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Thirty-four species of Triatominae (Hemiptera, Reduviidae) are recorded in Mexico, Triatoma Laporte, 1832 the most speciose genus in this country. Here, we describe Triatoma yelapensis sp. nov. from the Pacific coast of Jalisco (Mexico). The most similar species to T. yelapensis sp. nov. is T. recurva (Stål, 1868), but they differ in head longitude, the proportion of labial segments, coloration pattern of corium and connexivum, spiracles location, and male genitalia. To provide statistical support for the morphological distinctiveness of the new species, we performed a geometric morphometric analysis of T. yelapensis sp. nov., T. dimidiata s.s. (Latreille, 1811), T. gerstaeckeri (Stål, 1859), and T. recurva (Stål, 1868), considering head morphology. We also provide an updated key of the genus Triatoma for species recorded in Mexico.
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Chitinases are enzymes responsible for the hydrolysis of glycosidic linkages within chitin chains. In insects, chitinases are typically members of the multigenic glycoside hydrolase family 18 (GH18). They participate in the relocation of chitin during development and molt, and in digestion in detritivores and predatory insects, and they control the peritrophic membrane thickness. Chitin metabolism is a promising target for developing vector control strategies, and knowledge of the roles of chitinases may reveal new targets and illuminate unique aspects of their physiology and interaction with microorganisms. Rhodnius prolixus is an important vector of Chagas disease, which is caused by the parasite Trypanosoma cruzi. In this study, we performed annotation and structural characterization of nine chitinase and chitinase-like protein genes in the R. prolixus genome. The roles of their corresponding transcripts were studied in more depth; their physiological roles were studied through RNAi silencing. Phylogenetic analysis of coding sequences showed that these genes belong to different subfamilies of GH18 chitinases already described in other insects. The expression patterns of these genes in different tissues and developmental stages were initially characterized using RT-PCR. RNAi screening showed silencing of the gene family members with very different efficiencies. Based on the knockdown results and the general lack of information about subgroup VIII of GH18, the RpCht7 gene was chosen for phenotype analysis. RpCht7 knockdown doubled the mortality in starving fifth-instar nymphs compared to dsGFP-injected controls. However, it did not alter blood intake, diuresis, digestion, molting rate, molting defects, sexual ratio, percentage of hatching, or average hatching time. Nevertheless, female oviposition was reduced by 53% in RpCht7-silenced insects, and differences in oviposition occurred within 14-20 days after a saturating blood meal. These results suggest that RpCht7 may be involved in the reproductive physiology and vector fitness of R. prolixus.
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Phlebotomine sand flies are the main vectors of Leishmania genus species worldwide; therefore, the detection of some reproductive parasites, such as Wolbachia, has been considered a possible strategy for biological control. In Mexico, leishmaniasis cases have been recorded in 25 states, yet only two sand fly species have been related to Wolbachia spp. Although the state of Tabasco has a high number of leishmaniasis cases, only few studies have been done on sand fly species. The aim of this study was to analyze the diversity of sand fly species and to detect Wolbachia spp. and/or Leishmania spp. in the captured specimens. Sand flies were collected at the locality of Huimango, Tabasco, Mexico, during October 2019, using nine light traps (CDC) and two Shannon traps per night. The specimens were identified and females were analyzed by PCR for the DNA detection for pathogens. A total of 193 sand fly specimens belonging to five species were morphologically identified. Pintomyia ovallesi was the most abundant species (76.84%), followed by Micropygomyia cayennensis (6.40%). Furthermore, first records of four sand fly species were established for the state of Tabasco, thereby increasing the species richness in the state from four to eight. We observed a natural infection rate of 9.7% (10/103) for Leishmania and 0.91% (1/103) for Wolbachia. The importance of conducting entomological surveys in endemic areas of leishmaniasis in Mexico is highlighted, to determine whether other sand fly species may be potential vectors of Leishmania spp., and if some Wolbachia strains could be relevant for the control of leishmaniasis.
Assuntos
Leishmania , Leishmaniose Cutânea , Psychodidae , Wolbachia , Animais , DNA , Feminino , Insetos Vetores , Leishmania/genética , México , Psychodidae/genética , Wolbachia/genéticaRESUMO
Heloderma horridum is one of the few known venomous lizards in the world. Their populations are in decline due to habitat destruction and capture for the pet trade. In México, many zoos have decided to take care of this species, most of them at altitudes greater than the natural altitudinal distribution. However, we know little about the capacity of the reptiles to face high-altitude environments. The objective of this study was to compare hematological traits of H. horridum in captivity in high and low altitude environments. Our findings show that H. horridum does not respond to hypoxic environments, at least in blood traits, and that the organisms appear to be in homeostasis. Although we cannot know if individual H. horridum housed in high-altitude environments are completely comfortable, it appears hypoxia can be avoid without modifications of blood parameters. We suggest that future work should address changes in metabolic rates and in behavioral aspects to understand how to maintain the health and comfort of the reptiles native to low altitude when they are housed in high-altitude environments.
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Altitude , Bem-Estar do Animal , Hipóxia/sangue , Lagartos/fisiologia , Animais , Animais de Zoológico/fisiologia , Contagem de Eritrócitos , Hematócrito , Hemoglobinas/fisiologia , MéxicoRESUMO
Insect ß-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 ß-1,3-glucan binding protein involved in the activation of innate immune responses. Additionally, Ae. aegypti larvae contain significant ß-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 ß-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 ß-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.
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The 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 (IgG2aâ¯+â¯IgG2bâ¯>â¯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.
Assuntos
Doença de Chagas/prevenção & controle , Eletroporação/métodos , Vacinas Protozoárias/administração & dosagem , Vacinação/métodos , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Imunidade Celular , Imunização Secundária , Imunoglobulina G/sangue , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/imunologia , Absorção Cutânea , Trypanosoma cruzi/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
BACKGROUND: The sand fly Lutzomyia longipalpis is the main vector of American visceral leishmaniasis, a disease caused by parasites of the genus Leishmania. Adults of this insect feed on blood (females only) or sugar from plant sources, but their digestion of carbohydrates is poorly studied. Beta-glycosides as esculin and amygdalin are plant compounds and release toxic compounds as esculetin and mandelonitrile when hydrolyzed. Beta-glucosidase and trehalase are essential enzymes in sand fly metabolism and participate in sugar digestion. It is therefore possible that the toxic portions of these glycosides, released during digestion, affect sand fly physiology and the development of Leishmania. RESULTS: We tested the oral administration to sand flies of amygdalin, esculin, mandelonitrile, and esculetin in the sugar meal. These compounds significantly decreased the longevity of Lutzomyia longipalpis females and males. Lutzomyia longipalpis adults have significant hydrolytic activities against esculin and feeding on this compound cause changes in trehalase and ß-glucosidase activities. Female trehalase activity is inhibited in vitro by esculin. Esculin is naturally fluorescent, so its ingestion may be detected and quantified in whole insects or tissue samples stored in methanol. Mandelonitrile neither affected the amount of sugar ingested by sand flies nor showed repellent activity. Our results show that mandelonitrile significantly reduces the viability of L. amazonensis, L. braziliensis, L. infantum and L. mexicana, in a concentration-dependent manner. Esculetin caused a similar effect, reducing the number of L. infantum and L. mexicana. Female L. longipalpis fed on mandelonitrile had a reduction in the number of parasites and prevalence of infection after seven days of infection with L. mexicana, either by counting in a Neubauer chamber or by qPCR assays. CONCLUSIONS: Glycosides have significant effects on L. longipalpis longevity and metabolism and also affect the development of parasites in culture and inside the insect. These observations might help to conceptualize new vector control strategies using transmission blocking sugar baits.
Assuntos
Glicosídeos/toxicidade , Controle de Insetos/métodos , Insetos Vetores/enzimologia , Insetos Vetores/parasitologia , Leishmania/crescimento & desenvolvimento , Psychodidae/enzimologia , Psychodidae/parasitologia , Acetonitrilas/toxicidade , Amigdalina/toxicidade , Animais , Esculina/toxicidade , Feminino , Glicosídeos/administração & dosagem , Leishmaniose/prevenção & controle , Leishmaniose/transmissão , Masculino , Trealase/efeitos dos fármacos , Umbeliferonas/administração & dosagem , Umbeliferonas/toxicidade , beta-Glucosidase/efeitos dos fármacosRESUMO
PCR amplification and sequencing of Trypanosoma cruzi (T. cruzi) spliced-leader intergenic region of the mini-exon gene intergenic region (SL-IR) fragment was performed on intestinal tissue and fecal content DNA extracted from 19 Meccus pallidipennis (M. pallidipennis) specimens collected in the southern region of the State of Mexico. DNA sequence analysis from 49 bp T. cruzi SL-IR showed that all 19 samples corresponded to haplotype TcIa, and all of them were identical to GenBank sequence JQ028863. When extending the analysis to the whole 256 bp amplified sequence of the SL-IR, we found six sequences with a C insertion at position 10, one of which also presented a mutation (T/C) at position 54. One more sequence had an insertion (T) at position 223. Our findings suggest that two dominating TcIa clones are present in M. pallidipennis in the southern region of the State of Mexico. Interestingly, the SL-IR region of the dominating genotype was 100% identical to a circulating clone from Costa Rica present in humans, dogs, Triatoma dimidiata, and Panstrongylus rufotuberculatus. Future regional studies should explore the presence of this haplotype in humans and domestic animals.
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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.
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In this work, we developed a general perturbation theory and machine learning method for data mining of proteomes to discover new B-cell epitopes useful for vaccine design. The method predicts the epitope activity εq(cqj) of one query peptide (q-peptide) under a set of experimental query conditions (cqj). The method uses as input the sequence of the q-peptide. The method also uses as input information about the sequence and epitope activity εr(crj) of a peptide of reference (r-peptide) assayed under similar experimental conditions (crj). The model proposed here is able to classify 1â¯048â¯190 pairs of query and reference peptide sequences from the proteome of many organisms reported on IEDB database. These pairs have variations (perturbations) under sequence or assay conditions. The model has accuracy, sensitivity, and specificity between 71 and 80% for training and external validation series. The retrieved information contains structural changes in 83â¯683 peptides sequences (Seq) determined in experimental assays with boundary conditions involving 1448 epitope organisms (Org), 323 host organisms (Host), 15 types of in vivo process (Proc), 28 experimental techniques (Tech), and 505 adjuvant additives (Adj). Afterward, we reported the experimental sampling, isolation, and sequencing of 15 complete sequences of Bm86 gene from state of Colima, Mexico. Last, we used the model to predict the epitope immunogenic scores under different experimental conditions for the 26â¯112 peptides obtained from these sequences. The model may become a useful tool for epitope selection toward vaccine design. The theoretical-experimental results on Bm86 protein may help the future design of a new vaccine based on this protein.
Assuntos
Mineração de Dados/métodos , Epitopos de Linfócito B , Glicoproteínas de Membrana/genética , Proteoma/análise , Proteínas Recombinantes/genética , Vacinas/genética , Sequência de Aminoácidos , Animais , Aprendizado de Máquina , México , Modelos TeóricosRESUMO
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.
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Insetos Vetores , Rhodnius/fisiologia , Animais , Carboidratos , Corantes/análise , DNA de Plantas/análise , Comportamento Alimentar , Solanum lycopersicum , Coloração e RotulagemRESUMO
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 ß-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive ß-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.
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Phlebotomine sand flies are the vectors of medically important Leishmania. The Leishmania protozoa reside in the sand fly gut, but the nature of the immune response to the presence of Leishmania is unknown. Reactive oxygen species (ROS) are a major component of insect innate immune pathways regulating gut-microbe homeostasis. Here we show that the concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens but not after feeding on the Leishmania that uses the sand fly as a vector. Moreover, the Leishmania is sensitive to ROS either by oral administration of ROS to the infected fly or by silencing a gene that expresses a sand fly ROS-scavenging enzyme. Finally, the treatment of sand flies with an exogenous ROS scavenger (uric acid) altered the gut microbial homeostasis, led to an increased commensal gut microbiota, and reduced insect survival after oral infection with S. marcescens. Our study demonstrates a differential response of the sand fly ROS system to gut microbiota, an insect pathogen, and the Leishmania that utilize the sand fly as a vehicle for transmission between mammalian hosts.
Assuntos
Imunidade/imunologia , Leishmania mexicana/imunologia , Psychodidae/imunologia , Espécies Reativas de Oxigênio/imunologia , Serratia marcescens/imunologia , Sequência de Aminoácidos , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Catalase/classificação , Catalase/genética , Catalase/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/parasitologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Peróxido de Hidrogênio/metabolismo , Imunidade/efeitos dos fármacos , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/imunologia , Insetos Vetores/microbiologia , Insetos Vetores/parasitologia , Leishmania mexicana/fisiologia , Dados de Sequência Molecular , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Filogenia , Psychodidae/enzimologia , Psychodidae/genética , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de Aminoácidos , Serratia marcescens/fisiologia , Superóxido Dismutase/classificação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Ácido Úrico/administração & dosagem , Ácido Úrico/farmacologiaRESUMO
Here we describe clinical and pathologic evidence of Chagas disease caused in dogs by circulating Trypanosoma cruzi from a newly recognized endemic area in Mexico. We show that the Zumpahuacan isolate, although less virulent than the Sylvio-X10 reference strain that caused acute myocarditis and death, was pathogenic in dogs. Dogs infected with the Zumpahuacan isolate exhibited electrocardiographic alterations, left- and right-ventricle dilation, and hydropericardium. Histologically, diffused perimysial and endomysial lymphoplasmacytic cell infiltration, cardiomyocyte necrosis, and amastigote nests were noted in Zumpahuacan-infected dogs. These findings suggest that the risk of T. cruzi infection and Chagas disease is present in the State of Mexico, and further research is needed to identify the T. cruzi bio-types circulating in southern State of Mexico.
Assuntos
Doença de Chagas/veterinária , Doenças do Cão/parasitologia , Trypanosoma cruzi/patogenicidade , Animais , Cardiomiopatia Chagásica/epidemiologia , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/veterinária , Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Doença de Chagas/patologia , Modelos Animais de Doenças , Reservatórios de Doenças/veterinária , Doenças do Cão/epidemiologia , Doenças do Cão/patologia , Cães , México/epidemiologia , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/patologia , VirulênciaRESUMO
We used 5 diagnostic tests in a cross-sectional investigation of the prevalence of Trypanosoma cruzi in Tejupilco municipality, State of Mexico, Mexico. Our findings showed a substantial prevalence of immunoglobulin G (IgG) and IgM antibodies to T. cruzi in human (n = 293, IgG 2.05%, IgM 5.5%, both 7.1%) and dog (n = 114, IgG 15.8%, IgM 11.4%, both 21%) populations. We also found antibodies to T. cruzi (n = 80, IgG 10%, IgM 15%, both 17.5%) in dogs from Toluca, an area previously considered free of T. cruzi. Our data demonstrate the need for active epidemiologic surveillance programs in these regions. A direct correlation (r2 = 0.955) of seropositivity between humans and dogs suggests that seroanalysis in dogs may help identify the human prevalence of T. cruzi infection in these areas.