Influence of meteorological and seasonal parameters on the activity of Culicoides paraensis (Diptera: Ceratopogonidae), an annoying anthropophilic biting midge and putative vector of Oropouche Virus in Rondônia, Brazilian Amazon.

Biting midges of the genus Culicoides are insects of proven medical and veterinary importance, because of their role in the transmission of viruses, bacteria, protozoa and nematodes. Culicoides paraensis has been considered the main vector of the Oropouche Virus (OROV) in the urban cycle of the disease in the neotropics. Due to the great abundance of Culicoides spp. in the State of Rondônia and its epidemiological history of OROV, we investigated the biting activity in humans, the abundance as a function of meteorological parameters and seasonality, and the detection of OROV. Entomological collections occurred in three municipalities from Brazilian State of Rondônia: Porto Velho, Ariquemes, and Ouro Preto do Oeste. GLMM's were used to determine if Culicoides spp. abundance was predicted by seasonal, diurnal, and meteorological factors. Total RNA was extracted from insects and viral RNA detection was performed using the S segment as the target region of OROV via RT-qPCR. In total, 7315 individuals were captured and identified as C. paraensis. In the dry season, 1488 individuals (24.5%) were recorded, 4591 (75.5%) in the rainy season, with peaks of biting activity between 4pm and 6pm. All variables showed a significative effect on the midge abundance. The rainy season, temperature between 30 °C and 32 °C and relative air humidity between 75% and 85% were the main predictive parameters for capturing the highest average number of insects. Our results confirm diurnal activity of C. paraensis and its greatest abundance in rainy periods. No sample was positive for the OROV, which could be explained by the virus absence in local human populations, C. paraensis as a minor vector species in the sampled localities, and probable low rate of infection of biting midges. Our findings on hourly and seasonal biting activities can provide support to intervention actions regarding vector control and surveillance of this species. This was the first study to collect and analyze biting midges in a region where human OROV cases had already been detected, but without previous information on entomovirological surveillance.

Bioaccessibility of bioactive amines in dark chocolates made with different proportions of under-fermented and fermented cocoa beans.

The influence of under-fermented (UF) cocoa (0 to 65 %) on bioactive amines in chocolate and their in vitro bioaccessibility was investigated. The same amines were found in all treatments; however, treatments were divided into two groups regarding total amines [0 & 20 % UF (34 mg/kg) and 35 to 65 % UF (17 mg/kg)] and phenolic levels [lower and higher, respectively]. Serotonin, tyramine, putrescine, cadaverine, agmatine and phenylethylamine were higher in chocolate with ≤ 20 % UF cocoa. Histamine and spermidine were not affected. Digestibility studies indicated that low levels of amines were present in the oral phase. Gastric digestion was effective in releasing tyramine, spermidine and phenylethylamine from conjugates. Serotonin and agmatine were not detected after in vitro digestion of chocolate with ≥ 35 % UF cocoa. Histamine was released during in vitro intestinal digestion. By adding different proportions of UF cocoa during chocolate production, the levels and bioaccessibility of amines can be modulated.

DNA Barcoding of genus Culicoides biting midges (Diptera: Ceratopogonidae) in the Brazilian Amazon.

Culicoides biting midges are capable to transmit Oropouche virus, Bluetongue virus and Mansonella spp. This study aimed to assess the utility of DNA barcode as an alternative method in the Culicoides species identification. The study was conducted in Jamari National Forest. Biting midges were collected using HP light traps during four months, February, April, August and October 2018. Insects were morphologically identified to the species level, and rest of the body were subjected to DNA extraction and PCR targeting a fragment of the cytochrome c oxidase subunit I (COI) gene, which were analyzed and deposited in GenBank. A phylogenetic gene tree was reconstructed using RAxML software, and the sequences were assigned at Molecular Operational Taxonomic Unit (MOTU) level by species delimitation algorithms. According to morphological approach, 18 species of 2 subgenera and 7 species groups were identified. A total of 191 new COI barcodes from 18 species were generated. Of these, fifteen species have been deposited for the first time in all datasets in the world. These sequences allowed the correct identification of 188 and 187 specimens according to the BM and BCM criteria, respectively. The intraspecific genetic distances ranged from 0 to 16.5%, while the interspecific ones ranged from 2.1 to 27.1%. The nominal species Culicoides glabellus and C. tetrathyris splitted into three and two MOTUs, respectively, except for mPTP, indicating a cryptic diversity in these species. Also, sequences of C. pseudodiabolicus formed two MOTUs using all algorithms, except for PTP and ABGD, suggesting the existence of two potential species. In contrast, some barcodes of C. quasiparaensis and C. paraensis merged into a single MOTU, which can be explained by the complex characteristics of the paraensis group, since these species have similar morphological characters. Here, we provided the first COI barcodes for biting midges in Rondônia and Brazil, and demonstrates that these are sufficient to discriminate between some species.

DNA-based blood meal analysis of Culicoides (Diptera: Ceratopogonidae) species from Jamari National Forest, Southwestern Amazon, Brazil.

Culicoides biting midges are insects involved in the transmission of filarial nematodes, protozoans, and viruses. Greater knowledge of Culicoides blood meal sources could improve our understanding of parasite transmission cycles. Our study used molecular tools to evaluate the blood meal sources of Culicoides biting midges from sylvatic environments. This study was conducted in Jamari National Forest, Rondônia, Brazil. Culicoides were captured using HP light traps positioned at ground level (1.5 m above ground) and in canopy (15 m above ground). To identify blood meal sources, females were subjected to DNA extraction and PCR targeting the cytb gene fragment, and the obtained sequences were analyzed and compared with sequences from GenBank. DNA extraction and PCR were performed on 455 Culicoides females, and blood meal sources were identified in 186 females. Thirty Culicoides specimens were collected from the Potosi trail and 156 were collected from the Santa Maria trail. A total of 22 species were captured; all 22 species were collected in canopy (100%) but only three species were collected at ground level (13.6%). The cytb fragment was amplified in 162 of 186 samples. Sample sequencing identified cytb DNA from nine blood-meal sources: Pauxi sp., Psophia viridis, Ramphastos tucanus tucanus, Choloepus didactylus, Choloepus hoffmanni, Tamandua tetradactyla, Ateles chamek, Homo sapiens and Pithecia irrorata. We observed that several different blood meal sources were utilized by a high diversity of Culicoides species. The abundance of Culicoides in the canopy may be related to the fact that the majority of blood meal hosts feed in treetops. We observed that C. (Hoffmania) sp. and C. coutinhoi tend to be more generalist, feeding on a range of mammals and piciform, gruiform and galliform birds. This data improves our knowledge of the feeding profile of biting midges from forest environments and should serve as a future basis for defining zoonotic transmission cycles.