Extended in vitro culture of Microplitis croceipes teratocytes and secretion of TSP14 protein
Introduction
The presence of teratocytes, derived from the extra embryonic serosal membrane of the developing endoparasitic egg, has been reported in more than 40 hosts parasitized by nearly as many species of braconids and scelionids. Teratocytes are thought to have trophic, immunosuppressive and secretory roles in the host, each of which may have an impact on the growth and development of the host (Dahlman and Vinson, 1993). Even though teratocytes make important contributions to the ultimate success of a parasitoid, detailed studies of their specific contributions have been limited to only a few species.
We have extensively studied the Heliothis virescens: Microplitis croceipes system. Teratocytes are responsible for the developmental arrest observed in H. virescens parasitized by M. croceipes (Zhang and Dahlman, 1989). Teratocytes alter hormonal titers and reduce the titers of several abundantly expressed proteins found in the hemolymph, including arylphorins and juvenile hormone esterase (Zhang et al., 1992, Zhang et al., 1997). H. virescens larvae injected with secreted proteins obtained from in vitro cultures of teratocytes and parasitoid larvae developed more slowly, weighed less and many experienced incomplete larval-pupal ecdysis, similar to larvae injected with teratocytes (Schepers et al., 1998). Based on these results, it was concluded that teratocytes secreted the biologically active proteins and the mixture was called crude teratocyte secreted protein (TSP). A fraction of the crude TSP containing compounds with molecular sizes between 3 and 30 kDa inhibited protein synthesis in specific H. virescens tissue assays (Schepers et al., 1998; Dahlman, unpublished). A 13.9 kDa protein (TSP14) in this fraction was selected for purification, digestion with lys-C and determination of amino acid sequence. The cDNA sequence for TSP14 has been determined and a biologically active fraction has been produced using two different translation systems (Dahlman et al., unpublished). Both the natural and recombinant TSP14 have been shown to inhibit protein synthesis at the level of translation (Dahlman et al., unpublished).
We report here an in vitro system in which teratocytes were successfully cultured for at least 12 days in the absence of parasitoid larvae. Under these conditions teratocytes continue to secrete a biologically active component that was responsible for the developmental arrest of the host.
Section snippets
Insect colony
H. virescens larvae were reared at on an artificial diet in the laboratory under long day conditions (16L:8D). M. croceipes parasitoids were reared according to the methods described by Schepers et al. (1998). Premolt third instar H. virescens were parasitized for 1 h using M. croceipes at a ratio of 7:1. Parasitized host larvae were held in individual 22.2 ml plastic cups containing approximately 10 ml of diet. Parasitoid larvae emerged from the host 7–9 days after parasitization.
Teratocyte cultures
In vitro teratocyte viability
Exchange of medium every three days had a minor, but significant, effect on the viability of M. croceipes teratocytes held in the presence of parasitoid larvae (Fig. 1). Viability was lower in exchanged groups at the end of the second (day 6) and third (day 9) exchanges. By day 12 differences between unexchanged and exchanged cells were not significant but both were significantly lower than the percent viability observed in any of the other unexchanged cells (Fig. 1).
In vitro parasitoid larvae viability
Newly emerged first instar
Discussion
A continuous in vitro culture of M. croceipes teratocytes, in the presence of larvae, was maintained for at least 12 days when the medium was exchanged every three days. However, by the 12th day teratocytes had experienced 40% mortality, some of which could be associated with the exchange process which included cell washing and centrifugation. Nevertheless, the additional TSP obtained from medium exchanges more than compensated for the decreased teratocyte viability compared to TSP yield from
Acknowledgements
We gratefully acknowledge the support of National Science Foundation Grant (IBN-0004797). We express our appreciation to Deqing Zhang, Eric Schepers and Esther Fleming for their technical assistance during the work and a number of undergraduate student workers who faithfully assisted with insect colony maintenance. This is paper 01-08-127 of the Kentucky Agricultural Experiment Station, Lexington, KY.
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