Studies On The Properties Of Lectins Of Two Sibling Helicoverpa Species

In this study, two kinds of insects, Helicoverpa assulta (Guenée) and itssibling species Helicoverpa armigera (Hübner) were used. The physico-chemicalproperties of lectins and developmental characters and tissue-specific expression ofseven lectin genes of H. assulta and H. armigera were studied comparatively. Thehemagglutination activity of lectins in the hemolymph of H. assulta was identified,their physico-chemical properties were analysied. The properties of the lectin from theH. armigera were also investigated. Furthermore, the expression of seven lectin genesof two insects at different developmental stages and tissues were compared. Theresults were shown below:1. Hemagglutination activity of lectins in the hemolymph of H. assultaTo understand the relationship between lectins and development in H. assulta,and the sugar-binding specificity of H. assulta lectin, Hemagglutination assays wereused to determine the hemagglutinating activity (HA) of haemolymph extracted fromHelicoverpa assulta to the erythrocytes, and the changes of HA titer during differentdevelopmental stages of H. assulta. Furthermore, thirteen sugars were tested todetermine their inhibitory effect on H. assulta lectin. The results showed that lectinswere detected during all developmental stages of H. assulta. Hemagglutinationactivity (HA) titer fluctuated between and within the various developmental stages,particularly in different larval instars and pupal ages, female pupal H. assulta hadhigher HA titer than their male cohorts. These results indicated that HA of lectins maybe related to developmental stage, larval instar and molting process, and wasdependent on sex. The carbohydrate binding specificity of the lectins in pupalhemolymph from H. assulta and H. armigera was determined by comparing theinhibitory activity of different sugars. The carbohydrates, which inhibited HA ofH.assulta and H. armigera were similar. It showed that HA was obviously inhibitedby D-galactose, L-rhamnose, maltose and lactose, and partly inhibited by D-glucose,D-fructose, L-fucose and sucrose et al., However, only H. armigera lectins hadstronger binding specificity with L-arabinose, N-acetyl-glucosamine and L-sorbosecould inhibit HA of H. armigera, but did not inhibit HA of H. assulta. It wassuggested that lectins detected from H. assulta are different from that in H. armigera. 2. The physico-chemical properties of H. assulta hemagglutininThe effects of freeze and thaw cycling, temperature, EDTA, metal ions, trypsinand phenoloxidase (PO) on the hemagglutinating activity of Helicoverpa assultalectin was tested using erythrocytes of duck. The results showed that thehemagglutinating activity was not influenced after repeated cycles of freezing andthawing, but it declined with heat treatment temperature and time increasing. Itsactivity was completely destroyed at60℃for15min. The lectin displayed lowthermal stability. The hemagglutinating activity could be inhibited significantly by10mM EDTA, however it could recover after adding Ca2+, this indicated that thelectin of H. assulta was C-type. The agglutinin ability increased when duckerythrocytes were treated with trypsin. PO in the hemolymph did not influence thehemagglutinating activity of lectins.3Hemagglutination activity of lectins in the hemolymph of H. armigeraTo clarify the relationship between lectins and development in H. armigera, andthe sugar-binding specificity of H. armigera lectin, the hemagglutinating activity (HA)of haemolymph extracted from H. armigera, and the changes of HA titer duringdifferent developmental stages of H. Armigera were determined by theHemagglutination assays. Furthermore, thirteen kinds of sugars were tested todetermine their inhibitory effect on H.armigera lectin. The results showed that lectinswere detected during all developmental stages of H. armigera. By contrast, thehighest HA titer was obtained from larval hemolymph, low HA titers were detected inegg and adult insects. The HA titer obtained in old instar larvae was higher than thatin young. Hemagglutination activity (HA) titer was obviously different in differentlarval instars and pupal ages. Besides, female larval and pupal H. armigera had higherHA titer than their male cohorts. These results indicated that lectins may be related todevelopmental stage, larval instar, pupal ages, molting process, and sex. Thecarbohydrate binding specificity of the lectins in larval and pupal hemolymph from H.armigera was determined by comparing the inhibitory activity of different sugars. Itshowed that HA was obviously inhibited by Maltose, L-Arabinose, L-Rhamnose,Lactose and D-Galactose, and partly inhibited by D-Glucose, sucrose, mannose andL-sorbose. While D-fructose and N-acetyl-glucosamine could inhibit HA of pupal H.armigera, but did not inhibit HA of laval H. armigera. It suggested that lectinsdetected from larval are not exactly the same from pupal.4Comparatively study on the expression of lectin genes at differentdevelopmental stages and tissues in H. assulta and H. armigera The5th instar larvae, prepuae, and different age pupae were selected to analyzeexpression of two C-type lectins (ctlh1and ctlh2) and five C-type lectins (ctlf3、ctlf4、ctlf5、ctlf6and ctlf8) in different developmental stages of H. assulta and H.armigera by RT-PCR analysis. The transcripts of seven lectin genes were found fromH. assulta, they were almost same as those from H. armigera. There were significantdifferences in expression of seven lectin genes in different developmental stage of twosibling Helico-verpa species, and there were significant differences in expression ofthe same lectin gene in two sibling Helicoverpa species. Moreover, the haemocytesand fat bodies from H. assulta and H. armigera were selected to investigatetissue-specific expression of seven lectin genes by RT-PCR analysis. The resultsshowed that ctlh1mRNA was detected both in haemocyte and fat body. Thetissue-specific expression was not significant. But ctlh2mRNA was only detected infat body. Ctlf3and ctlf4were only detected in fat body of two insects. Ctlf8mRNAwas only detected in fat body of H. assulta, but was also detected both in haemocyteand fat body of H. armigera. The transcripts of ctlf5in fat body of H. assulta washigher than that in heamocyte. In contrast, the transcripts of ctlf5in heamocyte of H.armigera was higher than that in fat body. Ctlf6was expressed both in the heamocyteand fat body of H. assulta and H. armigera. All above results suggested that thetissue-spectific expression of seven lectin genes was not obvious.