Integrative taxonomy and phylogenetic systematics of the Triatoma rubrovaria subcomplex (Hemiptera, Triatominae).

Triatoma rubrovaria subcomplex consists of T. carcavalloi, T. circummaculata, T. klugi, T. limai, T. oliveirai, T. pintodiasi, T. rubrovaria, T. patagonica and T. guasayana, which can be vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease. In this study, morphological, morphometric, and genetic characters of T. circummaculata, T. pintodiasi, T. carcavalloi, T. klugi, and T. rubrovaria were analyzed in view of the integrative taxonomy and phylogeny of the T. rubrovaria subcomplex. Molecular studies were carried out through the sequencing and analysis of the mitochondrial genes COI and CytB, nuclear genes ITS I, ITS 2, 16S, and 28S from rDNA and rescued a monophyletic group. Furthermore, differential morphological characters were found among the five species in the pronotum, scutellum, stridulatory sulcus, male genitalia, and external female genitalia. Finally, morphometric analyses made it possible to differentiate the five species. Phylogenetic analyzes rescued the relationship of T. pintodiasi with members of the T. rubrovaria subcomplex and demonstrated that this subcomplex is a monophyletic group composed of the species T. carcavalloi, T. circummaculata, T. klugi, T. guasayana, T. limai, T. oliveirai, T. patagonica, T. pintodiasi, and T. rubrovaria. Furthermore, through integrative taxonomy, it was possible to confirm the specific status of the species T. carcavalloi, T. circummaculata, T. pintodiasi, T. klugi, and T. rubrovaria, offering new useful morphological characters for the differentiation and characterization of these potential vectors and distributed in Southern Brazil.

Growth Curve, Morphological and Molecular Characterization of Two Strains of Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) isolated from Triatoma sherlocki (Hemiptera, Reduviidae, Triatominae).

BACKGROUND: Trypanosoma cruzi presents great variability in morphology, virulence, pathogenicity, avoidance of the host immune system, and antigenic constitution, associated with different clinical manifestations of the disease. METHODS: Two strains of T. cruzi were cultivated in liver infusion tryptose to determine growth kinetics, morphometry and molecular characterization using restriction fragment length polymorphism polymerase chain reaction. RESULTS: The biological parameters showed sharp growth by the 7th day. Morphologically, both strains showed short and thin forms and were classified as Group I. CONCLUSION: Group TcI presents cardiac manifestations and T. sherlocki is adapting to the home environment, requiring attention to future problems.

Characterization of Female External Genitalia and Eggs of Four South American Species of the Triatoma Laporte, 1832 Genus (Hemiptera: Reduviidae: Triatominae).

Triatoma is the most diversified and one of the most important genera from an epidemiological perspective. Given the difficulty in identifying some species of the Triatoma genus, morphological, histological, and morphometric studies were performed to provide new characters that make it possible to differentiate T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto, triatomines that overlap geographically and have vector potential. Through the external female genitalia, as well as morphology, morphometry, and histology of eggshells, it was possible to discriminate the four species. In addition, this study reinforces the taxonomic validity of T. garciabesi and provides new data for discussion on systematic issues of T. guasayana and T. patagonica.

Effects of sodium valproate on the chromatin of Triatoma infestans (Klug, 1834) (Hemiptera, Reduviidae) under in vitro culture conditions.

Sodium valproate (VPA) is a classic anticonvulsive, a histone deacetylase inhibitor, and a chromatin remodeling inducer. When injected into specimens of Triatoma infestans, a vector of Chagas disease, VPA affects the chromatin supraorganization of chromocenter heterochromatin in only a few cells of the Malpighian tubules. To test whether this result was explained by the inaccessibility of all of the organ's cells to the drug, we investigated the nuclear phenotypes and global acetylation of lysine 9 in histone H3 (H3K9ac) in Malpighian tubules cultivated in vitro for 1-24 h in the presence of 0.05 mM-1 mM VPA. The present results revealed that the chromatin decondensation event in the chromocenter body, which was detected only under low VPA concentrations up to a 4-h treatment, was not frequent during organ culture, similar to the results for injected insects. Cultivation of T. infestans Malpighian tubules in vitro for 24 h revealed inadequate for cell preservation even in the absence of the drug. Immunofluorescence signals for H3K9ac following VPA treatment showed a slightly increased intensity in the euchromatin, but were never detected in the chromocenter bodies, except with great intensity at their periphery, where the 18S rDNA is located. In conclusion, when VPA affects the chromocenter heterochromatin in this animal cell model, it occurs through a pathway that excludes a classic global H3K9ac mark. Investigation of nonhistone proteins associated with histone methylation marks is still required to further explain the differential response of T. infestans chromatin to VPA.

Taxonomical over splitting in the Rhodnius prolixus (Insecta: Hemiptera: Reduviidae) clade: Are R. taquarussuensis (da Rosa et al., 2017) and R. neglectus (Lent, 1954) the same species?

The use of subtle features as species diagnostic traits in taxa with high morphological similarity sometimes fails in discriminating intraspecific variation from interspecific differences, leading to an incorrect species delimitation. A clear assessment of species boundaries is particularly relevant in disease vector organisms in order to understand epidemiological and evolutionary processes that affect transmission capacity. Here, we assess the validity of the recently described Rhodnius taquarussuensis (da Rosa et al., 2017) using interspecific crosses and molecular markers. We did not detect differences in hatching rates in interspecific crosses between R. taquarussuensis and R. neglectus (Lent, 1954). Furthermore, genetic divergence and species delimitation analyses show that R. taquarussuensis is not an independent lineage in the R. prolixus group. These results suggest that R. taquarussuensis is a phenotypic form of R. neglectus instead of a distinct species. We would like to stress that different sources of evidence are needed to correctly delimit species. We consider this is an important step in understanding vectorial Chagas disease spread and transmission.

Biological and Molecular Characterization of Trypanosoma cruzi Strains from Four States of Brazil.

Chagas disease affects between six and seven million people. Its etiological agent, Trypanosoma cruzi, is classified into six discrete typing units (DTUs). The biological study of 11 T. cruzi strains presented here included four parameters: growth kinetics, parasitemia curves, rate of macrophage infection, and serology to evaluate IgM, total IgG, IgG1, IgG2a, and IgG3. Sequencing of small subunit of ribosomal RNA (SSU rRNA)was performed and the T. cruzi strains were classified into three DTUs. When their growth in liver infusion tryptose medium was represented in curves, differences among the strains could be noted. The parasitemia profile varied among the strains from the TcI, TcII, and TcIII groups, and the 11 T. cruzi strains produced distinct parasitemia levels in infected BALB/c. The TcI group presented the highest rate of macrophage infection by amastigotes, followed by TcII and TcIII. Reactivity to immunoglobulins was observed in the TcI, TcII, and TcIII; all the animals infected with the different strains of T. cruzi showed anti-T. cruzi antibodies. The molecular study presented here resulted in the classification of the T. cruzi strains into the TcI (Bolivia, T lenti, Tm, SC90); TcII (Famema, SC96, SI8, Y); and TcIII (QMM3, QMM5, SI5) groups. These biological and molecular results from 11 T. cruzi strains clarified the factors involved in the biology of the parasite and its hosts. The collection of triatomine (vector) species, and the study of geographic distribution, as well as biological and molecular characterization of the parasite, will contribute to the reporting and surveillance measures in Brazilian states.

Third record of Rhodnius amazonicus and comparative study with R. pictipes (Hemiptera, Reduviidae, Triatominae).

Rhodnius amazonicus Almeida et al. (1973) is a triatomine of rare occurrence. This species was found for the third time in Breves city, Pará state, Brazil. Morphometric and morphological studies were carried out on one male and one female. Lent and Wygodzinsky (1979) considered this species as a synonym of R. pictipes, until its revalidation after 23 years by Bérenger and Pluot-Sigwalt (2002). Considering the synonym mentioned above, a comparative study between these two species was performed in order to report the third encounter of this species, and increase the number of morphological characters that distinguish R. amazonicus from R. pictipes. The dorsal side of the head, the dorsal and ventral portions of the thorax, the dorsal, ventral and posterior sides of the female genitalia, eggs and the median process of the pygophore were examined by scanning electronic microscopy (SEM). The head, thorax, abdomen and egg parameters of these two species were also measured. The identification of characters on the head, stridulatory sulcus, mesosternum, metasternum, scutellum, process of urotergite I, external female genitalia, pygophore and eggs made the distinction between R. amazonicus and R. pictipes possible. This study has shown the new characters recorded and described for the first time for R. amazonicus and R. pictipes by SEM here made it possible to confirm the morphological separation between these two species. The morphometric analysis also confirmed that the above two taxa are different at specific level.

Morphology of the spermathecae of twelve species of Triatominae (Hemiptera, Reduviidae) vectors of Chagas disease.

Trypanosoma cruzi, the etiological agent of Chagas disease, is transmitted by triatomines that have been described in a large number of studies. Most of those studies are related to external morphology and taxonomy, but some biochemical, genetic and physiological studies have also been published. There are a few publications in the literature about the internal organs of Triatominae, for instance the spermathecae, which are responsible for storing and maintaining the viability of the spermatozoids until the fertilization of the oocytes. This work aims to study the spermathecae of twelve species of triatomines obtained from the Triatominae Insectarium of the Faculty of Pharmaceutical Sciences, UNESP, Araraquara, using optical microscopy and scanning electron microscopy. The spermathecae of the twelve species studied showed three morphological patterns: a) P. herreri sn, P. lignarius, P. megistus, Triatoma brasiliensis, T. juazeirensis, T. sherlocki and T. tibiamaculata have spermathecae with a thin initial portion and an oval-shaped final portion; b) R. montenegrensis, R. nasutus, R. neglectus, R. pictipes and R. prolixus have tubular and winding spermathecae; c) T. infestans has oval spermathecae. In addition to the three morphological patterns, it was noted that each of the twelve species has particular features that differentiate them.

A new species of Rhodnius from Brazil (Hemiptera, Reduviidae, Triatominae).

A colony was formed from eggs of a Rhodnius sp. female collected in Taquarussu, Mato Grosso do Sul, Brazil, and its specimens were used to describe R. taquarussuensissp. n. This species is similar to R. neglectus, but distinct characters were observed on the head, thorax, abdomen, female external genitalia and male genitalia. Chromosomal differences between the two species were also established.

Insecticidal activity of an essential oil of Tagetes patula L. (Asteraceae) on common bed bug Cimex lectularius L. and molecular docking of major compounds at the catalytic site of ClAChE1.

Emerging resistance to insecticides has influenced pharmaceutical research and the search for alternatives to control the common bed bug Cimex lectularius. In this sense, natural products can play a major role. Tagetes patula, popularly known as dwarf marigold, is a plant native to North America with biocide potential. The aim of this work was to evaluate the biological activity of T. patula essential oil (EO) against adult common bed bugs via exposure to dry residues by the Impregnated Paper Disk Test (IPDT) using cypermethrin as a positive control. We selected the enzyme acetylcholinesterase as a target for modeling studies, with the intent of investigating the molecular basis of any biological activity of the EO. Chemical analysis of the EO was performed using gas chromatography coupled to mass spectrometry (GC-MS). Additionally, oral and dermal acute toxicity tests were performed according to Organization for Economic Cooperation and Development (OECD) guidelines. The sulforhodamine B assay (SRB) was performed to verify the cytotoxicity of EO to HaCaT cells. The EO eliminated 100 % of the bed bugs at 100 mg mL-1 with an LC50 value of 15.85 mg mL-1. GC-MS analysis identified α-terpinolene, limonene, piperitenone, and piperitone as major components of the mixture. Molecular modeling studies of these major compounds suggested that they are acetylcholinesterase inhibitors with good steric and electronic complementarity. The in vitro cytotoxicity evaluation revealed a LC50 = 37.06 µg mL-1 and in vivo acute toxicity showed an LC50 >4000 mg kg-1, indicating that the EO presents low risk of toxic side effects in humans. The T. patula essential oil components provide a promising strategy for controlling bed bug populations with low mammalian toxicity. These findings pave the way for further in vivo studies aimed at developing a safe and effective insecticide.

Description of Rhodnius marabaensis sp. n. (Hemiptera, Reduviidae, Triatominae) from Pará State, Brazil.

Rhodnius marabaensissp. n. was collected on 12 May 2014 in the Murumurú Environmental Reserve in the city of Marabá, Pará State, Brazil. This study was based on previous consultation of morphological descriptions of 19 Rhodnius species and compared to the identification key for the genus Rhodnius. The examination included specimens from 18 Rhodnius species held in the Brazilian National and International Triatomine Taxonomy Reference Laboratory in the Oswaldo Cruz Institute in Rio de Janeiro, Brazil. The morphological characteristics of the head, thorax, abdomen, genitalia, and eggs have been determined. Rhodnius prolixus and Rhodnius robustus were examined in more detail because the BLAST analysis of a cyt-b sequence shows they are closely related to the new species, which also occurs in the northern region of Brazil. The most notable morphological features that distinguish Rhodnius marabaensissp. n. are the keel-shaped apex of the head, the length of the second segment of the antennae, the shapes of the prosternum, mesosternum and metasternum, the set of spots on the abdomen, the male genitalia, the posterior and ventral surfaces of the external female genitalia, and the morphological characteristics of the eggs. Rhodnius jacundaensis Serra, Serra & Von Atzingen (1980) nomen nudum specimens deposited at the Maraba Cultural Center Foundation - MCCF were examined and considered as a synonym of Rhodnius marabaensissp. n.

Trypanosoma cruzi isolated from a triatomine found in one of the biggest metropolitan areas of Latin America.

INTRODUCTION: To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. METHODS: The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in São Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. RESULTS: After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. CONCLUSIONS: Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite.

Trypanosoma cruzi isolated from a triatomine found in one of the biggest metropolitan areas of Latin America

Abstract: INTRODUCTION: To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. METHODS: The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in São Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. RESULTS: After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. CONCLUSIONS: Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite.

Cytogenetic and morphologic approaches of hybrids from experimental crosses between Triatoma lenti Sherlock & Serafim, 1967 and T. sherlocki Papa et al., 2002 (Hemiptera: Reduviidae).

The reproductive capacity between Triatoma lenti and Triatoma sherlocki was observed in order to verify the fertility and viability of the offspring. Cytogenetic, morphological and morphometric approaches were used to analyze the differences that were inherited. Experimental crosses were performed in both directions. The fertility rate of the eggs in crosses involving T. sherlocki females was 65% and 90% in F1 and F2 offspring, respectively. In reciprocal crosses, it was 7% and 25% in F1 and F2 offspring, respectively. The cytogenetic analyses of the male meiotic process of the hybrids were performed using lacto-acetic orcein, C-banding and Feulgen techniques. The male F1 offspring presented normal chromosome behavior, a finding that was similar to those reported in parental species. However, cytogenetic analysis of F2 offspring showed errors in chromosome pairing. This post-zygotic isolation, which prevents hybrids in nature, may represent the collapse of the hybrid. This phenomenon is due to a genetic dysregulation that occurs in the chromosomes of F1. The results were similar in the hybrids from both crosses. Morphological features, such as color and size of connexive and the presence of red-orange rings on the femora, were similar to T. sherlocki, while wins size was similar to T. lenti in F1 offspring. The eggshells showed characteristics that were similar to species of origin, whereas the median process of the pygophore resulted in intermediate characteristics in the F1 and a segregating pattern in F2 offspring. Geometric morphometric techniques used on the wings showed that both F1 and F2 offspring were similar to T. lenti. These studies on the reproductive capacity between T. lenti and T. sherlocki confirm that both species are evolutionarily closed; hence, they are included in the brasiliensis subcomplex. The extremely reduced fertility observed in the F2 hybrids confirmed the specific status of the species that were analyzed.

Study of the external female genitalia of 14 Rhodnius species (Hemiptera, Reduviidae, Triatominae) using scanning electron microscopy.

BACKGROUND: Among the vectors of Chagas disease (Hemiptera: Reduviidae:Triatominae), there are eighteen Rhodnius species described and some are difficult to identify. The aim of this article is to contribute to the specific identification of fourteen Rhodnius spp. through morphological characters of the external female genitalia. METHODS: Female abdomens were cut transversely. The specimens were then prepared for examination by using scanning electron microscopy. RESULTS: The careful examination of the dorsal, posterior and ventral sides revealed characteristics that allowed the identification of each of the fourteen species. CONCLUSION: The use of external female genitalia as characteristics are proposed as a tool for specifically identifying Rhodnius species, and an identification key for these species is presented.