Comparing iDNA from mosquitoes and flies to survey mammals in a semi-controlled Neotropical area.

Ingested-derived DNA (iDNA) from insects represents a powerful tool for assessing vertebrate diversity because insects are easy to sample, have a diverse diet and are widely distributed. Because of these advantages, the use of iDNA for detecting mammals has gained increasing attention. Here we aimed to compare the effectiveness of mosquitoes and flies to detect mammals with a small sampling effort in a semi-controlled area, a zoo that houses native and non-native species. We compared mosquitoes and flies regarding the number of mammal species detected, the amount of mammal sequence reads recovered, and the flight distance range for detecting mammals. We also verified if the combination of two mini-barcodes (12SrRNA and 16SrRNA) would perform better than either mini-barcode alone to inform local mammal biodiversity from iDNA. To capture mosquitoes and flies, we distributed insect traps in eight sampling points during 5 days. We identified 43 Operational Taxonomic Units from 10 orders, from the iDNA of 17 mosquitoes and 46 flies. There was no difference in the number of species recovered per individual insect between mosquitoes and flies, but the number of flies captured was higher, resulting in more mammal species recovered by flies. Eight species were recorded exclusively by mosquitoes and 20 by flies, suggesting that using both samplers would allow a more comprehensive screening of the biodiversity. The maximum distance recorded was 337 m for flies and 289 m for mosquitoes, but the average range distance did not differ between insect groups. Our assay proved to be efficient for mammal detection, considering the high number of species detected with a reduced sampling effort.

Publishing data to support the fight against human vector-borne diseases.

Vector-borne diseases are responsible for more than 17% of human cases of infectious diseases. In most situations, effective control of debilitating and deadly vector-bone diseases (VBDs), such as malaria, dengue, chikungunya, yellow fever, Zika and Chagas requires up-to-date, robust and comprehensive information on the presence, diversity, ecology, bionomics and geographic spread of the organisms that carry and transmit the infectious agents. Huge gaps exist in the information related to these vectors, creating an essential need for campaigns to mobilise and share data. The publication of data papers is an effective tool for overcoming this challenge. These peer-reviewed articles provide scholarly credit for researchers whose vital work of assembling and publishing well-described, properly-formatted datasets often fails to receive appropriate recognition. To address this, GigaScience's sister journal GigaByte partnered with the Global Biodiversity Information Facility (GBIF) to publish a series of data papers, with support from the Special Programme for Research and Training in Tropical Diseases (TDR), hosted by the World Health Organisation (WHO). Here we outline the initial results of this targeted approach to sharing data and describe its importance for controlling VBDs and improving public health.

Culicidae (Diptera: Culicomorpha) in the southern Brazilian collection "Ana Leuch Lozovei" with notes on distribution and diversity

Biological collections are of extreme importance in acquiring knowledge of the biodiversity of a specific environment. In this article, we organise, list and catalogue the adult specimens belonging to the family Culicidae housed in the Parasitology Collection of the Basic Pathology Department at the Federal University of Paraná, southern Brazil. To this end, a data bank was created, containing information on the taxonomy and collecting of each sample. The culicids were collected using different methodologies in 18 municipalities in the state of Paraná, between 1967 and 1999. There are 5,739 catalogued specimens of which 4,704 (81.96%) are identified at a specific level, with a diversity of 205 species. Of these, 18 are new recorded samples for the state of Paraná and 3 for Brazil. This being the case, we propose, in honour of the 30 years dedicated to the study of culicids in the state of Paraná, the creation of the Ana Leuch Lozovei Entomological Collection, which is composed of insect vectors or potential vectors, of agents that cause diseases in humans. 

Culicidae (Diptera: Culicomorpha) in the southern Brazilian 'Ana Leuch Lozovei' collection, with notes on distribution and diversity.

Biological collections are important for acquiring knowledge of the biodiversity of a specific environment. Here, we organize, list and catalog adult specimens of Culicidae from the Parasitology Collection of the Basic Pathology Department at the Federal University of Paraná, Brazil, and present a databank of taxonomic and collection information for each sample. Culicids were collected using different methodologies in 18 municipalities in Paraná state, between 1967 and 1999. There are 5,739 catalogued specimens, of which 4,703 (81.94%) are identified at species level, with a diversity of 100 species. Of these, 18 are new recorded samples for Paraná, and three are new for Brazil. This collection is named the Ana Leuch Lozovei Entomological Collection in honor of the 30 years Prof Lovozei dedicated to culicid study in Paraná. The collection comprises insect vectors or potential vectors, of agents causing human diseases.

VRILLE shows high divergence among Higher Diptera flies but may retain role as transcriptional repressor of clock.

In the circadian system, the clock gene vrille (vri) is an essential component of the second feedback loop, being responsible in Drosophila for the rhythmicity of the Clock (Clk) gene transcription by its repression. Here we studied vri in a fruit fly pest, the Tephritidae Anastrepha fraterculus, aimingtoinvestigate its molecular evolution and expression patterns from whole-head extracts. We used a combination of transcriptomic, genomic and gene walking strategies to sequence and characterize Afravri in male and female head transcriptomes of A. fraterculus and detected two putative isoforms that may correspond to A and D vri isoforms of Drosophila. Both isoforms produced a full-length sequence that translates to 842 amino acids. While the protein sequence showed significant divergence to orthologous sequences from other organisms, the bZIP domain was highly conserved. Molecular evolutionary analyses showed that vri in higher Diptera flies has been evolving under positive selection. A more detailed analysis showed positive selection also in Tephritidae with 29 sites evolving under positive selection in comparison with Drosophilidae. Real time expression analysis in LD and DD conditions showed cyclic expression of Afravri mRNA with oscillation opposite to AfraClk, suggesting that VRI may also behave in Anastrepha as a transcriptional repressor of Clk, providing another indication that higher Diptera might share common interlocked transcript-translation feedback loops (TTFLs) mechanisms that differ from other insects in target genes.

Effects of habitat suitability for vectors, environmental factors and host characteristics on the spatial distribution of the diversity and prevalence of haemosporidians in waterbirds from three Brazilian wetlands.

BACKGROUND: Wetlands are ecosystems in which vectors of avian haemosporidians live and reproduce and where waterbirds join to breed in colonies. Brazil has wetlands at different latitudes, which enables testing the influence of the ecological factors on the prevalence and diversity of haemosporidians. We identified avian haemosporidians in waterbird species in three wetlands and investigated the effects of vector habitat suitability, landscape and host characteristics on the diversity and prevalence of these parasites. METHODS: We created a map with the probability of occurrence of avian haemosporidian vectors using maximum-entropy modelling based on references addressing species known to be vectors of haemosporidians in birds in Brazil. We determined the prevalence and diversity index of haemosporidians in the great egret (Ardea alba) (n = 129) and roseate spoonbill (Platalea ajaja) (n = 180) and compared the findings to data for the wood stork (Mycteria americana) (n = 199). RESULTS: We report the first record of Plasmodium in the family Threskiornithidae: four lineages in the roseate spoonbill, which also presented one lineage of Haemoproteus. In the family Ardeidae, we found three Plasmodium lineages in the great egret. The similar habitat suitability for vectors found in three wetlands explains the pattern of haemosporidian diversity determined for great egret and wood stork populations. Comparisons of haemosporidian diversity within each waterbird species and between regions showed a higher level in the central-western roseate spoonbill population than in the northern population (P = 0.021). Removing the host effect, we discussed the results obtained in terms of characteristics of the Pantanal region. Comparisons of Plasmodium spp. prevalence among waterbird species within the same wetland showed higher level in roseate spoonbill (74%) than those found in the great egret (21%) and wood stork (11%). Excluding the environmental effect, we interpreted result focusing host characteristics that favour infection: time required for nestlings to be covered by feathers and migratory behaviour. CONCLUSIONS: The map of habitat suitability showed that wetlands located in a 30° latitudinal range offer similar conditions for avian vectors species and diversity of haemosporidians. The lineages described in waterbirds were previously identified in birds of prey as Plasmodium paranucleophilum.

Evidence of Adaptive Evolution and Relaxed Constraints in Sex-Biased Genes of South American and West Indies Fruit Flies (Diptera: Tephritidae).

Several studies have demonstrated that genes differentially expressed between sexes (sex-biased genes) tend to evolve faster than unbiased genes, particularly in males. The reason for this accelerated evolution is not clear, but several explanations have involved adaptive and nonadaptive mechanisms. Furthermore, the differences of sex-biased expression patterns of closely related species are also little explored out of Drosophila. To address the evolutionary processes involved with sex-biased expression in species with incipient differentiation, we analyzed male and female transcriptomes of Anastrepha fraterculus and Anastrepha obliqua, a pair of species that have diverged recently, likely in the presence of gene flow. Using these data, we inferred differentiation indexes and evolutionary rates and tested for signals of selection in thousands of genes expressed in head and reproductive transcriptomes from both species. Our results indicate that sex-biased and reproductive-biased genes evolve faster than unbiased genes in both species, which is due to both adaptive pressure and relaxed constraints. Furthermore, among male-biased genes evolving under positive selection, we identified some related to sexual functions such as courtship behavior and fertility. These findings suggest that sex-biased genes may have played important roles in the establishment of reproductive isolation between these species, due to a combination of selection and drift, and unveil a plethora of genetic markers useful for more studies in these species and their differentiation.

Evolutionary coincidence of adaptive changes in exuperantia and the emergence of bicoid in Cyclorrhapha (Diptera).

The great radiation in the infraorder Cyclorrhapha involved several morphological and molecular changes, including important changes in anterior egg development. During Drosophila oogenesis, exuperantia (exu) is critical for localizing bicoid (bcd) messenger RNA (mRNA) to the anterior region of the oocyte. Because it is phylogenetically older than bcd, which is exclusive to Cyclorrhapha, we hypothesize that exu has undergone adaptive changes to enable this new function. Although exu has been well studied in Drosophila, there is no functional or transcriptional information about it in any other Diptera. Here, we investigate exu in the South American fruit fly Anastrepha fraterculus, a Cyclorrhapha of great agricultural importance that have lost bcd, aiming to understand the evolution of exu in this infraorder. We assessed its pattern of gene expression in A. fraterculus by analyzing transcriptomes from cephalic and reproductive tissues. A combination of next-generation data with classical sequencing procedures enabled identification of the structure of exu and its alternative transcripts in this species. In addition to the sex-specific isoforms described for Drosophila, we found that not only exu is expressed in heads, but this is mediated by two transcripts with a specific 5'UTR exon-likely a result from usage of a third promoter. Furthermore, we tested the hypothesis that exu is evolving under positive selection in Cyclorrhapha after divergence from lower Diptera. We found evidence of positive selection at two important exu domains, EXO-like and SAM-like, both involved with mRNA binding during bcd mRNA localization in Drosophila, which could reflect its cooptation for the new function of bcd mRNA localization in Cyclorrhapha.

Differential transcriptome analysis supports Rhodnius montenegrensis and Rhodnius robustus (Hemiptera, Reduviidae, Triatominae) as distinct species.

Chagas disease is one of the main parasitic diseases found in Latin America and it is estimated that between six and seven million people are infected worldwide. Its etiologic agent, the protozoan Trypanosoma cruzi, is transmitted by triatomines, some of which from the genus Rhodnius. Twenty species are currently recognized in this genus, including some closely related species with low levels of morphological differentiation, such as Rhodnius montenegrensis and Rhodnius robustus. In order to investigate genetic differences between these two species, we generated large-scale RNA-sequencing data (consisting of four RNA-seq libraries) from the heads and salivary glands of males of R. montenegrensis and R. robustus. Transcriptome assemblies produced for each species resulted in 64,952 contigs for R. montenegrensis and 70,894 contigs for R. robustus, with N50 of approximately 2,100 for both species. SNP calling based on the more complete R. robustus assembly revealed 3,055 fixed interspecific differences and 216 transcripts with high levels of divergence which contained only fixed differences between the two species. A gene ontology enrichment analysis revealed that these highly differentiated transcripts were enriched for eight GO terms related to AP-2 adaptor complex, as well as other interesting genes that could be involved in their differentiation. The results show that R. montenegrensis and R. robustus have a substantial quantity of fixed interspecific polymorphisms, which suggests a high degree of genetic divergence between the two species and likely corroborates the species status of R. montenegrensis.

Head Transcriptomes of Two Closely Related Species of Fruit Flies of the Anastrepha fraterculus Group Reveals Divergent Genes in Species with Extensive Gene Flow.

Several fruit flies species of the Anastrepha fraterculus group are of great economic importance for the damage they cause to a variety of fleshy fruits. Some species in this group have diverged recently, with evidence of introgression, showing similar morphological attributes that render their identification difficult, reinforcing the relevance of identifying new molecular markers that may differentiate species. We investigated genes expressed in head tissues from two closely related species: A. obliqua and A. fraterculus, aiming to identify fixed single nucleotide polymorphisms (SNPs) and highly differentiated transcripts, which, considering that these species still experience some level of gene flow, could indicate potential candidate genes involved in their differentiation process. We generated multiple libraries from head tissues of these two species, at different reproductive stages, for both sexes. Our analyses indicate that the de novo transcriptome assemblies are fairly complete. We also produced a hybrid assembly to map each species' reads, and identified 67,470 SNPs in A. fraterculus, 39,252 in A. obliqua, and 6386 that were common to both species. We identified 164 highly differentiated unigenes that had a mean interspecific index ([Formula: see text]) of at least 0.94. We selected unigenes that had Ka/Ks higher than 0.5, or had at least three or more highly differentiated SNPs as potential candidate genes for species differentiation. Among these candidates, we identified proteases, regulators of redox homeostasis, and an odorant-binding protein (Obp99c), among other genes. The head transcriptomes described here enabled the identification of thousands of genes hitherto unavailable for these species, and generated a set of candidate genes that are potentially important to genetically identify species and understand the speciation process in the presence of gene flow of A. obliqua and A. fraterculus.

Reference genes for accessing differential expression among developmental stages and analysis of differential expression of OBP genes in Anastrepha obliqua.

The West Indian fruit fly, Anastrepha obliqua, is an important agricultural pest in the New World. The use of pesticide-free methods to control invasive species such as this reinforces the search for genes potentially useful in their genetic control. Therefore, the study of chemosensory proteins involved with a range of responses to the chemical environment will help not only on the understanding of the species biology but may also help the development of environmentally friendly pest control strategies. Here we analyzed the expression patterns of three OBP genes, Obp19d_2, Obp56a and Obp99c, across different phases of A. obliqua development by qPCR. In order to do so, we tested eight and identified three reference genes for data normalization, rpl17, rpl18 and ef1a, which displayed stability for the conditions here tested. All OBPs showed differential expression on adults and some differential expression among adult stages. Obp99c had an almost exclusive expression in males and Obp56a showed high expression in virgin females. Thereby, our results provide relevant data not only for other gene expression studies in this species, as well as for the search of candidate genes that may help in the development of new pest control strategies.

Analysis of cycle gene expression in Aedes aegypti brains by in situ hybridization.

Even though the blood-sucking mosquito Aedes aegypti is one of the most important disease vectors, relatively little is known about the molecular mechanisms underlying processes involved in the temporal pattern of its activity and host seeking behavior. In this study, we analyzed the expression of the cycle (cyc) gene, one of the core components of the circadian clock, in Ae. aegypti brains by in situ hybridization at two different time points in light-dark conditions and compared the results with those obtained using a quantitative PCR assay (qPCR). Within the brain, differential labeling was detected according to distinct areas empirically pre-defined. Six out of seven of these areas showed significantly higher staining at ZT3 (three hours after light-on) compared to ZT11 (one before light-off), which is consistent with the qPCR data. Predominant staining was observed in three of those areas which correspond to positions of the optical and antennal lobes, as well as the region where the neurons controlling activity rhythms are presumably localized.

Differential testicular gene expression in seasonal fertility.

Spermatogenesis is an essential precursor for successful sexual reproduction. Recently, there has been an expansion in the knowledge of the genes associated with particular stages of normal, physiological testicular development and pubertal activation. What has been lacking, however, is an understanding of those genes that are involved in specifically regulating sperm production, rather than in maturation and elaboration of the testis as an organ. By using the reversible (seasonal) fertility of the Syrian hamster as a model system, the authors sought to discover genes that are specifically involved in turning off sperm production and not involved in tissue specification and/or maturation. Using gene expression microarrays and in situ hybridization in hamsters and genetically infertile mice, the authors have identified a variety of known and novel factors involved in reversible, transcriptional, translational, and posttranslational control of testicular function, as well those involved in cell division and macromolecular metabolism. The novel genes uncovered could be potential targets for therapies against fertility disorders.

Reproductive and post-embryonic daily rhythm patterns of the malaria vector Anopheles (Kerteszia) cruzii: aspects of the life cycle.

Females of Anopheles (Kerteszia) cruzii, a sporadic malaria vector in some areas of the Atlantic Forest in south and southeastern Brazil, were captured and studied under controlled conditions. In the laboratory, daily observations were conducted in natural light-dark cycles at 25.1+/-0.6 degrees C and relative humidity 57-81%. Post-embryonic development, which comprises four larval instars and the pupa, was continuously observed, and its cycles, as well as temporal components of reproduction, were registered. A preliminary study on female longevity was also performed. Oviposition, ecdysis from the third and fourth instars larvae, and pupation were visually monitored over three consecutive days and the emergence of adults over four consecutive days. Results were analyzed by circular statistics, and the null hypothesis of the absence of rhythm was assessed by Rayleigh's test at the 5% significance level. From a total of 141 females captured, 113 (80.14%) survived and 79 (69.91%) were successfully fed on blood, offered at one of two time intervals, 09:00-10:30 h (morning) or 18:30-20:00 h (evening). A total of 36 females laid 1063 eggs in 65 oviposition episodes, and 18 females presented fragmented oviposition. The average duration from egg-laying until adult emergence was 30.71+/-3.57 days, the larval stage being the longest in the post-embryonic development. Egg-laying showed a daily rhythm, with a peak at 23:24+/-3:47 h, 2 to 5 h after sunset. The time of the blood meal did not shift the phase of the egg-laying rhythm. The last larval ecdysis, pupation, and adult emergence did not follow a 24 h rhythmic pattern. A description of temporal patterns of post-embryonic development, particularly in the case of vectors, can be an important tool in research to determine methods of control.

Development of bioprocess for the production of purified protein derivative with Brazilian strains of Mycobacterium tuberculosis for diagnosis use.

Tuberculin, a purified protein derivative (PPD), when diluted in adequate concentration it is utilized for an early detection and to control tuberculosis. In Brazil, the PPD is imported and distributed by the Health System in all Brazilian regions. This, in addition to eventual delay in delivery caused by the legal import process, makes difficult the access of the product to poor communities from distant places as Brazil is a geographically a large country. Thus, indigenous production of PPD would be very beneficial for the society. This work was undertaken with a view to initiate studies towards the development of an indigenous technology for PPD production, using the strains of Mycobacterium tuberculosis isolated from patients during the course of disease from several regions of Brazil. The strain selection criteria for PPD production were the sequencing of three immunodominant proteins and the genetic differentiation by DNA fingerprinting and grouped by UPGMA program, the capacity of protein production in liquid medium, and finally the intradermal injection tests used on animal model. Compared to gold standard, the PPD showed similar indurations when tested individually, and better results were obtained when products were combined in pool. These strategies are discussed in detail in this research.

Eggs fertility and biological aspects of the life cycle in natural populations of Simulium (Diptera, Simuliidae)

Eggs fertility rate and some biological aspects of the immature forms were studied on six Simulium species from ovipositions collected in natural breeding places located in rural area. The fertility was considerably high. For a total of 213 ovipositions containing 74,906 eggs, the average fertility rate was 99.13 percent with variation from 94 to 100 percent. The developmental cycle of the immature forms was observed from egg eclosion to adult emergence under temperature from 16.5 to 28°C. In S. orbitale the life cycle duration was 30 to 32 days, for S. perflavum 32 to 35 days, for S. subnigrum 33 to 39 days, for S. inaequale 41 to 47 days, for S. pertinax 42 to 48 days, and finally, the longest duration was observed for S. distinctum to range 48 to 49 days.

Foram estudados o índice de fertilidade de ovos de espécies de Simulium e o ciclo biológico das fases aquáticas em posturas trazidas de criadouro natural localizado na área rural. A fertilidade de ovos mostrou-se altíssima, das 213 posturas coletadas, contendo 74.906 ovos, o índice médio percentual geral foi de 99,13 por cento. Entre as diferentes posturas, a variação oscilou de 94 por cento a 100 por cento. O ciclo biológico a partir da incubação do ovo até a emergência de adulto em Simulium orbitale foi de 30 a 32 dias; Simulium perflavum de 32 a 35 dias; Simulium subnigrum de 33 a 39 dias, Simulium inaequale de 41 a 47 dias; Simulium pertinax de 42 a 48 dias e Simulium distinctum de 48 a 49 dias em temperaturas com variação de 16,5°C a 28°C.