Leishmaniasis is one of the most diverse and complex of all vector-borne diseases worldwide. It is caused by parasites of the genus Leishmania, obligate intramacrophage protists characterised by ...diversity and complexity. Its most severe form is visceral leishmaniasis (VL), a systemic disease that is fatal if left untreated. In Latin America VL is caused by Leishmania infantum chagasi and transmitted by Lutzomyia longipalpis. This phlebotomine sandfly is only found in the New World, from Mexico to Argentina. In South America, migration and urbanisation have largely contributed to the increase of VL as a public health problem. Moreover, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases.
An inventory of the microbiota associated with insect vectors, especially of wild specimens, would aid in the development of novel strategies for controlling insect vectors. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. Previous studies on wild and laboratory reared female Lu. longipalpis have described gut bacteria using standard bacteriological methods. In this study, total RNA was extracted from the insects and submitted to high-throughput pyrosequencing. The analysis revealed the presence of sequences from bacteria, fungi, protist parasites, plants and metazoans.
This is the first time an unbiased and comprehensive metagenomic approach has been used to survey taxa associated with an infectious disease vector. The identification of gregarines suggested they are a possible efficient control method under natural conditions. Ongoing studies are determining the significance of the associated taxa found in this study in a greater number of adult male and female Lu. longipalpis samples from endemic and non-endemic locations. A particular emphasis is being given to those species involved in the biological control of this vector and to the etiologic agents of animal and plant diseases.
Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas' disease. This is a neglected disease affecting approximately 8 million people in Latin ...America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas' disease.
The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas' disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion.
Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas' disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.
The study of developmental processes in Rhodnius prolixus has recently advanced with the sequencing of the genome. In this work, we analyze the maternal gene expression driving oogenesis and early ...embryogenesis in R. prolixus. We examined the transcriptional profile of mRNAs to establish the genes expressed across the ovary, unfertilized eggs and different embryonic stages of R. prolixus until the formation of the germ band anlage (0, 12, 24, and 48 h post egg laying). We identified 81 putative maternal and ovary-related genes and validated their expression by qRT-PCR. We validate the function of the ortholog gene Bicaudal-D (Rp-BicD) by in situ hybridization and parental RNAi. Consistent with a role in oogenesis and early development of R. prolixus, we show that lack of Rp-BicD does not significantly affect oogenesis but impairs the formation of the blastoderm. Based on our findings, we propose three times of action for maternal genes during oogenesis and embryogenesis in R. prolixus.
We show a sensitive and straightforward off-line nano-LC-MALDI-MS/MS workflow that allowed the first comprehensive neuropeptidomic analysis of an insect disease vector. This approach was applied to ...identify neuropeptides in the brain of Rhodnius prolixus, a vector of Chagas disease. This work will contribute to the annotation of genes in the ongoing R. prolixus genome sequence project. Peptides were identified by de novo sequencing and comparisons to known neuropeptides from different organisms by database search. By these means, we were able to identify 42 novel neuropeptides from R. prolixus. The peptides were classified as extended FMRF-amide-related peptides, sulfakinins, myosuppressins, short neuropeptide F, long neuropeptide F, SIF-amide-related peptides, tachykinins, orcokinins, allatostatins, allatotropins, calcitonin-like diuretic hormones, corazonin, and pyrokinin. Some of them were detected in multiple isoforms and/or truncated fragments. Interestingly, some of the R. prolixus peptides, as myosuppressin and sulfakinins, are unique in their characteristic C-terminal domain among insect neuropeptides identified so far.
The morphology and physiology of the oogenesis have been well studied in the vector of Chagas disease
Rhodnius prolixus
. However, the molecular interactions that regulate the process of egg ...formation, key for the reproductive cycle of the vector, is still largely unknown. In order to understand the molecular and cellular basis of the oogenesis, we examined the function of the gene
Bicaudal C
(
BicC
) during oogenesis and early development of
R. prolixus
. We show that
R. prolixus BicC
(
Rp-BicC
) gene is expressed in the germarium, with cytoplasmic distribution, as well as in the follicular epithelium of the developing oocytes. RNAi silencing of
Rp-BicC
resulted in sterile females that lay few, small, non-viable eggs. The ovaries are reduced in size and show a disarray of the follicular epithelium. This indicates that
Rp-BicC
has a central role in the regulation of oogenesis. Although the follicular cells are able to form the chorion, the uptake of vitelline by the oocytes is compromised. We show evidence that the polarity of the follicular epithelium and the endocytic pathway, which are crucial for the proper yolk deposition, are affected. This study provides insights into the molecular mechanisms underlying oocyte development and show that
Rp-BicC
is important for de developmental of the egg and, therefore, a key player in the reproduction of this insect.
Anadenanthera colubrina var. cebil is a native South American tree species inhabiting seasonally dry tropical forests (SDTFs). Its current disjunct distribution presumably represents fragments of a ...historical much larger area of this forest type, which has also been highly impacted by human activities. In this way the hypothesis of this study is that the natural populations of A. colubrina var. cebil from Northern Argentina represent vestiges of ancient fragmentation, but they are additionally influenced by a certain degree of gene flow among them. We aimed to analyze the genetic structure of both nuclear and chloroplast DNA to evaluate the relative role of ancient and recent fragmentation on intraspecific diversity patterns. Sixty-nine individuals of four natural populations were analyzed using eight nuclear microsatellites (ncSSR) and four chloroplast microsatellite loci (cpSSR). The level and distribution of genetic variation were estimated by standard population genetic parameters and Neighbor Joining as well as Bayesian analyses. The eight ncSSR loci were highly polymorphic, while genetic diversity of cpSSRs was low. Nuclear SSRs displayed lower genetic differentiation among populations than cpSSR haplotypes (F ST 0.11 and 0.95, respectively). However, high differentiation between phytogeographic provinces was observed in both genomes. The high genetic differentiation detected emphasizes the role of ancient fragmentation. However, the Paranaense province also shows the effects of recent fragmentation on genetic structure, whereas gene flow by pollen preserves the effects of genetic drift in the Yungas province.
mRNP remodeling events required for the transition of an mRNA from active translation to degradation are currently poorly understood. We identified protein factors potentially involved in this ...transition, which are present in mammalian P bodies, cytoplasmic foci enriched in 5' --> 3' mRNA degrading enzymes. We demonstrate that human P bodies contain the cap-binding protein eIF4E and the related factor eIF4E-transporter (eIF4E-T), suggesting novel roles for these proteins in targeting mRNAs for 5' --> 3' degradation. Furthermore, fluorescence resonance energy transfer (FRET) studies indicate that eIF4E interacts with eIF4E-T and the putative DEAD box helicase rck/p54 in the P bodies in vivo. RNAi-mediated knockdowns revealed that a subset of P body factors, including eIF4E-T, LSm1, rck/p54, and Ccr4 are required for the accumulation of each other and eIF4E in P bodies. In addition, treatment of HeLa cells with cycloheximide, which inhibits translation, revealed that mRNA is also required for accumulation of mRNA degradation factors in P bodies. In contrast, knockdown of the decapping enzyme Dcp2, which initiates the actual 5' --> 3' mRNA degradation did not abolish P body formation, indicating it first functions after mRNPs have been targeted to these cytoplasmic foci. These data support a model in which mRNPs undergo several successive steps of remodeling and/or 3' trimming until their composition or structural organization promotes their accumulation in P bodies.
Insects operate complex humoral and cellular immune strategies to fend against invading microorganisms. The majority of these have been characterized in Drosophila and other dipterans. Information on ...hemipterans, including Triatominae vectors of Chagas disease remains incomplete and fractionated.
We identified putative immune-related homologs of three Triatominae vectors of Chagas disease, Triatoma pallidipennis, T. dimidiata and T. infestans (TTTs), using comparative transcriptomics based on established immune response gene references, in conjunction with the predicted proteomes of Rhodnius prolixus, Cimex lecticularis and Acyrthosiphon pisum hemimetabolous. We present a compressive description of the humoral and cellular innate immune components of these TTTs and extend the immune information of other related hemipterans. Key homologs of the constitutive and induced immunity genes were identified in all the studied hemipterans.
Our results in the TTTs extend previous observations in other hemipterans lacking several components of the Imd signaling pathway. Comparison with other hexapods, using published data, revealed that the absence of various Imd canonical components is common in several hemimetabolous species.
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