Behaviour and distribution of Aedes aegypti mosquitoes and their relation to dengue incidence in two transmission hotspots in coastal Ecuador.

BACKGROUND: Dengue (DENV) transmission is endemic throughout coastal Ecuador, showing heterogeneous incidence patterns in association with fine-scale variation in Aedes aegypti vector populations and other factors. Here, we investigated the impact of micro-climate and neighbourhood-level variation in urbanization on Aedes abundance, resting behaviour and associations with dengue incidence in two endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: Aedes aegypti were collected in Quinindé and Portoviejo, two urban cantons with hyperendemic dengue transmission in coastal Ecuador. Aedes vectors were sampled in and around houses within urban and peri-urban neighbourhoods at four time periods. We tested for variation in vector abundance and resting behaviour in relation to neighbourhood urbanization level and microclimatic factors. Aedes abundance increased towards the end of the rainy season, was significantly higher in Portoviejo than in Quinindé, and in urban than in peri-urban neighbourhoods. Aedes vectors were more likely to rest inside houses in Portoviejo but had similar abundance in indoor and outdoor resting collections in Quinindé. Over the study period, DENV incidence was lower in Quinindé than in Portoviejo. Relationships between weekly Ae. aegypti abundance and DENV incidence were highly variable between trapping methods; with positive associations being detected only between BG-sentinel and outdoor Prokopack collections. CONCLUSIONS/SIGNIFICANCE: Aedes aegypti abundance was significantly higher in urban than peri-urban neighbourhoods, and their resting behaviour varied between study sites. This fine-scale spatial heterogeneity in Ae. aegypti abundance and behaviour could generate site-specific variation in human exposure and the effectiveness of indoor-based interventions. The trap-dependent nature of associations between Aedes abundance and local DENV incidence indicates further work is needed to identify robust entomological indicators of infection risk.

Microarray Gene Expression Analysis of Lesional Skin in Canine Pemphigus Foliaceus.

Canine pemphigus foliaceus (PF) is considered the most common autoimmune skin disease in dogs; the mechanism of PF disease development is currently poorly understood. Therefore, this study aimed to characterize the molecular mechanisms and altered biological pathways in the skin lesions of canine PF patients. Using an RNA microarray on formalin-fixed, paraffin-embedded samples, we analyzed the transcriptome of canine PF lesional skin (n = 7) compared to healthy skin (n = 5). Of the 800 genes analyzed, 420 differentially expressed genes (DEGs) (p < 0.05) were found. Of those, 338 genes were significantly upregulated, including pro-inflammatory and Th17-related genes. Cell type profiling found enhancement of several cell types, such as neutrophils, T-cells, and macrophages, in PF skin compared to healthy skin. Enrichment analyses of the upregulated DEGs resulted in 78 statistically significant process networks (FDR < 0.05), including the Janus kinase signal transducer and activator of transcription (JAK-STAT) and mitogen-activated protein kinase (MAPK) signaling. In conclusion, canine PF lesional immune signature resembles previously published changes in human pemphigus skin lesions. Further studies with canine PF lesional skin using next-generation sequencing (e.g., RNA sequencing, spatial transcriptomics, etc.) and the development of canine keratinocyte/skin explant PF models are needed to elucidate the pathogenesis of this debilitating disease.

Characterisation of the pruritus responses and pruritic behaviours in an interleukin 31-induced canine model of pruritus.

BACKGROUND: Intravenous administration of interleukin (IL)-31 in healthy dogs has been used as a model to assess antipruritic drugs. However, there is no known in-depth characterisation of pruritic behaviours, and the repeatability of the IL-31-induced pruritus in the individual dogs is currently unknown. OBJECTIVES: To evaluate the immediate/delayed pruritus responses and the pruritic behaviours observed in the IL-31-induced pruritic model in healthy dogs after repeated IL-31 injections. ANIMALS: Fifteen healthy laboratory beagles. METHODS: All dogs were video-recorded for 270 min after two intravenous recombinant IL-31 injections (1.75 µg/kg) and vehicle (phosphate-buffered saline, control) injections, respectively; interventions were randomised and performed with a 2 week wash-out period. Two blinded investigators reviewed the pruritic behaviours of all video recordings. RESULTS: Both canine IL-31 (IL-31_01, IL-31_02) injections significantly increased pruritic seconds and categorical minutes ('YES'/'NO' behaviour per discrete 1 min interval) in healthy dogs compared with both vehicle groups (Vehicle_01, Vehicle_02). The second intravenous canine IL-31 (IL-31_02) administered 14 days after the first IL-31 injection induced a significant increase in pruritic seconds (p = 0.021) and not pruritic categorical minutes (p = 0.231). An increase in pruritic seconds was observed in both IL-31 groups in the first 30 min post-administration, while there was no significant difference between IL-31 and vehicle groups. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, intravenous IL-31 reproducibly induces itch responses in dogs. Future evaluations of the canine IL-31 pruritic model should assess total pruritic behaviours in seconds rather than using a biased 'YES/NO' behaviour per 1 min scoring system.

Characterization of the serum and skin inflammatory profile in canine pemphigus foliaceus using multiplex assay and quantitative real-time polymerase chain reaction (qRT-PCR).

Canine pemphigus foliaceus (PF) is a common autoimmune skin disease characterized by autoantibodies binding to epithelial adhesion molecules resulting inflammatory response. The immune network of cytokine and chemokine abnormalities that characterize the immune response in canine PF are poorly explored. This study evaluated serum and lesional skin cytokine and chemokine profiles of dogs diagnosed with PF compared to healthy control dogs. Serum samples obtained from 11 PF dogs and 16 healthy control dogs were analyzed using commercially available canine multiplex assay for 13 biomarkers (Canine Milliplex assay). Eight lesional skin samples from seven PF dogs and five healthy site-matched samples from five healthy dogs were evaluated for 20 immune markers using quantitative real-time PCR. Immunomodulating medications were suspended for at least four weeks in all dogs before obtaining serum and skin samples. PF patients showed significantly higher serum concentrations of tumor necrosis factor-α, interleukin (IL)- 6, IL-8, IL-18, CCL2, KC-like, and granulocyte-macrophages colony-stimulating factor when compared to healthy controls (Mann-Whitney U test; p < 0.05 for all). Lesional PF skin exhibited significant expression and upregulation of pro-inflammatory/T helper (Th1) 1 markers IL-1ß, MX1, GZMB, OAS1, and IFN-γ as well as Th2 cytokines IL-13, IL-33, TSLP, IL-31 and Th17/22 markers IL-17A and IL-22 (Mann-Whitney U test; p < 0.05 for all). Taken together, the findings from this study describe the role of numerous cytokines and chemokines associated with immune response in the skin and serum of canine PF patients. Further larger-sample proteomics and RNA-sequencing transcriptomics studies are needed to understand the immune pathogenesis of canine PF skin lesions.

Establishment of an Intradermal Canine IL-31-Induced Pruritus Model to Evaluate Therapeutic Candidates in Atopic Dermatitis.

Pruritic models in healthy dogs utilizing intravenous administration of interleukin 31 (IL-31) bypass the "natural" itch sensation in AD, which is initiated by pruriceptive primary afferent neurons in the skin. This study aimed to evaluate the immediate/delayed pruritus responses and the pruritic behaviors observed in an intradermal IL-31-induced pruritic model of healthy dogs and the anti-pruritic effect of oclacitinib on said model. In Phase 1, all the dogs were randomized and video-recorded for 300 min after intradermal canine recombinant IL-31 injections (1.75 µg/kg) and vehicle (phosphate-buffered saline) injections. In Phase 2, all the dogs received oral oclacitinib (0.4-0.6 mg/kg, twice daily for 4 consecutive days and once daily on day 5), with the intradermal IL-31 injection performed on day 5. Two blinded investigators reviewed the pruritic behaviors in all the video recordings. Intradermal IL-31 administration to healthy dogs caused a significant increase in the total (p = 0.0052) and local (p = 0.0003) seconds of pruritic behavior compared to the vehicle control. Oral oclacitinib administration significantly reduced the total (p = 0.0011) and local (p = 0.0156) intradermal IL-31-induced pruritic seconds; there was no significant difference in pruritic seconds between the vehicle and oclacitinib within the IL-31 groups. Significant delayed pruritic responses at 150-300 min after IL-31 injections were observed, and intradermal IL-31 failed to induce acute itch (first 30 min). Intradermal injection of IL-31 induces delayed itch responses in dogs that are diminished by the effect of oclacitinib, an oral JAK inhibitor.

An updated list of the Culicoides (Diptera, Ceratopogonidae) fauna from Ecuador.

An updated list of biting midges of the genus Culicoides inhabiting Ecuador is provided. Entomological investigations were carried out from July 2010 to May 2019 using CDC light traps in three Ecuadorian regions (Amazon basin, Andean (foothills and highlands) and Pacific Coast). A total of 12,073 Culicoides specimens from seven subgenera and nine species groups were collected. More species and higher variation were found in the Amazon basin than in either of the Andes regions or coastal sites. A total of 53 species were identified. Of these, 15 are herein reported as new species records for Ecuador: Culicoides acotylus Lutz, C. aitkeni Wirth & Blanton, C. benarrochi Ortiz & Mirsa, C. carvalhoi Wirth & Blanton, C. freitasi Wirth & Blanton, C. ginesi Ortíz, C. lopesi Barretto, C. lyrinotatus Wirth & Blanton, C. profundus Santarém, Felippe-Bauer & Trindade, C. pseudoreticulatus Santarém, Felippe-Bauer & Castellón, C. quasiparaensis Clastrier, C. vernoni Wirth & Blanton, C. youngi Wirth & Barreto and two new species. Our results show that the updated list of the Ecuadorian Culicoides fauna comprises 70 species. This inventory highlights the presence of species that have been incriminated as vectors of disease elsewhere in animals and humans, mainly C. insignis and C. paraensis.

Title: Une liste actualisée de la faune des Culicoides (Diptera, Ceratopogonidae) de l'Équateur. Abstract: Une liste actualisée des Culicoides vivant en Équateur est fournie. Des enquêtes entomologiques ont été menées de juillet 2010 à mai 2019 à l'aide de pièges lumineux CDC dans trois régions équatoriennes (le bassin amazonien, les Andes (piémont et hautes terres) et la côte Pacifique). Au total, 12 073 spécimens de Culicoides de sept sous-genres et neuf groupes d'espèces ont été collectés. Plus d'espèces et une variation plus élevée ont été trouvées dans le bassin amazonien que dans les régions des Andes ou les sites côtiers. Au total, 53 espèces ont été identifiées. Parmi celles-ci, 15 sont signalées ici comme nouvelles espèces pour l'Équateur : Culicoides acotylus Lutz, C. aitkeni Wirth & Blanton, C. benarrochi Ortiz & Mirsa, C. carvalhoi Wirth & Blanton, C. freitasi Wirth & Blanton, C. ginesi Ortíz, C. lopesi Barretto, C. lyrinotatus Wirth & Blanton, C. profundus Santarém, Felippe-Bauer & Trindade, C. pseudoreticulatus Santarém, Felippe-Bauer & Castellón, C. quasiparaensis Clastrier, C. vernoni Wirth & Blanton, C. youngi Wirth & Barreto et deux nouvelles espèces. Nos résultats montrent que la liste actualisée de la faune culicoïde équatorienne comprend 70 espèces. Cet inventaire met en évidence la présence d'espèces qui ont été incriminées ailleurs comme vecteurs de maladies chez l'animal et l'homme, principalement C. insignis et C. paraensis.

Seasonal Investigation of Anaplasma marginale Infection in Pakistani Cattle Reveals Hematological and Biochemical Changes, Multiple Associated Risk Factors and msp5 Gene Conservation.

Bovine anaplasmosis is a tick-borne disease caused by an obligate intercellular Gram-negative bacterium named Anaplasma (A.) marginale. In this study, we report the seasonal prevalence, potentially associated risk factors and phylogeny of A. marginale in cattle of three different breeds from Multan District, Southern Punjab, Pakistan. A total of 1020 blood samples (crossbred, n = 340; Holstein Friesian, n = 340; and Sahiwal breed, n = 340) from apparently healthy cattle were collected on a seasonal basis from March 2020 to April 2021. Based on PCR amplification of the msp5 partial sequence, overall, the A. marginale prevalence rate was estimated at 11.1% (113/1020) of the analyzed cattle samples. According to seasons, the highest prevalence rate was observed in autumn (16.5%), followed by winter (10.6%) and summer (9.8%), and the lowest was recorded in the spring (7.5%). The crossbred and Sahiwal cattle were the most susceptible to A. marginale infection, followed by Holstein Friesian cattle (7.9%). Analysis of epidemiological factors revealed that cattle reared on farms where dairy animals have tick loads, dogs coinhabit with cattle and dogs have tick loads have a higher risk of being infected with A. marginale. In addition, it was observed that white blood cell, lymphocyte (%), monocyte (%), hematocrit, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentrations were significantly disturbed in A. marginale-positive cattle compared with non-infested cattle. Genetic analysis of nucleotide sequences and a phylogenetic study based on msp5 partial sequencing demonstrated that this gene appears to be highly conserved among our isolates and those infecting apparently healthy cattle from geographically diverse worldwide regions. The presented data are crucial for estimating the risk of bovine anaplasmosis in order to develop integrated control policies against bovine anaplasmosis and other tick-borne diseases infecting cattle in the country.

Morphological description of Pintomyia (Pifanomyia) veintemillasi n. sp., a new sand fly species from the sub-Andean region of Bolivia.

BACKGROUND: Most sand fly species are located in the Americas; some act as vectors of leishmaniasis and other human diseases. In Bolivia, about 25% of Neotropical species have been identified, and only a few have been implicated as vectors of cutaneous and visceral leishmaniasis. A new species of anthropophilic sand fly from the sub-Andean region of Alto Beni is described herein. METHODS: A large systematic entomological survey was carried out in a subtropical humid forest located in the Marimonos mountain range, at around 900 m altitude, in the municipality of Palos Blancos, Sud Yungas Province, Department of La Paz, Bolivia. Sand flies were captured over a period of 26 months between January 1982 and February 1984, at the ground and canopy level, using both CDC light traps and protected human bait. A total of 24,730 sand flies were collected on the ground, distributed in 16 species, and 3259 in the canopy, with eight species. One of these species was labeled as Pintomia (Pifanomyia) nevesi, although certain morphological features allowed us to doubt that it was that taxon. To define the identity of this sand fly, a re-evaluation (this work) was recently carried out through morphological analyses and measurements of the available specimens mounted on Euparal, previously labeled as Pi. (Pif.) nevesi. RESULTS: Based on the morphological traits and measurements, the re-evaluated specimens were definitively identified as a new sand fly species, Pintomyia (Pifanomyia) veintemillasi, closely related to Pi. (Pif.) nevesi and Pintomyia (Pifanomyia) maranonensis within the Evansi series. This new sand fly was the third most numerous anthropophilic species at the floor (6.2%) and the second most numerous anthropophilic at the canopy (35.1%). CONCLUSIONS: A new anthropophilic sand fly species is described as Pi. (Pif.) veintemillasi n. sp. This sand fly species was caught at about 900 m altitude in the Marimonos mountain range, a highly endemic area for cutaneous and mucosal leishmaniasis due to Leishmania (Viannia) braziliensis. Therefore, this species could be involved in the leishmaniasis transmission in the sub-Andean foothills of Alto Beni, Department of La Paz, Bolivia.

Risk factor analysis for the prevalence of gastrointestinal parasites found in large ruminants in Lower Dir Khyber Pakhtunkhwa Pakistan.

The present study was designed to investigate the prevalence of gastrointestinal (GI) parasites in cattle and buffaloes of Lower Dir Khyber Pakhtunkhwa, Pakistan. The presence of the eggs, cysts, and oocysts of GI parasites in fecal samples were detected using direct smear methods and concentration techniques including floatation, centrifugation, and sedimentation. Identification of recovered fecal stages were determined by morphology using size and appearance of the recovered eggs, cysts, and oocysts. A total of 314 fecal samples were collected from the different Tehsils (Administrative Districts) and analyzed through microscopy. A higher prevalence was observed in the buffalo than the cow population. A total of 184 samples were positive for GI parasites of which 109/196 (55.61%) were from cattle, whereas 75/118 (63.55%) were from buffaloes. The minimum number of strongyle eggs detected in all the samples were 136.39 eggs/g (EPG). The mean EPG in cattle was 143.30 and 122.56 in buffaloes. The open-source water prevalence of GI parasites was higher than the other sources in cattle and the second highest after tap water in buffaloes. The seasonal prevalence of GI parasites ranged from 32.39% (23/71), in spring to 68.8% (86/125) in summer in cattle. In was For buffaloes the infection prevalence was 52.94% (27/51) and 71.64% (48/67) in spring and summer, respectively. Gastrointestinal parasites are a serious problem in cattle and buffaloes in the lower district of Dir Khyber Pakhtunkhwa Pakistan. In general, the burden of parasitic infection was low in most animals that received previous anti-parasitic treatment.

Epidemiology and molecular characterization of Theileria annulata in cattle from central Khyber Pakhtunkhwa, Pakistan.

Theileria annulata is a tick-borne hemoprotozoan parasite responsible for tropical theileriosis in the bovine population, which causes substantial economic losses to the livestock sector. The present study has investigated, characterized, and shaped epidemiologic and phylogenetic profiles of T. annulata infection in the cattle population of central Khyber Pakhtunkhwa, Pakistan. A total of 600 blood samples were collected from cattle. Microscopy and PCR (18S rRNA taxonomic marker) assays were performed to detect T. annulata infection in cattle from the study area. The overall relative prevalence rates of T. annulata in the examined cattle population were 12.8% (microscopy) and 23.7% (PCR). District-wise analysis (microscopy/PCR) showed that cattle from district Mardan were found more infected (16.0%/28.0%), as compared to cattle from district Charsadda (13.5%/25.5%) and district Peshawar (9.0%/17.5%). Based on host demographic and ecological parameters analysis, theileriosis was found to be higher in young, female, crossbred, freely grazing, tick-infested, and irregular/no acaricides treated cattle. The univariate logistic analysis showed that host age, tick infestation, acaricides use, and feeding method were significant risk factors (P<0.05) whereas multivariate analysis indicated that host age, gender, tick infestation, acaricidal application, and feeding method were potential risk factors (P<0.05) for tropical theileriosis in the cattle population. Phylogenetic and sequence analysis showed that T. annulata 18S rRNA isolates shared homology and phylogeny with other isolates from Asia and Europe. This study has addressed the epidemiology and phylogeny of T. annulata circulating in bovid in the study area where gaps were still present. These findings will serve as a baseline and will facilitate future large-scale epidemiological investigations on tropical theileriosis in the cattle population at a national level.

Biosafety at Home: How to Translate Biomedical Laboratory Safety Precautions for Everyday Use in the Context of COVID-19.

Population adoption of social distancing measures during the COVID-19 pandemic is at times deficient, increasing the risk of SARS-CoV-2 transmission. Healthcare workers and those living in areas of intense transmission may benefit from implementing biosafety measures in their daily lives. A mixed-methods approach, combining components of single negotiation text and the Delphi method, was used to create a COVID-19 biosafety-at-home protocol. A consensus building coordinator liaised with 12 experts to develop the protocol over 11 iterations. Experts had more than 200 years of combined experience in epidemiology, virology, infectious disease prevention, and public health. A flyer, created from the final protocol, was professionally designed and initially distributed via social media and institutional websites/emails in Ecuador beginning on May 2, 2020. Since then, it has been distributed in other countries, reaching ∼7,000 people. Translating research laboratory biosafety measures for the home/street environment might be challenging. The biosafety-at-home flyer addresses this challenge in a user-friendly format.

The mosquito electrocuting trap as an exposure-free method for measuring human-biting rates by Aedes mosquito vectors.

BACKGROUND: Entomological monitoring of Aedes vectors has largely relied on surveillance of larvae, pupae and non-host-seeking adults, which have been poorly correlated with human disease incidence. Exposure to mosquito-borne diseases can be more directly estimated using human landing catches (HLC), although this method is not recommended for Aedes-borne arboviruses. We evaluated a new method previously tested with malaria vectors, the mosquito electrocuting trap (MET) as an exposure-free alternative for measuring landing rates of Aedes mosquitoes on people. Aims were to (i) compare the MET to the BG-sentinel (BGS) trap gold standard approach for sampling host-seeking Aedes vectors; and (ii) characterize the diel activity of Aedes vectors and their association with microclimatic conditions. METHODS: The study was conducted over 12 days in Quinindé (Ecuador) in May 2017. Mosquito sampling stations were set up in the peridomestic area of four houses. On each day of sampling, each house was allocated either a MET or a BGS trap, which were rotated amongst the four houses daily in a Latin square design. Mosquito abundance and microclimatic conditions were recorded hourly at each sampling station between 7:00-19:00 h to assess variation between vector abundance, trapping methods, and environmental conditions. All Aedes aegypti females were tested for the presence of Zika (ZIKV), dengue (DENV) and chikungunya (CHIKV) viruses. RESULTS: A higher number of Ae. aegypti females were found in MET than in BGS collections, although no statistically significant differences in mean Ae. aegypti abundance between trapping methods were found. Both trapping methods indicated female Ae. aegypti had bimodal patterns of host-seeking, being highest during early morning and late afternoon hours. Mean Ae. aegypti daily abundance was negatively associated with daily temperature. No infection by ZIKV, DENV or CHIKV was detected in any Aedes mosquitoes caught by either trapping method. CONCLUSION: We conclude the MET performs at least as well as the BGS standard and offers the additional advantage of direct measurement of per capita human-biting rates. If detection of arboviruses can be confirmed in MET-collected Aedes in future studies, this surveillance method could provide a valuable tool for surveillance and prediction on human arboviral exposure risk.

Socio-Ecological Factors Associated with Dengue Risk and Aedes aegypti Presence in the Galápagos Islands, Ecuador.

Dengue fever is an emerging infectious disease in the Galápagos Islands of Ecuador, with the first cases reported in 2002 and subsequent periodic outbreaks. We report results of a 2014 pilot study conducted in Puerto Ayora (PA) on Santa Cruz Island, and Puerto Baquerizo Moreno (PB) on San Cristobal Island. To assess the socio-ecological risk factors associated with dengue and mosquito vector presence at the household level, we conducted 100 household surveys (50 on each island) in neighborhoods with prior reported dengue cases. Adult mosquitoes were collected inside and outside the home, larval indices were determined through container surveys, and heads of households were interviewed to determine demographics, self-reported prior dengue infections, housing conditions, and knowledge, attitudes, and practices regarding dengue. Multi-model selection methods were used to derive best-fit generalized linear regression models of prior dengue infection, and Aedes aegypti presence. We found that 24% of PB and 14% of PA respondents self-reported a prior dengue infection, and more PB homes than PA homes had Ae. aegypti. The top-ranked model for prior dengue infection included several factors related to human movement, household demographics, access to water quality issues, and dengue awareness. The top-ranked model for Ae. aegypti presence included housing conditions, mosquito control practices, and dengue risk perception. This is the first study of dengue risk and Ae. aegypti presence in the Galápagos Islands.

Declining Prevalence of Disease Vectors Under Climate Change.

More than half of the world population is at risk of vector-borne diseases including dengue fever, chikungunya, zika, yellow fever, leishmaniasis, chagas disease, and malaria, with highest incidences in tropical regions. In Ecuador, vector-borne diseases are present from coastal and Amazonian regions to the Andes Mountains; however, a detailed characterization of the distribution of their vectors has never been carried out. We estimate the distribution of 14 vectors of the above vector-borne diseases under present-day and future climates. Our results consistently suggest that climate warming is likely threatening some vector species with extinction, locally or completely. These results suggest that climate change could reduce the burden of specific vector species. Other vector species are likely to shift and constrain their geographic range to the highlands in Ecuador potentially affecting novel areas and populations. These forecasts show the need for development of early prevention strategies for vector species currently absent in areas projected as suitable under future climate conditions. Informed interventions could reduce the risk of human exposure to vector species with distributional shifts, in response to current and future climate changes. Based on the mixed effects of future climate on human exposure to disease vectors, we argue that research on vector-borne diseases should be cross-scale and include climatic, demographic, and landscape factors, as well as forces facilitating disease transmission at fine scales.

Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae), the Cayenne tick: phylogeography and evidence for allopatric speciation.

BACKGROUND: Amblyomma cajennense F. is one of the best known and studied ticks in the New World because of its very wide distribution, its economical importance as pest of domestic ungulates, and its association with a variety of animal and human pathogens. Recent observations, however, have challenged the taxonomic status of this tick and indicated that intraspecific cryptic speciation might be occurring. In the present study, we investigate the evolutionary and demographic history of this tick and examine its genetic structure based on the analyses of three mitochondrial (12SrDNA, d-loop, and COII) and one nuclear (ITS2) genes. Because A. cajennense is characterized by a typical trans-Amazonian distribution, lineage divergence dating is also performed to establish whether genetic diversity can be linked to dated vicariant events which shaped the topology of the Neotropics. RESULTS: Total evidence analyses of the concatenated mtDNA and nuclear + mtDNA datasets resulted in well-resolved and fully congruent reconstructions of the relationships within A. cajennense. The phylogenetic analyses consistently found A. cajennense to be monophyletic and to be separated into six genetic units defined by mutually exclusive haplotype compositions and habitat associations. Also, genetic divergence values showed that these lineages are as distinct from each other as recognized separate species of the same genus. The six clades are deeply split and node dating indicates that they started diverging in the middle-late Miocene. CONCLUSIONS: Behavioral differences and the results of laboratory cross-breeding experiments had already indicated that A. cajennense might be a complex of distinct taxonomic units. The combined and congruent mitochondrial and nuclear genetic evidence from this study reveals that A. cajennense is an assembly of six distinct species which have evolved separately from each other since at least 13.2 million years ago (Mya) in the earliest and 3.3 Mya in the latest lineages. The temporal and spatial diversification modes of the six lineages overlap the phylogeographical history of other organisms with similar extant trans-Amazonian distributions and are consistent with the present prevailing hypothesis that Neotropical diversity often finds its origins in the Miocene, after the Andean uplift changed the topology and consequently the climate and ecology of the Neotropics.

NEW RECORDS OF PHLEBOTOMINE SAND FLIES (DIPTERA: PSYCHODIDAE) FROM ECUADOR.

The number of recorded phlebotomine sand fly species in Ecuador has nearly doubled during the past 20 years as a result of surveys. In 2005, a sand fly survey of two localities, Tiputini in the Amazon rain forest and Paraiso Escondido in the Pacific coastal lowland forest, resulted in the capture of 25 species. New records for Ecuador consisted of five species from the Amazonian region and one from Paraiso Escondido. The Amazonian species were Nyssomyia richardwardi (Ready and Fraiha), Psathyromyia dreisbachi (Causey and Damasceno), Psathyromyia runoides (Fairchild and Hertig), Trichophoromyia pabloi (Barretto, Burbano and Young), and Trichopygomyia witoto (Young and Morales). The Pacific coastal lowland species was Psathyromyia punctigeniculata (Floch and Abonnenc).

flhDC, but not fleQ, regulates flagella biogenesis in Azotobacter vinelandii, and is under AlgU and CydR negative control.

Azotobacter vinelandii is a nitrogen-fixing soil bacterium that undergoes differentiation to form cysts resistant to desiccation. Upon encystment, this bacterium becomes non-motile. As in enteric bacteria, motility in A. vinelandii occurs through the use of peritrichous flagella. Pseudomonas aeruginosa, a phylogenetically close relative of A. vinelandii, possesses a single polar flagellum. The FlhDC proteins are the master regulators of flagella and motility in enterobacteria, whereas FleQ is the master regulator in P. aeruginosa, and it is under AlgU (sigmaE) negative control. At present, nothing is known about the organization and expression of flagella genes in A. vinelandii. Here, we identified the flagella gene cluster of this bacterium. Homologues of the master regulatory genes flhDC and fleQ are present in A. vinelandii. Inactivation of flhDC, but not fleQ, impaired flagella biogenesis and motility. We present evidence indicating that a negative effect of the AlgU sigma factor on flhDC expression causes loss of motility in A. vinelandii, and that CydR (a homologue of Fnr) is under AlgU control and has a negative effect on flhDC expression. Taken together, these results suggest the existence of a cascade consisting of AlgU and CydR that negatively controls expression of flhDC; the results also suggest that the block in flagella synthesis under encystment conditions centres on flhDC repression by the AlgU-CydR cascade.

Meconial peritrophic matrix structure, formation, and meconial degeneration in mosquito pupae/pharate adults: histological and ultrastructural aspects.

The noncellular peritrophic matrix (PM) that forms around the food bolus in the midgut of many arthropod species may influence the fate of ingested microbes. In mosquitoes, PMs have been identified in the pupal as well as larval and adult stages. In pupae, the PMs surround the meconium, the sloughed larval midgut epithelium. Meconial PM1 (MPM1) forms early in the pupal stadium, and a second meconial PM (MPM2) sometimes forms around the time of adult emergence. A recent study suggests that MPMs contribute to the sterilization of the adult midgut by sequestering microorganisms ingested during the larval stage, which, along with remaining meconial material, are egested after adult emergence. We have compared MPM1 formation and patterns of meconial degeneration in representative species in five mosquito genera and identified a temporal association between MPM1 formation, meconial degeneration, and apolysis. Ultrastructural study of MPM1 and MPM2 in Aedes aegypti (L.) revealed that MPM1 seems to be structurally different from either the larval or adult PMs, whereas MPM2 more closely resembles PM formed around a bloodmeal in adult females. Our results are consistent with the microbial sequestration role.