Under the light: high prevalence of haemoparasites in lizards (Reptilia: Squamata) from Central Amazonia revealed by microscopy.

Blood samples from 330 lizards of 19 species were collected to investigate the occurrence of haemoparasites. Samplings were performed in areas of upland (terra-firme) forest adjacent to Manaus municipality, Amazonas, Brazil. Blood parasites were detected in 220 (66%) lizards of 12 species and comprised four major groups: Apicomplexa (including haemogregarines, piroplasms, and haemosporidians), trypanosomatids, microfilarid nematodes and viral or bacterial organisms. Order Haemosporida had the highest prevalence, with 118 (35%) animals from 11 species. For lizard species, Uranoscodon superciliosus was the most parasitised host, with 103 (87%; n = 118) positive individuals. This species also presented the highest parasite diversity, with the occurrence of six taxa. Despite the difficulties attributed by many authors regarding the use of morphological characters for taxonomic resolution of haemoparasites, our low-cost approach using light microscopy recorded a high prevalence and diversity of blood parasite taxa in a relatively small number of host species. This report is the first survey of haemoparasites in lizards in the study region. It revealed a high diversity of lizard haemoparasites and highlights the need to understand their impacts on hosts.

An overview of malaria transmission from the perspective of Amazon Anopheles vectors.

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Anopheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.

Rhabdoviral Endogenous Sequences Identified in the Leishmaniasis Vector Lutzomyia longipalpis Are Widespread in Sandflies from South America.

Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions.

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.

Experimental Plasmodium vivax infection of key Anopheles species from the Brazilian Amazon.

BACKGROUND: Anopheles darlingi is the major malaria vector in countries located in the Amazon region. Anopheles aquasalis and Anopheles albitarsis s.l. are also proven vectors in this region. Anopheles nuneztovari s.l. and Anopheles triannulatus s.l. were found infected with Plasmodium vivax; however, their status as vectors is not yet well defined. Knowledge of susceptibility of Amazon anopheline populations to Plasmodium infection is necessary to better understand their vector capacity. Laboratory colonization of An. darlingi, the main Amazon vector, has proven to be difficult and presently An. aquasalis is the only available autonomous colony. METHODS: Larvae of An. darlingi, An. albitarsis s.l., An. nuneztovari s.l. and An. triannulatus s.l. were collected in the field and reared until adult stage. Adults of An. aquasalis were obtained from a well-established colony. Mosquitoes were blood-fed using a membrane-feeding device containing infected blood from malarial patients.The infection of the distinct Anopheles species was evaluated by the impact variance of the following parameters: (a) parasitaemia density; (b) blood serum inactivation of the infective bloodmeal; (c) influence of gametocyte number on infection rates and number of oocysts. The goal of this work was to compare the susceptibility to P. vivax of four field-collected Anopheles species with colonized An. aquasalis. RESULTS: All Anopheles species tested were susceptible to P. vivax infection, nevertheless the proportion of infected mosquitoes and the infection intensity measured by oocyst number varied significantly among species. Inactivation of the blood serum prior to mosquito feeding increased infection rates in An. darlingi and An. triannulatus s.l., but was diminished in An. albitarsis s.l. and An. aquasalis. There was a positive correlation between gametocyte density and the infection rate in all tests (Z = -8.37; p < 0.001) but varied among the mosquito species. Anopheles albitarsis s.l., An. aquasalis and An. nuneztovari s.l. had higher infection rates than An. darlingi. CONCLUSION: All field-collected Anopheles species, as well as colonized An. aquasalis are susceptible to experimental P. vivax infections by membrane feeding assays. Anopheles darlingi, An. albitarsis s.l. and An. aquasalis are very susceptible to P. vivax infection. However, colonized An. aquasalis mosquitoes showed the higher infection intensity represented by infection rate and oocyst numbers. This study is the first to characterize experimental development of Plasmodium infections in Amazon Anopheles vectors and also to endorse that P. vivax infection of colonized An. aquasalis is a feasible laboratory model.

Morphological aspects of immature stages of Migonemyia migonei (Diptera: Psychodidae, Phlebotominae), an important vector of Leishmaniosis in South America, described by scanning electron microscopy.

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the etiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of the egg, all instar larvae, and the pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy have been highlighted. Different sensillary subtypes-trichoidea, basiconica, coelonica and campanoformia-were observed in the larval stages. The results presented herein contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important vector species.

Sand Fly Fauna Associated With Dwellings and Forest Habitats Along the BR-319 Highway, Amazonas, Brazil.

Roads and highways can affect the spread of insect-borne diseases by limiting or amplifying the spatiotemporal distribution of vectors, pathogens, and hosts, which can, in turn, lead to the creation of a nidus of infection. The aim of this study was to compare the diversity (richness and abundance) of phlebotomine sand flies in household and forest edge environments found along two different segments of an Amazonian highway. Sampling was conducted along the northern and southern portions of highway BR-319, in Amazonas State, Brazil. At each sampling point, Hoover Pugedo traps were set in indoor and outdoor habitats, and at forests edges, and captures were made between 06:00 pm and 06:00 am. A total of 1,189 sand flies were captured and 48 species were identified. As expected, a greater number of species and individuals were captured in forest edge environments. Permutational Multivariate Analyses of Variance (PERMANOVA) and Permutational Analyses of Multivariate Dispersions (PERMDISP) analyses showed that sand fly fauna differed significantly among habitats, but no variance in species composition was observed between the two road segments. Some of the captured species were species that have been implicated as vectors of Leishmania spp. Ross, 1903 (Kinetoplastida: Trypanosomatidae).

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification.

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.

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.