Physiological And Molecular Research Of Vitellogenesis And Oogenesis In The Endoparasitoid Pteromalus Puparum

Vitellogenesis is a heterosynthetic process in insects and other arthropods. It involves the production of yolk protein precursors, vitellogenins (Vgs), from an extra ovarian source, such as the fat body, and the uptake of Vgs by ovaries. In most insects, Vgs are usually transported from the fat body by the hemolymph to the ovary and are specifically sequestered by the developing oocytes via receptor-mediated endocytosis and deposited as vitellins (Vts), which act as a nutritional reserve of amino acids for the embryo. So far, Vg cDNA sequences of about forty insects have been cloned and analyzed, and involved five Hymenopterns. The overall objective of this thesis was to analysis the cDNA sequence of vitellogenin and determine the endocrine regulation of yolk synthesis (vitellogenesis) and yolk accumulation by developing oocytes and process of oogenesis in the endoparasitoid Pteromalus puparum.1. Identification and biochemical characterization of Vg/Vt in P. puparum.Using gel electrophoresis, the main yolk protein, vitellin (Vt) and its hemolymph-borne precursor, vitellogenin (Vg) were identified from P. puparum ovaries and female hemolymph, respectively. The yolk protein had a native molecular mass of 350 kilodaltons and was composed of two polypeptides which molecular mass was 205 and 165 kilodaltons. Carbohydrates, lipids, and phosphorus were found associated with Vt as revealed by the respective stain ability to periodic acid-Schiff's reagent, Sudan Black B and Methyl Green Solution after gel electrophoresis. The soluble protein fractions of crude egg extracts eluted from the Sephadex-G100 gel in two peaks. With a combination of SDS-PAGE and Western blotting, the first peak was identified as the Vt with reference to the molecular mass of the Vt subunits. The fractions from the first peak were pooled and subsequently placed onto a UNOTMQ1 ion exchange column. The pooled sample eluted as two peaks from the column using a linear NaCl gradient of 0.0- 1.0 M, and fractions from the second peak were designated as the Vt by the same method as the first purification step. The purified Vt was composed of two subunits, Vt1 and Vt2. 2. Development of monoclonal antibodies to the vitellin in P. puparum and itsapplication methodsUsing hybridoma techniques, four monoclonal antibodies were developed to P. puparum soluble yolk proteins, and named as PpVt mAb1, PpVt mAb2, PpVt mAb3, PpVt mAb4, respectively. The four antibodies, being consisted of a lgG1 heavy chain andκlight chain, not only have high specificity and affinity to the ovarian vitellin, but also to female hemolymph vitellogenin. However, they act no immunological reaction with the male substances. The indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) is regarded as the most sensitive and accurate to measure Vg/Vt in the wasps by comparison with other three methods of ELISA, namely, direct, indirect and double antibody sandwich ELISA. This ELISA method is capable of detecting the Vg/Vt titer in a single wasp with the sensitivity of 20 ng/ml.3. Ovarian development and vitellogenesis in P. puparum.The ovary occurred pairly and each ovary was composed of three ovarioles. Length of ovariole increased significantly during the period from early pupal stage to 48 h after eclosion, and then decreased gradually within the following 72 h. At early pupal stage, no egg chamber could be observed. At the late pupal stage and pharate adult, egg chambers had been occurred, while yolk had not been deposited yet. At 12 h after eclosion, egg chambers with yolk protein could be observed, and within the following 36 h oocytes developed quickly and most of the oocytes completed egg maturation.Using the indirect double antibody sandwich ELISA, changes in Vg levels in the hemolymph at different developmental stages of the parasitoid after eclosion were dtermined. The Vg in the hemolymph were detected immediately after eclosion and increased to 0.58μg/female 24 h after eclosion. It declined slightly afterwards, and then increased to 0.57μg/female 72 h after eclosion. Shortly after eclosion (0-6 h), Vt in the ovaries was barely detectable. Thereafter, Vt levels in the ovaries increased gradually to 4.51μg/female 48 h after eclosion, and declined to 3.9μg/female 72 h after eclosion. There are significant positive linear relationship among Vg/Vt levels in ovaries and the ovariole length, and the percentage of mature oocytes during the pupal and adult stages. The Vg synthesis or uptake by ovaries was associated with ovarian development. 4. Oogenesis and apoptosis of nurse cells and follicular cells in P. puparum.The ovaries of P. puparum was of the meroistic polytrophic type. They were composed of three to five pair of tube-like ovarioles. Each ovariole was differentiated into an anterior germarium and a posterior vitellarium that contains 8-10 linearly arranged egg chambers. Oogenesis in P. puparum can be divided into five stages according to the proportional size of the nurse cells and oocytes. An individual egg chamber was composed of 1 oocytes and 15 nurse cells which were surrounded by a layer of follicular cells. Each nurse cell nucleus and follicular cell was surrounded by a thick three-dimensional cage of F-actin that was engaged in maintaining the position of the nuclei in the cell centres. The absorption of Vg from the hemolymph by the developing oocytes was initiated from late stage 2 of oogenesis and the yolk spheres can be detected from stage 3. Apoptosis of nurse cells was initiated from early stage 4 by double staining with propidium iodide and TUNEL, and apoptosis of follicular cells can be detected during late stage 4 of oogenesis.5. Vitellin degradation in developing embryos.Vt could be detected in wasp embryos at different developmental stages after the eggs were deposited in the host pupa. Vt levels remained more or less constant within the first 6 h after parasitism, increased sharply at 12 h after parasitism, but declined gradually 18 h after parasitism and thereafter.Soluble protein samples from the wasp embryos were examined by SDS-PAGE and Western blotting to determine if Vt utilization entailed changes in polypeptide compositions during embryonic development. The analyses by SDS-PAGE and Western blotting showed that the levels of both Vt subunits (Vt1 and Vt2) in the eggs declined progressively from 0 to 18 h after parasitism and became a mere trace 24 h after parasitism. Meanwhile, 9 new polypeptides showing marked immunoreactions with the monoclonal antibody against Vt ( PpVtmAb1), were found at different embryonic stages, and were referred to as EP1, EP2, EP3, EP4, EP5, EP6, EP7, EP8 and EP9, successively. These bands were probably derived from Vt during embryonic development and had molecular mass of 151, 135, 126, 114, 92, 87.6, 68.4, 62.5 and 59.2 kDa, respectively. Among these polypeptides, EP1, EP2, EP3, EP4, EP6 and EP8 occurred within 6 h after parasitism. In contrast, EP5 and EP7 were visualized 12 h after parasitism, and EP9 was observed 18 h after parasitism. All these polypeptides, however, became virtually undetectable 24 h after parasitism.6. Endocrine regulation of vitellogenesis in P. puparum.The titer of ecdysteroid, 20-hydroxyecdysone (20E) and JH were measured using the method of radioimmunoassay and GC-MS, respectively. The hemolymph profiles of Vg, Vg mRNA in fat bodies and ovarian Vt, were then measured. The result showed that the 20E titer and fat body Vg mRNA had the same dynamics tendency, the peak titers was at just adult eclosion and then decreased. The titer of JH III and ovarian Vt have the similar dynamics tendency, the peak titers was at 48h after adult eclosion and then decreased. The hemolymph profiles of Vg, Vg mRNA in fat bodies and ovarian Vt, were also measured in the wasps after various hormone treatments: 20E and juvenile hormone (JH)-III. The results showed that 20E was the only hormone that stimulated Vg-synthesis by fat body and its release into the hemolymph, and the JH III can accelerate the absorption of Vg by the oocytes. Allatectomy, which was believed to terminate the synthesis of JH in insects, could not inhibited the vitellogenesis and oocyte maturation in P. puparum, and application of JH III or 20E to the allatectomated female could improve the hemolymph Vg and accelerate the Vg uptake. So in P. puparum, Vg synthesis was dependent on ecdysteroids, and the ecdysteroids were the dominant hormone in regulation of vitellogenesis. JH III accelerated the accumulation of Vg by the developing oocytes, and postponed the oosorption.7. Effect of nutrition and mating on vitellogesis and egg maturing.Feeding with 20% honey water as a factor markedly increased hemolymph Vg level and ovarian Vt level, and also markedly increased the total egg numbers and percentage of mature eggs; but feeding with 20% sucrose water as a factor had no marked effect on both hemolymph Vg and ovarian Vt level. However, both adult feeding with 20% honey water and 20% sucrose water could significantly increase life expectancy of the wasps.8. Construction and analysis of cDNA expression library in P. puparum.A cDNA expressing library was constructed from the poly (A)~+ RNA prepared from the adult female fat body cells in theλZAP III phage vector. The original titration of library was about 1×10~5 pfu/ml with recombinant rate of 95.82%, and the titration of the amplified library was about 1×10~8pfu/ml. Tthe average length of insert cDNA fragment was between 0.5 kb and 2.5 kb. About one hundred Expressed sequence tag (ESTs) with an average length of 600.74 bp were obtained. After analyzed by BLASTn and BLASTx arithmetic, 40% of the ESTs showed significant matches with known sequences in database, mainly encoding the proteins with housekeeping functions (65.5%), signal transduction (12.5%) and gene transcription and regulation (18.6%).9. cDNA cloning and expression analysis of viteliogenin in P. puparum.By screening the library containing 1×10~5 recombinant phages with the anti-Vt monoclonal antibodies, eight clones that gave positive immunoreaction were isolated and cloned. Among these clones, five clones beard 2530 bp and three clones beard 1027 bp. Following the 5 RACE method, yielded a sequence of 5634 nucleotides long, and the deduced amino acid sequence had 1803 amino acids. Twenty N-terminal amino acid residues chemically determined for the 205 kD polypeptide matched exactly the deduced amino acid sequence starting from just the 18th residue, indicating that the first 17 amino acids was the signal peptide.A real time PCR was established using the cloned Vg sequence and housekeeping gene 18S rRNA to relatively quantify the expression of Vg mRNA. The Vg mRNA was first detected during pupae time in the female fat body cells, but neither expressed in ovaries nor in male fat body cells. The Vg mRNA of the female fat body cells in different stages was significantly different. A high Vg mRNA level is associated with a high titer of Vg in female wasps, but the high level of Vg in female hemolymph was postponed with about 24 h from that. A phylogenetic analysis (neighbour-joining) was made using vitellogenin sequences available in insects and the tree is congruent with the currently accepted insect phylogenetic schemes.