BackgroundThe insect gut harbors a variety of microorganisms that probably exceed the number of cells in insects themselves. These microorganisms can live and multiply in the insect, contributing to digestion, nutrition, and development of their host.Recent studies have shown that midgut bacteria appear to strengthen the mosquito's immune system and indirectly enhance protection from invading pathogens. Nevertheless, the physiological significance of these bacteria for mosquitoes has not been established to date. In this study, oral administration of antibiotics was employed in order to examine the contribution of gut bacteria to blood digestion and fecundity in Aedes aegypti.ResultsThe antibiotics carbenicillin, tetracycline, spectinomycin, gentamycin and kanamycin, were individually offered to female mosquitoes. Treatment of female mosquitoes with antibiotics affected the lysis of red blood cells (RBCs), retarded the digestion of blood proteins and reduced egg production. In addition, antibiotics did not affect the survival of mosquitoes. Mosquito fertility was restored in the second gonotrophic cycle after suspension of the antibiotic treatment, showing that the negative effects of antibiotics in blood digestion and egg production in the first gonotrophic cycle were reversible.ConclusionsThe reduction of bacteria affected RBC lysis, subsequently retarded protein digestion, deprived mosquito from essential nutrients and, finally, oocyte maturation was affected, resulting in the production of fewer viable eggs. These results indicate that Ae. aegypti and its midgut bacteria work in synergism to digest a blood meal.Our findings open new possibilities to investigate Ae. aegypti-associated bacteria as targets for mosquito control strategies.

In this work we show that the lumen of Aedes aegypti midgut is highly colonized by bacteria that were identified by culture-dependent and culture-independent methods. rDNA sequences obtained were compared with those from GenBank and the main bacterial genera identified were: Serratia, Klebsiella, Asaia, Bacillus, Enterococcus, Enterobacter,Kluyvera and Pantoea. All genera were identified in midgut except Enterobacter that was observed only in eggs. Asaia and Pantoea were also identified in eggs and ovary, respectively. In addition two yeast genera were observed in A. aegypti: Pichia isolated from midgut and Candida identified in midgut and ovary. The genus Serratia was dominant in all isolation assays representing 54.5% of the total of microorganisms. Thirty-nine and 24 bacterial clones were successfully obtained from midguts 24 and 48h after blood feeding (ABF), respectively. The majority of clones obtained were from Serratia sp. (48.7% and 50% for 24 and 48h ABF, respectively). Light microscopy showed that bacteria were located preferentially in the posterior midgut, around the blood meal and associated with peritrophic matrix. Scanning electron microscopy images showed a high number of bacteria in midgut during blood digestion and the peak of bacterial enumeration was reached 48h ABF, stage in which lumen was almost totally occupied by bacteria that were also interacting with epithelial microvilli. Our results show the dynamics of microbial colonization and their distribution in midgut during blood digestion.

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fedAedes aegypti (Diptera: Culicidae, Aedini) is the main urban vector for the human diseases yellow fever and dengue fever (Nasci & Miller 1996). The vector control, mainly by insecticide application and elimination of oviposition sites, has been used as the best solution to decrease the diseases incidence. Explore new strategies for blocking the insect transmitted diseases such as dengue fever is urgent in Tropical countries (Sperança & Capurro 2007). Microorganisms associated with the insect may have an important role for human infectious diseases epidemiology. Once a key role bacterium is discovered, it may be modified in order to affect the pathogen development and, consequently, the disease transmission (Azambuja et al. 2005, Riehle & Jacobs-Lorena 2005.The alimentary canal of the mosquito is composed by the foregut, midgut and hindgut. The foregut is involved primarily with ingestion, conduction and storage of food (Romoser 1996). In the alimentary canal of the mosquito three diverticula arise near the posterior end of the esophagus: two from the dorso-lateral and one from the ventral wall of the gut, all surrounded by a thin impermeable cuticle. The ventral diverticulum (VD), or crop, is large and may extend into the abdomen (Dapples & Lea 1974). Usually all three diverticula are filled with air bubbles and are used as food reservoirs (Thompson 1905, Consoli & Lourenço-de-Oliveira 1994. The sugar meal, such as floral nectar, is stored in the diverticula and passes slowly to the midgut, where it is digested (Thompson 1905).Microorganisms play important and often essential roles in the growth and development of many insect species. Despite the importance of these microbial associations with insects there are relatively few studies that elucidate their components and their roles in the interactions. Insects that rely on nutritionally poor diets tend to possess bacterial endosymbionts. Aphids, for example, which subsist solely on plant sap, harbor Buchnera spp., believed to provide amino acids and vitamins to their hosts (Douglas 1989). Blood is known to be severely deficient in essential B vitamins and some amino acids. Bloodsucking arthropods such as ticks, lice, bedbugs, reduviid bugs, and tsetse flies usually harbor symbiotic microorganisms (Buchner 1965). Symbiotic relationships have already been reported for blood feeding insects and some are very well established, as in the case of Rhodnius prolixus that maintains an association with the actinomycete bacteria Rhodococcus rhodnii (Dasch et al. 1984); and tsetse flies genus Glossina, which harbor three distinct symbiotic microorganisms: Wigglesworthia glossinidia, Sodalis glossinidius (commensal), and the parasitic microbe Wolbachia pipientis (Aksoy et al. 1997, Chen et al. 1999.Many key questions about bacteria within the mosquito's midgut remain largely unanswered, and ob...

BackgroundHematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.Methodology/Principal FindingsHz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.Conclusions/SignificanceHerein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

The kinetic and general growth features of Bacillus thuringiensis var. israelensis were evaluated. Initial glucose concentration (S0) in fermentation media varied from 10 to 152 g/l. The results afforded to characterize four morphologically and physiologically well-defined culture phases, independent of S0 values: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; and Phase IV, spores maturation and cell lysis. Important process parameters were also determined. The maximum specific growth rates (microX,m) were not affected with S0 up to 75 g/l (1.0-1.1 per hour), but higher glucose concentrations resulted in growth inhibition by substrate, revealed by a reduction in microX,m values. These higher S0 values led to longer Phases III and IV and delayed sporulation. Similar biomass concentrations (Xm=15.2-15.9 g/l) were achieved with S0 over 30.8 g/l, with increasing residual substrate, suggesting a limitation in some other nutrients and the use of glucose to form other metabolites. In this case, with S0 from 30.8 to 152 g/l, cell yield (YX/S) decreased from 0.58 to 0.41 g/g. On the other hand, with S0=10 g/l growth was limited by substrate, and YX/S has shown its maximum value (0.83 g/g).