Immune responsiveness in vector insects

Insects have been particularly successful in evolution, and current estimates are that they represent three-quarters of all extant animal species. With the marked exception of the seas, insects occupy nearly all ecological niches on earth and hence are confronted to innumerable potential pathogenic bacteria, viruses, fungi, and protozoan and helminth parasites. Not surprisingly therefore, insects have developed efficient host defense mechanisms. The current view is that the insect host defense is multifaceted and involves cellular reactions, namely phagocytosis and capsule formation by blood cells and a systemic response evidenced by the rapid and transient synthesis of a battery of potent, small cationic antimicrobial peptides. After septic injury, these molecules predominantly are produced in the fat body, an equivalent of the mammalian liver, and in some blood cells. They accumulate in the blood of infected insects where they oppose the development of invading microorganisms. Additional defense reactions in insects are blood coagulation and melanization, which occur at the sites of injury as a result of almost immediate activation of proteolytic cascades. It is speculated that some of the products of these cascades can activate the synthesis of antimicrobial peptides in the fat body and blood cells. Remarkably, the signaling cascades that lead to antimicrobial peptide gene expression in these responsive tissues show significant structural and functional similarities with those involved in the cytokine-induced expression of mammalian acute phase proteins (see refs. 1–4 for review).

The view summarized above essentially was obtained by studying a limited number of model insects amenable to biochemical and genetic analysis, namely the Cecropia moth, silkworms, fleshflies, and Drosophila. With few exceptions, these studies were centered on antibacterial and antifungal resistance.

Whether and how vector insects respond to invading protozoan parasites so far has remained elusive. Mosquitoes, in particular, have not been the subject of molecular and genetic investigations in this area until very recently, although they are the principal vectors for several major diseases affecting humans and lifestock, such as malaria and sleeping sickness. Our lack of information in this area is obviously detrimental for vector control programs relying on the genetic manipulation of vectors, as such programs require an understanding of basic physiological and biochemical processes that govern parasite-vector interactions.

A breakthrough in the molecular analysis of such interactions is reported in a study by the Kafatos laboratory in this issue of the Proceedings (5). The strategy of the authors was to identify several markers of an inducible immune response in Anopheles gambiae, the major African vector of human malaria. The markers include the well established antibacterial peptide insect defensin, a putative recognition protein for bacterial surfaces, a galactose binding lectin, and serine-proteases. All were shown to respond to an experimental infection by bacteria, or bacterial cell-wall components, with specific temporal and tissue-specific characteristics, as well in larvae and adults of A. gambiae as in an established cell line of this species. With these markers at hand, the team went on to demonstrate a significant role of the midgut in A. gambiae innate immunity. All the markers were found to be expressed in the midgut, with a particularly strong expression of Anopheles defensin in the anterior midgut. The most striking result was observed when A. gambiae adult females were fed with blood containing the rodent parasite Plasmodium berghei: a significant immune response was induced, as evidenced by the up-regulated expression of the markers, when the parasites were present in the blood meal. Defensin and a putative bacteria binding protein were particularly strong reactants. The parasite-induced expression of the markers was observed both locally, in the midgut, and systemically, in the rest of the body. As the authors note, these reactions were observed at a time when the parasites were physically constrained within the gut lumen or within the midgut epithelial layer, but well before they were released into the blood of the insects as sporozoites. This suggests that the midgut, in addition to its own transcriptional response to the parasite, conveys a message to other tissues, signaling them to mount an immune response. The authors shortly refer to additional experiments of their group (15) that indicate that the presence of asexual parasites in the blood meal is not sufficient to induce an immune reaction in the midgut, and that formation of the ookinetes leading to penetration of the midgut epithelium is required. This result raises the question whether the midgut expresses receptors that specifically recognize ookinete surface patterns or whether the penetrating ookinete leads to a localized injury, which in turn triggers the immune response.

I consider the paper by Dimopoulos and associates (5) as a milestone in the field primarily in that it shows for the first time with adequate molecular tools that A. gambiae reacts to the presence of a Plasmodium parasite within ingested blood meal by mounting an immune response. I agree with the authors that none of their markers may as yet be directly involved in antiparasitic reactions, all the more so as they respond similarly both in resistant and susceptible strains of A. gambiae. However, the stage is now set for a molecular and cellular analysis of the parasite-induced immune reaction of the midgut and its concomitant extension to the rest of the insect body. It is a fair guess that deciphering the mechanisms of induction and analyzing the genes that they turn on will lead to an understanding about what makes an Anopheles strain resistant or susceptible to Plasmodium. As stated in a recent issue of Nature (6), it is time to put malaria control on the global agenda, and understanding the molecular basis of refractoriness vs. susceptibility in insect vector strains is certainly one of the golden apples of the Hesperides.

The inference that the midgut is an immunocompetent tissue in blood-sucking insects is further substantiated by the studies of Lehane and associates (7), which also appear in this issue of the Proceedings. These authors worked on the stable-fly Stomoxys, a facultatively hematophagous dipteran insect and a transmitter of a variety of diseases predominantly among lifestock. The authors first noted that antibacterial activity was present in crude homogenates of fly midguts and that it peaked 24–36 hr after a blood meal. The activity increased when lipopolysaccharide was added to the blood meal. Subsequently the authors isolated from midgut extracts and identified two novel antibacterial peptides, which clearly belong to the large family of insect defensins, as is the case for Anopheles defensin used as one of the markers for immune-induction in the Dimopoulos et al. study (5). Defensins are widespread among invertebrates and are characterized by a cysteine array that accounts for the stabilization of an amphipathic α-helix to a strongly twisted antiparallel β-sheet (8). This structural arrangement also is found in some plant defensins (9), but is distinct from the three-dimensional organization of mammalian defensins, which are all β-sheet (see review in ref. 10). Insect defensins are mostly antibacterial, but have been reported to exhibit antifungal and antiparasitic activities. The precise functions of these molecules in the host defense of Stomoxys and Anopheles [and in Aedes aegypti, which synthesize large amounts of defensin in the fat body in response to bacterial challenge (11)] await further investigations. Of relevance in the present context is the observation by the Lehane group (7) that Stomoxys midgut defensins are constitutively expressed and that their expression is markedly up-regulated in response to sterile as well as lipopolysaccharide-containing blood meal. Interestingly, injection of lipopolysaccharide into the body cavity of this insect also up-regulates defensin expression in the midgut, indicating that this tissue can react in Stomoxys to stimuli from both the exterior (gut lumen) and interior (haemocoel). Although occasional reports in the literature have pointed to the expression of antimicrobial proteins in the insect gut (e.g., cecropin expression in histolysing gut cells in Drosophila pupae, ref. 12; lysozyme, ref. 13), a significant role of the midgut as an immune-responsive organ had not been fully appreciated so far. In view of the compelling evidence presented by the Dimopoulos and Lehane papers, and the paucity of reports on midgut antimicrobial peptide synthesis in other species, the question even arises whether blood-sucking insects have not evolved a particularly marked immune function for their midgut, which is a crucial interface between potential vector insects and the parasites they ingest.

The production of defensins by gut cells inevitably will evoke among immunologists the release of α-defensins (cryptdins) by the Paneth cells at the base of the Lieberkühn crypts of the small intestine in many mammalian species (14). It generally is assumed that these, and other antimicrobial peptides, mediate innate immunity in the hostile environment of the intestinal lumen. It is striking to note that a similar protective strategy against microbes has been adopted by at least some insect species, as shown here, and by mammals. Indeed, the study of the insect antimicrobial defense has revealed a surprisingly large array of similarities with mammalian innate, nonadaptive immunity, which extend from the recognition of some microbial patterns by homologous receptors to signaling pathways in immune-responsive cells. We can anticipate that exciting new insights will arise from the study of the host defense against protozoan parasites in vector insects, as already illustrated in this issue of the Proceedings.