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Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Triatominae is a subfamily of the order Hemiptera whose species are able to feed in the vertebrate blood (i.e., hematophagy). This feeding behavior presents a great physiological challenge to insects, especially in Hemipteran species with a digestion performed by lysosomal-like cathepsins instead of the more common trypsin-like enzymes. With the aim of having a deeper understanding of protease involvement in the evolutionary adaptation for hematophagy in Hemipterans, we screened peptidases in the Rhodnius prolixus genome and characterized them using common blast (NCBI) and conserved domain analyses (HMMER/blast manager software, FAT, plus PFAM database). We compared the results with available sequences from other hemipteran species and with 18 arthropod genomes present in the MEROPS database. Rhodnius prolixus contains at least 433 protease coding genes, belonging to 71 protease families. Seven peptidase families in R. prolixus presented higher gene numbers when compared to other arthropod genomes. Further analysis indicated that a gene expansion of the protease family A1 (Eukaryotic aspartyl protease, PF00026) might have played a major role in the adaptation to hematophagy since most of these peptidase genes seem to be recently acquired, are expressed in the gut and present putative secretory pathway signal peptides. Besides that, most R. prolixus A1 peptidases showed high frequencies of basic residues at the protein surface, a typical structural signature of Cathepsin D-like proteins. Other peptidase families expanded in R. prolixus (i.e., C2 and M17) also presented significant differences between hematophagous (higher number of peptidases) and non-hematophagous species. This study also provides evidence for gene acquisition from microorganisms in some peptidase families in R. prolixus : (1) family M74 (murein endopeptidase), (2) family S29 (Hepatitis C virus NS3 protease), and (3) family S24 (repressor LexA). This study revealed new targets for studying the adaptation of these insects for digestion of blood meals and their competence as vectors of Chagas disease.

Aedes aegypti larvae ingest several kinds of microorganisms. In spite of studies regarding mosquito digestion, little is known about the nutritional utilization of ingested cells by larvae. We investigated the effects of using yeasts as the sole nutrient source for A. aegypti larvae. We also assessed the role of beta-1,3-glucanases in digestion of live yeast cells. Beta-1,3-glucanases are enzymes which hydrolyze the cell wall beta-1,3-glucan polyssacharide. Larvae were fed with cat food (controls), live or autoclaved Saccharomyces cerevisiae cells and larval weight, time for pupation and adult emergence, larval and pupal mortality were measured. The presence of S. cerevisiae cells inside the larval gut was demonstrated by light microscopy. Beta-1,3-glucanase was measured in dissected larval samples. Viability assays were performed with live yeast cells and larval gut homogenates, with or without addition of competing beta-1,3-glucan. A. aegypti larvae fed with yeast cells were heavier at the 4th instar and showed complete development with normal mortality rates. Yeast cells were efficiently ingested by larvae and quickly killed (10% death in 2 h, 100% in 48 h). Larvae showed beta-1,3-glucanase in head, gut and rest of body. Gut beta-1,3-glucanase was not derived from ingested yeast cells. Gut and rest of body activity was not affected by the yeast diet, but head homogenates showed a lower activity in animals fed with autoclaved S. cerevisiae cells. The enzymatic lysis of live S. cerevisiae cells was demonstrated using gut homogenates, and this activity was abolished when excess beta-1,3-glucan was added to assays. These results show that live yeast cells are efficiently ingested and hydrolyzed by A. aegypti larvae, which are able to fully-develop on a diet based exclusively on these organisms. Beta-1,3-glucanase seems to be essential for yeast lytic activity of A. aegypti larvae, which possess significant amounts of these enzyme in all parts investigated.

Background: Triatomines, which are the vectors of Trypanosoma cruzi, have been considered to be exclusive blood feeders for more than 100 years, since the discovery of Chagas disease., Methods: We offered artificial sugar meals to the laboratory model-insect Rhodnius prolixus, which is considered a strict haematophagous insect. We registered feeding by adding colorant to sugar meals. To assess putative phytophagy, fruits of the tomato Solanum lycopersicum were offered to R. prolixus and the presence of tomato DNA was assessed in the insects using PCR. We also assessed longevity, blood feeding and urine production of fruit-exposed triatomines and control insects., Results: All instars of R. prolixus ingested sugar from artificial sugar meals in laboratory conditions. First instar R. prolixus ingested plant tissue from S. lycopersicum fruits, and this increased the amount of blood ingested and urine excreted. Decreased mortality was also observed after blood feeding. Exposure to S. lycopersicum increased longevity and reduced weight loss caused by desiccation., Conclusions: We describe here the first report of sugar feeding and phytophagy in a species that was considered to be a strict blood-feeder for over a century. We suggest that local plants might be not merely shelters for insects and vertebrate hosts as previously described, but may have a nutritional role for the maintenance of the triatomine vectors. The description of sugar and plant meals in triatomines opens new perspectives for the study and control of Chagas Disease.

Background: Phlebotomine sand flies transmit the haemoflagellate Leishmania, the causative agent of human leishmaniasis. The Leishmania promastigotes are confined to the gut lumen and are exposed to the gut microbiota within female sand flies. Here we study the colonisation resistance of yeast and bacteria in preventing the establishment of a Leishmania population in sand flies and the ability of Leishmania to provide colonisation resistance towards the insect bacterial pathogen Serratia marcescens that is also pathogenic towards Leishmania., Methods: We isolated microorganisms from wild-caught and laboratory-reared female Lutzomyia longipalpis, identified as Pseudozyma sp. Asaia sp. and Ochrobactrum intermedium. We fed the females with a sugar meal containing the microorganisms and then subsequently fed them with a bloodmeal containing Leishmania mexicana and recorded the development of the Leishmania population. Further experiments examined the effect of first colonising the sand fly gut with L. mexicana followed by feeding with, Serratia marcescens, an insect bacterial pathogen. The mortality of the flies due to S. marcescens was recorded in the presence and absence of Leishmania., Results: There was a reduction in the number of flies harbouring a Leishmania population that had been pre-fed with Pseudozyma sp. and Asaia sp. or O. intermedium. Experiments in which L. mexicana colonised the sand fly gut prior to being fed an insect bacterial pathogen, Serratia marcescens, showed that the survival of flies with a Leishmania infection was significantly higher compared to flies without Leishmania infection., Conclusions: The yeast and bacterial colonisation experiments show that the presence of sand fly gut microorganisms reduce the potential for Leishmania to establish within the sand fly vector. Sand flies infected with Leishmania were able to survive an attack by the bacterial pathogen that would have killed the insect and we concluded that Leishmania may benefit its insect host whilst increasing the potential to establish itself in the sand fly vector. We suggest that the increased ability of the sand fly to withstand a bacterial entomopathogen, due to the presence of the Leishmania, may provide an evolutionary pressure for the maintenance of the Leishmania-vector association.

Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.

Phlebotomine sand flies are vectors of Leishmania that are acquired by the female sand fly during blood feeding on an infected mammal. Leishmania parasites develop exclusively in the gut lumen during their residence in the insect before transmission to a suitable host during the next blood feed. Female phlebotomine sand flies are blood feeding insects but their life style of visiting plants as well as animals, and the propensity for larvae to feed on detritus including animal faeces means that the insect host and parasite are exposed to a range of microorganisms. Thus, the sand fly microbiota may interact with the developing Leishmania population in the gut. The aim of the study was to investigate and identify the bacterial diversity associated with wild adult female Lutzomyia sand flies from different geographical locations in the New World. The bacterial phylotypes recovered from 16S rRNA gene clone libraries obtained from wild caught adult female Lutzomyia sand flies were estimated from direct band sequencing after denaturing gradient gel electrophoresis of bacterial 16 rRNA gene fragments. These results confirm that the Lutzomyia sand flies contain a limited array of bacterial phylotypes across several divisions. Several potential plant-related bacterial sequences were detected including Erwinia sp. and putative Ralstonia sp. from two sand fly species sampled from 3 geographically separated regions in Brazil. Identification of putative human pathogens also demonstrated the potential for sand flies to act as vectors of bacterial pathogens of medical importance in addition to their role in Leishmania transmission.

The gut bacterial community from four species of feral locusts and grasshoppers was determined by denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rRNA gene fragments. The study revealed an effect of phase polymorphism on gut bacterial diversity in brown locusts from South Africa. A single bacterial phylotype, consistent with Citrobacter sp. dominated the gut microbiota of two sympatric populations of Moroccan and Italian locusts in Spain. There was evidence for Wollbachia sp. in the meadow grasshopper caught locally in the UK. Sequence analysis of DGGE products did not reveal evidence for unculturable bacteria and homologies suggested that bacterial species were principally Gammaproteobacteria from the family Enterobacteriaceae similar to those recorded previously in laboratory reared locusts.

Fungus garden material from recently established Atta sexdens rubropilosa colonies (6-12 months old) was sampled to detect antibiotic producing microorganisms that inhibited the growth of pathogens of insects and of the fungus gardens but did not affect their mutualistic fungus. A bacterium with activity against the entomopathogenic fungus Beauveria bassiana was isolated from 56% of the gardens tested (n=57) and identified from its biochemical profile and from 16S and 23S ribosomal DNA sequences as a member of the genus Burkholderia. The ant-associated Burkholderia isolates secreted a potent, anti-fungal agent that inhibited germination of conidia of the entomopathogenic fungi B. bassiana, Metarhizium anisopliae, of the saprophytic Verticillium lecanii, and also of a specialist fungus garden Escovopsis weberi. Growth of the ant's mutualist fungus was unaffected.

The diversity of the Insecta is reflected in the large and varied microbial communities inhabiting the gut. Studies, particularly with termites and cockroaches, have focused on the nutritional contributions of gut bacteria in insects living on suboptimal diets. The indigenous gut bacteria, however, also play a role in withstanding the colonization of the gut by non-indigenous species including pathogens. Gut bacterial consortia adapt by the transfer of plasmids and transconjugation between bacterial strains, and some insect species provide ideal conditions for bacterial conjugation, which suggests that the gut is a "hot spot" for gene transfer. Genomic analysis provides new avenues for the study of the gut microbial community and will reveal the molecular foundations of the relationships between the insect and its microbiome. In this review the intestinal bacteria is discussed in the context of developing our understanding of symbiotic relationships, of multitrophic interactions between insects and plant or animal host, and in developing new strategies for controlling insect pests.

A Gram-positive, anaerobic, moderate thermophile, strain Wv6T, capable of reducing indigo dye, was isolated from a fermenting woad vat prepared essentially as in medieval Europe. Strain Wv6T formed rod-shaped cells, which occurred singly, in pairs or in chains and produced terminal oval endospores. Strain Wv6T was saccharolytic. Growth occurred at pH 5.9-9.9 (initial pH) with an optimum at 50 degrees C of pH 7.2 +/- 0.2 (constant pH). At pH 7.8, the temperature range for growth was 30-55 degrees C with the optimum at 49-52 degrees C. Comparative 16S rRNA gene sequence analysis demonstrated that the bacterium represents a hitherto unknown subline within rRNA cluster I Clostridium. Based on the results of the phylogenetic analysis and phenotypic criteria, it is proposed that the unknown moderate thermophile should be classified as Clostridium isatidis sp. nov., a new species of the genus Clostridium. The type strain of Clostridium isatidis is strain Wv6T (= NCFB 3071T).

Levels of cyanide in two varieties of malted millet, spent grain (Machicha) and opaque beer (Mbege) were determined. Protein content and amino acid composition of the malt, Mbege and Machicha were determined. Mbege was made in the laboratory using an improved method. The cyanide content of millet, malt, spent grain and Mbege were 40.6, 513.4, 18.9 and 8.1 ppm, respectively for the Moshi local millet variety. For Sumbawanga-2 millet variety the cyanide content was found to be 41.2, 489.2, 17.8 and 6.8 ppm for the millet, malt, spent grain and Mbege, respectively. The cyanide content increased linearly as the number of days of germination of the millet grain increased and the highest values of cyanide were attained on the third day of germination. Cyanogenic glucosides in the millet were enzymetically hydrolysed to respective cyanohydrins and volatile hydrogen cyanide due to low pH level of the Mbege which was 4. Malting of the millet increased the protein content by 5%. Lysine, the most limiting amino acid in millet, increased by 20%. It was concluded that the fermentation process of the millet malt into Mbege is efficient in reducing the levels of cyanogenic glucosides below levels considered toxic and therefore rendering the product safe.

A typical munkoyo beverage was made by fermenting Rhynchosia heterophylla root extract-cooked maize meal mixture with Lactobacillus confusus LZI and Saccharomyces cerevisiae YZ20. The fermented munkoyo beverage had a pH of 3.3, lactic acid content of 60 mmol/l, ethanol 320-410 mmol/l and a characteristic 'munkoyo' aroma. L. confusus, used alone, produced a beverage with a faint munkoyo flavour note whilst beverage produced with S. cerevisiae alone seemed not to have a typical munkoyo note. R. heterophylla root extract converted 75% of the starch in sterile cooked maize meat to maltose (80% of total sugars), maltotriose (17%) and glucose (3%) in I h at 45 degrees C. During fermentation by the mixed culture or the yeast alone most of the maltose was utilised but little or none of the maltotriose. The ratio of yeast to lactic acid bacteria in the starter culture affected the rate of production of ethanol but had no effect on the growth or acid production by the bacterium. To obtain a munkoyo beverage with the desired low alcohol concentration (< 100 mmol/l), the ratio of yeast concentration (cfu/ml) to Lactobacillus concentration in the starter culture should be 1:1000 or less and the beverage should be fermented for 24 h only.

The sulfite tolerance of meat yeasts was shown to be determined by pH, sulfite concentration, substrate availability, and the composition of the preincubation medium. Acetaldehyde production by Candida norvegica was sulfite-induced and occurred during the exponential growth phase in sulfited (500 micrograms SO2 ml-1) lab lemco glucose broth cultures buffered at pH 5, 6, or 7. Growth at pH 4, however, was inhibited by sulfite. Acetaldehyde production occurred in sulfited medium containing fructose or ethanol but not lactate nor a range of other assimilable substrates. A non-acetaldehyde-producing yeast, Candida vini, grew in sulfited (500 micrograms SO2 ml-1) lab lemco broth containing glucose or lactate buffered at pH 6 or 7 but not at pH 4 or 5.

The literature dealing with the role of glucose in the microbiological changes of meat and certain meat products is reviewed. Discussion is centered on two aspects. First, glucose plays a part in the selection of the dominant spoilage organisms, Pseudomonas fragi, Ps. lundensis, and Ps. fluorescens, on red meat stored aerobically under chill (2-7 degrees C) conditions. It is concluded that the pseudomonads flourish because they convert glucose to the less commonly used substrate, gluconate. The latter serves as an extracellular energy store. With its depletion, the pseudomonads utilize amino acids, thereby producing the characteristic off-odors of spoiled meat. Storage of meat in a modified atmosphere (viz., 20% CO2:80% O2) selects Gram-positive flora (lactobacilli and Brochothrix thermosphacta) which impart a "cheesy odor" through acid production from glucose and volatile fatty acids from amino acids. The first mentioned organisms produce the same off-odors in "acid" meat (pH 5.5) from which oxygen is excluded. So too does the less acid-tolerant Br. thermosphacta in less acid meat (pH greater than 5.8), especially if trace amounts of O2 are present. Such meat may be colonized by Shewanella putrefaciens also, with green discoloration resulting from the release of H2S from amino acids. The addition of glucose and NO2- to, and the exclusion of oxygen from, comminuted meat selects a flora dominated by Lactobacillus spp. and staphylococci such as Staphylococcus carnosus. Second, sulfite, the preservative of British-style sausages, has a sparing action on glucose. As a consequence of its curtailed breakdown there is only a meager acid drift with storage even though a fermentative flora of lactobacilli and Br. thermosphacta is selected. Yeasts also contribute to the microbial association in sausages; members of four of the six commonly occurring genera bind sulfite through acetaldehyde production. Glucose appears to be essential for acetaldehyde synthesis. The role of glucose in spoilage and the conditions which select particular groups of spoilage organisms are considered in the context of chemical probes and/or instrumental methods for routine assessment of the "freshness" of meat and meat products.