Effects of Carbohydrate Intake on Anopheles darlingi and Anopheles deaneorum Fitness under Lab-Reared Conditions.

The maintenance of a highly productive colony of anopheline mosquitoes requires standardized methods in order to obtain a sufficient number of homogeneous individuals for malaria research. In this context, nutritional status may affect survival, fecundity, and the capacity to support pathogen development. Here we assess the effects of carbohydrate sources on fecundity, survival, and susceptibility to Plasmodium vivax infection in colonies of Anopheles darlingi and Anopheles deaneorum mosquitoes. Newly emerged females from each species were fed either 10% sugar or 15% honey solutions until the end of each experiment. The type of carbohydrate meal did not impact any entomological parameters for An. deaneorum, except for survival. For both species, honey meal significantly increased median survival post-emergence by three to four days, probably due to its nutritional value. For An. darlingi fed with honey, a higher mean frequency in stage 5 was observed at 48 h post-blood-meal, which could indicate a delay in the digestion process. However, no effects on fecundity parameters were observed. Regarding susceptibility, An. darlingi fed with sugar exhibited a low intensity of sporozoites, although any negative effects of sucrose on sporozoites invasions in the salivary glands are unknown. Based on the increase in mosquito survival, a carbohydrate source composed of 15% honey solution could be better for maintaining An. darlingi and An. deaneorum in the lab-rearing context.

Optimization of Plasmodium vivax infection of colonized Amazonian Anopheles darlingi.

Obtaining Plasmodium vivax sporozoites is essential for in vitro culture of liver stage parasites, not only to understand fundamental aspects of parasite biology, but also for drug and vaccine development. A major impediment to establish high-throughput in vitro P. vivax liver stage assays for drug development is obtaining sufficient numbers of sporozoites. To do so, female anopheline mosquitoes have to be fed on blood from P. vivax-infected patients through an artificial membrane-feeding system, which in turns requires a well-established Anopheles colony. In this study we established conditions to provide a robust supply of P. vivax sporozoites. Adding a combination of serum replacement and antibiotics to the membrane-feeding protocol was found to best improve sporozoite production. A simple centrifugation method appears to be a possible tool for rapidly obtaining purified sporozoites with a minimal loss of yield. However, this method needs to be better defined since sporozoite viability and hepatocyte infection were not evaluated.

Transmission-blocking activity of antimalarials for Plasmodium vivax malaria in Anopheles darlingi.

Malaria is caused by parasite of the genus Plasmodium and is still one of the most important infectious diseases in the world. Several biological characteristics of Plasmodium vivax contribute to the resilience of this species, including early gametocyte production, both of which lead to efficient malaria transmission to mosquitoes. This study evaluated the impact of currently used drugs on the transmission of P. vivax. Participants received one of the following treatments for malaria: i) chloroquine [10 mg/kg on day 1 and 7.5 mg/kg on day 2 and 3] co-administered with Primaquine [0.5 mg/kg/day for 7 days]; ii) Chloroquine [10 mg/kg on day 1 and 7.5 mg/kg on day 2 and 3] co-administered with one-dose of Tafenoquine [300 mg on day 1]; and iii) Artesunate and Mefloquine [100 mg and 200 mg on day 1, 2 and 3] co-administered with Primaquine [0.5 mg/kg/day for 14 days]. Patient blood was collected before treatment and 4 h, 24 h, 48 h and 72 h after treatment. The blood was used to perform a direct membrane feeding assay (DMFA) using Anopheles darlingi mosquitoes. The results showed 100% inhibition of the mosquito infection after 4 h using ASMQ+PQ, after 24 h for the combination of CQ+PQ and 48 h using CQ+TQ. The density of gametocytes declined over time in all treatment groups, although the decline was more rapid in the ASMQ+PQ group. In conclusion, it was possible to demonstrate the transmission-blocking efficacy of the malaria vivax treatment and that ASMQ+PQ acts faster than the two other treatments.

Novel Transmission-Blocking Antimalarials Identified by High-Throughput Screening of Plasmodium berghei Ookluc.

Safe and effective malaria transmission-blocking chemotherapeutics would allow a community-level approach to malaria control and eradication efforts by targeting the mosquito sexual stage of the parasite life cycle. However, only a single drug, primaquine, is currently approved for use in reducing transmission, and drug toxicity limits its widespread implementation. To address this limitation in antimalarial chemotherapeutics, we used a recently developed transgenic Plasmodium berghei line, Ookluc, to perform a series of high-throughput in vitro screens for compounds that inhibit parasite fertilization, the initial step of parasite development within the mosquito. Screens of antimalarial compounds, approved drug collections, and drug-like molecule libraries identified 185 compounds that inhibit parasite maturation to the zygote form. Seven compounds were further characterized to block gametocyte activation or to be cytotoxic to formed zygotes. These were further validated in mosquito membrane-feeding assays using Plasmodium falciparum and P. vivax. This work demonstrates that high-throughput screens using the Ookluc line can identify compounds that are active against the two most relevant human Plasmodium species and provides a list of compounds that can be explored for the development of new antimalarials to block transmission.

Evaluation of sustainable susceptibility to Plasmodium vivax infection among colonized Anopheles darlingi and Anopheles deaneorum.

BACKGROUND: The colonization of mosquitoes susceptible to Plasmodium vivax via direct membrane feeding assay (DMFA) has the potential to significantly advance our knowledge of P. vivax biology, vector-parasite interaction and transmission-blocking vaccine research. Anopheles darlingi and Anopheles deaneorum are important vectors of malaria in the Western Brazilian Amazon. Since 2018, well-established colonies of these species have been maintained in order to mass produce mosquitoes destined for P. vivax infection. Plasmodium susceptibility was confirmed when the colonies were established, but susceptibility needs to be maintained for these colonies to remain good models for pathogen transmission. Thus, the susceptibility was assessed of colonized mosquitoes to P. vivax isolates circulating in the Western Amazon. METHODS: Laboratory-reared mosquitoes from F10-F25 generations were fed on P. vivax blood isolates via DMFA. Susceptibility was determined by prevalence and intensity of infection as represented by oocyst load seven days after blood feeding, and sporozoite load 14 days after blood feeding. The effect of infection on mosquito survival was evaluated from initial blood feeding until sporogonic development and survival rates were compared between mosquitoes fed on infected and uninfected blood. Correlation was calculated between gametocytaemia and prevalence/intensity of infection, and between oocyst and sporozoite load. RESULTS: Significant differences were found in prevalence and intensity of infection between species. Anopheles darlingi showed a higher proportion of infected mosquitoes and higher oocyst and sporozoite intensity than An. deaneorum. Survival analysis showed that An. deaneorum survival decreased drastically until 14 days post infection (dpi). Plasmodium vivax infection decreased survival in both species relative to uninfected mosquitoes. No correlation was observed between gametocytaemia and prevalence/intensity of infection, but oocyst and sporozoite load had a moderate to strong correlation. CONCLUSIONS: Colonized An. darlingi make excellent subjects for modelling pathogen transmission. On the other hand, An. deaneorum could serve as a model for immunity studies due the low susceptibility under current colonized conditions. In the application of DMFA, gametocyte density is not a reliable parameter for predicting mosquito infection by P. vivax, but oocyst intensity should be used to schedule sporozoite experiments.

Anopheles darlingi polytene chromosomes: revised maps including newly described inversions and evidence for population structure in Manaus.

Salivary gland polytene chromosomes of 4th instar Anopheles darlingi Root were examined from multiple locations in the Brazilian Amazon. Minor modifications were made to existing polytene photomaps. These included changes to the breakpoint positions of several previously described paracentric inversions and descriptions of four new paracentric inversions, two on the right arm of chromosome 3 and two on the left arm of chromosome 3 that were found in multiple locations. A total of 18 inversions on the X (n = 1) chromosome, chromosome 2 (n = 7) and 3 (n = 11) were scored for 83 individuals from Manaus, Macapá and Porto Velho municipalities. The frequency of 2Ra inversion karyotypes in Manaus shows significant deficiency of heterozygotes (p < 0.0009). No significant linkage disequilibrium was found between inversions on chromosome 2 and 3. We hypothesize that at least two sympatric subpopulations exist within the An. darlingi population at Manaus based on inversion frequencies.

Anopheles darlingi polytene chromosomes: revised maps including newly described inversions and evidence for population structure in Manaus

Salivary gland polytene chromosomes of 4th instar Anopheles darlingi Root were examined from multiple locations in the Brazilian Amazon. Minor modifications were made to existing polytene photomaps. These included changes to the breakpoint positions of several previously described paracentric inversions and descriptions of four new paracentric inversions, two on the right arm of chromosome 3 and two on the left arm of chromosome 3 that were found in multiple locations. A total of 18 inversions on the X (n = 1) chromosome, chromosome 2 (n = 7) and 3 (n = 11) were scored for 83 individuals from Manaus, Macapá and Porto Velho municipalities. The frequency of 2Ra inversion karyotypes in Manaus shows significant deficiency of heterozygotes (p < 0.0009). No significant linkage disequilibrium was found between inversions on chromosome 2 and 3. We hypothesize that at least two sympatric subpopulations exist within the An. darlingi population at Manaus based on inversion frequencies.

A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes.

BACKGROUND: Psorophora mosquitoes are exclusively found in the Americas and have been associated with transmission of encephalitis and West Nile fever viruses, among other arboviruses. Mosquito salivary glands represent the final route of differentiation and transmission of many parasites. They also secrete molecules with powerful pharmacologic actions that modulate host hemostasis, inflammation, and immune response. Here, we employed next generation sequencing and proteome approaches to investigate for the first time the salivary composition of a mosquito member of the Psorophora genus. We additionally discuss the evolutionary position of this mosquito genus into the Culicidae family by comparing the identity of its secreted salivary compounds to other mosquito salivary proteins identified so far. RESULTS: Illumina sequencing resulted in 13,535,229 sequence reads, which were assembled into 3,247 contigs. All families were classified according to their in silico-predicted function/ activity. Annotation of these sequences allowed classification of their products into 83 salivary protein families, twenty (24.39%) of which were confirmed by our subsequent proteome analysis. Two protein families were deorphanized from Aedes and one from Ochlerotatus, while four protein families were described as novel to Psorophora genus because they had no match with any other known mosquito salivary sequence. Several protein families described as exclusive to Culicines were present in Psorophora mosquitoes, while we did not identify any member of the protein families already known as unique to Anophelines. Also, the Psorophora salivary proteins had better identity to homologs in Aedes (69.23%), followed by Ochlerotatus (8.15%), Culex (6.52%), and Anopheles (4.66%), respectively. CONCLUSIONS: This is the first sialome (from the Greek sialo = saliva) catalog of salivary proteins from a Psorophora mosquito, which may be useful for better understanding the lifecycle of this mosquito and the role of its salivary secretion in arboviral transmission.

Importância do método de gota espessa de sangue no diagnóstico de filárias simpátricas no Amazonas, Brasil / The importance of thick blood film method for sympatric filarial diagnosis in Amazon basin

Esse trabalho relata os métodos de diagnósticos para filárias humanas no sangue, referindo-se a importância do método de gota espessa em relação aos métodos de filtração em membrana de policarbonato e Knott na diferenciação das espécies de microfilárias, principalmente em áreas de ocorrência de mais de uma espécie, como em regiões do Amazonas. Lâminas com microfilárias de Mansonella ozzardi foram montadas e fotografadas pelos diferentes métodos de diagnósticos. O método da gota espessa de sangue é o mais confiável, pois permite visualizar com nitidez o espaço cefálico e caudal, disposição dos núcleos caudais e formato da cauda, que são características morfológicas que diferenciam as espécies de microfilárias sanguíneas que ocorrem no Amazonas.

In this work, the visual diagnostic methods for human filarias in the blood are evaluated. The thick blood film method is more faithful to identify the microfilariae species if compared to policarbonate membrane filtration and Knott methods, especially in areas where more than one species occur, as in Amazon basin. Slides with Mansonella ozzardi microfilariae were mounted and photographed by different diagnosis methods. The thick blood film method is much easier to see the diagnostic morphological characters as cephalic and caudal space, pattern of nucleation of the tail and tail shape that can separate the blood Amazonian microfilariae species.

Larval ontogeny and morphological variations of Psaroniocompsa incrustata (Lutz) (Diptera: Simuliidae) in the State of Rio Grande do Norte, Northeastern Brazil.

Psaroniocompsa incrustata (Lutz) is an antropophilic species widely distributed in Central and South America. It is the vector of Onchocerca volvulus in a Brazilian focus and has been considered a plague in several areas of this country. The objective of this study was to determine the number of larval instars and to describe the morphological variations and teratologies of a population of P. incrustata from the Pium river, Rio Grande do Norte State. The number of larval instars was determined measuring the head capsule lateral length of 3,164 larvae. The larval instars were determined using the measurement frequency distribution, Student's t-test, the Dyar and Crosby growth rules. Eight larval instars were determined for P. incrustata. A high rate of teratologies (9.6%) in the hypostomium and variations in the lateral serrations and the latero-mandibular process were found.

Larval ontogeny and morphological variations of Psaroniocompsa incrustata (Lutz) (Diptera: Simuliidae) in the State of Rio Grande do Norte, Northeastern Brazil / Ontogenia larval e variações morfológicas de Psaroniocompsa incrustata (Lutz) (Diptera: Simuliidae) do Rio Grande do Norte

Psaroniocompsa incrustata (Lutz) is an antropophilic species widely distributed in Central and South America. It is the vector of Onchocerca volvulus in a Brazilian focus and has been considered a plague in several areas of this country. The objective of this study was to determine the number of larval instars and to describe the morphological variations and teratologies of a population of P. incrustata from the Pium river, Rio Grande do Norte State. The number of larval instars was determined measuring the head capsule lateral length of 3,164 larvae. The larval instars were determined using the measurement frequency distribution, Student's t-test, the Dyar and Crosby growth rules. Eight larval instars were determined for P. incrustata. A high rate of teratologies (9.6 percent) in the hypostomium and variations in the lateral serrations and the latero-mandibular process were found.

Psaroniocompsa incrustata (Lutz) é uma espécie antropofílica amplamente distribuída na América Central e do Sul. No foco brasileiro de oncocercose é considerada vetora de Onchocerca volvulus, sendo praga em outras regiões do país. Este estudo teve por objetivos determinar o número de estádios larvais e descrever as variações morfológicas e teratologias de uma população de P. incrustata do rio Pium, Rio Grande do Norte. O número de estádios larvais dessa espécie foi determinado medindo o comprimento lateral da cápsula cefálica de 3.164 larvas. Os estádios larvais foram determinados usando distribuição de frequências, teste-t de Student, regra de Dyar e de crescimento de Crosby. Foram identificados oito estádios larvais para P. incrustata. Foi encontrada uma elevada taxa de teratologias (9,6 por cento) no hipostômio, variações nas serrações laterais e no processo látero-mandibular.

[First record of simuliidae (Diptera) with pollinaria of asclepiadoideae (Apocynaceae) attached]. / Primeiro registro de simuliidae (Diptera) com polinários de asclepiadoideae (Apocynaceae).

The presence of pollinaria of two species of Asclepiadoideae (Apocynaceae), possibly Tassadia cf. martiana Decne. and T. cf. obovata Decne., attached to the mouth parts of simulid black flies [Cerqueirellum amazonicum (Goeldi), C. argentiscutum (Shelley & Luna Dias), C. oyapockense (Floch & Abonnenc), and Cerqueirellum sp.] are reported for the first time. The frequency and distribution of simulids recorded with pollinaria suggest that removal of pollinaria by these flies is not casual. Simulids probably use nectar in flowers of Asclepiadoideae as source of sugar, being able to remove their pollinaria. This finding demonstrates that simulids are not only vector of pathogenic parasites, but also carry pollinaria, and thus may represent a group of pollinators for species of Asclepiadoideae with small flowers.

Primeiro registro de simuliidae (Diptera) com polinários de asclepiadoideae (Apocynaceae) / First record of simuliidae (Diptera) with pollinaria of asclepiadoideae (Apocynaceae) attached

Polinários de duas espécies de Asclepiadoideae (Apocynaceae), possivelmente de Tassadia cf. martiana Decne. e T. cf. obovata Decne., foram observados pela primeira vez presos ao aparelho bucal de simulídeos [Cerqueirellum amazonicum (Goeldi), C. argentiscutum (Shelley & Luna Dias), C. oyapockense (Floch & Abonnenc) e Cerqueirellum sp.]. A frequência e distribuição dos insetos observados com polinários sugerem que esse tipo de evento não é casual. Os simulídeos devem buscar néctar nas flores de Asclepiadoideae, sendo capazes de remover seus polinários. Essa descoberta demonstra que os simulídeos não carregam apenas parasitas patogênicos, mas também polinários, e assim podem representar um grupo de polinizadores de espécies de Asclepiadoideae com flores pequenas.

The presence of pollinaria of two species of Asclepiadoideae (Apocynaceae), possibly Tassadia cf. martiana Decne. and T. cf. obovata Decne., attached to the mouth parts of simulid black flies [Cerqueirellum amazonicum (Goeldi), C. argentiscutum (Shelley & Luna Dias), C. oyapockense (Floch & Abonnenc), and Cerqueirellum sp.] are reported for the first time. The frequency and distribution of simulids recorded with pollinaria suggest that removal of pollinaria by these flies is not casual. Simulids probably use nectar in flowers of Asclepiadoideae as source of sugar, being able to remove their pollinaria. This finding demonstrates that simulids are not only vector of pathogenic parasites, but also carry pollinaria, and thus may represent a group of pollinators for species of Asclepiadoideae with small flowers.

Calliphoridae (Diptera) coletados em cadáveres humanos no Rio Grande do Norte / Calliphoridae (Diptera) from human cadavers in Rio Grande do Norte State, northeastern Brazil

Este artigo relata um estudo sobre Calliphoridae no Rio Grande do Norte. Foram coletados Calliphoridae associados a cadáveres em diferentes estados de decomposição, de várias regiões do estado, recém-levados ao Instituto Técnico e Científico de Polícia de Natal. Foram coletadas as espécies: Chrysomya megacephala (Fabr.), Chrysomya albiceps (Wied.), Cochliomyia macellaria (Fabr.), Lucilia eximia (Wied.), Lucilia cuprina (Wied.) e Lucilia sp.

This paper reports a study on Calliphoridae associated to human corpus in Rio Grande do Norte State, Brazil. Collections of Calliphoridae were made in cadavers in different stages of decomposition taken to the Instituto Técnico e Científico de Polícia / Natal, from different regions of the state. Thefollowing species were collected: Chrysomya megacephala (Fabr.), Chrysomya albiceps (Wied.), Cochliomyia macellaria (Fabr.), Lucilia eximia (Wied.), Lucilia cuprina (Wied.) and Lucilia sp.