Parasitic Behavior Of The Parasitoid Macrocentrus Cingulum And Immune Defense Of The Host Ostrinia Furnacalis

The polyembryonic wasp, Microcentrus cigululm is a parasite of the larvae ofOstrinia furnacalis (Asian corn borer). As one of the important factors that allowparasitoids to avoid destruction by the immune system of host, materials on thesurface of wasps's eggs and embryos help parasitoids avoid immune reactionsfrom the host. This dissertation includes research on the physiology, biochemstryand parasitic behavior in the fields of cellular recognition of different surfacecharacteristics of foreign bodies, kinetics of encapsulation and melanization byhemocytes of host O. furnacalis larvae, how to choose host ages and strategiesthat the parasitoids M. cigulum rely on extraembryonic membrane to avoid theimmune reaction of host O. furnacalis larvae. The aim of this work is to providesome theoretical viewpoints for biological control of O. furnacalis by usingwasps.The main results are as follows:1. Encapsulation by haemocytes in O. furnacalis larvae was biphasic. WhenDEAE-Sephadex A-25 silicon beads were introduced into the hemocoele of O.furnacalis larvae, granular cells first attached to the surface of the beads within 3minutes, which was followed by the attachment of multiple layers ofplasmatocytes, eventually forming a smooth capsule comprised of overlappinglayers of cells until 48 h post injection. The complete capsule showed threedistinct regions. The role of the initial identification of foreign bodies within thehemocoele was played by the granular cells. Once the beads were identified asforeign bodies by granular cells, some putative materials released by lysedgranular cells would induce plasmatocytes to recognize the foreign bodies, andthen encapsulation was immediately triggered. Collectively, our resultssuggested that encapsulation in O. furnacalis larvae depends on a cooperativeresponse between haemocytes.2. Prior studies indicated that granular cells are usually the first hemocyte typeto attach to foreign targets in O. furnacalis larvae. Thereafter, large numbers ofplasmotocytes attach to the target and form a smooth capsule. To identify surfacefeatures of foreign targets that induce an encapsulation response, 12 types ofchromatography beads that differed in matrix composition, functional groups,and charge were tested using in vitro and in vivo bioassays. We first conducted invitro assays using hemocytes with no plasma components present. Theseexperiments indicated that bead types having diethylaminoethyl (DEAE),quaternary aminoethy (QAE), and quaternary amine (Q) functional groups wereencapsulated significantly more often than beads with other functional groups (P< 0.01). Charge also significantly affected encapsulation with positively chargedbeads being encapsulated more often than negatively charged or neutral beads (P< 0.05). Bead types that were encapsulated under these in vitro conditions werealways rapidly encapsulated when injected into O. furnacalis larvae. However,some beads types, like CM-Sephadex, not encapsulated by hemocytes in vitrowere encapsulated if preincubated in plasma or encapsulated in vivo if left in theO. furnacalis hemocoel for a longer period of time (at least 5 h). Basiccharacterization studies suggest these humoral recognition molecules areproteins or small peptides. Collectively, these results reveal that O. furnacalisrecognizes some targets as foreign by pattern recognition receptors on granularcells, whereas others are recognized by pattern recognition molecules in plasma.3. The hymenopteran M. cingulum is a polyembryonic endoparasitoid oflepidopteran O. furnacalis larvae. Previous studies indicated that the adultparasitoids do not have polydnaviruses (PDVs) associated with oviposition, buttheir mature eggs can avoid encapsulation by host's immune attack depending onthe surface fibrous layer of the eggs. However, M. cingulum can not alwayssuccessfully oviposit into the host at different ages, and usually they prefere tooviposit only into younger hosts. One or more other mechanisms may contributeto prevent encapsulaiton of M. cingulum eggs in O. furnacalis. Here, weproposed that O. furnacalis's parasitoids have been selected to infest the kind ofhost in which they can easily evade the host's encapsulation responses and aremost likely to successfully develop. We measured the kinetics of encapsulationand melanization elicited by DEAE-Sephadex A-25 in the haemocoel of M.cingulum larvae at different ages. It was found that the kinetics of encapsulationor melanization increased with the age of M. cingulum larvae, the total hemocytecounts (THC) of circulating hemocytes also increased with the age of the larvae,and the THC of O. furnacalis larvae were negatively correlated with theparasitism rate by M. cingulum. The results suggested that encapsualtion ormelanization of M. cingulum eggs was age-dependent. Yong O. furnacalis larvaepossess a weak cellular defense, and the survival probability of the eggsdecreased in hosts carrying a larger number of circulating hemocytes. It seemslikely that a correlation between the kinetics of encapsulation or melanizationand host choice in the O. furnacalis/M. cingulum system may exist.4. Prior studies showed that the surface fibrous layer of M. cingulum matureeggs may play a role in protecting eggs from host's immune attack. However,how the parasitoid embryos avoid encapsulation by the host's immune responseremains unknown. Because extraembryonic membrane was present throughoutembryonic development, we hypothesized that a second function of themembrane is protection of M. cingulum from the host immune response. Wereport here another kind of possible mechanism on passive evasion by M.cingulum embryos of encapsulaiton response of host O. furnacalis larvae. In vivoor in vitro incubation of M. cingulum embryos using O. furnacalis as permissivehosts and Helicoverpa armigera as non-permissive hosts. It was found thatpermissive hosts have a functional cellular immune system but fail toencapsulate M. cingulum embryos because of the presence of the extraembryonicmembrane. In contrast, the non-permissive host always encapsulate M. cingulumembryos. This is circumstantial evidence that M. cingulum embryos passivelyavoid encapsulation via surface features rather than by introducing factors thatactively suppress the host cellular immune system. Furthermore, it was foundthat a 97 kDa protein extracted from M. cingulum eggs and embryos may existand demonstrated that M. cingulum extraembryonic membrane was stronglylabeled with fluorescein-isothiocyanate (FITC)-conjugated H.P. lectin under thefluorescence microscope. The result suggested that the biochemical or molecularnature of the extraembryonic membrane of M. cingulum embryos maybe ahomologue of Drosophila hemomucin.5. Morphology and ultrastructure of the venom apparatus and ovary ofendoparasitoid M. cingulum, were observed by using light and electronmicroscopes. The venom apparatus of M. cingulum was composed of one venomreservoir and two gland filaments. The gland filaments joined together at the topof the venom reservoir. The gland filaments consisted of an outer single layer ofsecretory cells, a layer of degenerated ectodermal cells and an inner intimawhich lined the lumen. Secretory cells contained a distinct nucleus and a bigvesicular organelle, which secreted the components of venom. The reservoirconsisted of a muscular sheath and squamous cells, but no secretory cells. M.cingulum has a pair of ovaries and each one has approximately ten ovariolesconnected with a lateral oviduct via a small calyx region. Two lateral oviductsformed a common oviduct and opened into the ovipositor. The venom apparatusjoin-together with the common oviduct by its venom duct. The significance ofvenom apparatus of parasitic wasps in biology, cytology, taxonomy andevolution of the parasitoids are discussed.