Elimination of Wolbachia from Urolepis rufipes (Hymenoptera: Pteromalidae) with Heat and Antibiotic Treatments: Implications for Host Reproduction

Molecular analyses using ftsZ and wsp primers identified infections of type-A Wolbachia bacteria in populations of Urolepis rufipes (Ashmead) (Hymenoptera: Pteromalidae), a potential biocontrol agent for pest flies (Diptera: Muscidae) in livestock confinements. Incidence of infections ranged from 10 to 45% for three field populations and 100% in a laboratory colony. Provision of adult wasps with sugar water containing 50 μg mL^–1 tetracycline hydrochloride, or continuous rearing at 34±0.5°C eliminated Wolbachia from experimental populations after four and six generations, respectively. Results were similar for experimental crosses between infected parents, between uninfected parents, and between infected female and uninfected male parents. Embryonic mortality was less than 5% for the F₁ generation, which had an adult sex ratio of 2♀:1♂. In contrast, experimental crosses between uninfected female and infected male parents were associated with an embryonic mortality of about 20% and produced 37% fewer F₁ adults. However, because of an F₁ sex ratio of almost 0♀:1♂,?this latter cross produced an overall higher number of F₁ males. These combined results reflect elements of both male development (MD) type cytoplasmic incompatibility (CI) and female mortality (FM) type CI Wolbachia. MD type CI Wolbachia in incompatible crosses causes haploidization of fertilized (i.e., female) eggs. The resultant haploid eggs develop into males so that more males are produced in incompatible versus compatible crosses. FM type CI produces fewer offspring and a male-biased F₁ generation caused by enhanced mortality of female embryos. We speculate that the fate of fertilized eggs - haploidization versus mortality - may reflect differences in bacterial densities.     

Sequence and phylogenetic analysis of the SNF4/AMPK gamma subunit gene from Drosophila melanogaster.

To optimize gene expression under different environmental conditions, many organisms have evolved systems which can quickly up- and down-regulate the activity of other genes. Recently, the SNF1 kinase complex from yeast and the AMP-activated protein kinase complex from mammals have been shown to represent homologous metabolic sensors that are key to regulating energy levels under times of metabolic stress. Using heterologous probing, we have cloned the Drosophila melanogaster homologue of SNF4, the noncatalytic effector subunit from this kinase complex. A sequence corresponding to the partial genomic sequence as well as the full-length cDNA was obtained, and shows that the D. melanogaster SNF4 is encoded in a 1944-bp cDNA representing a protein of 648 amino acids (aa). Southern analysis of Drosophila genomic DNA in concert with a survey of mammalian SNF4 ESTs indicates that in metazoans, SNF4 is a duplicated gene, and possibly even a larger gene family. We propose that one gene copy codes for a short (330 aa) protein, whereas the second locus codes for a longer version (<410 aa) that is extended at the carboxy terminus, as typified by the Drosophila homologue presented here. Phylogenetic analysis of yeast, invertebrate, and multiple mammalian isoforms of SNF4 shows that the gene duplication likely occurred early in the metazoan lineage, as the protein products of the different loci are relatively divergent. When the phylogeny was extended beyond the SNF4 gene family, SNF4 shares sequence similarity with other cystathionine-beta-synthase domain-containing proteins, including IMP dehydrogenase and a variety of uncharacterized Methanococcus proteins.     

A rapid PCR-based test for the endogenous viral element ev3 of chickens

A short fragment of chicken genomic DNA encompassing the insertion site of the endogenous avian leucosis viral element ev 3 was isolated using the inverse polymerase chain reaction (inverse PCR) technique. The nucleotide sequence of the unoccupied site was used to design PCR primers that can be used to unambiguously determine the genetic status of any chicken, with respect to ev 3. Screening of a small number of individuals from exotic breeds of chickens suggested that the frequency of ev 3 is highly variable. The ev 3 integration site shows a high degree of sequence homology with the macrophage-specific tyrosine kinase gene, bmk , in mice.     

Robust expression in yeast cells of a reporter gene driven by rumen protozoal promoter sequences

The objectives of this study were the identification of genes that show relatively strong levels of expression in the rumen protozoan, Isotricha intestinalis, and the demonstration that promoters from such genes can be used in the construction of recombinant expression vectors. In order to identify highly expressed genes, a cDNA library was constructed for I. intestinalis, and RNA expression analysis conducted on 62 clones using a filter array hybridization assay. Expression levels for individual clones ranged from easily detectable to below the detection threshold of the technique. Eleven cDNAs showed relatively intense hybridization signals, and the gene for one of these clones, I87, was characterized in detail. The ability of the I87 promoter to drive the expression of recombinant genes was tested by linking it to the luciferase reporter gene in a yeast shuttle vector and transforming Saccharomyces cerevisiae cells for expression analysis. The results showed that a rumen protozoal gene promoter is capable of directing the expression of a reporter gene in S. cerevisiae. Accession numbers: I87 gene, AY247961, Isotricha sp. BBF-2003 ESTs, CB305319–CB305329