Mary S. Cupp

Simulidin: a black fly (Simulium vittatum) salivary gland protein with anti-thrombin activity

Abstract A protein purified from the salivary gland lysate of female Simulium vittatum was found to inhibit bovine α-thrombin. This protein is stable to heat, has a mass of 11,334 Da and is rich in threonine. Based on N-terminal sequencing for the first 35 amino acids, no significant sequence similarity with other proteins was detected, indicating that this salivary component may be unique in structure. Because of its source and its anti-hemostatic properties, this protein has been named simulidin.

Salivary gland apyrase in black flies (Simulium vittatum)

Abstract Apyrase enzyme activity was demonstrated in the salivary glands of a colonized strain of Simulium vittatum . Activity was maximum (8.5 ± 0.7 mU/pair of gland equivalents) at pH 8.0, with ADP as substrate and Ca 2+ as the divalent cation. Activity was minimal in newly emerged females (1.6 ± 0.5 mU/pair of gland equivalents) but increased by 48 h. Activity in male salivary glands was marginally detectable (0.7 ± 0.8 mU/pair of gland equivalents), even 72 h after emergence. When newly emerged females were maintained at 4°C, salivary apyrase activity accumulated at a slow rate. Transferring females to warmer temperatures increased the rate of apyrase accumulation, but 27°C did not yield greater activity than 20°C. Apyrase activity was decreased when females engorged on whole bovine blood or on a simulated blood meal. Activity remained low 6 h after feeding, but increased to prefeeding levels by 48 h. During the second, anautogenous gonotrophic cycle, apyrase activity was not greater than during the first, autogenous gonotrophic cycle. Apyrase activity was not related to long term colonization as total salivary gland apyrase activity and pH profile in wild S. vittatum was not different from colonized S. vittatum .

Perspectives on the in vitro culture of filariae.

SummaryA primary constraint in the culture of human filariae is in obtaining starting material—either microfilariae (mfs), which infect invertebrates, or third stage larvae (L3s), which are infective to humans. Cryopreservation methods which partially overcome this difficulty have been developed for both mfs and L3s. Complete development of mfs to L3s outside an intact host was obtained recently when mosquito thoraces infected byBrugia malayi (24 h after the bloodmeal) were maintained in vitro. In another recent study in which no host tissues were present, a semidefined culture medium was used to investigate the properties of reduced glutathione (GSH) that stimulate early development ofOnchocerca lienalis mfs. An extended cysteinyl backbone and a free sulfhydryl were identified as the key structural elements provided by GSH. Stimulation also required the presence of low and high molecular weight components of serum as well as oxygen. Molting ofOnchocerca spp. L3s to the fourth stage at the rate of 50 to 70% has been reported by several researchers. Key factors identified in those successes have been temperature and serum lot. Improved long-term viability occurred with cellular co-culture. Beneficial effects of co-culture were shown to be due both to cellular conditioning of the medium as well as to lowered dissolved oxygen levels as a result of cellular metabolism. With the use of cell-conditioned medium and decreased incubator oxygen levels, long-term viability ofOnchocerca larvae in culture exceeded that previously reported. Recently,Brugia malayi adults of both sexes were cultured from L3s using a semidefined medium supplemented with human serum. Many of these sexually matured adults mated and produced viable microfilariae.