The impact of ZIKV infection on gene expression in neural cells over time.

Zika virus (ZIKV) outbreak caused one of the most significant medical emergencies in the Americas due to associated microcephaly in newborns. To evaluate the impact of ZIKV infection on neuronal cells over time, we retrieved gene expression data from several ZIKV-infected samples obtained at different time point post-infection (pi). Differential gene expression analysis was applied at each time point, with more differentially expressed genes (DEG) identified at 72h pi. There were 5 DEGs (PLA2G2F, TMEM71, PKD1L2, UBD, and TNFAIP3 genes) across all timepoints, which clearly distinguished between infected and healthy samples. The highest expression levels of all five genes were identified at 72h pi. Taken together, our results indicate that ZIKV infection greatly impacts human neural cells at early times of infection, with peak perturbation observed at 72h pi. Our analysis revealed that all five DEGs, in samples of ZIKV-infected human neural stem cells, remained highly upregulated across the timepoints evaluated. Moreover, despite the pronounced inflammatory host response observed throughout infection, the impact of ZIKV is variable over time. Finally, the five DEGs identified herein play prominent roles in infection, and could serve to guide future investigations into virus-host interaction, as well as constitute targets for therapeutic drug development.

Use of light emitting diodes (LEDs) are effective and useful for sand fly ecoepidemiology studies in an Amazonian environment.

One strategy to identify transmission foci is based on vector monitoring, and efficient methods are important for vector control. Our study evaluated different light sources (red, green, ultraviolet, blue, and incandescent light) to capture sand fly in Porto Velho, Brazil. We also evaluated Leishmania and blood meal sources in females. A total of 1,943 individuals were identified in 45 species level-taxa, with Trichophoromyia ubiquitalis (Mangabeira) (n = 364), Nyssomyia antunesi (n = 241), Bichromomyia flaviscutellata (Mangabeira) (n = 222), and Psychodopygus davisi (Root) (n = 148) being the most abundant. Incandescent light captured most individuals (n = 589), followed by blue (n = 471), green (n = 452), ultraviolet (n = 281) and red (n = 150). No significant difference was observed between the species composition and lights (PERMANOVA: Pseudo F = 1.29, p = 0.14, NMDS: Stress 0.18). The Shannon and Simpson indices demonstrated a high diversity captured using all lights. Our data demonstrated that LEDs are alternative devices for sand fly capture, with blue and green LEDs presenting similar results to incandescent light. 53 pools were analyzed, only one pool was positive for kDNA and hsp70 targets [Nyssomyia fraihai (Martins, Falão & Silva)] and identified as Endotrypanum spp., suggesting that other trypanosomatids may circulate in the locality. Choloepus hoffmanni (two-toed sloth) Peters, Homo sapiens Linnaeus, Proechimys gardneri (Gardner's spiny rat) Da Silva, and Tamandua tetradactyla (lesser anteater) (Linnaeus) were blood meal sources identified in females, increasing the knowledge about sources used by these insects.

Sand fly fauna and molecular detection of Leishmania species and blood meal sources in different rural environments in western Amazon.

Cutaneous Leishmaniasis (CL) is a disease caused by Leishmania species and transmitted by the bites of infected female sand fly species. The diversity of these insects in Rondônia State (where CL is the predominant form) is large but unexplored, and consequently the vector species are unknown. The purpose of this study was to evaluate the sand fly fauna in two environments (forest fragment and peridomicile) in rural areas of four municipalities of the state, DNA amplification of Leishmania species and the presence of blood meal sources for these insects. After identifying the species, sample coverage was applied to estimate the fauna coverage in each environment. Females were used to amplify and detect Leishmania DNA and blood meal sources, then posteriorly identified by sequencing. A total of 1706 individuals were included in 61 species, which was a sample coverage of 97% for the forest fragments (56 species), whereas 98% was observed (32 species) in peridomiciles. Next, 41 pools were prepared from 1227 females, and none were positive for Leishmania DNA. We observed 160 engorged females (forest fragments: 21, peridomiciles: 139) belonging to females of the Antunesi complex and another eight species. Two of these females were positive for Leishmania braziliensis-DNA: one in the Antunesi complex and one in Psychodopygus hirsutus. Seven blood meal sources were identified by sequencing analysis: Bos taurus and Sus scrofa from the peridomiciles, and Dasypus novemcinctus, Pecari tajacu, Philander canus, Plecturocebus bernhardi, and Tamandua tetradactyla from the forest fragments. Our data confirmed the feeding behavior of field-caught sand flies and could contribute to our understanding about local vectors and possible reservoirs in the transmission of Leishmania spp.

Redescription of Two Psathyromyia Species (Diptera: Psychodidae), Including Description of the Female of Psathyromyia pradobarrientosi Using Molecular and Morphological Approaches.

The taxonomic identity of two species of sand flies, Psathyromyia pradobarrientosi (Le Pont, Matias, Martinez & Dujardin, 2004) and Psathyromyia runoides (Fairchild & Hertig, 1953) (Diptera, Psychodidae), was evaluated morphologically and molecularly based upon specimens collected in Brazilian states. The morphological component compared collected specimens with paratypes of Pa. runoides and Pa. pradobarrientosi and their descriptions. Phylogenetic analysis of coI sequences of Pa. pradobarrientosi showed a well-supported group distinct from Pa. runoides. Morphologically, Psathyromyia runoides and Pa. pradobarrientosi males are distinguished by characteristics of the aedeagal ducts and parameral sheath in the genitalia; females are distinguished by the number and shape of the teeth in the cibarium and by the shape of the spermathecae. Given the morphological similarity between the males and the absence of the description of the female of Pa. pradobarrientosi, it is possible that specimens previously identified as Pa. runoides in Brazil are in fact Pa. pradobarrientosi.

Detection of Leishmania species (Kinetoplastida, Trypanosomatidae) in phlebotomine sand flies (Diptera, Psychodidae) from Porto Velho, Northern Brazil.

Cutaneous Leishmaniasis (CL) is a vector-borne disease caused by Leishmania species and transmitted by infected female sand flies. CL is widely distributed in Brazil, but knowledge about vectors and transmission cycles could be complex according to localities. The sand fly fauna in Rondônia State is extensive, diverse, and largely unexplored. Although the state records a mean of 1,000 CL cases per year, the vectors of CL are unknown. The aim of this study was to assess phlebotomine sand fly fauna composition using diversity indexes (Shannon [H'] and Simpson [1/D]) and to detect the prevalence of Leishmania infection to verify potential vectors in three ecotopes: (i) forest fragment (FF), (ii) forest edge (FE), and (iii) peridomicile (PE). Captures were performed in four rural districts in the municipality of Porto Velho. A total of 7,026 specimens were captured comprising 72 species, and individuals classified in subgenus level. Overall, the most abundant species were Lutzomyia davisi (n: 1,105), Lutzomyia melloi (n: 760), Lutzomyia auraensis (n: 738) and Lutzomyia antunesi (n: 479). Fauna was most diverse in the FF ecotope (H' = 20.2, 1/D = 11.2), followed by the FE (H' = 18.0, 1/D = 10.1) and PE (H' = 16.6, 1/D = 10.1) ecotopes. Leishmania DNA was detected in 24 of 232 pools. In every ecotope, Leishmania naiffi DNA was identified in the following sand fly species: Lu. antunesi, Lu. davisi, Lu. hirsuta hirsuta, Lu. shawi, Lu. sordellii and Lu. (Trichophoromyia) spp. This observation may indicate that a Le. naiffi transmission focus is present in the study localities. In addition, Leishmania lainsoni was detected in Lutzomyia (Trichophoromyia) spp. Our findings show that sand fly fauna in the study localities is diverse, that Leishmania parasites are circulating in all three ecotopes, and that some sand fly species may be implicated in the transmission of Leishmania to humans in localities evaluated.

First Observation of Experimental Plasmodium vivax Infection of Three Malaria Vectors from the Brazilian Amazon.

Although malaria is endemic to the Amazon region, little is known about the susceptibility of potential parasite vectors in Brazil. Assessing the vector susceptibility of Anopheles mosquitoes will increase our understanding of parasite-vector interactions and aid the design of vector control strategies. This study assessed the susceptibility of three Anopheles species to midgut infection by Plasmodium vivax, the predominant malaria species in Rondônia State, Brazil. Blood from P. vivax infected patients was fed to Anopheles aquasalis, Anopheles darlingi, and Anopheles deaneorum mosquitoes using a membrane feeding assay (MFA). Gametocytemia was estimated by microscopic examination of blood smears and oocyst prevalence, and infection intensity was assessed. The presence of oocysts was determined by microscopy, and the infection rates and infection intensity were determined for all species. Data from six MFAs showed that An. darlingi and An. deaneorum exhibited the highest infection rates (97% and 90%, respectively) and developed a similar median number of P. vivax oocysts (142 and 123, respectively), while An. aquasalis exhibited the smallest infection rates (77%) and the median number of oocysts (88). Established laboratory colonies of An. darlingi and An. deaneorum and susceptibility to plasmodial infection would be beneficial for modeling P. vivax vector-parasite interactions in Brazil.

New Records of Phlebotomine Sand Flies (Diptera: Psychodidae) From the Western Brazilian Amazon and the Description of the Female of Pintomyia fiocruzi.

An entomological survey was conducted in several localities of Rondônia State, and new records were obtained for seven sand fly species: Evandromyia apurinan Shimabukuro, Figueira & Silva, 2013, Evandromyia carmelinoi (Ryan, Fraiha, Lainson & Shaw, 1986), Micropygomyia echinatopharynx Andrade-Filho, Galati, Andrade & Falcão, 2004, Nyssomyia urbinattii Galati & Galvis, 2012, Pintomyia duckei Oliveira, Alencar & Freitas, 2018, Psathyromyia pradobarrientosi (Le Pont, Matias, Martinez & Dujardin, 2004), and Sciopemyia vattierae (Le Pont & Desjeux, 1992). Herein, we also describe the female of Pintomyia fiocruzi Pereira Júnior, Pessoa, Marivalva & Medeiros, 2019. The female has spermathecae with a detached apical ring similar to the spermathecae of Pintomyia serrana (Damasceno & Arouck, 1949), Pintomyia odax (Fairchild & Hertig, 1961), Pintomyia ottolinai (Ortiz & Scorza, 1963), and Pintomyia robusta (Galati, Cáceres & Le Pont, 1995). Here, we provide characters to separate Pi. fiocruzi from Pi. serrana and Pi. odax. Pintomyia ottolinai has a trans-Andean distribution and Pi. robusta has not been found in Brazil and in our studies, we associated the females found in this study with Pi. fiocruzi males found at the same collection sites. Our findings highlight the importance of entomological fauna surveys and demonstrate the need for taxonomists to study species diversity in Rondônia. This study increases the number of sand fly species recorded in Rondônia to 143.

Biological Aspects of Rhodnius montenegrensis (Hemiptera, Reduviidae, Triatominae) Under Laboratory Conditions.

Triatominae are insects notorious as vectors of Trypanosoma cruzi, which is the etiologic agent of Chagas disease, and other trypanosomatids. Triatomines of the genus Rhodnius are primarily sylvatic, nevertheless the occurrence of native species that invade households suggests their possible role in the transmission of Chagas disease. Rhodnius montenegrensis was first described in 2012, but the biological aspects of this species are still unknown. This study aimed to analyze the biological aspects of R. montenegrensis under laboratory conditions. The emergence rate was 63.0%, the mean time required for the emergence was 13.9 ± 1.7 days, the biological cycle from egg to adult phase occurred in 105.2 ± 9.2 days, the number of bloodmeals required for each nymphal stage to reach the next stage varies between a minimum of two and a maximum of seven. The weight gained after a bloodmeal varied between 10.6 times on 1st-instar nymphs and 3.9 times on 5th-instar nymphs. The adult specimens had the lowest gain of weight, reaching 2.2 times on females and 1.6 times on males. The sex ratio observed was 1:1. These data are relevant to understand the life cycle of this new described species and to elaborate more effective vector control strategies.

Composition and Vertical Stratification of Phlebotomine Sand Fly Fauna and the Molecular Detection of Leishmania in Forested Areas in Rondônia State Municipalities, Western Amazon, Brazil.

This study examined sand fly diversity and the natural infection of sand flies by Leishmania in three localities in Rondônia State: Santo Antônio and São Vicente farm, which are anthropized environments, and the Jamari FLONA, which has not been anthropized. Sand flies were collected in canopies and at ground level using CDC light traps and BG-Sentinel CO2 traps. Female sand flies were grouped into pools that were subjected to DNA extraction and the samples were subjected to PCR to detect and identify Leishmania. A total of 6696 specimens were examined and 52 species were identified. Abundance was greater in canopies than at ground level (n = 5657 and n = 1039, respectively); however, fauna composition was more diverse at ground level. A total of 4870 specimens were collected using CDC light traps and 1826 specimens were collected using BG-Sentinel traps. The presence of Leishmania DNA was detected in 12 pools, and Leishmania species profiles were identified in three of these pools: one pool of Psychodopygus amazonensis and one pool of Psychodopygus chagasi infected with Le. (Vi.) braziliensis, and one pool of Psychodopygus davisi infected with Le. (Le.) amazonensis. Sand flies are sensitive to environmental modification and species diversity is greater in regions that have experienced less anthropization. Although the annual infection rate of cutaneous leishmaniasis is high in Rondônia State, the role that sand flies play in the leishmaniasis transmission cycle remains poorly understood. The high abundance of Ps. davisi and Trichophoromyia auraensis and the presence in these species of natural infection by Leishmania spp. suggest that these species are acting as vectors of tegumentary leishmaniasis in Rondônia.