Ratio changes in blood-feeding and voltinism in black flies (Diptera: Simuliidae) over a latitudinal gradient in North America north of Mexico.

We explored how the ratio of blood-feeding behaviour-nonfeeding, bird feeding (ornithophily) and mammal feeding (mammalophily)-and voltinism (univoltine and multivoltine) vary over a latitudinal gradient from Alaska to Florida. These two fixed species traits were divided into five mutually exclusive combinations of trait states-nonfeeding/univoltine, ornithophilic/univoltine, mammalophilic/univoltine, ornithophilic/multivoltine and mammalophilic/multivoltine-within each of three datasets (north, east and west). We found a significant association between location (north, east and west) and trait state, which was driven by the large percentage of nonfeeding females in the north. When this trait state was removed, no significant differences were found for the remaining trait states and locations. Although the distribution of trait states did not differ between east and west datasets, the distribution with relation to 1° changes in latitude within each of these datasets showed distinct differences. In the east, both ornithophilic/univoltine and mammalophilic/univoltine species significantly increased with latitude, in proportion to the total species present. In contrast, the proportion of mammalophilic/multivoltine species decreased as latitude increased. Ornithophilic/multivoltine species in the east and the trait states in the west did not show any significant relationship to latitude.

Lipid Metabolism in Insect Vectors of Diseases.

According to the World Health Organization vector-borne diseases account for more than 17% of all infectious diseases, causing more than 700,000 deaths annually. Vectors are organisms that are able to transmit infectious pathogens between humans, or from animals to humans. Many of these vectors are hematophagous insects, which ingest the pathogen from an infected host during a blood meal, and later transmit it into a new host. Malaria, dengue, African trypanosomiasis, yellow fever, leishmaniasis, Chagas disease, and many others are examples of diseases transmitted by insects.Both the diet and the infection with pathogens trigger changes in many metabolic pathways, including lipid metabolism, compared to other insects. Blood contains mostly proteins and is very poor in lipids and carbohydrates. Thus, hematophagous insects attempt to efficiently digest and absorb diet lipids and also rely on a large de novo lipid biosynthesis based on utilization of proteins and carbohydrates as carbon source. Blood meal triggers essential physiological processes as molting, excretion, and oogenesis; therefore, lipid metabolism and utilization of lipid storage should be finely synchronized and regulated regarding that, in order to provide the necessary energy source for these events. Also, pathogens have evolved mechanisms to hijack essential lipids from the insect host by interfering in the biosynthesis, catabolism, and transport of lipids, which pose challenges to reproduction, survival, fitness, and other insect traits.In this chapter, we have tried to collect and highlight the current knowledge and recent discoveries on the metabolism of lipids in insect vectors of diseases related to the hematophagous diet and pathogen infection.

New artificial hematophagy system with attractive polymeric biofilm for maintenance of Culex quinquefasciatus (Diptera: Culicidae) in the laboratory.

BACKGROUND: Maintaining mosquito colonies in the laboratory requires a blood supply so that females' oocytes can mature and oviposition can take place. In this study, a new artificial hematophagy system for colonization and maintenance of Culex quinquefasciatus in the laboratory was developed and tested. METHODS: We developed an attractive polymeric biofilm including 25% L-lactic acid for use as a membrane in an artificial hematophagy system and compared the feeding rate of females with Parafilm-M®. We also evaluated the oviposition rate, larval survival and adult emergence of females fed through the attractive biofilm. RESULTS: The average percentage of female Cx. quinquefasciatus fed through the attractive biofilm was 87%, while only 20% became engorged with Parafilm-M® (p < 0.0001). Feeding through the attractive biofilm developed in this study produced high levels of evaluated biological parameters; the percentage of egg laying by females that underwent artificial hematophagy through the biofilm was 90%, with an average of 158 eggs per raft. From these eggs, 97% of the larvae hatched, of which 95% reached the pupal stage. The adult emergence rate corresponded to 93% of pupae. CONCLUSIONS: Insects fed with attractant through the biofilm system had a higher engorgement rate compared to those fed through Parafilm-M®. Our study is preliminary and suggests that polymeric biofilm has great potential for artificially feeding mosquitoes in the laboratory. Based on this research, new studies will be carried out with biofilm and different systems.

Western diet consumption by host vertebrate promotes altered gene expression on Aedes aegypti reducing its lifespan and increasing fertility following blood feeding.

BACKGROUND: The high prevalence of metabolic syndrome in low- and middle-income countries is linked to an increase in Western diet consumption, characterized by a high intake of processed foods, which impacts the levels of blood sugar and lipids, hormones, and cytokines. Hematophagous insect vectors, such as the yellow fever mosquito Aedes aegypti, rely on blood meals for reproduction and development and are therefore exposed to the components of blood plasma. However, the impact of the alteration of blood composition due to malnutrition and metabolic conditions on mosquito biology remains understudied. METHODS: In this study, we investigated the impact of whole-blood alterations resulting from a Western-type diet on the biology of Ae. aegypti. We kept C57Bl6/J mice on a high-fat, high-sucrose (HFHS) diet for 20 weeks and followed biological parameters, including plasma insulin and lipid levels, insulin tolerance, and weight gain, to validate the development of metabolic syndrome. We further allowed Ae. aegypti mosquitoes to feed on mice and tracked how altered host blood composition modulated parameters of vector capacity. RESULTS: Our findings identified that HFHS-fed mice resulted in reduced mosquito longevity and increased fecundity upon mosquito feeding, which correlated with alteration in the gene expression profile of nutrient sensing and physiological and metabolic markers as studied up to several days after blood ingestion. CONCLUSIONS: Our study provides new insights into the overall effect of alterations of blood components on mosquito biology and its implications for the transmission of infectious diseases in conditions where the frequency of Western diet-induced metabolic syndromes is becoming more frequent. These findings highlight the importance of addressing metabolic health to further understand the spread of mosquito-borne illnesses in endemic areas.

You are what you eat: a systematic review exploring the interaction between Brazilian sand flies and their vertebrate food sources

Sand flies play a crucial role as vectors of bacteria, viruses, and protists, with Leishmania being the most notable among them, transmitted to vertebrate hosts during blood feeding. Understanding the feeding behaviours of sand flies is imperative for gaining insights into their eco-epidemiological roles in the transmission of these infectious agents. This systematic review aimed to answer the question 'What are the blood-feeding sources identified in Brazilian sand flies?' to provide an analysis of their blood-feeding habits. The diverse range of at least 16 vertebrate orders identified as blood sources for 54 sand fly species across different geographic regions was summarised, and the factors potentially associated with the risk of bias in the included studies were analysed. The findings broaden the discussion concerning methods used to identify blood meal sources and shed light on the implications of sand fly feeding behaviours for the transmission dynamics of Leishmania.

Evaluation of insemination, blood feeding, and Plasmodium vivax infection effects on locomotor activity patterns of the malaria vector Anopheles darlingi (Diptera: Culicidae).

Circadian behavioral patterns in mosquitoes can be observed through their locomotor activity, which includes fundamental behaviors such as foraging, mating, and oviposition. These habits, which are fundamental to the life cycle of Anopheles mosquitoes, are closely related to pathogen transmission to humans. While rhythmic cycles of locomotor activity have been described in Anopheles species, no studies have been conducted on Anopheles darlingi species, the main malaria vector in the Amazon region. The aim of this study was to investigate how insemination status, blood meal, and Plasmodium vivax infection affect the locomotor activity of An. darlingi. The experiments were performed with 3- to 10-day-old An. darlingi females, which had been fed with 15% honey solution. These mosquitoes were obtained from the Malaria Vector Production and Infection Platform (PIVEM)/FIOCRUZ-RO. The experimental groups were divided into four categories: virgin vs. inseminated, unfed virgin vs. blood-fed virgin, unfed inseminated vs. blood-fed inseminated, and infected blood vs. uninfected blood. Locomotor activity was monitored using the Flybox equipment, capturing images that were subsequently converted into video to measure the insect activity, using PySoLo software. The periodicity and rhythmicity of mosquito locomotor activity were analyzed using MatLab® software. The locomotor activity of An. darlingi females showed a nocturnal and bimodal pattern under LD conditions. When comparing the insemination states and blood meal, there was a reduction in the locomotor activity in inseminated and blood-fed females. However, the P. vivax+ infection did not increase locomotor activity of An. darlingi species.

Host-mosquito interactions in rural and urban equestrian facilities from temperate Argentina.

Targeted vector surveillance informed by data on mosquito biting patterns can help limit arboviral zoonotic diseases. To characterise host-biting networks in rural and urban equestrian facilities from temperate Argentina, adult resting mosquitoes were collected (December 2018-April 2019) with a battery-powered aspirator. Engorged females were sorted to species, and their blood source was identified using molecular techniques. Bipartite network analysis was performed for rural and urban matrices. A total of 177 bloodmeals from 11 mosquito species of Aedes and Culex were identified, with seven mammal and 17 bird species recognised as hosts. Mammals represented 61% of the total feeds, mainly horse, dog and sheep; the best represented avian hosts were Columbiformes. Aedes species and Culex maxi fed only on mammals, while most other Culex species presented a wide range of hosts. The rural network had more nodes and interactions than its urban counterpart, both with some degree of host selection and aggregated patterns according to network indices. Culex quinquefasciatus was the strongest species in both networks, whereas Culex apicinus and Culex dolosus had a prominent role in the rural network. Bipartite network analysis will contribute to understanding the effects of urbanisation in the dynamics of vector-borne diseases.

First Record of Forcipomyia (Lasiohelea) Kieffer (Diptera: Ceratopogonidae) in Uruguay.

During an insect surveillance in the Santa Lucía Wetlands, Montevideo, it was captured three specimens of Forcipomyia (Lasiohelea) stylifera (Lutz), being the first record for this subgenus from Uruguay. Lasiohelea comprise species of veterinary and medical concern, due to the hematophagous feeding nature of adult females, which cause diverse degrees of nuisance on humans and animals. In the last decade, suspicion was raised involving this taxon in the transmission of Leishmania parasites, but there is still no conclusive evidence.

High Parasitic Loads Quantified in Sylvatic Triatoma melanica, a Chagas Disease Vector.

Triatoma melanica is a sylvatic vector species in Brazil. In We aimed to characterize the Trypanosoma cruzi discrete typing units (DTUs), the parasitic loads, and the blood meal sources of insects collected in rocky outcrops in rural areas in the state of Minas Gerais. An optical microscope (OM) and kDNA-PCR were used to examine natural infection by T. cruzi, and positive samples were genotyped by conventional multilocus PCR. Quantification of the T. cruzi load was performed using qPCR, and the blood meal sources were identified by Sanger sequencing the 12S rRNA gene. A total of 141 T. melanica were captured. Of these, ~55% (61/111) and ~91% (63/69) were positive by OM and KDNA-PCR, respectively. We genotyped ~89% (56/63) of the T. cruzi-positive triatomines, with TcI (~55%, 31/56) being the most prevalent DTU, followed by TcIII (~20%, 11/56) and TcII (~7%, 4/56). Only TcI+TcIII mixed infections were detected in 10 (~18%) specimens. A wide range of variation in the parasitic loads of T. melanica was observed, with an overall median value of 104 parasites/intestine, with females having higher T. cruzi loads than N2, N4, and N5. TcII showed lower parasitic loads compared to TcI and TcIII. The OM positive diagnosis odds ratio between T. cruzi infection when the parasite load is 107 compared to 103 was approximately 29.1. The most frequent blood meal source was Kerodon rupestris (~58%), followed by Thrichomys apereoides (~18%), Wiedomys cerradensis (~8%), Galactis cuja (~8%) and Gallus gallus (~8%). Our findings characterize biological and epidemiological aspects of the sylvatic population of T. melanica in the study area, highlighting the need to extend surveillance and control to this vector.

Treatment of dogs with fluralaner reduced pyrethroid-resistant Triatoma infestans abundance, Trypanosoma cruzi infection and human-triatomine contact in the Argentine Chaco.

BACKGROUND: Triatomine elimination efforts and the interruption of domestic transmission of Trypanosoma cruzi are hampered by pyrethroid resistance. Fluralaner, a long-lasting ectoparasiticide administered to dogs, substantially reduced site infestation and abundance of pyrethroid-resistant Triatoma infestans Klug (Heteroptera: Reduviidae) in an ongoing 10-month trial in Castelli (Chaco Province, Argentina). We assessed the effects of fluralaner on vector infection with T. cruzi and blood meal sources stratified by ecotope and quantified its medium-term effects on site infestation and triatomine abundance. METHODS: We conducted a placebo-controlled, before-and-after efficacy trial of fluralaner in 28 infested sites over a 22-month period. All dogs received either an oral dose of fluralaner (treated group) or placebo (control group) at 0 month post-treatment [MPT]. Placebo-treated dogs were rescue-treated with fluralaner at 1 MPT, as were all eligible dogs at 7 MPT. Site-level infestation and abundance were periodically assessed by timed manual searches with a dislodging aerosol. Vector infection was mainly determined by kDNA-PCR and blood meal sources were determined by enzyme-linked immunosorbent assay. RESULTS: In fluralaner-treated households, site infestation dropped from 100% at 0 MPT to 18-19% over the period 6-22 MPT while mean abundance plummeted from 5.5 to 0.6 triatomines per unit effort. In control households, infestation dropped similarly post-treatment. The overall prevalence of T. cruzi infection steadily decreased from 13.8% at 0-1 MPT (baseline) to 6.4% and subsequently 2.3% thereafter, while in domiciles, kitchens and storerooms it dropped from 17.4% to 4.7% and subsequently 3.3% thereafter. Most infected triatomines occurred in domiciles and had fed on humans. Infected-bug abundance plummeted after fluralaner treatment and remained marginal or nil thereafter. The human blood index of triatomines collected in domiciles, kitchens and storerooms highly significantly fell from 42.9% at baseline to 5.3-9.1% over the period 6-10 MPT, increasing to 36.8% at 22 MPT. Dog blood meals occurred before fluralaner administration only. The cat blood index increased from 9.9% at baseline to 57.9-72.7% over the period 6-10 MPT and dropped to 5.3% at 22 MPT, whereas chicken blood meals rose from 39.6% to 63.2-88.6%. CONCLUSION: Fluralaner severely impacted infestation- and transmission-related indices over nearly 2 years, causing evident effects at 1 MPT, and deserves larger efficacy trials.

Towards the Laboratory Maintenance of Haemagogus janthinomys (Dyar, 1921), the Major Neotropical Vector of Sylvatic Yellow Fever.

Haemagogus (Haemagogus) janthinomys (Dyar, 1921), the major neotropical vector of sylvatic yellow fever virus, is notoriously difficult to maintain in captivity. It has never been reared beyond an F1 generation, and almost no experimental transmission studies have been performed with this species since the 1940s. Herein we describe installment hatching, artificial blood feeding, and forced-mating techniques that enabled us to produce small numbers of F3 generation Hg. janthinomys eggs for the first time. A total of 62.8% (1562/2486) F1 generation eggs hatched during ≤10 four-day cycles of immersion in a bamboo leaf infusion followed by partial drying. Hatching decreased to 20.1% (190/944) in the F2 generation for eggs laid by mosquitoes copulated by forced mating. More than 85% (79/92) female F2 mosquitoes fed on an artificial blood feeding system. While we were unable to maintain a laboratory colony of Hg. janthinomys past the F3 generation, our methods provide a foundation for experimental transmission studies with this species in a laboratory setting, a critical capacity in a region with hyper-endemic transmission of dengue, Zika, and chikungunya viruses, all posing a risk of spillback into a sylvatic cycle.

Characterization of the reproductive tract bacterial microbiota of virgin, mated, and blood-fed Aedes aegypti and Aedes albopictus females.

BACKGROUND: Aedes aegypti and Ae. albopictus are vectors of numerous arboviruses that adversely affect human health. In mosquito vectors of disease, the bacterial microbiota influence several physiological processes, including fertility and vector competence, making manipulation of the bacterial community a promising method to control mosquito vectors. In this study, we describe the reproductive tract tissue microbiota of lab-reared virgin Ae. aegypti and Ae. albopictus males, and virgin, mated, and mated + blood-fed females of each species, comparing the bacterial composition found there to the well-described gut microbiota. METHODS: We performed metabarcoding of the 16S rRNA isolated from the gut, upper reproductive tract (URT; testes or ovaries), and lower reproductive tract (LRT; males: seminal vesicles and accessory glands; females: oviduct, spermathecae, and bursa) for each species, and evaluated the influence of host species, tissue, nutritional status, and reproductive status on microbiota composition. Finally, based on the identified taxonomic profiles of the tissues assessed, bacterial metabolic pathway abundance was predicted. RESULTS: The community structure of the reproductive tract is unique compared to the gut. Asaia is the most prevalent OTU in the LRTs of both Ae. aegypti and Ae. albopictus. In the URT, we observed differences between species, with Wolbachia OTUs being dominant in the Ae. albopictus URT, while Enterobacter and Serratia were dominant in Ae. aegypti URT. Host species and tissue were the best predictors of the community composition compared to reproductive status (i.e., virgin or mated) and nutritional status (i.e., sugar or blood-fed). The predicted functional profile shows changes in the abundance of specific microbial pathways that are associated with mating and blood-feeding, like energy production in mated tissues and siderophore synthesis in blood-fed female tissues. CONCLUSIONS: Aedes aegypti and Ae. albopictus have distinct differences in the composition of microbiota found in the reproductive tract. The distribution of the bacterial taxonomic groups indicates that some bacteria have tissue-specific tropism for reproductive tract tissue, such as Asaia and Wolbachia. No significant differences in the taxonomic composition were observed in the reproductive tract between virgin, mated, and mated + blood-fed females, but changes in the abundance of specific metabolic pathways were found in the predicted microbial functional profiles in mated and blood-fed females.

Triatomine and Trypanosoma cruzi discrete typing units distribution in a semi-arid area of northeastern Brazil.

The occurrence of triatomine species, their bloodmeal sources and the discrete typing units (DTUs) of Trypanosoma cruzi isolated from them were determined in different municipalities of the state of Rio Grande do Norte, Brazil. Triatomine captures were carried out in the rural areas of 23 municipalities. The genotyping of T. cruzi isolates was performed using the mitochondrial cytochrome c oxidase subunit 2 (coii) gene, the D7 region of the 24Sα rDNA, and the spliced leader intergenic region (SL-IR). Five triatomine species were captured, and the most frequent was Triatoma brasiliensis (84.3%; 916/1086), which was found in 16 of the 23 municipalities surveyed, and infested all types of environment investigated. The TcI DTU was found in all mesoregions surveyed in 51.5% (17/33) of the culture-positive samples. In contrast, TcII (9.1%; 3/33) was detected in the Central mesoregion, while TcIII (27.3%; 9/33) was found in all mesoregions. The geographic distribution and spatial overlap of different DTUs was inferred using the superposition of the radius of occurrence of isolates and using ecological niche distribution modelling. Triatoma brasiliensis was found infected in all mesoregions and with all three T. cruzi DTUs, including mixed infections. With regard to bloodmeal sources, the DNA of rodents was found in triatomines infected with either TcI or TcIII, while that of domestic animals and humans was associated with both single and mixed infections. Our findings demonstrate that different DTUs of T. cruzi are widely dispersed among triatomines in our study area. The association of T. brasiliensis with several different mammalian hosts, as well as overlapping areas with different DTUs, suggests that this triatomine species may have an important role as a vector in both anthropic and sylvatic environments.

Non-immune Traits Triggered by Blood Intake Impact Vectorial Competence.

Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector-pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host-parasite relationships.

Trypanosoma cruzi infection in Triatoma infestans and high levels of human-vector contact across a rural-to-urban gradient in the Argentine Chaco.

BACKGROUND: Peri-urban and urban settings have recently gained more prominence in studies on vector-borne transmission of Trypanosoma cruzi due to sustained rural-to-urban migrations and reports of urban infestations with triatomines. Prompted by the finding of Triatoma infestans across the rural-to-urban gradient in Avia Terai, an endemic municipality of the Argentine Chaco, we assessed selected components of domestic transmission risk in order to determine its variation across the gradient. METHODS: A baseline vector survey was conducted between October 2015 and March 2016, following which we used multistage random sampling to select a representative sample of T. infestans at the municipal level. We assessed T. cruzi infection and blood-feeding sources of 561 insects collected from 109 houses using kinetoplast DNA-PCR assays and direct enzyme-linked immunosorbent assays, respectively. We stratified triatomines according to their collection site (domestic or peridomestic), and we further categorized peridomestic sites in ecotopes of low- or high-risk for T. cruzi infection. RESULTS: The overall adjusted prevalence of T. cruzi-infected T. infestans was 1.8% (95% confidence interval [CI] 1.3-2.3) and did not differ between peri-urban (1.7%) and rural (2.2%) environments. No infection was detected in bugs captured in the urban setting; rather, infected triatomines were mainly collected in rural and peri-urban domiciles, occurring in 8% of T. infestans-infested houses. The main blood-feeding sources of domestic and peridomestic triatomines across the gradient were humans and chickens, respectively. The proportion of triatomines that had fed on humans did not differ between peri-urban (62.5%) and rural (65.7%) domiciles, peaking in the few domestic triatomines collected in urban houses and decreasing significantly with an increasing proportion of chicken- and dog- or cat-fed bugs. The relative odds ratio (OR) of having a T. cruzi infection was nearly threefold higher in bugs having a blood meal on humans (OR 3.15), dogs (OR 2.80) or cats (OR: 4.02) in a Firth-penalized multiple logistic model. CONCLUSIONS: Trypanosoma cruzi transmission was likely occurring both in peri-urban and rural houses of Avia Terai. Widespread infestation in a third of urban blocks combined with high levels of human-triatomine contact in the few infested domiciles implies a threat to urban inhabitants. Vector control strategies and surveillance originally conceived for rural areas should be tailored to peri-urban and urban settings in order to achieve sustainable interruption of domestic transmission in the Chaco region.

The influence of different sources of blood meals on the physiology of Aedes aegypti harboring Wolbachia wMel: mouse blood as an alternative for mosquito rearing.

BACKGROUND: Aedes aegypti control programs have failed to restrain mosquito population expansion and, consequently, the spread of diseases such as dengue, Zika, and Chikungunya. Wolbachia infection of mosquitoes is a new and promising complementary tool for the control of arbovirus transmission. The use of Wolbachia-infected mosquitoes, mass reared using human blood, is currently being tested in several countries. However, the use of human blood for mass rearing mosquitoes, and thus expansion of this strategy, is problematic. With the aim of overcoming this problem, we tested the effect of different types of blood source on the fitness parameters of female Ae. aegypti and the Wolbachia titer over generations to be able to guarantee the suitability of an alternative source to human blood for mass rearing Wolbachia-infected mosquitoes. METHODS: We investigated and compared essential parameters of the vector capacity of laboratory strains of Ae. aegypti with and without Wolbachia that fed on blood of different types of host (human, guinea pig, and mouse). The parameters analyzed were fecundity, fertility, pupation dynamics, and adult survival. Also, we tested whether it is possible to maintain mosquitoes with Wolbachia on mouse blood over generations without losing the bacterium titer. RESULTS: The average number of eggs per female, egg viability and pupation dynamics in the Wolbachia-infected mosquito (wMelBr) strain were similar, regardless of the blood source. The F1 progenies of females that fed on mouse blood or human blood were analyzed. The longevity of males was lower than that of females. F1 female survival differed depending on the presence of Wolbachia in the mother. In subsequent generations analyzed up until F35, the relative Wolbachia density was even higher when mosquitoes fed on mouse blood in comparison to human blood. CONCLUSIONS: Taken together, our results provide no evidence that the different types of blood influenced the fitness of the Wolbachia-infected mosquitoes. The presence of the bacterium in the colonies of Wolbachia-infected Ae. aegypti after 35 generations under the conditions evaluated indicates that they can be maintained on mouse blood. Based on these results, we show that it is possible to use mouse blood to feed female mosquitoes when using human blood for this purpose is problematic.

An Integrative Sialomic Analysis Reveals Molecules From Triatoma sordida (Hemiptera: Reduviidae).

Triatomines have evolved salivary glands that produce versatile molecules with various biological functions, including those leading their interactions with vertebrate hosts' hemostatic and immunological systems. Here, using high-throughput transcriptomics and proteomics, we report the first sialome study on the synanthropic triatomine Triatoma sordida. As a result, 57,645,372 reads were assembled into 26,670 coding sequences (CDS). From these, a total of 16,683 were successfully annotated. The sialotranscriptomic profile shows Lipocalin as the most abundant protein family within putative secreted transcripts. Trialysins and Kazal-type protease inhibitors have high transcript levels followed by ubiquitous protein families and enzyme classes. Interestingly, abundant trialysin and Kazal-type members are highlighted in this triatomine sialotranscriptome. Furthermore, we identified 132 proteins in T. sordida salivary gland soluble extract through LC-MS/MS spectrometry. Lipocalins, Hemiptera specific families, CRISP/Antigen-5 and Kazal-type protein inhibitors proteins were identified. Our study provides a comprehensive description of the transcript and protein compositions of the salivary glands of T. sordida. It significantly enhances the information in the Triatominae sialome databanks reported so far, improving the understanding of the vector's biology, the hematophagous behaviour, and the Triatominae subfamily's evolution.

Host feeding patterns of Nyssorhynchus darlingi (Diptera: Culicidae) in the Brazilian Amazon.

Nyssorhynchus darlingi (Root) is the dominant malaria vector in the Brazilian Amazon River basin, with additional Anophelinae Grassi species involved in local and regional transmission. Mosquito blood-feeding behavior is an essential component to define the mosquito-human contact rate and shape the transmission cycle of vector-borne diseases. However, there is little information on the host preferences and blood-feeding behavior of Anophelinae vectors in rural Amazonian landscapes. The barrier screen sampling (BSS) method was employed to sample females from 34 peridomestic habitats in 27 rural communities from 11 municipalities in the Brazilian Amazon states of Acre, Amazonas, Pará and Rondônia, from August 2015 to November 2017. Nyssorhynchus darlingi comprised 97.94% of the females collected resting on barrier screens, and DNA sequence comparison detected 9 vertebrate hosts species. The HBI index ranged from 0.03-1.00. Results revealed the plasticity of Ny. darlingi in blood-feeding on a wide range of mainly mammalian hosts. In addition, the identification of blood meal sources using silica-dried females is appropriate for studies of human malaria vectors in remote locations.

A simple and reliable method for longitudinal assessment of untethered mosquito induced flight activity.

Aedes aegypti adult females are key vectors of several arboviruses and flight activity plays a central role in mosquito biology and disease transmission. Available methods to quantify mosquito flight usually require special devices and mostly assess spontaneous locomotor activity at individual level. Here, we developed a new method to determine longitudinal untethered adult A. aegypti induced flight activity: the INduced FLight Activity TEst (INFLATE). This method was an adaptation of the "rapid iterative negative geotaxis" assay to assess locomotor activity in Drosophila and explore the spontaneous behavior of mosquitoes to fly following a physical stimulus. Insects were placed on a plastic cage previously divided in four vertical quadrants and flight performance was carried out by tapping cages towards the laboratory bench. After one minute, the number of insects per quadrant was registered by visual inspection and categorized in five different scores. By using INFLATE, we observed that flight performance was not influenced by repeated testing, sex or 5% ethanol intake. However, induced flight activity was strongly affected by aging, blood meal and inhibition of mitochondrial complex I. This simple and rapid method allows the longitudinal assessment of induced flight activity of multiple untethered mosquitoes and may contribute to a better understanding of A. aegypti dispersal biology.

Dinner and a show: The role of male copulatory courtship song and female blood-feeding in the reproductive success of Lutzomyia longipalpis from Lapinha, Brazil.

Lutzomyia longipalpis is the main vector of visceral Leishmaniasis in the Americas and is composed of a species complex. Males of this sand-fly produce acoustic signals during copulation and different patterns are observed among Brazilian populations. Such acoustic signals are commonly involved in species recognition. However, since the song is only produced during copulation it is not clear how it affects mating success or contributes to sexual isolation. Another aspect that may affect reproductive success is the presence of food. Since hematophagy is such an important aspect of L. longipalpis biology, we wanted to test if blood-feeding can influence the reproductive behaviour of this insect. We performed crossing experiments removing males' wings (silencing them) and playing back either the homo-specific or the hetero-specific song to either unfed or blood-fed females. Our results showed that both songs and blood-feeding affect insemination success, but not the frequency of copulation. In trials where females were not blood-fed song clearly affected insemination; males with wings, and males with homo-specific song playback had a higher insemination success than wingless males (no song) and trials with hetero-specific song. Blood-feeding females prior to the trials increased insemination in all groups including the control group which suggests that mating happens simultaneously with, or immediately after, the blood meal. Blood-fed females also seemed to discriminate less against the wrong song or the lack of song (wingless) one day after feeding, however trials with the correct song still had higher insemination rates. Altogether, our results show that both the male copulatory courtship songs and female blood-feeding are important for reproductive success and as such are important components of the sexual behaviour of L. longipalpis.