Characterization of acetylcholinesterase purified from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae).

Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the lesser grain borer (Rhyzopertha dominica) was significantly inhibited by higher concentrations of the substrates acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (A beta MTC) and propionylthiocholine (PTC). 2. The efficiency of AChE for hydrolyzing different substrates was ATC > A beta MTC > PTC > S-butyrylthiocholine. The enzyme activity was completely inhibited by 10(-5) M eserine or BW284C51, but was only partially inhibited by ethopropazine at the same concentration. These results confirmed that the purified enzyme was an typical insect AChE. 3. Non-denaturing and SDS polyacrylamide gel electrophoresis (PAGE) showed only one major molecular form in the purified AChE with a molecular weight of about 107,000 prior to reduction and about 56,000 after reduction, suggesting the homodimer of AChE linked with disulfide bonds.

Development and partial characterization of monoclonal antibodies to venom of the parasitoid Microplitis demolitor.

The venom of Microplitis demolitor consists of a mixture of proteins. On native PAGE gels three major proteins designated a, b, and g were detected, while on SDS-PAGE gels two major proteins of M(r) 64.5 and 30.8 kD and several minor proteins were detected. No proteins smaller than M(r) 30.8 kD were present. Murine monoclonal antibodies were generated against different venom components. Analysis by Western blot of venom proteins separated on native and SDS-PAGE gels confirmed that antibodies from seven hybridoma lines recognized venom components. Two of the seven hybridoma lines reacted specifically with protein g on native PAGE gels and the M(r) 30.8 k protein on SDS-PAGE gels, while four other lines cross-reacted with these and other venom proteins. The final hybridoma line reacted with protein a when venom was separated on native PAGE gels and an array of proteins when venom was separated on SDS-PAGE gels. Using an enzyme-immunoassay and specific monoclonal antibodies, M. demolitor females were estimated to inject 0.02-0.05 venom gland reservoir equivalents into its host, Pseudoplusia includens, at oviposition. Venom proteins persisted in host hemolymph for 6-12 h before dropping to undetectable levels.

Ecdysteroid induction of embryonic morphogenesis in a parasitic wasp.

Development of the parasitic wasp Copidosoma floridanum (Hymenoptera: Encyrtidae) is unusual in two ways. As many as 3000 embryos are formed from a single egg and embryonic morphogenesis is closely synchronized with the onset of the metamorphosis of its host. Given this extreme synchrony between parasite and host development, we undertook a series of experiments to determine whether host endocrine factors regulate C. floridanum embryonic morphogenesis. Here we report that C. floridanum embryos must develop for 9 days before acquiring the competence to undergo morphogenesis. Furthermore, several pieces of evidence suggest that ecdysteroids of host origin regulate induction of C. floridanum morphogenesis. First, competent embryos initiated morphogenesis when transplanted into host larvae and pupae, host stages possessing elevated ecdysteroid titers, but not when transplanted into adult moths. Second, morphogenesis was arrested by ablation of the host's source of ecdysone, but could be rescued by injection of 20-hydroxyecdysone in a dose-dependent manner. Finally, a segment of DNA encoding a zinc finger nearly identical in sequence to a portion of the ecdysone receptor of Drosophila melanogaster was isolated and characterized from C. floridanum. This putative ecdysone receptor probe indicated that expression of this gene was correlated with the initiation of C. floridanum embryonic morphogenesis. The temporal pattern of putative receptor RNA accumulation increased in association with the onset of morphogenesis, while the spatial pattern of expression was associated with the invagination of cells forming the gastrula. Together, these data suggest that ecdysone of host origin is directly involved in the induction of C. floridanum embryonic morphogenesis.

Persistence and expression of Microplitis demolitor polydnavirus in Pseudoplusia includens.

Persistence and expression of Microplitis demolitor polydnavirus (MdPDV) was examined in parasitized and virus-injected Pseudoplusia includens larvae. Viral DNA persisted in P. includens larvae for 6 days, but no increase in the amount of viral DNA present was detected. Viral transcripts were observed in parasitized and virus-injected larvae 4 h post-parasitism and expression continued for 6 days. When specific host tissues were examined, more viral DNA and RNA was detected in haemocytes than in the gut, nervous system and fat body. 32P-labelled MdPDV DNA hybridized to approximately six different size classes of mRNAs on Northern blots of RNA from haemocytes of parasitized larvae. MdPDV transcription was first detected in haemocytes at 4 h post-parasitism and continued for 6 days. Similar transcripts were observed in haemocytes from larvae that had been injected with calyx fluid or MdPDV plus venom. First-strand cDNA probes of haemocyte-specific MdPDV transcripts hybridized to only certain MdPDV viral DNAs, suggesting that only part of the MdPDV genome is expressed in this host cell type.

Developmental disruption of Pseudoplusia includens and Heliothis virescens larvae by the calyx fluid and venom of Microplitis demolitor.

Calyx fluid and venom from the braconid parasitoid Microplitis demolitor differentially affected the development of Pseudoplusia includens and Heliothis virescens. P. includens exhibited delays in larval development, supernumerary instars, and formed larval-pupal intermediates when injected with 0.01-0.10 wasp equivalents of calyx fluid. In contrast, H. virescens was relatively unaffected by calyx fluid regardless of dose. Venom did not affect the development of either host species, but appeared to synergize the activity of calyx fluid. This was particularly evident in H. virescens, where injection of 0.10-0.20 wasp equivalents of calyx fluid and venom induced the formation of a large number of intermediates while the same amount of calyx fluid did not. The particulate portion of M. demolitor calyx fluid was the only component that caused developmental delays and the formation of intermediates in both host species. Purified virus caused developmental alterations in P. includens, while trioxsalen treated calyx fluid did not affect development of P. includens or H. virescens. These data suggest the requirement for venom in parasitism may differ between host species, and that dosage plays an important role in interpreting the interaction between calyx and venom components.