Although some novel antimicrobial peptides (AMP) have been successfully isolated from Bactrocera dorsalis Hendel, the mechanisms underlying the induction of these peptides are still elusive. The homolog of NF-κB transcription factor Relish, designated as BdRelish, was cloned from B. dorsalis. The full length cDNA of BdRelish is 3954 bp with an open reading frame that encodes 1013 amino acids. Similar to Drosophila Relish and the mammalian p100, it is a compound protein containing a conserved Rel homology domain, an IPT (Ig-like, plexins, transcription factors) domain and an IκB-like domain (four ankyrin repeats), the nuclear localization signal RKRRR is also detected at the residues 449–453, suggesting that it has homology to Relish and it is a member of the Rel family of transcription activator proteins. Reverse transcription quantitative polymerase chain reaction analysis reveals that BdRelish mRNAs are detected in different quantities from various tissues and the highest transcription level of BdRelish is determined in fat body. The injection challenge of Escherichia coli and Staphylococcus aureas significantly upregulated the expression of BdRelish. The injection of BdRelish dsRNA markedly reduced the expression of BdRelish and decreased the transcription magnitude of antimicrobial peptides. Individuals injected BdRelish dsRNA died at a significantly faster rate compared with the control groups. Therefore, BdRelish is vital for the transcription of AMPs to attack the invading bacteria.

Because immune defenses are often costly employed, insect immunocompetence cannot be always maintained at its maximum level. Here, the oriental fruit fly, Bactrocera dorsalis (Hendel), was used as a study object to investigate how its immune defenses varied with the developmental stage change and mating access. Our data indicated that both phenoloxidase (PO) activity and antibacterial activity significantly increased from new larvae to pupae but decreased in adults after emergence. Furthermore, both the PO activity and antibacterial activity in the hemolymph of copulated male and female adults were dramatically higher than that of virgin male and female ones, respectively. It provided the evidence that copulation could increase the magnitude of immune defense in hemolymph of B. dorsalis. Together, these results suggest that B. dorsalis possess a flexible investment strategy in immunity to meet its specific needs based on the endo- and exogenous factors, such as their distinct food source and living environments.

Rhynchophorus ferrugineus (Coleoptera, Curculionidae) is the most destructive pest of palm trees worldwide containing it invasive areas, such as the southern part of China. It is always emphasized to develop integrated pest management based on biological agents, but their success is not very exciting. Presently, the immune defenses of this pest against biological agents attract scarce attention. It is still unclear whether immune priming also generally occurs in insect pests and in response to different pathogens. Our results indicated that previous challenge of bacteria pathogen enhanced the magnitude of phenoloxidase activity and antibacterial activity in R. ferrugineus larvae against the secondary infection. Furthermore, trans-generational immune priming was also determined in this pest, and only challenged R. ferrugineus mothers transferred the immune protection to their offspring which suggested males and females of this pest might have evolved different strategies on the investment of delivering immune protection to their offspring. Importantly, our data provide the evidence to suggest that different kinds of biological control agents might be used alternatively or in combination to fight against R. ferrugineus because of the existence of immune priming with low species-specific level. On the other hand, for this invasive pest, the immune priming may also facilitate its adaptation and dispersal in the new regions.

The field of ecological immunology currently relies on using a number of immune effectors or markers. These markers are usually used to infer ecological trade-offs (via conflicts in resource allocation), though physiological nature of these markers remains elusive. Here, we review markers frequently used in insect evolutionary ecology research: cuticle darkening, haemocyte density, nodule/capsule formation, phagocytosis and encapsulation/melanization via use of nylon filaments and beads, phenoloxidase activity, nitric oxide production, lysozyme and antimicrobial peptide production. We also provide physiologically based information that may shed light on the probable trade-offs inferred when these markers are used. In addition, we provide a number of methodological suggestions to improve immune marker assessment.

Photorhabdus sp. are entomopathogenic bacteria which, upon experimental infection, interact with the insect immune system, but little is known about the roles of their symbiotic nematode partners Heterorhabditis sp. in natural infections. Here, we investigated the respective contributions of nematodes and bacteria by examining humoral and cellular immune reactions of the model lepidopteran insect Manduca sexta against Heterorhabditis carrying Photorhabdus, nematodes free of bacteria (axenic nematodes) and bacteria alone. Insect mortality was slower following infection with axenic nematodes than when insects were infected with nematodes containing Photorhabdus, or the bacteria alone. Nematodes elicited host immune responses to a lesser extent than bacteria. Transcription of certain recognition and antibacterial genes was lower when insects were naturally infected with nematodes carrying no bacteria compared to insects that received bacteria, either with or without nematodes. Axenic nematodes also did not elicit such high levels of phenoloxidase activity and haemocyte aggregates as did treatments involving Photorhabdus. By contrast, the phagocytic capability of host haemocytes was decreased by both axenic and bacteria-associated nematodes, but not by Photorhabdus alone. These results imply that both bacteria and nematodes contribute separately to the pathogenic modulation of host immune responses during natural infections by the mutualistic Heterorhabdus-Photorhabdus complex.

Trials were carried out to study the humoral immune response of blackflies to filariae following infection using the intrathoracic injection technique. An induced 66 kDa protein was abundant in the haemolymph of the European species Simulium ornatum following infection with bovine Onchocerca lienalis. This protein was apparently at higher concentrations in the haemolymph of sham-inoculated flies, i.e. flies that received sterile medium without the parasites. A molecule of the same size was also observed in the haemolymph of infected S. damnosum s.l. following infection with human O. volvulus or bovine O. ochengi. However, the level of this protein was lower in blackflies injected with microfilariae of bovine O. dukei. Unlike O. volvulus and O. ochengi this species is not transmitted by S. damnosum s.l. under natural conditions. No such reaction was observed if the African blackflies had received a sham inoculation. Feeding experiments with wild-caught nulliparous S. damnosum sl. on Onchocerca-infected cattle supported the results of the injection trials. The 66 kDa protein could only be found in the haemolymph of specimens infected via a blood meal. This 66 kDa molecule was identified as phenoloxidase. It appeared in the haemolymph due to the activation of the prophenoloxidase system following the filarial infection and we hypothesize that it may be sequestered by the parasites, as part of a non-self recognition system.

A recombinant cysteine protease inhibitor, onchocystatin of the parasitic nematode Onchocerca volvulus, was tested for its role in microfilarial development in the simuliid vector. Onchocystatin was found to be present in female adults and skin microfilariae of the bovine parasite O. ochengi, the closest relative of O. volvulus. In addition the inhibitor could be detected as an excretory–secretory (E–S) product of the microfilariae. Co-injection of onchocystatin and the O. ochengi microfilariae into the surrogate vector Simulium ornatum s.l. significantly enhanced the recovery rates of the parasite within 24 h into the infection (P>0·001). The findings suggest a possible role of onchocystatin in the evasion by the parasite of the immune response of its vector.

A specific extracellular proteinase, degrading selectively the cecropin-based defence system of insects, is secreted into the larval body during parasitism of the greater wax moth by the Heterorhabditis bacteriophora/Photorhabdus luminescens complex and by phase 1 of P. luminescens. The proteolytic digestion of insect inducible cecropin-like immune molecules was demonstrated by the disappearance of the Galleria mellonella cecropins and purified Hyalophora cecropin B peptide PAGE bands upon exposure to infected extracts, and a similar abrogation of antibacterial activity using an agar diffusion assay. Proteolytic activity of infected extracts produced by nematode/bacterial complex and phase 1 variant of P. luminescens was shown to be correlated with cecropin-inhibitory activity, suggesting that this anti-cecropin agent may be responsible for the ability of bacteria to establish infection and the insecticidal nature of H. bacteriophora. Antibacterial activity of Galleria lysozyme and that of chicken egg-white lysozyme to which P. luminescens is insensitive, was unaffected by H. bacteriophora proteinase.