The pharyngeal taste organ of a blood-feeding insect functions in food recognition.

BACKGROUND: Obligate blood-feeding insects obtain the nutrients and water necessary to ensure survival from the vertebrate blood. The internal taste sensilla, situated in the pharynx, evaluate the suitability of the ingested food. Here, through multiple approaches, we characterized the pharyngeal organ (PO) of the hematophagous kissing bug Rhodnius prolixus to determine its role in food assessment. The PO, located antero-dorsally in the pharynx, comprises eight taste sensilla that become bathed with the incoming blood. RESULTS: We showed that these taste sensilla house gustatory receptor neurons projecting their axons through the labral nerves to reach the subesophageal zone in the brain. We found that these neurons are electrically activated by relevant appetitive and aversive gustatory stimuli such as NaCl, ATP, and caffeine. Using RNA-Seq, we examined the expression of sensory-related gene families in the PO. We identified gustatory receptors, ionotropic receptors, transient receptor potential channels, pickpocket channels, opsins, takeouts, neuropeptide precursors, neuropeptide receptors, and biogenic amine receptors. RNA interference assays demonstrated that the salt-related pickpocket channel Rproppk014276 is required during feeding of an appetitive solution of NaCl and ATP. CONCLUSIONS: We provide evidence of the role of the pharyngeal organ in food evaluation. This work shows a comprehensive characterization of a pharyngeal taste organ in a hematophagous insect.

Vitellogenin genes are transcribed in Culex quinquefasciatus ovary

BACKGROUND Culex quinquefasciatus, a cosmopolitan, domestic, and highly anthropophilic mosquito, is a vector of pathogenic arboviruses such as West Nile virus and Rift Valley virus, as well as lymphatic filariasis. The current knowledge on its reproductive physiology regarding vitellogenin expression in different tissues is still limited. OBJECTIVES In this study, we analysed the transcriptional profiles of vitellogenin genes in the fat body and ovaries of C. quinquefasciatus females during the first gonotrophic cycle. METHODS C. quinquefasciatus ovaries and/or fat bodies were dissected in different times during the first gonotrophic cycle and total RNA was extracted and used for reverse transcription polymerase chain reaction, quantitative real time-PCR, and in situ hybridisation. FINDINGS We confirmed the classical descriptions of the vitellogenic process in mosquitoes by verifying that vitellogenin genes are transcribed in the fat bodies of C. quinquefasciatus females. Using RNA in situ hybridisation approach, we showed that vitellogenin genes are also transcribed in developing ovaries, specifically by the follicle cells. MAIN CONCLUSIONS This is the first time that vitellogenin transcripts are observed in mosquito ovaries. Studies to determine if Vg transcripts are translated into proteins and their contribution to the reproductive success of the mosquito need to be further investigated.

PCR-based identification of Neospora caninum in the umbilical cord of a newborn calf in Brazil / Identificação de Neospora caninum por PCR em cordão umbilical de bezerros recém nascido no Brasil

ABSTRACT: This study was conducted at a beef cattle breeding farm in the far southern region of Brazil. The birth of a calf with unilateral corneal opacity was immediately reported to the Laboratory of Parasitology, in the Universidade Federal de Pelotas, Rio Grande do Sul, Brazil; blood was collected from the cow and calf before colostrum intake. The umbilical cords from this calf and from six other healthy animals were collected. Serological examination, utilizing an indirect fluorescent antibody test, was done using a cut-off point of 1:100. Polymerase chain reaction (PCR) was also performed using the umbilical cord samples. Serological tests showed that the calf was positive for Neospora caninum at birth, with a titer of 1:1600; a titer of 1:3200 was reported in the dam. PCR, using umbilical cord tissue from the affected animal, was positive for the presence of this parasite, and the molecular identity of the amplified product was confirmed by sequencing. Therefore, the detection of N. caninum DNA in the umbilical cord represents a novel alternative test for the diagnosis of this parasitic infection in newborn calves that are clinically suspected to have neosporosis.

RESUMO: O estudo foi realizado em uma fazenda de criação de gado de corte na região do extremo sul do Brasil. O nascimento de um bezerro com opacidade unilateral da córnea foi imediatamente relatado ao Laboratório de Parasitologia da Universidade Federal de Pelotas (RS) e coletou-se sangue da vaca e do bezerro antes da ingestão do colostro. Foram coletados sangue e cordão umbilical do bezerro com opacidade na córnea bem como de outros seis bezerros aparentemente saudáveis antes da ingestão de colostro. Foram realizados exames sorológicos utilizando o teste indireto de anticorpos fluorescentes (IFAT) com ponto de corte de 1: 100 e reação em cadeia da polimerase (PCR). O resultado dos exames sorológicos mostraram que o bezerro foi positivo para N. caninum ao nascer com titulação de 1:1600 e o de sua mãe foi de 1:3200 na IFAT. A PCR do cordão umbilical foi positiva para o agente no animal com opacidade córnea unilateral e a identidade molecular do produto amplificado foi confirmada por sequenciamento. A detecção de DNA de N. caninum no cordão umbilical é uma nova alternativa para o diagnóstico deste agente em bezerros recém-nascidos com suspeita clínica de neosporose.

Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands.

BACKGROUND: The circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration. METHODS: Peptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system. RESULTS: Transient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum. CONCLUSION: These results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.

São Paulo urban heat islands have a higher incidence of dengue than other urban areas

Urban heat islands are characterized by high land surface temperature, low humidity, and poor vegetation, and considered to favor the transmission of the mosquito-borne dengue fever that is transmitted by the Aedes aegypti mosquito. We analyzed the recorded dengue incidence in Sao Paulo city, Brazil, in 2010-2011, in terms of multiple environmental and socioeconomic variables. Geographical information systems, thermal remote sensing images, and census data were used to classify city areas according to land surface temper- ature, vegetation cover, population density, socioeconomic status, and housing standards. Of the 7415 dengue cases, a majority (93.1%) mapped to areas with land surface temperature >28 ◦ C. The dengue incidence rate (cases per 100,000 inhabitants) was low (3.2 cases) in high vegetation cover areas, but high (72.3 cases) in low vegetation cover areas where the land surface temperature was 29 ± 2 ◦ C. Interestingly, a multiple cluster analysis phenogram showed more dengue cases clustered in areas of land surface temperature >32 ◦ C, than in areas characterized as low socioeconomic zones, high population density areas, or slum-like areas. In laboratory experiments, A. aegypti mosquito larval development, blood feeding, and oviposition associated positively with temperatures of 28-32 ◦ C, indicating these temperatures to be favorable for dengue transmission. Thus, among all the variables studied, dengue incidence was most affected by the temperature.

Antiplasmodial activity study of angiotensin II via Ala scan analogs.

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid-phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide-capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile(5) or the His(6) residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala(6) ]-AII (79%), and [Ala(5) ]-AII (75%). Analogs [Ala(3) ]-AII, [Ala(1) ]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala(8) ]-AII, [Ala(7) ]-AII, [Ala(4) ]-AII, and [Ala(2) ]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a ß-fold conformation in different solutions. All AII analogs, except [Ala(4) ]-AII and [Ala(8) ]-AII, show contractile responses and interact with the AT1 receptor, [Ala(5) ]-AII and [Ala(6) ]-AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds.

Genetic lineages in the yellow fever mosquito Aedes (Stegomyia) aegypti (Diptera: Culicidae) from Peru

The yellow fever mosquito Aedes aegypti was introduced in Peru in 1852 and was considered to be eradicated in 1958. In 2001, Ae. aegypti had been recorded in 15 out of 24 Peruvian Departments. Peru has great ecological differences between the east and west sides of Andes. Because of this, we consider that Ae. aegypti populations of both east and west sides can have a genetically distinct population structure. In this study we examined genetic variability and genealogical relationships among three Ae. aegypti Peruvian populations: Lima, Piura (west Andes), and Iquitos (east Andes) using a fragment of the ND4 gene of the mitochondrial genome. Three haplotypes were detected among 55 samples. Lima and Iquitos showed the same haplotype (Haplotype I), whereas Piura has two haplotypes (Haplotype II and III). Haplotype II is four mutational steps apart from Haplotype I, while Haplotype III is 13 mutational steps apart from Haplotype I in the network. The analysis of molecular variation showed that mostly of the detected genetic variation occurs at interpopulational level. The significant value phist suggests that Piura population is structured in relation to Lima and Iquitos populations and the gene flow of the ND4 is restricted in Piura when compared to Lima and Iquitos. Genetic relationship between haplotype I and haplotype II suggests introduction of the same mtDNA lineage into those localities. However the existence of a genetically distant haplotype III also suggests introduction of at least two Ae. aegypti lineages in Peru.