Sex Pheromone of the Rare Click Beetle Betarmon bisbimaculatus.

The click beetle Betarmon bisbimaculatus (Fabricius, 1803) (Coleoptera: Elateridae) has a scattered distribution throughout a large area in Europe and the near East. Due to its scarcity, little is known about the ecology, biology, and development of this peculiar species. Here, we studied the composition of the female-released sex pheromone of B. bisbimaculatus. Neryl hexanoate, neryl octanoate, and neryl decanoate, in a ratio of approximately 3:1:6, were the only volatile compounds present in the extracts of pheromone glands. A synthetic mixture of all three compounds in the natural ratio was highly attractive to males in field traps. When the compounds were tested individually, only traps baited with neryl hexanoate were attractive, but they caught only a sixth of the males compared to the mixture. Based on the similarity of their sex pheromones, we propose that the tribe Pomachiliini with B. bisbimaculatus is closely related to the tribe Agriotini. This study shows the potential of sex pheromone studies for monitoring of rare and threatened insects as well as for elucidating phylogenetic relationships.

Cloning, heterologous expression, and characterization of three aquaglyceroporins from Trypanosoma brucei.

Trypanosoma brucei, causative for African sleeping sickness, relies exclusively on glycolysis for ATP production. Under anaerobic conditions, glucose is converted to equimolar amounts of glycerol and pyruvate, which are both secreted from the parasite. As we have shown previously, glycerol transport in T. brucei occurs via specific membrane proteins (Wille, U., Schade, B., and Duszenko, M. (1998) Eur. J. Biochem. 256, 245-250). Here, we describe cloning and biochemical characterization of the three trypanosomal aquaglyceroporins (AQP; TbAQP1-3), which show a 40-45% identity to mammalian AQP3 and -9. AQPs belong to the major intrinsic protein family and represent channels for small non-ionic molecules. Both TbAQP1 and TbAQP3 contain two highly conserved NPA motifs within the pore-forming region, whereas TbAQP2 contains NSA and NPS motifs instead, which are only occasionally found in AQPs. For functional characterization, all three proteins were heterologously expressed in yeast and Xenopus oocytes. In the yeast fps1Delta mutant, TbAQPs suppressed hypoosmosensitivity and rendered cells to a hyper-osmosensitive phenotype, as expected for unregulated glycerol channels. Under iso- and hyperosmotic conditions, these cells constitutively released glycerol, consistent with a glycerol efflux function of TbAQP proteins. TbAQP expression in Xenopus oocytes increased permeability for water, glycerol and, interestingly, dihydroxyacetone. Except for urea, TbAQPs were virtually impermeable for other polyols; only TbAQP3 transported erythritol and ribitol. Thus, TbAQPs represent mainly water/glycerol/dihydroxyacetone channels involved in osmoregulation and glycerol metabolism in T. brucei. This function and especially the so far not investigated transport of dihydroxyacetone may be pivotal for the survival of the parasite survival under non-aerobic or osmotic stress conditions.

GPI anchor transamidase of Trypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange.

GPI8 from Trypanosoma brucei was cloned and expressed in Escherichia coli. TbGPI8 encodes a 37 kDa protein (35 kDa after removal of the putative signal sequence) with a pI of 5.5. It contains one potential N-glycosylation site near the N-terminus but no C-terminal hydrophobic region. Enzyme activity assays using trypanosomal lysates or recombinant TbGpi8 exhibited cleavage of the synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine, indicating that TbGpi8 is indeed directly involved in the proteolytic processing of the GPI anchoring signal. Intracellular localization of TbGpi8 within tubular structures, such as the endoplasmic reticulum, was observed by using specific anti-TbGpi8 antibodies. The transamidase mechanism of GPI anchoring was studied in bloodstream forms of Trypanosoma brucei using media containing hydrazine or biotinylated hydrazine. In the presence of the latter nucleophile, part of the newly formed VSG was linked to this instead of the GPI anchor and was not transferred to the cell surface. VSG-hydrazine-biotin was detected by streptavidin in western blots and intracellularly in Golgi-like compartments.

A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae.

Metacaspases constitute a new group of cysteine proteases homologous to caspases. Heterologous expression of Trypanosoma brucei metacaspase TbMCA4 in the budding yeast Saccharomyces cerevisiae resulted in growth inhibition, mitochondrial dysfunction and clonal death. The metacaspase orthologue of yeast, ScMCA1 (YOR197w), exhibited genetic interaction with WWM1 (YFL010c), which encodes a small WW domain protein. WWM1 overexpression resulted in growth arrest and clonal death, which was suppressed by concomitant overexpression of ScMCA1. GFP-fusion reporters of WWM1, ScMCA1 and TbMCA4 localized to the nucleus. Taken together, we suggest that metacaspases may play a role in nuclear function controlling cellular proliferation coupled to mitochondrial biogenesis.