Extended in vitro culture of Microplitis croceipes teratocytes and secretion of TSP14 protein

Abstract

Teratocytes, cells which originate from the serosal membrane of some Braconidae and Scelionidae, can be found in the hemocoel of permissive hosts during part or all of the developmental time of the parasitoid larva. Teratocytes from Microplitis croceipes are known to secrete biologically active proteins, which contribute to developmental arrest and failure to pupate of Heliothis virescens larvae. One such protein, which has a molecular weight of approximately 14 kDa is called TSP14. The presence of parasitoid larvae is essential to maintain teratocytes under in vitro conditions with protein-free EX-CELL 400. The teratocyte viability was maintained in vitro for at least 12 days in the presence of larvae when medium was exchanged every three days. Western blots show that TSP14 was secreted during the entire period of exchanges. In the absence of parasitoid larvae, teratocyte viability was only 30% by day 6 and no TSP14 had been secreted. In the absence of parasitoid larvae, teratocytes maintained in vitro in EX-CELL 400 medium supplemented with 10% FBS remained viable for at least nine days and secreted TSP14 for at least six days. This suggests that parasitoid larval secretions are sufficient but not uniquely essential to maintain teratocyte viability. Parasitoid larvae maintained in the absence of teratocytes did not secrete TSP14 and their secretory products did not inhibit pupation of H. virescens larvae.

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.