals, distinct families of innate immune pattern recognition receptors (PRRs) initiate highly complex intracellular signaling cascades. Those cascades induce a variety of immune functions that restrain the spread of microbes in the host. Insect and mammalian innate immune receptors include molecules that recognize conserved microbial molecular patterns. Innate immune recognition leads to the recruitment of adaptor molecules forming multi-protein complexes that include kinases, transcription factors, and other regulatory molecules. Innate immune signaling cascades induce the expression of genes encoding antimicrobial peptides and other key factors that mount and regulate the immune response against microbial challenge. In this review, we summarize our current understanding of the bacterial and fungal PRRs for homologous innate signaling pathways of insects and mammals in an effort to provide a framework for future studies.