Gene expression in reproductive organs of tsetse females - initial data in an approach to reduce fecundity.

BACKGROUND: Tsetse flies are vectors of African trypanosomes, and their vectorial capacity results in a major public health emergency and vast economic losses in sub-Saharan Africa. Given the limited ability of trypanosome prevention and eradication, tsetse vectors remain major targets of control efforts. Larvae of all three instars are developed in mothers' uteri, nourished through milk, and 'larviposited' shortly before pupation. The past few years have witnessed the emergence of approaches based on knockdown of genes involved in milk production, resulting in a significant reduction of fecundity. RESULTS: In order to identify further genes applicable in the control of tsetse flies, we determined the expression of protein-coding genes in ovaries and uteri from both virgin and heavily pregnant Glossina morsitans morsitans females. Comparison of expression profiles allowed us to identify candidate genes with increased expression in pregnant individuals. Lists with the highest increases include genes involved in oocyte and embryonic development, or nourishment. Maximum ovarian fold change does not exceed 700, while the highest uterine fold change reaches to more than 4000. Relatively high fold changes of two neuropeptide receptors (for corazonin and myosuppressin) propose the corresponding genes alternative targets. CONCLUSIONS: Given the higher fold changes in the uterus, targeting gene expression in this tissue may result in a more evident reduction of fecundity. However, ovaries should not be neglected, as manifested by several genes with top fold changes involved in early developmental stages. Apart from focusing on the highest fold changes, neuropeptide receptors with moderate increases in expression should be also verified as targets, given their roles in mediating the tissue control. However, this data needs to be considered initial, and the potential of these genes in affecting female fecundity needs to be verified experimentally.

Crowding of Drosophila larvae affects lifespan and other life-history traits via reduced availability of dietary yeast.

Conditions experienced during development have often long-lasting effects persisting into adulthood. In Drosophila, it is well-documented that larval crowding influences fitness-related traits such as body size, starvation resistance and lifespan. However, the underlying mechanism of this phenomenon is not well understood. Here, we show that the effects of increased larval density on life-history traits can be explained by decreased yeast availability in the diet during development. Yeast-poor larval diet alters various life-history traits and mimics the effects of larval crowding. In particular, reduced amount of yeast in larval diet prolongs developmental time, reduces body size, increases body fat content and starvation resistance, and prolongs Drosophila lifespan. Conversely, the effects of larval crowding can be rescued by increasing the concentration of the dietary yeast in the diet during development. Altogether, our results show that the well-known effects of larval crowding on life-history traits are mainly caused by the reduced availability of dietary yeasts due to increased larval competition.

Antibacterial properties of lucifensin in Lucilia sericata maggots after septic injury.

OBJECTIVE: To investigate the antibacterial properties of lucifensin in maggots of Lucilia sericata after septic injury. METHODS: In our preliminary study we have shown that injuring the maggots with a needle soaked in lipopolysaccharide solution induced within 24 h lucifensin expression in the fat body and in the grease coupler of the salivary glands. It is assumed that lucifensin is secreted solely from this tissue into the haemolymph (similar to other insect defensins) and not into secreted/excreted products. We used high-performance liquid chromatography fractionation and radial diffusion assay to investigate the antibacterial properties of haemolymph extracted from larvae after septic injury. RESULTS: After septic injury, production of lucifensin in the haemolymph is increased. This led to higher antibacterial activity of such haemolymph in comparison to non-stimulated larvae. COCLUSIONS: These results suggest that beside the previously demonstrated role of lucifensin in the debridement therapy, lucifensin is simultaneously important as a part of the systematic immune response.

Honeybee glucose oxidase--its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys.

Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2), which is generated by glucose oxidase (GOX)-mediated conversion of glucose in diluted honey. However, honeys exhibit considerable variation in their antibacterial activity. Therefore, the aim of the study was to identify the mechanism behind the variation in this activity and in the H2O2 content in honeys associated with the role of GOX in this process. Immunoblots and in situ hybridization analyses demonstrated that gox is solely expressed in the hypopharyngeal glands of worker bees performing various tasks and not in other glands or tissues. Real-time PCR with reference genes selected for worker heads shows that the gox expression progressively increases with ageing of the youngest bees and nurses and reached the highest values in processor bees. Immunoblot analysis of honey samples revealed that GOX is a regular honey component but its content significantly varied among honeys. Neither botanical source nor geographical origin of honeys affected the level of GOX suggesting that some other factors such as honeybee nutrition and/or genetic/epigenetic factors may take part in the observed variation. A strong correlation was found between the content of GOX and the level of generated H2O2 in honeys except honeydew honeys. Total antibacterial activity of most honey samples against Pseudomonas aeruginosa isolate significantly correlated with the H2O2 content. These results demonstrate that the level of GOX can significantly affect the total antibacterial activity of honey. They also support an idea that breeding of novel honeybee lines expressing higher amounts of GOX could help to increase the antibacterial efficacy of the hypopharyngeal gland secretion that could have positive influence on a resistance of colonies against bacterial pathogens.

Methylglyoxal may affect hydrogen peroxide accumulation in manuka honey through the inhibition of glucose oxidase.

Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H2O2 accumulation in natural non-manuka honeys. H2O2 concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H2O2 producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H2O2 concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8±9.1 µM H2O2 in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9±6.8 and 495.8±9.1 µM H2O2, respectively. Levels of H2O2 increased significantly over time in both honey solutions. Contrary to this, the MGO-treated honeys generated significantly lower amounts of H2O2 (P<.001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H2O2 generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.

A complete sequence of Saccharomyces paradoxus mitochondrial genome that restores the respiration in S. cerevisiae.

We determined the complete sequence of 71 355-bp-long mitochondrial genome from Saccharomyces paradoxus entirely by direct sequencing of purified mitochondrial DNA (mtDNA). This mtDNA possesses the same features as its close relative Saccharomyces cerevisiae - A + T content 85.9%, set of genes coding for the three components of cytochrome oxidase, cytochrome b, three subunits of ATPase, both ribosomal subunits, gene for ribosomal protein, rnpB gene, tRNA package (24) and yeast genetic code. Genes are interrupted by nine group I and group II introns, two of which are in positions unknown in S. cerevisiae, but recognized in Saccharomyces pastorianus. The gene products are related to S. cerevisiae, and the identity of amino acid residues varies from 100% for cox2 to 83% for rps3. The remarkable differences from S. cerevisiae are (1) different gene order (translocation of trnF-trnT1-trnV-cox3-trnfM-rnpb-trnP and transposition of trnW-rns), (2) occurrence of two unusual GI introns, (3) eight active ori elements, and (4) reduced number of GC clusters and divergent intergenic spacers. Despite these facts, the sequenced S. paradoxus mtDNA introduced to S. cerevisiae was able to support the respiratory function to the same extent as the original mtDNAs.

Mitochondrial genome from the facultative anaerobe and petite-positive yeast Dekkera bruxellensis contains the NADH dehydrogenase subunit genes.

The progenitor of the Dekkera/Brettanomyces clade separated from the Saccharomyces/Kluyveromyces clade over 200 million years ago. However, within both clades, several lineages developed similar physiological traits. Both Saccharomyces cerevisiae and Dekkera bruxellensis are facultative anaerobes; in the presence of excess oxygen and sugars, they accumulate ethanol (Crabtree effect) and they both spontaneously generate respiratory-deficient mutants (petites). In order to understand the role of respiratory metabolism, the mitochondrial DNA (mtDNA) molecules of two Dekkera/Brettanomyces species were analysed. Dekkera bruxellensis mtDNA shares several properties with S. cerevisiae, such as the large genome size (76 453 bp), and the organization of the intergenic sequences consisting of spacious AT-rich regions containing a number of hairpin GC-rich cluster-like elements. In addition to a basic set of the mitochondrial genes coding for the components of cytochrome oxidase, cytochrome b, subunits of ATPase, two rRNA subunits and 25 tRNAs, D. bruxellensis also carries genes for the NADH dehydrogenase complex. Apparently, in yeast, the loss of this complex is not a precondition to develop a petite-positive, Crabtree-positive and anaerobic nature. On the other hand, mtDNA from a petite-negative Brettanomyces custersianus is much smaller (30 058 bp); it contains a similar gene set and has only short intergenic sequences.