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.

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.

Spawning behavior of Aedini (Diptera: Culicidae) in a remnant of Atlantic Forest in the state of Rio de Janeiro.

BACKGROUND: Mosquito assemblages are organized along an ecological gradient, including small habitats where interspecific competition predominates and large permanent habitats where predation predominates. This study aimed to analyze the oviposition behavior of mosquitoes regarding the preference for traps installed at two different heights with regard to ground level and the tendency to share spawning sites in an Atlantic Forest fragment in Nova Iguaçu, State of Rio de Janeiro, Brazil. METHODS: The eggs were collected from April 2018 to March 2019. Twelve ovitraps were used, randomly distributed in trees at ground level and at a height of 3 m in a forest environment. RESULTS: They were sequentially numbered, monitored, and replaced every 2 weeks. Among the 5818 eggs collected, 3941 hatched, 3756 reached the pupa stage, and 2370 reached the adult stage. The most abundant species were Aedes albopictus (63%) and Haemagogus leucocelaenus (35%), followed by Aedes terrens (2%) and Haemagogus janthinomys (1%). Analyses showed a significant difference of (P = 0.02) between the number of mosquito species collected in the palettes at ground level and the number collected at the height of 3 m. Cluster analysis of species abundance showed that the eggs collected in the palettes at ground level were more abundant than those collected in the palettes at the height of 3 m. We detected co-occurrence of species in the oviposition palettes; according to the null model, such species distribution was not random. CONCLUSIONS: The exploitation of oviposition sites by mosquito species can represent an event forced by population density facilitated by the ecological valence of individuals of one species. Understanding the aggregate distribution of larvae at the oviposition site allows us to conduct more in-depth studies of the oviposition behavior of female mosquitoes.

Bionomy of Mosquitoes in Bamboo Internodes in an Atlantic Forest Remnant of the State of Rio De Janeiro, Brazil.

Living bamboo stalks are one of the most specialized habitats for mosquito oviposition and immature development. Most of the mosquito species that breed in these habitats are sylvatic, and some are of importance for public health as possible vectors of pathogens. Perforated internodes are a very specialized environment due to the difficulty of access. Furthermore, due to their relatively simple fauna, they represent a valuable model for ecological studies that may be applicable to more complex environments. This study aims to assess the mosquito bionomics of species raised in bamboo internodes. Therefore, the diversity of mosquito species and the influence of abiotic variables (pH and temperature) on the distribution of mosquitoes that breed in this habitat were analyzed. The study area is a fragment of Atlantic Forest within the Association of da Armada (ATA) in Nova Iguaçu, state of Rio de Janeiro, Brazil. Immature mosquitoes were sampled with suction tubes (mouth aspirators) between August 2017 and July 2018. A total of 3,170 larvae were collected in 5 bamboo plants, each with 8 stalks perforated. Of these, 688 larvae reached the adult stage, representing 10 genera and 19 species. The most common species were Culex neglectus (43%), Trichoprosopon digitatum (22%), Culex iridescens (8%), Sabethes identicus (7%), and Orthopodomyia albicosta (7%). The richness of the immatures collected in the ATA was 19 species, with a diversity of 1.10 and Shannon evenness of 0.57. A diverse composition of Culicidae in bamboo stalks was found, although dominance was low.

External female genitalia of Triatoma jatai, Triatoma costalimai and Triatoma williami (Hemiptera: Reduviidae: Triatominae).

BACKGROUND: Taxonomic identification of triatomines is generally performed based on aspects of their external morphology. However, the use of a multidisciplinary approach, considering morphological aspects of the external genitalia, morphometry, genetics, and phylogeography has been suggested, especially for similar and/or cryptic species. The rupestral species Triatoma jatai Gonçalves et al., 2013, Triatoma costalimai Verano & Galvão, 1959 and Triatoma williami Galvão et al., 1965, which are morphologically similar, have been found naturally infected with Trypanosoma cruzi (Chagas, 1909) in wild, peridomestic, and intradomestic environments, representing a risk of new outbreaks of Chagas disease. This study presents morphological description complementation of these species, with an emphasis on the structures of the female external genitalia, using scanning electron microscopy. METHODS: The females of T. jatai and T. costalimai (n = 10 of each) were captured in the Brazilian municipalities of Paranã and Aurora do Tocantins and were identified with the use of a dichotomous key for the Matogrossensis subcomplex. Females of T. williami (n = 5), were obtained from a laboratory colony. The females were cut transversely at the sixth abdominal segment and examined under scanning electron microscopy (SEM) at the Oswaldo Cruz/Fiocruz Institute Electronic Microscopy Platform. RESULTS: It was possible to differentiate the three species based on the characteristics of urotergites VII, VIII and IX and urosternite VII, as well as the genital plaques, gonocoxites, and gonapophyses. To our knowledge, morphological differences in the spines present on gonapophysis 8 in triatomines are described here for the first time. CONCLUSIONS: The results show that external genitalia of females are useful structures to differentiate T. costalimai, T. jatai and T. williami. SEM analysis contributes to and corroborates, together with other tools morphological and molecular, the distinction of the three species.

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.

New sex-determination system in the genus Panstrongylus (Hemiptera: Reduviidae) revealed by chromosomal analysis of Panstrongylus lutzi.

BACKGROUND: Panstrongylus lutzi (Neiva & Pinto, 1923) is a triatomine species native to Caatinga habitats in north-eastern Brazil. It is considered an important vector of Chagas disease in this region, presenting high rates of natural infection with Trypanosoma cruzi Chagas, 1909, and readily invading houses by flight. This study describes a previously unknown chromosomal sex system in the genus Panstrongylus based on P. lutzi. METHODS: Fifth-instar and male adults of P. lutzi originating from municipality of Várzea Alegre, Ceará (Brazil) were analysed. Chromosomal analyses of male meiotic process were done by Giemsa staining. RESULTS: Chromosomal analyses of male meiosis reveal a diploid chromosome number of 24 chromosomes (20 autosomes plus X1X2X3Y). During meiotic prophase I, the sex chromosomes remained close together, forming four heteropycnotic chromocenters in zygotene, and a single chromocenter in pachytene and diplotene. Still at the diplotene stage, each one of the ten autosomal bivalents showed an evident chiasma. In metaphase I, the four sex chromosomes appeared clearly separated. The three X chromosomes were the smallest of the complement and isopycnotic with respect to the Y chromosome. Two bivalents appear larger, whereas the other eight showed no significant difference in size. CONCLUSION: Karyotype analysis of P. lutzi revealed a new sex system in the genus Panstrongylus. This result is of utmost importance to karyosystematics of P. lutzi, and demonstrates the need for further studies of this type in the subfamily Triatominae.

Mitochondrial Genes Reveal Triatoma jatai as a Sister Species to Triatoma costalimai (Reduviidae: Triatominae).

Triatoma jatai was described using a set of morphological structures from specimens collected in Paranã municipality of Tocantins State, Brazil. Under a Bayesian framework and using two mitochondrial genes (16S and COI), phylogenetic analysis recovered T. jatai as a sister species to Triatoma costalimai with higher genetic distances than between other well-recognized species. Our results agree with previous suggestions based on morphometric analysis. In the light of the non-monophyly of Matogrossensis subcomplex, the inclusion of T. jatai shall be considered for reevaluating this group.

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.

Ultrastructure of immature stages of Lucilia cuprina (Diptera: Calliphoridae) using scanning electron microscopy.

The blowfly Lucilia cuprina is distributed worldwide and is a mechanical vector of pathogens. It can cause myiasis in humans and is strongly related to forensic entomology, as it is frequently found on human and animal corpses. However, most of the L. cuprina found on corpses are the immature stages of this fly. Correct identification is very important for forensic entomology but at present only the identification keys of adult L. cuprina are available. Thus, the aim of this paper was to describe and analyze the morphological characteristics of all larval instars and the puparia of L. cuprina using scanning electron microscopy (SEM).

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.

Ultrastructure of larvae and puparia of the blowfly Chrysomya megacephala (Diptera: Calliphoridae).

Chrysomya megacephala is a forensic important fly, and its immature forms also cause myiasis. The adults are the first insects to reach a carcass and can oviposit just a few hours after arrival. Therefore, the knowledge of immature stages of flies is essential for correct identification of the species found on corpses. Scanning electron microscopy (SEM) gives detailed information on the morphological characters that can help identify the immature forms of flies. The aim of this study is to identify larvae and puparia of C. megacephala using SEM. The larval instar body of C. megacephala is similar at all instars. The integument is smooth with small spines located at the limit of all segments. The cephalic region has a group of robust spines with one or two tips. The puparia are very similar to third instar larvae, except for the cephalic structures that are retracted. The integument shows the wrinkles from the third instar larvae and posterior spiracle disc with three spiracular openings localized on the top of an elevation. In conclusion, SEM provides some characteristics to distinguish among Chrysomya species that could help entomologists to identify immature found on corpses.

Ultrastructure of immature stages of the blowfly Chrysomya putoria (Wiedemann, 1818) (Diptera: Calliphoridae).

Forensic entomology is an area of science that serves as a tool in crime scene investigations. Usually, flies are the first insects to reach a carcass and can oviposit just a few hours after arrival. Therefore, the knowledge of immature stages is essential for correct identification of the species found on corpses. Scanning electron microscopy (SEM) gives detailed information about morphological characters helping to identify the immature forms of flies. Chrysomya putoria (Wiedemann) is a very important fly for forensic entomology, because it has high population densities and is easily found in colonizing carcasses, moreover, it is also a possible causative agent for secondary myiasis. The aim of this study is to identify larvae and puparia of C. putoria using SEM. The first instar larvae were composed of 12 smooth segments separated by spines. Antennae and maxillary palps were visible. Anterior spiracle was absent and only one spiracular opening could be seen at the posterior spiracle. Second and third larval instars were similar to first instar, except for the presence of anterior spiracle that is composed by 11-12 spiracular ramifications. At the anal segment, two spiracular openings were found in second instars and three openings in third instar larvae. Puparia showed a retracted cephalic region and none of the head structures were visible.

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi.

Semiconductor nanoparticles, such as quantum dots (QDs), were used to carry out experiments in vivo and ex vivo with Trypanosoma cruzi. However, questions have been raised regarding the nanotoxicity of QDs in living cells, microorganisms, tissues and whole animals. The objective of this paper was to conduct a QD nanotoxicity study on living T. cruzi protozoa using analytical methods. This was accomplished using in vitro experiments to test the interference of the QDs on parasite development, morphology and viability. Our results show that after 72 h, a 200 µM cadmium telluride (CdTe) QD solution induced important morphological alterations in T. cruzi, such as DNA damage, plasma membrane blebbing and mitochondrial swelling. Flow cytometry assays showed no damage to the plasma membrane when incubated with 200 µM CdTe QDs for up to 72 h (propidium iodide cells), giving no evidence of classical necrosis. Parasites incubated with 2 µM CdTe QDs still proliferated after seven days. In summary, a low concentration of CdTe QDs (2 µM) is optimal for bioimaging, whereas a high concentration (200 µM CdTe) could be toxic to cells. Taken together, our data indicate that 2 µM QD can be used for the successful long-term study of the parasite-vector interaction in real time.

Scanning electron microscopy of the eggs of Aedes scapularis from southern South America.

The eggs of Aedes scapularis analyzed by scanning electron microscopy are black and elliptical in outline, measuring approximately 620.4 +/- 16.74 microm long and 163.7 +/- 16.90 microm (n = 10) wide, with an egg index (length/width ratio) of 3.79. The anterior extremity tapered abruptly from a width of 51.6 microm, while such tapering was more gradual at the posterior extremity, from a width of 61.4 microm. The ventral surface of the chorionic coating presented cells with a tubular aspect containing tubercles in rows at a density of 5 to 9 per cell with 2 different sizes, the largest measuring 7.23 +/- 0.98 microm in a longitudinal diameter and the smallest 4.15 +/- 0.53 microm (n = 30). In the dorsal region, the external chorionic reticulum had a porous appearance, and its thickness ranged from 2.5 to 4.1 microm. Isolated tubercles presented wide variation per cell. In the central region of some chorionic cells were tubercles of greater diameter, characterized as central tubercles of 8.45 +/- 0.67 microm, and around them 3 to 5 smaller tubercles measuring 2.57 +/- 0.26 microm. The micropylar apparatus presented a collar with a very evident molding and edges with defined margins for the transition area and a thickness of around 11.1 microm. The micropyle disc margins were raised, measuring around 17.8 microm in diameter and 229 microm in circumference. The micropyle orifice was very evident, with a diameter of 1.41 microm.

Ultramorphological characteristics of immature stages of Chrysomya albiceps (Wiedemann 1819) (Diptera: Calliphoridae), a fly specie of forensic importance.

Forensic entomology is an area of science that serves as a tool in crime scene investigations. Usually, flies are the first insects to reach a dead body and can oviposit just a few hours after arrival. Therefore the knowledge of immature stages is essential for correct identification of the species found on corpses. Scanning electron microscopy gives detailed information about morphological characters helping to identify the immature forms and consequently serves as a tool in crime scene investigations. C. albiceps is a very important fly for forensic entomologists because its larvae are almost always present on a dead body and it is facultative predators and therefore can alter the composition of species present at the carcass. The aim of this study is to identify eggs, larvae, and puparia of C. albiceps using SEM. Eggs were elongated with the anterior region ending in a "Y" shape and the posterior end was tapered. The micropyle was a well-adorned orifice with some projections around it. The first instar larva was composed of 12 segments separated by spines. Only one spiracular opening could be seen at the posterior spiracle. Body tegument was smooth and tubercles were not seen. Antennae and maxillary palps were visible. Second and third larval instars were very similar to first instar, except for the presence of anterior spiracle. However, body tegument was composed of net-like patches and tubercles were visible. Tubercles present at the third instar larvae were robust and erect. Puparia showed a retracted cephalic region and curved tubercles.

Structural study of the salivary glands of Anocentor nitens (Acari: Ixodidae) during the feeding cycle.

The salivary glands of Anocentor nitens (Neumann,1897) occur in pairs and are located in the anterolateral region of the general cavity, with milky white color and approximately equal sizes. They consist of a secretory portion and an excretion duct. In some glandular acini, all the cells had a basophilic appearance they were stained by hematoxylin, whereas others presented cells with different staining affinities. In this work, we describe the variations observed in these glands during the feeding cycle of ticks [after feeding (0 h) and successively at 24, 48, 72, 96, 120, and 144 h]. The cells stained by hematoxylin were shown to be more reactive to Alcian blue, thus demonstrating the presence of acid glycosaminoglycans, whereas those stained using eosin presented weak or no reaction. A strong reaction was found by the use of the periodic acid-Schiff (PAS) technique, thereby suggesting the presence of glycogen and/or glycoconjugates containing hexose, confirmed by using salivary amylase before PAS, with partial destaining of the slides. Continuing presence of residual staining in these cells suggests the presence of glycoconjugates containing hexose. Cells with nuclei of circular outline and few granules (of different sizes) were found in type II acini, 72 h after collection. Type I acini presented wide lumina and walls composed of larger numbers of cells of cubic to cylindrical shape. The pronounced degranulation shown in this study over the course of the feeding cycle was associated with the release of substances for oviposition.

Histology and ultrastructure of Aedes albopictus larval midgut infected with Bacillus thuringiensis var. israelensis.

Histological and ultrastrucutural alterations in the midgut of Aedes albopictus larvae infected with Bacillus thuringiensis var. israelensis (Bti) were observed by light and transmission electron microscopy (TEM). Two formulations of Bti were used: granulated and powder, with 0.2% active ingredient in 90 larvae of Ae. albopictus distributed in three containers containing 30 larvae each (one control group and two test groups). The midgut epithelium of the control group presented flattened and elongated cells with mace-shape with a narrow base. Midgut epithelium cells' surface was convex and had a large circular nucleus located in the median-apical portion of the cell. These cells also presented a basal lamina with a small accumulation of extracellular fibrous matrix, thus characterizing a basal membrane, with a muscle layer and a peritoneal membrane more externally. After Bti ingestion, the larvae stopped/slowed their natural movements down in 5 min. After 30 min approximately, the swimming movements stopped completely. Internally, the intestinal cells showed a disorganization of the basal processes, dilatation and fragmentation of the rough endoplasmic reticulum, with intense cytoplasmic vacuolization. There were concentric dense laminas accumulated in the cytoplasm, and these residual membranous bodies were seen greatly increased in size after 60 min. Mitochondria, fragments of rough endoplasmic reticulum and other remainder organelles were surrounded and segregated from the cytoplasm by exocytosis. This article reports the histopathological alterations in the midgut of Ae. albopictus after infection with Bti and contributes to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against mosquito larvae.

Scanning electron microscopy of the egg of Haemagogus tropicalis.

Haemagogus tropicalis is strictly a forest-dwelling species from the fertile valley area of the Amazônia forest. It is a diurnal mosquito, and the oviposition sites for the species include tree holes. The eggs of Hg. tropicalis used in this study were from females captured on Combú Island, situated across from the city of Belém, Guajará Bay, state of Pará, at 1 degrees 25'S latitude and 48 degrees 25'W longitude. The eggs are elliptical and approximately 575 microm long with a width of approximately 144 microm. The ventral surface of the chorionic reticulum has regular chorionic cells with hexagonal and sometimes pentagonal ornamentation. Each chorionic cell has a thick external chorionic reticulum with regular borders. The interior of the chorionic cells have small, evenly distributed tubercles, and the dorsal external chorionic reticulum appears porous. The micropylar apparatus, located on the anterior area of the egg, was formed by a collar with a well-developed frame. Centrally, the micropylar disc had a diameter of approximately 20 microm and the micropylar orifice is 2.1 microm in diameter. These data may enable construction of taxonomic keys for identifying eggs of Haemagogus species.