Effects Of Heavy Metal Cd²⁺ On The Immune Function Of The Flesh Fly, Boettcherisca(Sarcophage) Peregrina And Screening Of Antibacterial Peptides From The CDNA Library Of The Fly

I studied the effect of heavy metals of Cd 2+on Boettcherisca peregrine celluar immunity, humoral immunity, and the ultrastructure of haemocytes. We screen of antimicrobial genes from the cDNA library. Be summarized the main content as follows:1. Effects of cadmium on morphology of hemocytes from the larve of Boettcherisca peregrineThere was no difference in haemocytes morphology observed under the optical microscope between the control group and treated group, plasmatocytes and granulocytes can still spreaded and formed pseudopod. Oenocytoids, prohemocytes, plasmatocytes and granulocytes have been some changes when observed under transmission electron microscope. There is some electronic density of particles in the cytoplasm of prohemocytes of the larve and we can see the prohemocytes division occasionally. The cytoplasm of the prohemocytes is shrinkage, chromatin condensation and the membrane invagination in the treatment group. Similarly, the nucleolus of plasmatocytes is not obvious and chromatin also condensation, it was full of numerous vesicle which is often filled with inclusions in cytoplasmic. The rough endoplasmic reticulum (rER) showed dilation and vesiculation Whole cell of it has inflation trend. There is a gap in the granulocytes membrane, some inclusions leaked.2. Effects of cadmium on population, survival, spreading and encapsulation of hemocytes from the larve of Boettcherisca peregrineThe determination of Cd on the flies sub-letnal concentration LC25 is approximately 100μg/g, and LC50 is about 150μg/g. With these two concentrations of Cd2+treated the larvae of flies separately. After be treated the total numbers of haemocytes of the larvae significantly reduced compared with the control. As for the extending of the haemocytes, the extension rate was no difference compared with the control in 30mins after treatment 72 and 96 hours. However the rate is decreased after 24,48 and 96 hours, the rate is decreased significantly after culture 3 hours. After Cd-treated, the encapsulation rate of pupae was significantly lower than the control. It's note that Cd has a certain degree inhibition effect on the pupae and larvae of the fly.3. Effects of cadmium on hemolymph PO and LO activity from the larve of Boettcherisca peregrineThe larvae after treated with 100μg/g Cd2+and 150μg/g Cd2+, because the processing time and processing of different concentrations, the PO activity was significantly affected. The high concentration of Cd ions enhanced the PO activity at 24 and 48 hours, but suffered inhibition at 72 and 96 hours, and had no significant changes at 120 h.The results showed that Cd ions has distinct inhibited effected on the LO activity of hemolymph. The inhibition effect significantly increased with the concentration at 24 h, and decreased significantly compared with the control group at other times, but there was no difference between the two concentrations. It was noted that the poisoning effect of Cd2+100μg/g and 150μg/g Cd2+on hemolymph of larvae of Boettcherisca fly was not very different.4. Screening of antibacterial peptides from the cDNA library of Boettcherisca peregrineUse high throughput screening method for identifying insect antibacterial peptide, based on cDNA expression library. Through screening and testing repeatedly, some positive cloning with certain anti-bacterial activity were obtained. After sequencing their nucleotide sequence, comparing the results in the data base of Genbank and AMP, we found that there were some new anti-bacterial genes such as:Insect cuticle proteins, cytochrome b and ferritin also some usual anti-bacterial genes for as lectin, attacin, acid protease inhibitors, chitinase. Therefore, our next research direction will be for these new genes for further validation of antimicrobial activity.