Salivary proteins electrophoretic patterns enabled differentiating Colombian Rhodnius Trans-Andean and Cis-Andean groups / [Los patrones electroforéticos de proteínas salivales permiten diferenciar los grupos transandino y cisandino de las especies de Rhodnius de Colombia].

Introduction: Rhodnius (Hemiptera: Reduviidae: Triatominae) species are made up of haematophagous insect vectors for Trypanosoma cruzi (Chagas' disease aetiological agent) and T. rangeli, an infective parasite that is not pathogenic for vertebrate hosts. The study of their salivary protein diversity enables the obtention of characteristic one-dimensional electrophoretic profiles of some triatomine species; however, few reports have dealt with Rhodnius species salivary proteins electrophoretic patterns. Objective: To compare R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus' salivary proteins one-dimensional electrophoretic profiles. Materials and methods: SDS-PAGE was used for obtaining electrophoretic profiles of saliva from the species under study. The unweighted pair group method with arithmetic mean (UPGMA) was used for constructing a phenogram. Results: Electrophoretic profiles of soluble saliva had protein bands ranging from 15 to 45 kDa, thereby enabling the five species studied to be differentiated. The phenogram revealed two main groups, one formed by the Pictipes and Prolixus cis-Andean groups and another consisting of the Pallescens trans-Andean group. Conclusion: Differences were revealed regarding R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus electrophoretic profiles of salivary proteins; their variability facilitated constructing a phenogram which was taxonomically congruent with the groups from the genus Rhodnius.

Introducción. Las especies Rhodnius (Hemiptera: Reduviidae: Triatominae) están conformadas por insectos hematófagos vectores de Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, y T. rangeli, parásito infectivo pero no patógeno para el vertebrado. El estudio de la diversidad proteica de la saliva de estos insectos permite la obtención de perfiles electroforéticos unidimensionales característicos de algunas especies de triatominos. Sin embargo, el reporte de los patrones electroforéticos de proteínas salivales de las especies de Rhodnius ha sido escaso. Objetivo. Hacer un análisis comparativo de los perfiles electroforéticos unidimensionales de las proteínas salivales de R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus. Materiales y métodos. Se obtuvieron los perfiles electroforéticos de la saliva de las especies en estudio mediante electroforesis en gel de poliacrilamida con dodecilsulfatosódico (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) y se construyó un fenograma mediante el método UPGMA (Unweighted Pair Group Method Using Arithmetic Averages). Resultados. Los perfiles electroforéticos de las proteínas solubles de saliva presentaron bandas en un rango de masa aproximado de 15 a 45 kDa, los cuales permitieron diferenciar las cinco especies estudiadas. El fenograma reveló la existencia de dos grupos principales: uno conformado por los grupos cisandinos Pictipes y Prolixus y otro constituido por el grupo transandino Pallescens. Conclusiones. Existen diferencias en los perfiles electroforéticos de las proteínas salivales entre R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus, cuya variabilidad permitió construir un fenograma congruente con los grupos del género Rhodnius.

The Pharmacopea within Triatomine Salivary Glands.

Triatomines are blood-feeding insects that prey on vertebrate hosts. Their saliva is largely responsible for their feeding success. The triatomine salivary content has been studied over the past decades, revealing multifunctional bioactive proteins targeting the host´s hemostasis and immune system. Recently, sequencing of salivary-gland mRNA libraries revealed increasingly complex and complete transcript databases that have been used to validate the expression of deduced proteins through proteomics. This review provides an insight into the journey of discovery and characterization of novel molecules in triatomine saliva.

Evidence suggesting interactions between immunodominant membrane protein Imp of Flavescence dorée phytoplasma and protein extracts from distantly related insect species.

AIMS: In this study, binding between the immunodominant membrane protein Imp of the 16SrV-D phytoplasma associated with Flavescence dorée disease (FD-Dp) and insect proteins of vectors and non-vectors of FD-Dp was tested. METHODS AND RESULTS: Six Auchenorrhyncha species, from distantly related groups were selected: Scaphoideus titanus, Euscelidius variegatus, Macrosteles quadripunctulatus, Zyginidia pullula (Cicadomorpha), Ricania speculum and Metcalfa pruinosa (Fulgoromorpha). The vector status of each species was retrieved from the literature or determined by transmission trials in this study. A His-tagged partial Imp protein and a rabbit polyclonal antibody were synthesized and used for Western and Far-Western dot Blot (FWdB) experiments. Total native and membrane proteins (MP) were extracted from entire bodies and organs (gut and salivary glands) of each insect species. FWdB showed decreasing interaction intensities of Imp fusion protein with total proteins from entire bodies of S. titanus, E. variegatus (competent vectors) and M. quadripunctulatus (non-vector), while no interaction signal was detected with the other three species (non-vectors). A strong signal detected upon interaction of FD-D Imp and MP from guts of closely related insects supports the role of this organ as the first barrier to ensure successful transmission. CONCLUSIONS: Our results showed that specific Imp binding, correlated with vector status, is involved in interactions between FD-Dp and insect proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Integrating knowledge on host-pathogen protein-protein interactions and on insect phylogeny would help to identify the actual range of vectors of phytoplasma strains of economic importance.

Identification of phlebotomine sand flies through MALDI-TOF mass spectrometry and in-house reference database.

Phlebotomine sand flies are vectors for many pathogens responsible for human and animal diseases worldwide. Their identification at species level is of importance in epidemiological studies and control programmes. MALDI-TOF MS has been increasingly investigated as an alternative approach to the conventional identification of arthropods species. To establish an in-house protein spectra database for a quick and reliable species identification of phlebotomine sand flies, 166 field-caught sand fly specimens, morphologically identified as Phlebotomus perniciosus (no = 56; 26 males and 30 females), Phlebotomus neglectus (no = 4 males), Phlebotomus sergenti (no = 6; 4 males and 2 females) and Sergentomyia minuta (no = 100; 45 males and 55 females), were subjected to MALDI-TOF MS analyses. Out of 166, 149 specimens (89.8%) produced consistent species-specific protein spectra. Good quality database for P. perniciosus and S. minuta were generated; no databases have yet constructed for P. neglectus and P. sergenti due to the low number of specimens examined. The identification of 80 sand flies (no = 20 P. perniciosus; no = 60 S. minuta) were confirmed using the new generated SuperSpectra as validation test. The results reported support the use of MALDI-TOF MS for rapid, simple and reliable phlebotomine sand fly species identification suggesting its usefulness in accurate survey studies, ultimately improving biological and epidemiological knowledge on these important vectors of pathogens.

An insight into the sialotranscriptome and virome of Amazonian anophelines.

BACKGROUND: Saliva of mosquitoes contains anti-platelet, anti-clotting, vasodilatory, anti-complement and anti-inflammatory substances that help the blood feeding process. The salivary polypeptides are at a fast pace of evolution possibly due to their relative lack of structural constraint and possibly also by positive selection on their genes leading to evasion of host immune pressure. RESULTS: In this study, we used deep mRNA sequence to uncover for the first time the sialomes of four Amazonian anophelines species (Anopheles braziliensis, A. marajorara, A. nuneztovari and A. triannulatus) and extend the knowledge of the A. darlingi sialome. Two libraries were generated from A. darlingi mosquitoes, sampled from two localities separated ~ 1100 km apart. A total of 60,016 sequences were submitted to GenBank, which will help discovery of novel pharmacologically active polypeptides and the design of specific immunological markers of mosquito exposure. Additionally, in these analyses we identified and characterized novel phasmaviruses and anpheviruses associated to the sialomes of A. triannulatus, A. marajorara and A. darlingi species. CONCLUSIONS: Besides their pharmacological properties, which may be exploited for the development of new drugs (e.g. anti-thrombotics), salivary proteins of blood feeding arthropods may be turned into tools to prevent and/or better control vector borne diseases; for example, through the development of vaccines or biomarkers to evaluate human exposure to vector bites. The sialotranscriptome study reported here provided novel data on four New World anopheline species and allowed to extend our knowledge on the salivary repertoire of A. darlingi. Additionally, we discovered novel viruses following analysis of the transcriptomes, a procedure that should become standard within future RNAseq studies.

Laser-induced breakdown spectroscopy (LIBS) as a novel technique for detecting bacterial infection in insects.

To prevent the spread of diseases in humans, animals or plants, determining whether potential vectors are infected is crucial. For example, early detection of the citrus disease Huanglongbing, which has been a scourge on the citrus industries around the world, is a critical need. This vector-borne disease is transmitted by Diaphorina citri, the Asian citrus psyllid, which carries the putative bacterial phytopathogen, Candidatus Liberibacter asiaticus (CLas). In this investigation, we introduced Laser-Induced Breakdown Spectroscopy (LIBS) to reveal key biochemical differences between CLas-infected and non-infected psyllids. The emission spectra captured from laser ablation of CLas-infected and healthy psyllids were processed through the principal component analysis (PCA) method and compared. Thirteen peaks from seven different elements were detected in D. citri. The t-test showed that CLas-infected D. citri were deficients in zinc, iron, copper, magnesium, calcium, and nitrogen. The PCA showed that LIBS can successfully differentiate between CLas-infected and healthy D. citri by comparing their elemental profile. In this work, we demonstrated a method that allows for a fast and precise compositional microanalysis of an insect vector which can contribute to the early detection of citrus huanglongbing.

Human Antimicrobial Peptide Isolated From Triatoma infestans Haemolymph, Trypanosoma cruzi-Transmitting Vector.

The importance of antimicrobial peptides (AMPs) in relation to the survival of invertebrates is well known. The source and the mode of action on the insects' immune system of these molecules have been described from different perspectives. Insects produce their own AMPs as well as obtain these molecules from various sources, for example by absorption through the intestinal tract, as previously described for Boophilus microplus. Blood-sucking barber bug Triatoma infestans attracts social, economic and medical interest owing to its role in the transmission of Chagas disease. Despite new studies, descriptions of AMPs from this insect have remained elusive. Thus, the aims of this work were to characterize the antimicrobial potential of human fibrinopeptide A (FbPA) obtained from the T. infestans haemolymph and identify its natural source. Therefore, FbPA was isolated from the T. infestans haemolymph through liquid chromatography and identified by mass spectrometry. This peptide exhibited antimicrobial activity against Micrococcus luteus. Native FbPA from human blood and the synthetic FbPA also exhibited antimicrobial activity. The synthetic FbPA was conjugated with fluorescein isothiocyanate and offered to the insects. The haemolymph collected after 72 h exhibited fluorescence at the same wavelength as fluorescein isothiocyanate. Our experiments show that beyond intrinsic AMP production, T. infestans is able to co-opt molecules via internalization and may use them as AMPs for protection.

Characterization and expression of a long neuropeptide F (NPF) receptor in the Chagas disease vector Rhodnius prolixus.

In this study, a long neuropeptide F receptor of the blood-feeding hemipteran, Rhodnius prolixus (RhoprNPFR) has been cloned and characterized. Approximately 70% of the RhoprNPFR deduced protein sequence is identical to that of other hemipteran NPFRs. RhoprNPFR has seven highly-conserved transmembrane domains, two cysteine residues in the 2nd and 3rd extracellular loops that likely form a disulfide bond integral for maintaining the structure of the receptor, and a conserved DRY motif after the third transmembrane domain. All of these characteristics are typical of class A rhodopsin-like GPCRs. The receptor transcript is predominantly expressed in the central nervous system (CNS) and gut of both fifth instar and adult R. prolixus. Using fluorescent in situ hybridization (FISH), we identified six bilaterally-paired large median neurosecretory cells (approximately 30µm in diameter) in the brain that express the RhoprNPFR mRNA transcript. We also found RhoprNPFR transcript expression in endocrine cells in the anterior midgut of fifth instars, as well as in putative pre-follicular cells present in the germarium and between developing oocytes, and in the nutritive cord. These results suggest that RhoprNPFR may play a role within the CNS, and in digestion and possibly egg production and/or egg development in R. prolixus.

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection.

Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite Trypanosoma cruzi, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total T. cruzi parasite counts in R. prolixus were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

Lower galactosylation levels of the Lipophosphoglycan from Leishmania (Leishmania) major-like strains affect interaction with Phlebotomus papatasi and Lutzomyia longipalpis.

BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.

"Sweeter than a rose", at least to Triatoma phyllosoma complex males (Triatominae: Reduviidae).

BACKGROUND: The Triatoma phyllosoma complex of Trypanosoma cruzi vectors (Triatominae: Reduviidae) is distributed in both Neotropical and Nearctic bioregions of Mexico. METHODS: Volatile organic compounds emitted by disturbed Triatoma longipennis, Triatoma pallidipennis and Triatoma phyllosoma, and from their Brindley's and metasternal glands, were identified using solid-phase microextraction coupled with gas chromatography-mass spectrometry. RESULTS: Disturbed bugs and the metasternal glands from T. phyllosoma released or had significantly fewer compounds than T. longipennis and T. pallidipennis. Isobutyric acid was the most abundant compound secreted by disturbed bugs of the three species, while Brindley's glands of all species produced another four compounds: propanoic acid, isobutyric acid, pentyl butanoate, and 2-methyl hexanoic acid. Two novel compounds, both rose oxide isomers, were produced in MGs and released only by disturbed females of all three species, making this the first report in Triatominae of these monoterpenes. The principal compound in MGs of both sexes of T. longipennis and T. phyllosoma was 3-methyl-2-hexanone, while cis-rose oxide was the principal compound in T. pallidipennis females. The major components in male effluvia of T. pallidipennis were 2-decanol and 3-methyl-2-hexanone. CONCLUSION: Discriminant analysis of volatile organic compounds was significant, separating the three species and was consistent with morphological and genetic evidence for species distinctions within the complex.

Caracterização bioquímica e funcional da proteína XadA3 de Xylella fastidiosa / Biochemistry and functional characterization of Xylella fastidiosa XadA3 adhesin

Gram-negativas e é utilizado por diversos patógenos para colonizar seus hospedeiros, sendo o primeiro passo do processo de desenvolvimento do biolfilme. Uma variedade de apêndices celulares e proteínas está envolvida na adesão bacteriana, tais como pili, fimbrias, adesinas fimbriais e afimbriais. O fitopatógeno Xylella fastidiosa, agente causal de importantes doenças como a doença de Pierce de videiras, a clorose variegada dos citros e a síndrome do rápido declínio de oliveiras, possui em sua superfície várias dessas estruturas que são potencialmente responsáveis pela colonização eficiente de insetos-vetores e plantas hospedeiras. Entre as adesinas afimbriais codificadas no genoma dessa bactéria, três XadA (XadA1, Hsf/XadA2 e XadA3) são classificadas como autotransportadores triméricos. Dados da literatura sugerem que XadA1 e XadA2 são importantes para a formação do biofilme, porém a função de XadA3 ainda não havia sido investigada. Nesse trabalho, tivemos como objetivo caracterizar bioquímica e funcionalmente a proteína XadA3 e obter informações adicionais sobre o papel desempenhado por XadA1 e XadA2 na adesão e virulência de X. fastidiosa. Utilizando imunodetecção com um anticorpo policlonal anti-XadA3 por nós obtido, demonstramos que essa proteína localiza-se na superfície bacteriana e medeia a adesão intercelular. A caracterização dos fenótipos de mutantes de deleção de cada um dos genes das adesinas XadA revelou que o mutante ΔxadA3 tem reduzida capacidade de agregação celular e formação de biofilme quando comparado tanto aos mutantes ΔxadA1 e ΔxadA2 como à cepa selvagem Temecula. A deleção dos genes xadA afeta marginalmente o perfil de expressão gênica global avaliado através de RNAseq das cepas mutantes comparativamente à cepa selvagem, porém destaca-se, nas cepas mutantes, o aumento nos níveis dos transcritos de lipases/esterases. Já foi descrito que essas enzimas parecem atuar na degradação do tecido vegetal associada aos sintomas da doença de Pierce de videiras. A deleção de xadA3 resulta em um fenótipo de hipervirulência em videiras, mas também de deficiência de transmissão pelo inseto-vetor. O conjunto dos resultados obtidos nesse trabalho evidenciam o importante papel desempenhado pelas adesinas XadAs, particularmente XadA3, na adesão intercelular, no desenvolvimento do biofilme e na virulência de X. fastidiosa

Adhesion is a widely conserved mechanism of virulence among Gram-negative bacteria that is used by several pathogens to colonize their hosts, being the first step in biolfilm development. A variety of appendages and proteins are involved in bacterial adhesion, such as pili, fimbriae, fimbrial and afimbrials adhesins. The phytopathogen Xylella fastidiosa, causal agent of important diseases such as Pierce's disease of grapevines, citrus variegated chlorosis and olive quick decline syndrome, harbours on its surface several of these structures that are potentially responsible for efficient colonization of insect vectors and plant hosts. Among the afimbrial adhesins encoded in the genome of this bacterium, three XadAs (XadA1, Hsf/XadA2 and XadA3) are classified as trimeric autotransporters. Data from the literature suggest that XadA1 and XadA2 are important for biofilm formation, but XadA3 function has not been yet investigated. In this work, we aimed to biochemically and functionally characterize the XadA3 protein and gather additional information about the role played by XadA1 and XadA2 in X. fastidiosa adhesion and virulence. Using immunodetection with a polyclonal anti-XadA3 antibody, we have demonstrated that this protein localizes to the bacterial surface and mediates intercellular adhesion. Phenotypic characterization of the deletion mutants of XadA adhesins encoded genes revealed that the ΔxadA3 mutant has reduced cell aggregation capacity and biofilm formation when compared to both ΔxadA1 and ΔxadA2 mutants as well as to Temecula wild type strain. Deletion of the xadA genes marginally affects the global gene expression profile assessed by RNA-seq of the mutant strains compared to the wild-type strain, eventhough an increase in lipase/esterase transcripts levels was observed in the mutant strains. It has been reported that these enzymes appear to participate in the degradation of plant tissue that is associated with symptoms of Pierce's disease of grapevines. The deletion of xadA3 results in a phenotype of hypervirulence in grapevines but also of deficiency in insect-vector transmission. The results obtained in this work evidenced the important role played by XadAs adhesins, particularly XadA3, in X. fastidiosa intercellular adhesion, biofilm development and virulence

High-resolution structure of a Kazal-type serine protease inhibitor from the dengue vector Aedes aegypti.

Blood-feeding exoparasites are rich sources of protease inhibitors, and the mosquito Aedes aegypti, which is a vector of Dengue virus, Yellow fever virus, Chikungunya virus and Zika virus, is no exception. AaTI is a single-domain, noncanonical Kazal-type serine proteinase inhibitor from A. aegypti that recognizes both digestive trypsin-like serine proteinases and the central protease in blood clotting, thrombin, albeit with an affinity that is three orders of magnitude lower. Here, the 1.4 Šresolution crystal structure of AaTI is reported from extremely tightly packed crystals (∼22% solvent content), revealing the structural determinants for the observed inhibitory profile of this molecule.

Identification of the Aggregation-sex Pheromone Produced by Male Monochamus saltuarius, a Major Insect Vector of the Pine Wood Nematode.

In this study, we isolated and identified an aggregation-sex pheromone from Monochamus saltuarius, the major insect vector of the pine wood nematode in Korea. Adult males of M. saltuarius produce 2-undecyloxy-1-ethanol, which is known as an aggregation-sex pheromone in other Monochamus species. We performed field experiments to determine the attractiveness of the pheromone and other synergists. More M. saltuarius adult beetles were attracted to traps baited with the pheromone than to unbaited traps. Ethanol and (-)-α-pinene interacted synergistically with the pheromone. Traps baited with the pheromone + (-)-α-pinene +ethanol were more attractive to M. saltuarius adults than traps baited with the pheromone, (-)-α-pinene, or ethanol alone. Ipsenol, ipsdienol, and limonene were also identified as synergists of the aggregation-sex pheromone for M. saltuarius adults. In field experiments, the proportion of females was much higher in the beetles caught in traps than among the beetles emerging from naturally-infested logs in the laboratory. Our results suggest that a combination of aggregation-sex pheromone and synergists could be very effective for monitoring and managing M. saltuarius.

Seroconversion to Lutzomyia intermedia LinB-13 as a biomarker for developing cutaneous leishmaniasis.

Sand flies inject saliva while feeding in the vertebrate host and anti-saliva antibodies can be used as biomarkers of exposure to Leishmania vectors. We expressed recombinant salivary proteins from Lutzomyia intermedia, a vector of Leishmania braziliensis, and evaluated the seroreactivity in exposed individuals in search for exposure markers. We found a strong correlation among positive serology to recombinant proteins LinB-13, 26, 15, 21 and to salivary proteins: rLinB-13 was the top performing molecule; IgG4 was the most predominant antibody subclass and antibodies to rLinB-13 did not cross react with Lu. longipalpis salivary proteins. By evaluating a cohort of contacts of CL patients, we confirmed that rLinB-13, an antigen 5-related protein, is a marker of exposure to Lu. intermedia with high degree of accuracy. In a 5-year follow up, we determined that individuals who developed CL presented higher anti-rLinB13 IgG responses, before the appearance of clinical symptoms. They also presented a lower frequency of cellular responses to the parasite (DTH). Our results show that seroconversion to a salivary molecule, rLinB-13, is a marker of risk for CL development caused by Leishmania braziliensis. This highlight the possibility of developing tools based on vector molecules to manage the disease in endemic areas.

[Diversity of Triatominae (Hemiptera: Reduviidae) in Santander, Colombia: Epidemiological implications].

INTRODUCTION: Domestic and wild triatomines in the department of Santander have an epidemiological impact, as recently they have been linked to outbreaks of acute Chagas disease. The analysis of their diversity and temporal variation contributes to the understanding of their biology and ecology in one of the most endemic areas of the country. OBJECTIVES: To analyze triatominae diversity in two regions of Santander. MATERIALS AND METHODS: We analyzed the triatomine records for Santander contained in the CINTROP-UIS entomology lab database. We grouped the information for two regions: the Middle Magdalena area and the Andean region, and for each one we designed species accumulation and range-abundance curves, we calculated diversity and equality indices, and we analyzed colonization and temporal variation or persistence of the community. RESULTS: Ninety five percent of triatomines came from the Andean area and 4.57% from Magdalena Medio, with nine and ten species each. The dominant species in the Andean area were Rhodnius prolixus and Triatoma dimidiata while in Magdalena Medio they were Rhodnius pallescens and Panstrongylus geniculatus. We found a greater diversity and richness in Middle Magdalena compared to the Andean area. The temporal variation showed persistence of communities over time. CONCLUSIONS: Results revealed differences in the diversity of the two regions and the potential of wild species to occupy artificial ecotopes. Triatomines intrusion and the recent involvement of wild species in the transmission of Trypanosoma cruzi emphasize the need to further investigate the ecology of these vectors in order to guide population control strategies.

Maxadilan-simile expression in Nyssomyia neivai, a sandfly vector in an endemic region of Brazil, and its immunogenicity in patients with American tegumentary leishmaniasis.

BACKGROUND: Maxadilan (Max) is a salivary component in the sandfly Lutzomyia longipalpis (Lutz & Neiva 1912), a vector of visceral leishmaniasis. Max has a powerful vasodilatory effect and is a candidate vaccine that has been tested in experimental leishmaniasis. Nyssomyia neivai (Pinto 1926) is a vector of the pathogen responsible for American tegumentary leishmaniasis (ATL) in Brazil. OBJECTIVE: We searched for Max expression in Ny. neivai and for antibodies against Max in ATL patients. METHODS: cDNA and protein were extracted from the cephalic segment, including salivary glands, of Ny. neivai and analysed by polymerase chain reaction, DNA sequencing, and blotting assays. The results were compared with data obtained from Lu. longipalpis samples. We quantified antibodies against Max in serum samples from 41 patients with ATL (31 and 10 with the cutaneous and mucocutaneous forms, respectively) and 63 controls from the endemic northeastern region of São Paulo state, using enzyme-linked immunosorbent assay. FINDINGS: Recognition of a Max-simile peptide by specific antibodies confirmed expression of a Max sequence in Ny. neivai (GenBank EF601123.1). Compared to controls, patients with ATL presented higher levels of antibodies against Max (p = 0.004); 24.4% of the patients with ATL and 3.2% of the controls presented anti-Max levels above the cutoff index (p = 0.014). The anti-Max levels were not associated with the specific clinical form of ATL, leishmanin skin test response, absence or presence of amastigotes in histopathologic exam, results of indirect immunofluorescence testing for leishmaniasis, or duration of cutaneous form disease. MAIN CONCLUSION: High serum anti-Max levels did not protect patients against ATL, but confirmed previous natural exposure to Ny. neivai bites in this ATL endemic region.

Diversidad de Triatominae (Hemiptera: Reduviidae) en Santander, Colombia: implicaciones epidemiológicas / Diversity of Triatominae (Hemiptera: Reduviidae) in Santander, Colombia: Epidemiological implications

Resumen Introducción: Los triatominos domiciliados y silvestres constituyen un problema de impacto epidemiológico en el departamento de Santander, pues se han asociado recientemente con brotes agudos de la enfermedad de Chagas, por lo cual el análisis de su diversidad y variación temporal contribuye al conocimiento de su biología y ecología en una de las áreas más endémicas del país. Objetivo: Analizar la diversidad de triatominos en dos regiones de Santander. Materiales y métodos: Se analizó la información de la base de datos del Laboratorio de Entomología del Centro de Investigaciones en Enfermedades Tropicales de la Universidad Industrial de Santander (CINTROP-UIS), la cual contiene registros de triatominos en Santander. La información se separó en dos regiones, el Magdalena Medio y la zona andina, para cada una de las cuales se diseñaron curvas de acumulación de especies y de rango de abundancia, se calcularon los índices de diversidad y de igualdad, se analizó la colonización y se evaluó la variación temporal o persistencia de la comunidad. Resultados: El 95 % de los triatominos provenía de la zona andina y, el 4,57 %, del Magdalena Medio, con nueve y diez especies, respectivamente. Se encontró mayor diversidad y riqueza en el Magdalena Medio en comparación con la zona andina. Las especies dominantes en la zona andina fueron Rhodnius prolixus y Triatoma dimidiata, mientras que en Magdalena Medio fueron Rhodnius pallescens y Panstrongylus geniculatus. La variación temporal mostró persistencia de las comunidades en el tiempo. Conclusiones:. Los resultados evidenciaron diferencias en la diversidad de las dos regiones, además del potencial de las especies silvestres para ocupar ecótopos artificiales. La intrusión de triatominos y la reciente incriminación de especies silvestres en la transmisión de Trypanosoma cruzi, indican la necesidad de un mayor conocimiento de la ecología de estos vectores para orientar las estrategias de control.

Abstract Introduction: Domestic and wild triatomines in the department of Santander have an epidemiological impact, as recently they have been linked to outbreaks of acute Chagas disease. The analysis of their diversity and temporal variation contributes to the understanding of their biology and ecology in one of the most endemic areas of the country. Objectives: To analyze triatominae diversity in two regions of Santander. Materials and methods: We analyzed the triatomine records for Santander contained in the CINTROPUIS entomology lab database. We grouped the information for two regions: the Middle Magdalena area and the Andean region, and for each one we designed species accumulation and range-abundance curves, we calculated diversity and equality indices, and we analyzed colonization and temporal variation or persistence of the community. Results: Ninety five percent of triatomines came from the Andean area and 4.57% from Magdalena Medio, with nine and ten species each. The dominant species in the Andean area were Rhodnius prolixus and Triatoma dimidiata while in Magdalena Medio they were Rhodnius pallescens and Panstrongylus geniculatus. We found a greater diversity and richness in Middle Magdalena compared to the Andean area. The temporal variation showed persistence of communities over time. Conclusions: Results revealed differences in the diversity of the two regions and the potential of wild species to occupy artificial ecotopes. Triatomines intrusion and the recent involvement of wild species in the transmission of Trypanosoma cruzi emphasize the need to further investigate the ecology of these vectors in order to guide population control strategies.

The mitochondrial genomes of Culex tritaeniorhynchus and Culex pipiens pallens (Diptera: Culicidae) and comparison analysis with two other Culex species.

BACKGROUND: Culex tritaeniorhynchus and Culex pipiens pallens are the major vectors of the Japanese encephalitis virus and Wuchereria bancrofti, the causative agent of filariasis. The knowledge of mitochondrial genomes has been widely useful for the studies on molecular evolution, phylogenetics and population genetics. METHODS: In this study, we sequenced and annotated the mitochondrial (mt) genomes of Cx. tritaeniorhynchus and Cx. p. pallens, and performed a comparative analysis including four known mt genomes of species of the subgenus Culex (Culex). The phylogenetic relationships of Cx. tritaeniorhynchus, Cx. p. pallens and four known Culex mt genome sequences were reconstructed by maximum likelihood based on concatenated protein-coding gene sequences. RESULTS: Culex tritaeniorhynchus and Cx. p. pallens mt genomes are 14,844 bp and 15,617 bp long, both consists of 13 PCGs, 22 tRNAs, 2 rRNAs and 1 CR (not sequenced for Cx. tritaeniorhynchus). The initiation and termination codons of PCGs are ATN and TAA, respectively, except for COI starting with TCG, and COI and COII terminated with T. tRNAs have the typical clover-leaf secondary structures except for trnS ((AGN)) that is lacking the DHU stem. 16S rRNA and 12S rRNA secondary structures were drawn for the first time for mosquito mt genomes. The control region of Cx. p. pallens mt genome is 747 bp long and with four tandem repeat structures. Phylogenetic analyses demonstrated that the mt genome of Cx. tritaeniorhynchus was significantly separated from the remaining five mt genomes of Culex spp. Culex p. pipiens, Cx. p. pallens and Cx. p. quinquefasciatus formed a monophyletic clade with Cx. p. quinquefasciatus linked in the middle of the clade, and Cx. p. pallens should have the same taxonomic level as Culex p. pipiens and Cx. p. quinquefasciatus. CONCLUSIONS: The mt genomes of Cx. tritaeniorhynchus and Cx. p. pallens share the same gene composition and order with those of two other Culex species. Culex p. pallens of the Pipiens complex should have the same taxonomic level as Culex p. pipiens and Cx. p. quinquefasciatus investigated. We enriched the Culex mt genome data and provided a reference basis for further Culex mt genome sequencing and analyses.

Comparative proteomic analysis of the saliva of the Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri triatomines reveals a high interespecific functional biodiversity.

Triatomines are hematophagous arthropods that transmit Trypanosoma cruzi and Trypanosoma rangeli. Feeding behavior and pathogen transmission is known to vary between the different species, and this characteristic is directly or indirectly dependent on the bioactive molecules of the saliva that facilitate the vector-host-parasite interaction. Here, we identify, characterize and compare the sialoproteomic (from the Greek sialo: saliva) repertoire of important species of the main triatomine genera in the Americas (Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri) to better explain this interaction through two-dimensional electrophoresis and mass spectrometry. We identified 221 proteins, 69 from R. prolixus, 100 from T. lecticularia and 52 from P. herreri. We identified high abundance molecules with a great potential to modulate host defenses and homeostasis, highlighting Nitrophorin-4 (28.7%), Salivary lipocalin-5 (65.2%) and Putative triabin (20.5%) in R. prolixus, T. lecticularia and P. herreri, respectively. We also observed that only a single hypothetical protein is shared among three species, which was not functionally categorized. This study corroborates previous findings with R. prolixus, increasing the knowledge about this species with relevant proteomic information and comparisons with the other two targets of the study, T. lecticularia and P. herreri, for which no studies are available from a proteomics perspective.