Identification and Physicochemical Properties of the Novel Hemolysin(s) From Oral Secretions of Helicoverpa armigera (Lepidoptera: Noctuidae).

Hemolysins cause the lysis of invading organisms, representing major humoral immunity used by invertebrates. Hemolysins have been discovered in hemolymph of Helicoverpa armigera larvae as immune factors. As oral immunity is great important to clear general pathogens, we presumed that hemolysins may be present in oral secretions (OS). To confirm this hypothesis, we conducted four testing methods to identify hemolysin(s) in larval OS of H. armigera, and analyzed physicochemical properties of the hemolysin in comparison with hemolytic melittin of Apis mellifera (L.) (Hymenoptera: Apidae) venom. We found hemolysin(s) from OS of H. armigera for the first time, and further identified in other lepidopteran herbivores. It could be precipitated by ammonium sulfate, which demonstrates that the hemolytic factor is proteinaceous. Labial gland showed significantly higher hemolytic activity than gut tissues, suggesting that hemolysin of OS is mainly derived from saliva secreted by labial glands. Physicochemical properties of hemolysin in caterpillar's OS were different from bee venom. It was noteworthy that hemolytic activity of OS was only partially inhibited even at 100°C. Hemolytic activity of OS was not inhibited by nine tested carbohydrates contrary to bee venom melittin. Moreover, effects of metal ions on hemolytic activity were different between OS and bee venom. We conclude that there is at least a novel hemolysin in OS of herbivorous insects with proposed antibacterial function, and its hemolytic mechanism may be different from melittin. Our study enriches understanding of the potential role of hemolysins in insect immunity and provides useful data to the field of herbivorous insect-pathogen research.

Differential responses of Helicoverpa armigera C-type immunlectin genes to the endoparasitoid Campoletis chlorideae.

The C-type lectins mediate nonself recognition in insects. The previous studies focused on host immunlectin response to bacterial infection; however, the molecular basis of immunlectin reactions to endoparasitoids has not been elucidated. The present study investigated the effect of parasitization by Campoletis chlorideae on hemagglutination activity (HA; defined as the ability of lectin to agglutinate erythrocytes or other cells), and transcriptional expression of C-type immunlectin genes in the larval host, Helicoverpa armigera. Parasitization induced four- to eightfold higher HA in the parasitized larvae, compared to nonparasitized larvae at days 2 and 6 postparasitization (PP), however inhibited HA at other days PP. Eight C-type lectins were differentially expressed in different host developmental stages, from feeding to wandering stage. The mRNA levels of HaCTL1, HaCTL3, HaCTL4, and HaCTL5 were upregulated and HaCTL2 and HaCTL7 were downregulated. Tissue analysis showed that HaCTLs were mainly expressed in fat body or hemocytes, while HaCTL5 was highly expressed in testes. The effects of parasitization on the lectin expression patterns differed. Lectins except HaCTL6 or HaCTL5 were significantly down- or upregulated in parasitized larvae at day 4 or 6 PP compared with that of nonparasitized larvae. We infer from our results that C-type immunlectins are involved in host-parasitoid interactions, and parasitization alter host immunlectin levels both in inhibiting and promoting host immune defenses to endoparasitoids. These immunlectin genes indicated an altered physiological status of the host insect, depending on developmental stage, tissue, and parasitization.

THE ENDOPARASITOID Campoletis chlorideae INDUCES A HEMOLYTIC FACTOR IN THE HERBIVOROUS INSECT Helicoverpa armigera.

Although lysis of invading organisms is a major innate form of immunity used by invertebrates, it remains unclear whether herbivorous insects have hemolysin or not. To address this general question, we tested the hemolytic (HL) activity of the hemolymph and tissue extracts from various stages of the polyphagous insect Helicoverpa armigera (Hübner) against the erythrocytes from chicken, duck, and rabbit. An HL activity was identified in the hemolymph of H. armigera larvae. Further studies demonstrated that the HL activity is proteinaceous as it was precipitable by deproteinizing agents. Hemolysins were found in Helicoverpa egg, larva, pupa, and adult, but the activity was higher in feeding larvae than in molting or newly molted larvae. Hemolysins were distributed among a variety of larval tissues including salivary gland, fat body, epidermis, midgut, or testes, but the highest activity was found in salivary gland and fat body. Relative to nonparasitized larvae, parasitization of H. armigera larvae by the endoparasitoid Campoletis chlorideae Uchida induced a 3.4-fold increase in the HL activity in the plasma of parasitized host at day two postparasitization. The present study shows the presence of a parasitoid inducible HL factor in the parasitized insect. The HL activity increased significantly in H. armigera larvae at 12 and 24 h postinjection with Escherichia coli. We infer the HL factor(s) is inducible or due to de novo synthesis, which means that the HL factor(s) is associated with insect immune response by inhibiting or clearance of invading organisms.