Insect parasitic nematodes have acquired mechanisms to evade their host immune response for successful parasitism. Despite the importance of understanding of the evolution of evasion mechanisms from host immunity, insect immune response against non-parasitic nematodes has not been well studied. In our previous study, we demonstrated that a non-insect parasitic nematode Caenorhabditis elegans was not encapsulated by haemocytes in the larvae of the greater wax moth Galleria mellonella. To understand how nematodes influence insect haemocytes to escape encapsulation, we examined the effect of C. elegans on haemocytes in the haemocoel of G. mellonella larvae. Injection of nematodes resulted in the decrease of haemocyte density while mortality and spreading ability of haemocytes, the haematopoietic organs were not affected. In vitro co-incubation of haemocytes with nematodes resulted in a decrease of haemocyte density and we observed feeding on haemocytes by nematodes. Injection of C. elegans feeding-delay mutants into insects did not cause the decrease of haemocyte density. The decrease of haemocyte density was due to the nematode's ingestion of haemocytes. Furthermore, an entomopathogenic nematode and other bacterial feeding nematodes also showed similar feeding behaviour. The nematode's ability to feed on haemocytes may have played an important role in the evolution of nematode parasitism in bacterial-feeding nematodes.

Immunity to microbial infections is well understood; however, information regarding the immunity to parasitic multicellular organisms remains lacking. To understand innate host cellular immunity to nematodes, we compared the cellular response of the greater wax moth (Galleria mellonella) larvae against the non-parasitic, bacterial-feeding nematode Caenorhabditis elegans and pathogenic nematode Heterorhabditis bacteriophora. When intact first-instar or dauer larvae of C. elegans were injected into a G. mellonella larva, most of the nematodes were alive and not confined by the surrounding reaction by insect haemocytes (encapsulation), similarly as the pathogenic nematode, whereas most of the heat-killed nematodes of both species were severely encapsulated by 24 h after inoculation. Other non-parasitic nematodes were also not encapsulated. Surprisingly, C. elegans injected into the insect haemocoel grew and propagated in the live insect, resulting in death of the host insect. Our results suggest that C. elegans has some basic mechanisms to evade immunity of G. mellonenlla and grow in the haemocoel.

Triatomines are vectors that transmit the protozoan haemoflagellate Trypanosoma cruzi, the causative agent of Chagas disease. The aim of the current review is to provide a synthesis of the immune mechanisms of triatomines against bacteria, viruses, fungi and parasites to provide clues for areas of further research including biological control. Regarding bacteria, the triatomine immune response includes antimicrobial peptides (AMPs) such as defensins, lysozymes, attacins and cecropins, whose sites of synthesis are principally the fat body and haemocytes. These peptides are used against pathogenic bacteria (especially during ecdysis and feeding), and also attack symbiotic bacteria. In relation to viruses, Triatoma virus is the only one known to attack and kill triatomines. Although the immune response to this virus is unknown, we hypothesize that haemocytes, phenoloxidase (PO) and nitric oxide (NO) could be activated. Different fungal species have been described in a few triatomines and some immune components against these pathogens are PO and proPO. In relation to parasites, triatomines respond with AMPs, including PO, NO and lectin. In the case of T. cruzi this may be effective, but Trypanosoma rangeli seems to evade and suppress PO response. Although it is clear that three parasite-killing processes are used by triatomines – phagocytosis, nodule formation and encapsulation – the precise immune mechanisms of triatomines against invading agents, including trypanosomes, are as yet unknown. The signalling processes used in triatomine immune response are IMD, Toll and Jak-STAT. Based on the information compiled, we propose some lines of research that include strategic approaches of biological control.

The melanotic encapsulation response mounted by Drosophila melanogaster against macroparasites, which is based on haemocyte binding to foreign objects, is poorly characterized relative to its humoral immune response against microbes, and appears to be variable across insect lineages. The genus Zaprionus is a diverse clade of flies embedded within the genus Drosophila. Here we characterize the immune response of Zaprionus indianus against endoparasitoid wasp eggs, which elicit the melanotic encapsulation response in D. melanogaster. We find that Z. indianus is highly resistant to diverse wasp species. Although Z. indianus mounts the canonical melanotic encapsulation response against some wasps, it can also potentially fight off wasp infection using two other mechanisms: encapsulation without melanization and a non-cellular form of wasp killing. Zaprionus indianus produces a large number of haemocytes including nematocytes, which are large fusiform haemocytes absent in D. melanogaster, but which we found in several other species in the subgenus Drosophila. Several lines of evidence suggest these nematocytes are involved in anti-wasp immunity in Z. indianus and in particular in the encapsulation of wasp eggs. Altogether, our data show that the canonical anti-wasp immune response and haemocyte make-up of the model organism D. melanogaster vary across the genus Drosophila.

Parasitoids are parasites that invariably kill their host. Polydnaviruses are injected by parasitoid wasps into the body cavity of their insect host and cause immunosuppression, allowing the parasitoid to develop in the absence of encapsulation. One of the targets of the polydnaviruses are the haemocytes of the host, which undergo significant changes in response to entry of the virus. In some systems, haemocyte apoptosis is induced, or haemocyte clumping may be seen; in others, the cells round up and fail to adhere to a substrate. Effects on haemocytes may be transitory or permanent (cell death). Various polydnavirus gene products have been identified that interfere with normal haemocyte function. Phenoloxidase activity also is inhibited during parasitism, and the effect is inducible by polydnavirus. In some systems, venom components may act synergistically with polydnavirus in mediating the virally-induced effects on the host immune system. Polydnaviruses are powerful influences on the host immune system, which serve to permit successful development of the parasitoid without triggering the host immune response.

Introduction. The ectoparasitic honey bee mite Varroa destructor feeds on the haemolymph of the honey bee, Apis mellifera, through a single puncture wound that does not heal but remains open for several days. It was hypothesized that factors in the varroa saliva are responsible for this aberrant wound healing. Methods. An in vitro procedure was developed for collecting salivary gland secretions from V. destructor. Mites were incubated on balls of cotton wool soaked in a tissue culture medium (TC-100), and then induced to spit by topical application of an ethanolic pilocarpine solution. Results. Elution of secretions from balls of cotton wool, followed by electrophoretic analysis by SDS-PAGE and electroblotting indicated the presence of at least 15 distinct protein bands, with molecular weights ranging from 130 kDa to <17 kDa. Serial titration of V. destructor salivary secretions in TC-100 followed by an 18-h incubation with haemocytes from the caterpillar, Lacanobia oleracea, indicated that the secretions damage the haemocytes and suppresses their ability to extend pseudopods and form aggregates. Conclusion. We suggest that these secretions facilitate the ability of V. destructor to feed repeatedly off their bee hosts by suppressing haemocyte-mediated wound healing and plugging responses in the host.

The ability of pathogens to neutralize host defence mechanisms represents a fundamental requisite in the successful establishment of an infection. Host-pathogen interactions between quahog parasite unknown (QPX) and its hard clam host are poorly understood. Our prior in vivo investigations have shown that different QPX isolates display varying levels of pathogenicity toward clams. Similarly, field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. An in vitro approach was developed in this study to evaluate the toxicity of QPX cells and extracellular products toward haemocytes using a neutral red uptake assay. Results demonstrated that QPX produces virulence factors that are cytotoxic to M. mercenaria haemocytes. This cytotoxicity appears to be induced by clam factors, suggesting that it may play an important role in supporting QPX infection and proliferation within the host. Moreover, application of this technique to different QPX isolates and clam broodstocks indicates variations of QPX cytotoxicity in agreement with previous in vivo experiments, strengthening the existence of different QPX strains.

The number of circulating haemocytes, the size of the haematopoietic organ, and the size of haemocyte capsules around the parasite were studied in M-line Biomphalaria glabrata snails exposed to 100 or 400 first-stage larvae of Angiostrongylus cantonensis. The number of haemocytes in exposed snails increased significantly at 1 day post-exposure, decreased to control value, and then increased again. The decrease in number of circulating haemocytes is probably due to the removal of cells from the circulation to participate in encapsulation of larvae. The majority of circulating haemocytes in M-line B. glabrata are fully-spread granulocytes, which increase significantly in number in snails following exposure to A. cantonensis larvae. However, populations of partially-spread granulocytes, round cells, hyalinocytes and miscellaneous haemocytes were relatively constant. The size of capsules around the parasite increased during the 42-day interval of the experiment. The haematopoietic organ increased in size in response to infection.

Aiming to further characterize the haemocyte subsets in Biomphalaria snails, we have performed a detailed flow cytometric analysis of whole haemolymph cellular components using a multiparametric dual colour labelling procedure. Ethidium bromide/acridine orange fluorescence features were used to first select viable haemocytes followed by flow cytometric morphometric analysis based on the laser scatter properties (forward scatter-FSC and side scatter-SSC). Our findings demonstrated that B. glabrata (BG-BH, highly susceptible to S. mansoni) and 2 strains of B. tenagophila (BT-CF, moderately susceptible and BT-Taim, resistant to S. mansoni) have 3 major circulating haemocyte subsets, referred to as small, medium and large haemocytes. The frequency of small haemocytes was higher in BG-BH, while medium haemocytes were the most abundant cell-type in both B. tenagophila strains. Schistosoma mansoni infection resulted in early reduction of large and medium circulating haemocytes followed by an increase of small haemocytes. Although parasite infection induced haemocyte alterations in all Biomphalaria strains, the response was particularly intense in BT-Taim, the parasite-resistant snail. Interestingly, the trematode infection induces changes in haemocytes with less granular rather than in those with more granular profile. The results indicated that, in B. tenagophila of Taim strain, circulating haemocytes, especially the medium and high subset with less granular profile, are very reactive cells upon S. mansoni infection, suggesting that this cell subset would participate in the early parasite destruction observed in this snail strain.

The ability of the larval forms of Schistosoma mansoni to invade and parasitize their molluscan host, Biomphalaria glabrata, is determined by a multitude of factors. In this study we sought to elucidate the possible mechanisms by which the invading larvae are able to counteract the potentially harmful oxidative environment presented by the host upon initial miracidial infection. This was attempted by examining the gene expression profile of parasite antioxidant enzymes of the linked glutathione-(GSH) thioredoxin (Trx) redox pathway during early intramolluscan larval development. Three such enzymes, the peroxiredoxins (Prx1, Prx2 and Prx3) were examined as to their activity and sites of expression within S. mansoni miracidia and in vitro-cultured mother sporocysts. Results of these studies demonstrated that the H2O2-reducing enzymes Prx1 and 2 are upregulated during early mother sporocyst development compared to miracidia. Immunolocalization studies further indicated that Prx1 and Prx2 proteins are expressed within the apical papillae of miracidia and tegumental syncytium of sporocysts, and are released with parasite excretory-secretory proteins (ESP) during in vitro larval transformation. Removal of Prx1 and Prx2 from larval ESP by immunoabsorption significantly reduced the ability of ESP to breakdown exogenous H2O2, thereby directly linking ESP Prx proteins with H2O2-scavenging activity. Moreover, exposure of live sporocysts to exogenous H2O2 stimulated an upregulation of Prx1 and 2 gene expression suggesting the involvement of H2O2–responsive elements in regulating larval Prx gene expression. These data provide evidence that Prx1 and Prx2 may function in the protection of S. mansoni sporocysts during the early stages of infection.

Killing of intramolluscan schistosomes by host haemocytes is mediated by reactive oxygen metabolites. Hence, defence against oxidative damage is essential for the parasite to survive. In this study, expression of three key antioxidant enzymes, superoxide dismutase (EC 1.15.1.1), glutathione peroxidase (EC 1.11.1.9) and glutathione-S-transferase (EC 2.5.1.18) was determined in Schistosoma mansoni miracidia, sporocysts and cercariae. Stage-dependent expression of these enzymes was shown to be regulated at the transcriptional level. Second, the influence on enzyme expression of reactive oxygen species (ROS) and of haemocytes from schistosome-resistant and -susceptible host snails was determined. Generation of ROS by xanthine/xanthine oxidase resulted in increased transcript levels for all three enzymes. Addition of hydrogen peroxide induced a significantly increased expression of GPx and SOD but not GST. Snail haemocytes induced an up-regulation of SOD and GPx at 12 and 18 h post-exposure, respectively. Susceptible haemocytes elicited a stronger induction of transcript expression than resistant haemocytes. After 36–48 h, SOD remained up-regulated in sporocysts encapsulated by haemocytes from susceptible hosts, whereas a down-regulation of SOD and GPx occurred in schistosomes encapsulated by haemocytes from resistant snails. These observations indicate that schistosomes express elevated levels of antioxidant enzymes in interaction with haemocytes from susceptible snail hosts in which they survive. On the other hand, haemocytes of resistant snails may interfere with reactive oxygen detoxification via down-regulation of schistosome antioxidant enzymes, thus shifting the balance towards parasite killing.

In parallel with massive research efforts in human schistosomiasis over the past 30 years, persistent efforts have been made to understand the basis for compatibility and incompatibility in molluscan schistosomiasis. Snail plasma contains molecules that are toxic to trematodes, but these seem to kill only species that never parasitize the mollusc used as the source of plasma. A sporocyst will be killed actively by haemocytes alone if they are from a snail that is resistant to the trematode. Oxygen-dependent killing mechanisms play a major role. Enzymes such as NADPH oxidase, superoxide dismutase, myeloperoxidase and nitric oxide synthase are critical components of the putative killing pathways. Metabolic intermediates such as hydrogen peroxide and nitric oxide appear to be more important against trematodes than the shorter-lived intermediates that are more important in anti-microbial defences. Products secreted by trematode larvae influence the physiology of snail haemocytes, implying active counter-defences mounted by the parasite, but these remain largely unexplored. A possible molecular basis for the susceptibility/resistance dichotomy in molluscan schistosomiasis is suggested to be deficient forms of enzymes in the respiratory burst pathway, and a selective disadvantage for schistosome resistance is an integral component of this model.

Although the effects of trematode infection on snail host physiology or host responses on parasite development have been well described in the literature, very little is known regarding the underlying mechanisms and specific molecules responsible for mediating those effects. It is presumed that many host–parasite interactions are communicated through receptor-mediated events, in particular those involving haemocytic immune responses to invading parasites, larval motility and migration through host tissues, and larval acquisition of host molecules either as nutrients or critical developmental factors. The intent of this chapter is to review current knowledge of molecules (both receptors and their ligands or counter-receptors) involved in molecular communication at the interface between larval trematodes, especially the mother or primary sporocyst stage, and host cells/tissues in intimate proximity to developing larvae. Information to date suggests that the molecular exchange at this interface is a highly complex and dynamic process, and appears to be regulated in specific cases. Topics discussed will focus on snail cell receptor interactions with the sporocyst tegument and its secretions, host cell–cell and cell–substrate adhesion receptors and their related signal transduction pathways, and sporocyst tegumental surface receptors and ligands involved in the binding of soluble host molecules.