Regulation Of Lectin And Hemolysin In Helicoverpa Armigera By Parasitoid, And Identification Of The Host Novel Immune-related Factors

In the present paper, an important crop pest Helicoverpa armigera and its dominant larval endoparasitoid Campoletis chlorideae were chosen to study the regulation of immune factors lectin and hemolysin in host H. armigera by the parasitic wasp C. chlorideae, and the host novel immune-related factors were identified. Further, the physico-chemical properties of hemolysin(s) from H. armigera hemolymph and saliva were investigated, respectively, in order to explore the molecular structure and physiological function in future. The results were shown as below: 1. Effect of parasitism by the wasp Campoletis chlorideae on the hemagglutinin activity(HA) and transcriptional expression of the eight C-type immunlectin genes from Helicoverpa armigeraEndoparasitoids can survive and develop in their host insects due to their ability to overcome host immune systems. In order to understand the molecular bases of the host immune response to endoparasitoid wasps attack, we investigated the effect of parasitization by the endoparasitic wasp Campoletis chlorideae on host insect Helicoverpa armigera larval growth and development, protein and hemagglutination activity in host larval hemolymph, and transcriptional expression of the eight C-type immunlectin genes. The results showed that larvae of H. armigera parasitized by wasp experienced arrested development, and sharply reduced weight gain compared to non-parasitized larvae. The total protein content of hemolymph from parasitized larvae was higher than that of non-parasitized larvae. SDS-PAGE profile of hemolymph proteins revealed that several parasitism-specific proteins were induced, but some proteins were inhibited in parasitized larvae. Lectins were abundant at different developmental stages of H. armigera, and the higher larval instars had relatively higher HA titer. The hemagglutinating reaction showed that the parasitization induced 4- or 8-fold higher HA in the hemolymph of parasitized host larvae than that of non-parasitized larvae at day 2 and 6 post-parasitization. The expression of eight C-type lectin genes differed in their patterns at different developmental stages. From feeding to wandering stage, the m RNA levels of Ha CTL1, Ha CTL3, Ha CTL4 and Ha CTL5 were up-regulated, in contrast, Ha CTL2 and Ha CTL7 were down-regulated. This result indicated that these immunlectin genes were related to insect development. The eight C-type immunlectin genes were mainly expressed in the two main immunity-conferring tissues, hemocytes and fat body, also expressed in other tissues including midgut, epidermis, malpighion tubes, testes, and salivary gland. However, the expression pattern among them were different. The parasitism also regulated the expression of these eight immunlectin genes, but the regulative effect on different genes were absolutely different. The transcripts of these eight genes except Ha CTL6 were significantly down-regulated in parasitized larvae at day 4 post-parasitization compared to non-parasitized host larvae. The results suggest that C-type immunlectins are involved in host-parasitoid interactions. This study provides information on differential expression of immunlectin genes following parasitism and also improves our current understandings the roles played by parasitoids. These eight C-type immunlectin genes indicated an altered physiological status of the insect, depending on developmental stage, tissue and parasitism. 2. The endoparasitoid C. chlorideae induces a hemolytic factor in the herbivorous insect H. armigeraAlthough 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(H bner) against the erythrocytes from chicken, duck and rabbit. A 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 non-parasitized larvae, parasitization of H. armigera larvae by the endoparasitoid Campoletis chlorideae Uchida induced a 3.42-fold increase in the HL activity in the plasma of parasitized host at day two post-parasitization. The present study shows the presence of a parasitoid inducible HL factor(s) in the parasitized insect The HL activity increased significantly in H. armigera larvae at 12 and 24 h post-injection 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. 3. Identification of the novel lectin and hemolysin in saliva(regurgitant) of H. armigeraThere are aboundant lectins in hemolymph of H. armigera, but until now no lectin has ever been reported in insect saliva(regurgitant). In the present paper, the agglutination activity of H. armigera saliva was detected in V-bottom microtiter plates. The agglutination reaction of chicken erythrocytes treated with saliva was observed obviously under phase contrast microscope. By ammonium sulfate precipitation, the protein precipitated by 35% and 45% ammonium sulfate possessed obvious agglutination activity, this indicated that the chemical nature of the substance with agglutination activity in saliva is protein. The carbohydrate-inhibition assay discovered the difference between saliva and hemolymph in carbohydrate-binding profiles of lectin, this suggested that they were different lectins. Moreover, we detected the hemolytic activity of saliva by V-bottom microtiter plate, microscopy observation, lysis zone of agarose plate and spectrophotometry. This demonstrated that H. armigera saliva contains hemolytic activity substance, and its hemolytic activity was much higher than the same volume of hemolymph. The hemolytic activity of low-instar larva was slightly higher than that of high-instar larva. And the hemolytic activity of saliva was found in all tested lepidopteran insects, such as Ostrinia furnacalis, Spodoptera exigua, Proxenus lepigone, Pieris rapae and so on. But there were differences in hemolytic activity of saliva among these different last larval instar, for example, P. lepigone had the highest hemolytic activity, followed by H. armigera, while the hemolytic activity of O. furnacalis, S. exigua and P. rapae were relatively low. Diet also had an impact on hemolytic activity of saliva, for example, H. armigera larvae fed with artificial diet possessed the highest hemolytic activity, followed by capsicum, and then cabbage. Further, the source of saliva hemolysin was analysed. The results showed that the midgut fluid had the highest hemolytic activity, followed by saliva and foregut fluid, and the hemolytic activity of hindgut fluid was the lowest. Through the investigation of hemolytic activity of corresponding tissue grinding fluid, we found that salivary gland grinding fluid had the highest hemolytic activity, followed by foregut and mingut, and the hindgut only had very low hemolytic activity. From these we can conclude that the salivary glands or midgut is possible source. After treated with ammonium sulfate precipitation and dialysis, the saliva also showed hemolytic activity, this indicated that it is a protein. The result of cross-adsorption tests of different vertebrate erythrocyte showed that saliva had higher hemolytic activity for rabbit erythrocytes than other vertebrate erythrocytes, and hemolytic activity of saliva adsorbed with erythrocytes was inhibited significantly. Further, saliva sample adsorbed with red cell membrane could be used to found the hemolytic membrane-binding proteins by SDS-PAGE, by using this method, three different protein bands with molecular weight of 48 k Da, 28 k Da and 23 k Da were chosen. Through ESI mass spectrum identification, we screened out gram negative bacteria-binding protein and peptidoglycan recognition protein from a large amount of protein information, which has molecular weight of 42.0 k Da and 20.1 k Da, and p I of 5.85 and 5.71, respectively, also incuding some enzymes. It inferred that the hemolysin of H. armigera saliva may be related to the recognition and lysis of pathogenic bacteria. 4. The physico-chemical properties of hemolysin in H. armigera hemolymphIn order to explore the molecular structure and physiological function of a new immune-related factor hemolysin found in H. armigera hemolymph, we should understand its physico-chemical properties first. So the effect of temperature, p H, EDTA and metal ions on the hemolysin and carbohydrate-binding properties were researched, respectively. The results showed that the hemolytic activity was significantly reduced at 50℃, and completely destroyed at 60℃. The hemolysin displayed thermolability. The concentration of hemolymph hemolysin was not in direct proportion to its hemolytic activity. The hemolytic activity of 8-fold diluent was higher than that of 4-fold diluent, however, with the increase of dilution ratio, the hemolytic activity of hemolymph completely disappeared at 32-fold or more. Hemolymph and erythrocytes were in the reaction for 60 min, no obvious changes happened in the percentage of hemolysis, 2 h later, the rate of erythrocytes lysis was dramatically rapid, complete hemolysis reached after 6 h. The optimum p H for hemolymph hemolytic activity was at 7-8. The concentration of EDTA at 62.5, 125, 150 and 500 m M could promote hemolytic activity of hemolymph, While the metal ions Mn2+, Zn2+, Co2+ and Ca2+ possessed a very significant inhibition. The result of carbohydrate-inhibition assay indicated that hemolytic activity of hemolymph could not be inhibited by D-lactulose, raffinose, mannose, galactose, glucose, lactose, trehalose, p-nitrophenyl-β-D-galactopyranoside( PNPG) and sialic acid, this suggested that hemolymph hemolysin did not have the carbohydrate-binding properties with these carbohydrates. 5. The physico-chemical properties of hemolysin in H. armigera saliva(regurgitant)The research on physico-chemical properties of hemolysin from H. armigera saliva(regurgitant) could provide information on the intensive study of immune defense mechanism of H. armigera. The effect of temperature, concentration, reaction time, p H, EDTA and metal ions on the hemolytic activity of H. armigera saliva was tested using erythrocytes of chicken, and its binding properties was analysed by carbohydrate-inhibition assay. Meanwhile, the properties of melittin form apisin was as a comparative study. The result showed that hemolytic activity of H. armigera saliva increased at 40 and 50℃, decreased at 60-100℃, but still reached 67.5% of the maximum hemolytic activity. This indicated that the hemolysin of H. armigera saliva had a certain thermal stability. In contrast, thermal stability of apisin was extremely strong, its hemolytic activity remained unchanged at 100℃. The concentration of saliva hemolysin was not in direct proportion to its hemolytic activity, 16-fold diluent of saliva had the highest hemolytic activity, and it was 1.6 times hemolytic activity of the undiluted saliva. Hemolytic activity of apisin diluted between 1-32 times did not change significantly. When saliva and erythrocytes were in the reaction for 5-15 min, the speed of erythrocytes lysis reached the maximum. Apisin showed two rapid erythrocytes lysis at 35-1 h and 2-6 h. Saliva hemolysin had wide p H tolerance, hemolytic activity unchanged at p H 2-10. After treatment with 125, 250 and 500 m M EDTA, the hemolytic activity could be promoted significantly. While the hemolytic activity of apisin only significantly improved at 500 m M EDTA, and was significantly inhibited at 62.5, 125, 250 and 1000 m M EDTA. Mn2+, Zn2+, Co2+, Ca2+ and Ni2+ could inhibit hemolytic activity of saliva, while K+ and Mg2+ could promote the hemolytic activity. For apisin, Mn2+, Zn2+, Co2+ and Ni2+ could significantly inhibit its hemolytic activity. The result of carbohydrate-inhibition assay indicated that the tested D-lactulose, raffinose, mannose, galactose, glucose, lactose, trehalose, p-nitrophenyl-β-D-galactopyranoside(PNPG) and sialic acid did not have inhibition on hemolytic activity of saliva, but apisin’s hemolytic activity could be inhibited significantly by D-lactulose and lactose. This suggests that the binding properties of hemolysin in H. armigera are different from melittin of apisin.