| In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Much work has reported that variations in immunity during insects metamorphosis. However, the variation is taxa-related. In order to understand the changes in immunocompetence of three developmental stages (last instar larvae, pupae and adults) during insects metamorphosis, mealworm beetles (Tenebrio molitor) from each stage were chosen to be injected with live Bacillus subtilis. In this paper, the indicators of stage-related immune functions were quantified using standard immunocompetence assays. In addition, the level of immune response related genes mRNA across different stages were determned using RT-PCR. Our preliminary studies will provide theory evidences for the relationship between antimicrobial response and metabolic processes and reproduction during insects metamorphosis, and provided a valuable insight into the insect bioecology at molecular biology.Two main aspects are involved.1) Studies on the difference of immune response. The mealworm beetle T. molitor that newly formed last instar larvae(13th instar), pupae and adults were collected within one day to be infected by live B. subtilis. And we investigated the magnitude variation in phenoloxidase (PO) activity, lysozyme relative activity (LRA) , antimicrobial activity, total haemocytes count (THC), total haemocytes microaggregates count (THMC), haemocytes phagocytosis and haemocytes spreading competence in haemolymph after 24h.The results showed that last instar larvae kept the highest THC, haemocytes phagocytosis competence and haemocytes spreading competence compared to pupae and adults. In addition, last instar larvae assembled much more LRA and antimicrobial activity than pupae, but had the lowest PO activity in its haemolymph. At same time THMC and haemocytes spreading percentage was remarkably increased by immune insults, especially more significant in last instar larvae than in others. Although pupae possed high THC and haemocytes phagocytosis competence, its LRA and antimicrobial activity was much more lower than adults. There was the highest PO activity in adults, But THC and haemocytes phagocytosis competence and haemocytes spreading competence diminished towards older stages. So last instar larvae possess the most vigorous cellular immunity and high humoral immunity; pupae maintain a high cellular immunity; Whereas adults assemble the most pronounced humoral immunity in general.2) The variations of immunity related genes transcription. The level of related genes (MRA, PGRP, Tenecin 1, Tenecin 3, PPO) mRNA expression across different stages in immune response after three treaments were determned using RT-PCR.The results presented that the PGRP transcript in fat body of T. molitor declined with development in general. In Na?ve group, MRG didn't express in puape, and weakly in last instar larvae, while much in adults; Tenecin 3(antifungal protein gene) and Tenecin 1(antibacterial peptide gene) mRNA were not expressed in pupae and adult, but weakly in last instar larvae. In constrast, they were abundantly expressed in larvae and adults, while little was detected in pupae after immune challenge by B. subtilis. In Na?ve group, PPO was not detected in pupae, but a little in last instar larvae and adults. It increased from last instar larvae to adults by turns, namely, PPO transcript was the most in adults, next in puape, then in last instar larvae.Take together, all evidences were provided in this paper, suggesting that the immune responses of T. molitor is developmental stages-related, and this was shown by the developmental activity of the immune response effective factors and the stages-dependent immune response related genes transcript. |
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