Characterisation of an area of coexistent visceral and cutaneous leishmaniasis transmission in the State of Piauí, Brazil.

BACKGROUND: In Brazil, transmission of visceral and cutaneous leishmaniasis has expanded geographically over the last decades, with both clinical forms occurring simultaneously in the same area. OBJECTIVES: This study characterised the clinical, spatial, and temporal distribution, and performed entomological surveillance and natural infection analysis of a leishmaniasis-endemic area. METHODS: In order to characterise the risk of leishmaniasis transmission in Altos, Piauí, we described the clinical and socio-demographic variables and the spatial and temporal distribution of cases of American visceral leishmaniasis (AVL) and American cutaneous leishmaniasis (ACL) cases and identified potential phlebotomine vectors. FINDINGS: The urban area concentrated almost 54% of ACL and 86.8% of AVL cases. The temporal and spatial distribution of AVL and ACL cases in Altos show a reduction in the number of risk areas, but the presence of permanent disease transmission foci is observed especially in the urban area. 3,808 phlebotomine specimens were captured, with Lutzomyia longipalpis as the most frequent species (98.45%). Of the 35 females assessed for natural infection, one specimen of Lu. longipalpis tested positive for the presence of Leishmania infantum and Leishmania braziliensis DNA. MAIN CONCLUSION: Our results indicate the presence of risk areas for ACL and AVL in the municipality of Altos and highlight the importance of entomological surveillance to further understand a possible role of Lu. longipalpis in ACL transmission.

Entomopathogenic fungi and Schinus molle essential oil: The combination of two eco-friendly agents against Aedes aegypti larvae.

Aedes aegypti transmits arbovirus, which is a public health concern. Certain filamentous fungi have the potential to control the disease. Here, the effects of Metarhizium anisopliae s.l. CG 153, Beauveria bassiana s.l. CG 206 and Schinus molle L. were investigated against Aedes aegypti larvae. In addition, the effect of essential oil on fungal development was analyzed. Fungal germination was assessed after combination with essential oil at 0.0025 %, 0.0075 %, 0.005 %, or 0.01 %; all of the oil concentrations affected germination except 0.0025 % (v/v). Larvae were exposed to 0.0025 %, 0.0075 %, 0.005 %, or 0.01 % of the essential oil or Tween 80 at 0.01 %; however, only the essential oil at 0.0025 % achieved similar results as the control. Larvae were exposed to fungi at 107 conidia mL-1 alone or in combination with the essential oil at 0.0025 %. Regardless of the combination, M. anisopliae reduced the median survival time of mosquitoes more than B. bassiana. The cumulative survival of mosquitoes exposed to M. anisopliae alone or in combination with essential oil was 7.5 % and 2 %, respectively, and for B. bassiana, it was 75 % and 71 %, respectively. M. anisopliae + essential oil had a synergistic effect against larvae, whereas B. bassiana + essential oil was antagonistic. Scanning and transmission electron microscopy, and histopathology confirmed that the interaction of M. anisopliae was through the gut and hemocoel. In contrast, the mosquito's gut was the main route for invasion by B. bassiana. Results from gas chromatography studies demonstrated sabinene and bicyclogermacrene as the main compounds of S. molle, and the in-silico investigation found evidence that both compounds affect a wide range of biological activity. For the first time, we demonstrated the potential of S. molle and its interaction with both fungal strains against A. aegypti larvae. Moreover, for the first time, we reported that S. molle might be responsible for significant changes in larval physiology. This study provides new insights into host-pathogen interplay and contributes to a better understanding of pathogenesis in mosquitoes, which have significant consequences for biological control strategies.

Anti-Leishmania braziliensis activity of 1,10-phenanthroline-5,6-dione and its Cu(II) and Ag(I) complexes.

Leishmaniasis, included in the priority list of the WHO, remains as a neglected disease caused by parasites of the Leishmania genus. There is no vaccine available for human leishmaniasis, and the current treatment is based on old drugs that cause serious side effects. Herein, we initially studied the cellular distribution of the virulence factor gp63, the major metallopeptidase, in a virulent strain of Leishmania braziliensis, and then we measured the inhibitory effects of 1,10-phenanthroline-5,6-dione (phendione), and its metal complexes, [Cu(phendione)3](ClO4)2.4H2O and [Ag(phendione)2]ClO4, on both cellular and extracellular metallopeptidases produced by promastigotes. The action of the three compounds on parasite viability and on parasite-macrophage interaction was also determined. Gp63 molecules were detected in several parasite compartments, including the cytoplasm, the membrane lining the cell body and flagellum, and in the flagellar pocket, which explains the presence of gp63 in the culture medium. The test compounds inhibited parasite metallopeptidases in a typical dose-dependent manner, and they also caused a significant and irreversible inhibition of parasite motility. Moreover, the pre-treatment of promastigotes with the test compounds induced a decrease in the association index with macrophages. Collectively, phendione and its Cu(II) and Ag(I) complexes are excellent prototypes for the development of new anti-L. braziliensis drugs.

Laboratory evaluation of Brevibacillus laterosporus strains as biocidal agents against Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) larvae.

The biocidal activity of three strains of Brevibacillus laterosporus upon the post-embryonic developmental stages of Chrysomya megacephala was evaluated. Bioassays were performed to verify lethal and sub-lethal effects including ultra-structural changes in the midgut. Among the strains assayed, Shi3 presented the highest larval mortality rates, achieving 70% at a concentration of 1×108 spores/g of diet. Transmission electron microscopy revealed intestinal alterations caused by all strains tested. The findings of this study indicate that Shi3 represents a promising tool for use in the biocontrol of C. megacephala.

Larvicidal activity and effects on post embrionary development of laboratory reared Musca domestica (Linnaeus, 1758) (Diptera: Muscidae), treated with Brevibacillus laterosporus (Laubach) spore suspensions.

The application of a spore suspension of Brevibacillus laterosporus (Laubach) (strain Bon707), at a concentration of 1.94×10(9)CFU/mL in the diet, induced a level of 70% mortality in larvae of Musca domestica. No sublethal effects, upon feeding activity or development were recorded. However, electron microscopic examination of the digestive tract of larvae fed with B. laterosporus, revealed cellular vacuolization and cytoplasmic disorganization.

Evaluation of larvicidal activity and effects on post embrionary development of laboratory reared Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae), treated with Brevibacillus laterosporus.

The blowfly Lucilia cuprina is an economically important livestock pest that is also associated with human myiasis. To date, methods including the application of chemical pesticides, plant extracts, insect growth regulators and a range of Bacillus thuringiensis strains have been used, with varying degrees of success, to control this pest. The present study evaluated the larvicidal activity and the induction of sub lethal effects upon post embrionary development following ingestion of 12 strains of Brevibacillus laterosporus, presented individually in the diet as spores. All strains were shown to be larvicidal, with corrected mortality levels of 29 to 54%. No significant differences were observed, in terms of larval weight at the time of abandoning the diet, in the time taken for the initiation of the pupation process, in the duration of the pupation process, the period of adult emergence or cumulative mortality during the transition from larvae to adult. However, an influence upon sex ratio was observed. This study suggests that strains of B. laterosporus hold potential for development as a tool in the biological control of L. cuprina.

Cytotoxicity of piperamides towards Aedes aegypti (Diptera: Culicidae).

The effectiveness of the amides piplartine and piperlonguminine isolated from Piper species for controlling L3 and L4 of Aedes aegypti (L.) was assessed through bioassays at concentrations ranging from 1 to 300 g/l ml. Piplartine reduced the mosquito development period and caused larval mortality only at concentrations > 100 microg/ml, whereas piperlonguminine resulted in an extended period of mosquito development (10 microg/ml) and caused 100% larval mortality (30 microg/ml) within 24 h. The toxicity and cytotoxic effects of piperlonguminine on epithelial cells of the digestive system of Ae. aegypti were viewed using transmission electron microscopy, which indicated vacuolization of cytoplasm, mitochondrial swelling and leaking of nuclear material. Piperlonguminine was the more effective amide, showing toxic activity with LD50 of approximately 12 microg/ml against the larvae of Ae. aegypti.

Ultrastructure of immature stages of Cochliomyia macellaria (Diptera: Calliphoridae), a fly of medical and veterinary importance.

Cochliomyia macellaria (Diptera: Calliphoridae) is known as the secondary screwworm because it causes secondary or facultative myiasis when the larvae feed on necrotic tissues. This fly has a significant medical and veterinary importance since it has been reported to transport eggs of Dermatobia hominis (human botfly), which can cause significant economic losses to livestock. Since this screwworm has been collected colonizing both pig carcasses and human cadavers, it is considered one of the most important species for forensic entomology studies. Scanning electron microscopy (SEM) gives detailed information on the morphological characteristics which can help identify the immature forms of the flies. The aim of this study was to describe and analyze the morphological characteristics of the eggs, all the larval instars, and the puparia of Cochliomyia macellaria using SEM. The egg is ellipsoid and the dorsal surface is concave. The islands inside the median area had no anastomosis, but some perforations could be observed. From the second larval instar onwards, besides the intersegmental spines, other bands of spines were observed at the abdominal segments. Two spiracular openings were visible on the first and second larval instars, which were not expected. These characteristics are specific to Cochliomyia genus. The number and the general aspect of the spine tips in the cephalic region, the intersegmental bands on the abdomen, and the number of the spiracular openings could together help identify C. macellaria.

Scanning electron microscopy of eggs of Georgecraigius fluviatilis (Lutz) (Diptera: Culicidae, Aedini).

Scanning Electron Microscopy was used to describe the eggs of Georgecraigius fluviatilis (Lutz). Length is 722.8±39.6 µm and width is 177.1±9.8 µm. Diameter of the micropylar disk, surrounded by an irregular flattened collar, is 28 µm. The outer chorionic sculpture consists of cells of irregular shapes, containing tubercles with pitted surface. In the ventral region, tubercles of several diameters are irregularly distributed in chorionic cells, while in the dorsal region one larger tubercle is surrounded by several smaller ones. The eggs appear to lack structures for adhesion, certainly unnecessary due to the habit of laying eggs separately on water surfaces.

Development and intrapuparial characterization of Peckia (Euboettcheria) collusor (Curran and Walley, 1934) (Diptera: Sarcophagidae) for application in forensic entomology.

The family Sarcophagidae is very diverse in Brazil. Due to their living habits, they are the subject of many medical, veterinary, sanitary, and entomological studies. However, Sarcophagidae species are still poorly studied in forensic entomology, although they are frequently reported in carcasses and even human corpses. Thus, this study aims to identify and compare the developmental stages and intrapuparial morphological characteristics of Peckia (Euboettcheria) collusor to serve as an auxiliary tool in forensic entomology. The pupae collected after zero hour at 27 °C and 32 °C were sacrificed every three hours until the first 24 h and then every six hours until the emergence of the first adults, using 30 pupae each time, totaling 1560 for 27 °C and 1290 for 32 °C. The intrapuparial development time of this fly species under laboratory-controlled conditions was 288 h at 27 °C and 228 h at 32 °C. The 2820 pupae were analyzed according to temperature and classified into eight possible stages. This contributed to the selection of 16 key morphological characteristics to identify the age of the pupae. The identified intrapupal morphological characteristics have great potential to help researchers, experts, technical assistants, and forensic entomologists estimate the minimum post-mortem interval (minPMI) of cadavers.

Triatoma jatai sp. nov. in the state of Tocantins, Brazil (Hemiptera: Reduviidae: Triatominae).

Triatoma jatai sp. nov. is the first new species of triatomine to be described in the state of Tocantins, in the northern region of Brazil. It was caught on rock outcrops in the wild environment and, more recently, invading homes. While T. jatai sp. nov. is morphologically similar to Triatoma costalimai, it is distinguished by its general colouring, differences in the blotches on the connexivum, wing size in females and external structures of the male genitalia. The type series has been deposited in the Entomological Collection and Herman Lent Collection, Oswaldo Cruz Institute-Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.

Scanning electron microscopy of eggs of Sabethes cyaneus.

Mosquitoes of the Neotropical genus Sabethes, some species of which are yellow fever vectors, most often develop through the immature stages in tree holes. Sabethes eggs have not been previously characterized using scanning electron microscopy. Eggs of Sabethes cyaneus (length: 349.6 +/- 2.7 microm; width: 172.6 +/- 1.14 microm; n = 10) are almost biconical when examined from the top. From a lateral perspective 2 surfaces can be seen. One surface is smooth and more convex, whereas the other is less convex and partially covered by a network from which many fungiform tubercles arise. The micropyle is situated on the smooth surface of the pointed anterior tip and is surrounded by an irregular row of tubercles, some of which are leaf shaped. No structures possibly involved in adhesion to surfaces are visible. When hatching, the egg splits dorsoventrally approximately two-thirds of the length from the anterior end. The tubercles appear to be water repellent, and the more convex/smoother surface is downturned, and this position on water was confirmed by direct observation. The eggs float free on the water surface.

Ultrastructure and morphometry of eggs of Triatoma rubrovaria (Blanchard, 1843), Triatoma carcavalloi Juberg, Rocha & Lent, 1998 and Triatoma circummaculata (Stål, 1859) (Hemiptera-Reduviidae-Triatominae).

This study analyzed the body and the operculum of eggs of Triatoma rubrovaria, T. carcavalloi and T. circummaculata, considered sylvatic species that live in sympatry.Triatoma rubrovaria is currently considered the most important vector of Trypanosoma cruzi in the rural areas of the state of Rio Grande do Sul, followed by T.circummaculata. Significant differences other than morphometry have been observed in the egg structures of the three species using traditional microscopy and scanning electron microscopy. Triatoma circummaculata eggs are smaller than those of T.rubrovaria and T. carcavalloi. The average number of perforations in corionic cells in the egg body is higher for the T. rubrovaria. The average number of perforations in the operculum cell is higher in T. circummaculata. This is the first morpho-structural description of T. carcavalloi eggs. These results widen the concept of these three species and create new subsidies for the entomological monitoring in areas in which these vectors may infest human living quarters.

Monthly abundance and diversity of mosquitoes (Diptera: Culicidae) in an Atlantic Forest area of Rio de Janeiro, Brazil.

Several mosquito species in the Atlantic Forest are yellow fever vectors; therefore, this biome can represent a potential risk to the human population. Studies on mosquitoes from predominantly sylvatic areas produce valuable data for understanding the emergence of new epidemics. In addition, they can elucidate environmental components favoring or hindering biodiversity and species distribution. Our study aimed to evaluate the monthly distribution, composition, diversity, and influence of seasonal periods (dry and rainy) on the mosquito fauna. We used CDC light traps at different levels in a forest area bordering a Conservation Unit of Nova Iguaçu in the state of Rio de Janeiro, Brazil. Specimens were collected from August 2018 to July 2019 by installing traps in sampling sites under different vegetation covers. We detected some species of epidemiological importance in terms of arbovirus transmission. A total of 4,048 specimens representing 20 different species were collected. Among them, Aedes (Stg.) albopictus Skuse, 1894 showed recurrent association with the closest level to human residences and Haemagogus (Con.) leucocelaenus Dyar and Shannon, 1924 with the most distant levels. Since these mosquitoes are possible vectors of yellow fever, monitoring the area is extremely important. Under the studied conditions, the mosquito populations were directly influenced by dry and rainy periods, posing a risk to the nearby resident population.

Characterization of External Female Genitalia in Five Triatoma Laporte Species of South America (Hemiptera: Reduviidae: Triatominae).

Currently, there are 158 valid species of triatomines, all of which are potential vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. The correct taxonomic identification of triatomines is essential since each species hos a different epidemiological importance. The aim of the study is to compare five species of South American Triatoma. Here we present a comparative study of terminal abdominal segments in females by scanning electron microscopy (SEM) of the species Triatoma delpontei, T. jurbergi, T. infestans var. melanosoma, T. platensis, and T. vandae. The results showed diagnostic characters for the studied species. The dorsal view featured more valuable characters, with seven informative characters. Similarities were observed among T. delpontei, T. infestans var. melanosoma, and T. platensis, and between T. jurbergi and T. vandae, correlating with previous studies. Thus, female genital characters proved to be reliable and useful in the diagnosis of the Triatoma species studied here; additional studies, along with other sets of behavioral, morphological, and molecular data, helped to reinforce the hypotheses found here.

Species-specific differences in the egg exochorium of the sympatric taxa Triatoma costalimai and Triatoma jatai (Hemiptera: Reduviidae: Triatominae).

Triatoma costalimai and Triatoma jatai are related species, which occur in sympatry in Paranã, Tocantins, Brazil, in rocky outcrops and in peridomicile and intradomicile environments. This study compared morphologic and morphometric aspects of the eggs of these species using optical microscopy (OM) and scanning electron microscopy (SEM). Operculum cells (OP) and egg body (EB) were drawn and photographed, their surfaces were measured, and spots were quantified. Statistical analyses were performed using ANOVA and t-tests. OM showed an egg exochorium with spots in T. costalimai and a predominance of short lines in T. jatai. We found significant differences in egg length and width, which were larger in T. costalimai. SEM analysis showed that the operculum of both species had cells with straight and/or rounded rims, with a smooth aspect, random spots, and predominantly pentagonal shape. In the EB, hexagonal cells were predominant, with indices exceeding 60% in both species. Triatoma costalimai cells were flat, with a discrete definition of the rims, whereas T. jatai cells were smooth with well-defined rims. Statistical tests showed significant differences for EB, where T. costalimai cells were larger and have more spots than T. jatai. The eggs can thus be differentiated, thereby contributing to integrative taxonomy.

Association between the potential distribution of Lutzomyia longipalpis and Nyssomyia whitmani and leishmaniasis incidence in Piauí State, Brazil.

BACKGROUND: Leishmaniases are vector borne diseases caused by Leishmania spp. parasites transmitted by female sandflies (Diptera: Psychodidae) whose geographic distribution is influenced by environmental factors. Among the main tools for studying the distribution of vector species, modeling techniques are used to analyze the influence of climatic and environmental factors on the distribution of these insects and their association with human cases of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used a multiscale ecological niche modeling approach to assess the environmental suitability of sandfly vectors of the etiological agents of Visceral (VL) and American Cutaneous Leishmaniasis (ACL) in Piauí state, northeastern Brazil, and then evaluated their relationship with human disease incidence. For this, we obtained the geographic coordinates of the vector species Lutzomyia longipalpis and Nyssomyia whitmani through literature review, online databases and unpublished records. These data were used for the development of predictive models of the distribution of both sandflies species based on climatic and environmental variables. Finally, the environmental suitability for the presence of these vectors was compared with the incidence of both the diseases at the municipality level. The final models for each sandfly species showed good predictive powers with performance metric values of 0.889 for Lu. longipalpis and 0.776 for Ny. whitmani. The areas with greater environmental suitability for the presence of these species were concentrated in the central-north region of Piauí and coincide with the location of those municipalities presenting higher incidences of VL and ACL, situated in the central-north and extreme north of the state, respectively. The south and southeast regions of Piauí state have low incidence of these diseases and presented low environmental suitability for the presence of both vectors. CONCLUSIONS/SIGNIFICANCE: We discuss how predictive modeling can guide entomological and epidemiological surveillances and recommend an increased supervision and control activities in Teresina (capital of the state of Piaui), Altos and Pedro II, in addition to other municipalities with similar social and environmental characteristics.

A molecular and morphological study of Ascaris suum in a human-pig contact scenario in northeastern Brazil.

The aim of the present study was to assess morphologic and genetic data on ascariasis in swine (Sus scrofa domesticus) and humans in low-resource rural and periurban communities in the state of Piauí, Brazil. Our cross-sectional survey included 100 fecal samples obtained from swine and 682 samples from humans. Fifteen pigs were necropsied. Human and porcine fecal samples were examined to identify Ascaris eggs. Parasites obtained in the swine necropsies were studied using scanning electron microscopy (SEM), and the mitochondrial gene encoding the cytochrome oxidase 1 (cox1) enzyme was partially amplified and sequenced for molecular taxonomy and phylogenetic analyses. The overall prevalence of Ascaris eggs in the swine fecal samples was 16/100 (16%). No Ascaris eggs were identified in the human fecal samples. SEM of six worms recovered from pigs demonstrated morphological characteristics of A. suum. Cox1 sequences were compatible with A. suum reference sequences. Original and reference (GenBank) nucleotide sequences were organized into clusters that did not segregate the parasites by host species or and region. The largest haplogroups were dominated by haplotypes H01, H02 and H31. In the communities studied, there was no epidemiological evidence of the zoonotic transmission of ascariasis at the human-swine interface.

Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity.

BACKGROUND: Mosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated. METHODS: Larvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR. RESULTS: Propagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF. CONCLUSION: By 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae.

Study on Morphological Changes and Interference in the Development of Aedes aegypti Caused by Some Essential Oil Constituents.

Dengue, Chikungunya and Zika are arboviruses, transmitted by the mosquito Aedes aegypti, that cause high mortality and serious health consequences in human populations. Efforts to control Ae. aegypti are important for preventing outbreaks of these diseases. Essential oil constituents are known to exhibit many activities, such as their use as larvicides. Given their potential, the present study aimed to characterize the larvicidal effect of dihydrojasmone, p-cymene, carvacrol, thymol, farnesol and nerolidol on the larvae of Ae. aegypti and their interference over the morphology of the mosquitos. The essential oil constituents were dissolved in dimethylsulfoxide at concentrations of 1-100 µg/mL and were applied in the breeding environment of third-stage larvae. The larvae from bioassays were fixed, dehydrated and embedded. Ultrathin sections were contrasted using 5% uranyl acetate and 1% lead citrate for observation through transmission electron microscopy. The oil with the highest larvicidal efficiency was found to be nerolidol, followed by farnesol, p-cymene, carvacrol, thymol and dihydrojasmone, with an LC50 of 11, 21, 23, 40, 45 and 66 µg/mL, respectively. The treated Ae. aegypti larvae caused alteration to the tegument or internal portions of larvae. The present study demonstrated which of these oils-dihydrojasmone, farnesol, thymol, p-cymene, carvacrol and nerolidol-have effective larvicidal activity.