Everybody loves sugar:first report of plant feeding in triatomines

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

Is erythrocyte size a strategy to avoid hypoxia in Wiegmann’s Torquate Lizards (Sceloporus torquatus)? Field evidence

This study examined changes in certain hematological parameters in a reptilian model naturally exposed to altitude-associated hypoxia. Four populations of the Mexican lizard Sceloporus torquatus Wiegmann, 1828 (Wiegmann’s Torquate Lizard) from different altitudes were sampled to evaluate erythrocyte count (Erc), hematocrit (Hct), mean corpuscular hemoglobin concentration (MCHC), and erythrocyte size (Ers). Blood was also assayed to determine hemoglobin ([Hb]), glucose, lactate, and electrolyte concentrations. Erc was performed using a Neubauer hemocytometer. Hct was calculated as percentage of packed cell volume by centrifuging blood samples. [Hb] was determined using a Bausch and Lomb Spectronic colorimeter. MCHC was calculated with the formula 100 × [Hb]/Hct. Ers was calculated from blood smear microphotographs analyzed with the Sigma Scan Pro software. Values of serum electrolytes (sodium (Na+), potassium (K+), and calcium (Ca2+)), pH, glucose, and lactate from blood samples were obtained through a blood electrolyte analyzer. Highland populations of S. torquatus exhibited a significant increase in Erc, Hct, Ers, and [Hb]. In contrast, MCHC showed no correlation with altitude. Additionally, significant differences in lactate, Na+, K+, and Ca2+ were observed in highland populations. In general, we found that most hematological parameters were significantly different among lizard populations from different altitudes. This is the first study to report changes in Ers in relation to altitude, which could be a physiological response to hypoxia.CONACYT 17872

Digestion of yeasts and Beta-1, 3-Glucanases in mosquito larvae:physiological and biochemical considerations

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 2h, 100% in 48h). 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

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes

Non-invasive visualisation and identification of fluorescent Leishmania tarentolae in infected sand flies

Background: The leishmaniases are neglected diseases that affect some of the most vulnerable populations in the tropical and sub-tropical world. The parasites are transmitted by sand flies and novel strategies to control this neglected vector-borne disease are needed. Blocking transmission by targeting the parasite inside the phlebotomine vector offers potential in this regard. Some experimental approaches can be best performed by longitudinal study of parasites within flies, for which non-destructive methods to identify infected flies and to follow parasite population changes are required. Methods:Lutzomyia longipalpis were reared under standard insectary conditions at the Wellcome Centre for Molecular Parasitology. Flies were artificially infected with L. tarentolae expressing green fluorescent protein (GFP. Parasite counts were carried out 5 days post-infection and the percentage of infected flies and survival of infected females was established up to days 5 post-infection. Whole living females were visualised using an epifluorescence inverted microscope to detect the presence parasites inferred by a localised green fluorescent region in the upper thorax. Confirmation of infection was performed by localised-fluorescence of dissected flies and estimates of the parasite population. Results:Leishmania tarentolae was successfully transfected and expressed GFP in vitro. L. tarentolae-GFP Infected flies showed similar parasite populations when compared to non-transfected parasites ( L. tarentolae-WT). Survival of non-infected females was higher than L. tarentolae-infected groups, (Log-rank (Mantel-Cox) test, p<0.05). L. tarentolae-GFP infected females displayed an intense localised fluorescence in the thorax while other specimens from the same infected group did not. Localised fluorescent flies were dissected and showed higher parasite populations compared to those that did not demonstrate high concentrations in this region (t-test, p<0.005). Conclusion: These results demonstrate the feasibility of establishing a safe non-human infectious fluorescent Leishmania-sand fly infection model by allowing non-destructive imaging to signal the establishment of Leishmania infections in living sand flies

Genetic diversity of Bm86 sequences in Rhipicephalus (Boophilus) microplus ticks from Mexico: analysis of haplotype distribution patterns

Artículo científico derivado de Tesis doctoral de Saúl Gabriel Martínez ArzateBackground: Ticks are a problem for cattle production mainly in tropical and subtropical regions, because they generate great economic losses. Acaricides and vaccines have been used to try to keep tick populations under control. This has been proven difficult given the resistance to acaricides and vaccines observed in ticks. Resistance to protein rBm86-based vaccines has been associated with the genetic diversity of Bm86 among the ectoparasite’s populations. So far, neither genetic diversity, nor spatial distribution of circulating Bm86 haplotypes, have been studied within the Mexican territory. Here, we explored the genetic diversity of 125 Bm86 cDNA gene sequences from R. microplus from 10 endemic areas of Mexico by analyzing haplotype distribution patterns to help in understanding the population genetic structure of Mexican ticks. Results: Our results showed an average nucleotide identity among the Mexican isolates of 98.3%, ranging from 91.1 to 100%. Divergence between the Mexican and Yeerongpilly (the Bm86 reference vaccine antigen) sequences ranged from 3.1 to 7.4%. Based on the geographic distribution of Bm86 haplotypes in Mexico, our results suggest gene flow occurrence within different regions of the Mexican territory, and even the USA. Conclusions: The polymorphism of Bm86 found in the populations included in this study, could account for the poor efficacy of the current Bm86 antigen based commercial vaccine in many regions of Mexico. Our data may contribute towards designing new, highly-specific, Bm86 antigen vaccine candidates against R. microplus circulating in Mexico.SIEAE UAEM FES

Colonisation resistance in the sand fly gut:Leishmania protects Lutzomyia longipalpis from bacterial infection

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

Biochemical and Functional Characterization of Glycoside Hydrolase Family 16 Genes in Aedes aegypti Larvae: Identification of the Major Digestive β-1,3-Glucanase

Insect β-1,3-glucanases belong to Glycoside Hydrolase Family 16 (GHF16) and are involved in digestion of detritus and plant hemicellulose. In this work, we investigated the role of GHF16 genes in Aedes aegypti larvae, due to their detritivore diet. Aedes aegypti genome has six genes belonging to GHF16 (Aae GH16.1 – Aae GH16.6), containing two to six exons. Sequence analysis suggests that five of these GHF16 sequences (Aae GH16.1, 2, 3, 5, and 6) contain the conserved catalytic residues of this family and correspond to glucanases. All genomes of Nematocera analyzed showed putative gene duplications corresponding to these sequences. Aae GH16.4 has no conserved catalytic residues and is probably a β-1,3-glucan binding protein involved in the activation of innate immune responses. Additionally, Ae. aegypti larvae contain significant β-1,3-glucanase activities in the head, gut and rest of body. These activities have optimum pH about 5–6 and molecular masses between 41 and 150 kDa. All GHF16 genes above showed different levels of expression in the larval head, gut or rest of the body. Knock-down of AeGH16.5 resulted in survival and pupation rates lower than controls (dsGFP and water treated). However, under stress conditions, severe mortalities were observed in AeGH16.1 and AeGH16.6 knocked-down larvae. Enzymatic assays of β-1,3-glucanase in AeGH16.5 silenced larvae exhibited lower activity in the gut and no change in the rest of the body. Chromatographic activity profiles from gut samples after GH16.5 silencing showed suppression of enzymatic activity, suggesting that this gene codes for the digestive larval β-1,3-glucanase of Ae. aegypti. This gene and enzyme are attractive targets for new control strategies, based on the impairment of normal gut physiology

TcVac1 vaccine delivery by intradermal electroporation enhances vaccine induced immune protection against Trypanosoma cruzi infection in mice

Trabajo de investigación doctoral de Wael Hegazy Hassan Moustafa bajo la dirección de Juan Carlos Vázquez ChagoyánThe efforts for the development and testing of vaccines against Trypanosoma cruzi infection have increased during the past years. We have designed a TcVac series of vaccines composed of T. cruzi derived, GPI-anchored membrane antigens. The TcVac vaccines have been shown to elicit humoral and cellular mediated immune responses and provide significant (but not complete) control of experimental infection in mice and dogs. Herein, we aimed to test two immunization protocols for the delivery of DNA-prime/ DNA-boost vaccine (TcVac1) composed of TcG2 and TcG4 antigens in a BALB/c mouse model. Mice were immunized with TcVac1 through intradermal/electroporation (IDE) or intramuscular (IM) routes, challenged with T. cruzi, and evaluated during acute phase of infection. The humoral immune response was evaluated through the assessment of anti-TcG2 and anti-TcG4 IgG subtypes by using an ELISA. Cellular immune response was assessed through a lymphocyte proliferation assay. Finally, clinical and morphopathological aspects were evaluated for all experimental animals. Our results demonstrated that when comparing TcVac1 IDE delivery vs IM delivery, the former induced significantly higher level of antigen-specific antibody response (IgG2a + IgG2b > IgG1) and lymphocyte proliferation, which expanded in response to challenge infection. Histological evaluation after challenge infection showed infiltration of inflammatory cells (macrophages and lymphocytes) in the heart and skeletal tissue of all infected mice. However, the largest increase in inflammatory infiltrate was observed in TcVac1_IDE/Tc mice when compared with TcVac1_IM/Tc or non-vaccinated/infected mice. The extent of tissue inflammatory infiltrate was directly associated with the control of tissue amastigote nests in vaccinated/ infected (vs. non-vaccinated/infected) mice. Our results suggest that IDE delivery improves the protective efficacy of TcVac1 vaccine against T. cruzi infection in mice when compared with IM delivery of the vaccine.Universidad Autónoma de Estado de México (proyecto No. 3326/2012C), Consejo Nacional de Ciencia y Tecnología (Proyecto No. 156701) . Beca CONACyT a M.Sc. Wael Hegazy Hassan Moustafa (Beca numero No. 518232/291117)

Inhibition of trypsin expression in Lutzomyia longipalpis using RNAi enhances the survival of Leishmania

Background Leishmania parasites must overcome several barriers to achieve transmission by their sand fly vectors. One of the earliest threats is exposure to enzymes during blood meal digestion. Trypsin-like enzymes appear to be detrimental to parasite survival during the very early phase of development as amastigotes transform into promastigote stages. Here, we investigate whether parasites can affect trypsin secretion by the sand fly midgut epithelium and if inhibition of this process is of survival value to the parasites. Results Infections of Lutzomyia longipalpis with Leishmania mexicana were studied and these showed that infected sand flies produced less trypsin-like enzyme activity during blood meal digestion when compared to uninfected controls. RNA interference was used to inhibit trypsin 1 gene expression by micro-injection into the thorax, as trypsin 1 is the major blood meal induced trypsin activity in the sand fly midgut. Injection of specific double stranded RNA reduced trypsin 1 expression as assessed by RT-PCR and enzyme assays, and also led to increased numbers of parasites in comparison with mock-injected controls. Injection by itself was observed to have an inhibitory effect on the level of infection, possibly through stimulation of a wound repair or immune response by the sand fly. Conclusion Leishmania mexicana was shown to be able to modulate trypsin secretion by Lutzomyia longipalpis to its own advantage, and direct inhibition of trypsin gene expression led to increased parasite numbers in the midguts of infected flies. Successful application of RNA interference methodology to Leishmania-infected sand flies now opens up the use of this technique to study a wide range of sand fly genes and their role in the parasite-vector interaction