Search

Abstract Background Dengue is an emerging disease in Argentina due to the colonization of Aedes aegypti, the mosquito vector. Buenos Aires Province is the biggest and most populated district in Argentina, suffering dengue outbreaks of growing magnitude. During epidemic periods, pyrethroid insecticides are used in this country to control adult mosquitoes. Pyrethroid resistance in dengue vectors has been reported worldwide, making it necessary to implement resistance management strategies. The voltage-gated sodium channel is the target site of pyrethroids. Mutations in the gene encoding this protein, called kdr mutations, are usually the molecular cause of pyrethroid resistance in insects. In Ae. aegypti from the Americas, three kdr substitutions were described: V410L, V1016I, and F1534C. The diagnostic of kdr mutations is recommended for the early detection of pyrethroid resistance as well as the consequent planning of evidence-based control policies. Methods We distributed ovitraps across 16 localities in Buenos Aires Province, collecting 22,123 eggs. A total of 522 mosquitoes were genotyped in positions 1016 and 1534 of voltage-gated channel using multiplex high-resolution melting and/or TaqMan probe methods. A subset of 449 samples was also genotyped by a singleplex high-resolution melting method developed ad hoc and/or Sanger sequencing. Results We have documented, for the first time to our knowledge in the central region of Argentina, the presence of the 1016Ikdr + 1534Ckdr allele. Additionally, our study reports the first identification of the V410L mutation in central Argentina. These results underscore a growing trend of pyrethroid resistance in Ae. aegypti, fueled by the widespread use of these insecticides. Conclusions We detected 1016Ikdr + 1534Ckdr and 410Lkdr mutations in central Argentina for the first time and improved the processivity and accuracy of kdr genotyping methods. The results are both a tool for resistance monitoring and a sign of alarm to direct efforts towards finding sustainable methods for vector control to complement or replace pyrethroids. Joint efforts between academia and authorities to develop and implement public policies for vector control are a productive way to transfer scientific results for their application in public health. Graphical Abstract

Abstract 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.

Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.