A Cytotoxicity Assay as an Alternative to the Murine Model for the Potency Testing of Bothrops jararaca Venom and Antivenom: An Intralaboratory Pre-validation Study.

Antivenom therapy is the only specific treatment for snakebite envenomation, and antivenom potency determination is key in the efficacy assurance quality control process. Nowadays, this process relies on the in vivo murine model - thus, the development of alternative in vitro methods is imperative. In the current study, the principle of the proposed method is the ability of Bothrops venom to induce cytotoxic effects in Vero cells, and the capacity to evaluate the inhibition of this cytotoxicity by the respective antivenom. After exposure to the venom/antivenom, the relative proportions of adherent (viable) cells were evaluated by direct staining with Coomassie Blue. The optical density (OD) of the lysed cell eluate was directly proportional to the number of adherent cells. This cytotoxicity-based alternative method could represent a potential candidate for validation as a replacement for the current in vivo test. The in vitro-determined cytotoxicity of the Brazilian Bothrops reference venom (expressed as the 50% effective concentration; EC50) was 3.61 µg/ml; the in vitro-determined 50% inhibitory concentration (IC50) of the Brazilian Bothrops reference antivenom was 0.133 µl/ml. From these two values, it was possible to calculate the potency of the reference antivenom. The results from the assays exhibited a good linear response, indicating that the method could be a potential candidate replacement method for use in antivenom quality control prior to lot release, subject to further validation.

First record of Brachymeria amenocles (Hymenoptera: Chalcididae) associated with larvae of Peckia (Peckia) chrysostoma (Diptera: Sarcophagidae) in Brazil.

In this work, we report for the first time the occurrence of the parasitoid wasp Brachymeria amenocles (Walker, 1846) (Hymenoptera: Chalcididae) associated with the larvae of the flesh fly, Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae), in Brazil. This parasitoid species was collected together with the species Brachymeria podagrica (Fabricius, 1787), in the municipality of Rio de Janeiro, geographically located in the Southeast region of Brazil, in larvae of the same host species reared in the same attraction substrate, behaving as solitary parasitoids. A trap containing putrefying sardine was exposed for 48 h, in the upper part of a house, located in Vila Isabel, an urban area. The larvae collected were reared in the laboratory without control of environmental conditions. A total of three adult parasitoid wasps of the species B. podagrica, and nine adult parasitoid wasps of the species B. amenocles emerged from 12 host pupae, resulting in a prevalence of parasitoidism of 4.8% and 14.5%, respectively. Developing parasitoids at the pupal phase were observed in another 34 host pupae dissected.

Overview of paratransgenesis as a strategy to control pathogen transmission by insect vectors.

This article presents an overview of paratransgenesis as a strategy to control pathogen transmission by insect vectors. It first briefly summarises some of the disease-causing pathogens vectored by insects and emphasises the need for innovative control methods to counter the threat of resistance by both the vector insect to pesticides and the pathogens to therapeutic drugs. Subsequently, the state of art of paratransgenesis is described, which is a particularly ingenious method currently under development in many important vector insects that could provide an additional powerful tool for use in integrated pest control programmes. The requirements and recent advances of the paratransgenesis technique are detailed and an overview is given of the microorganisms selected for genetic modification, the effector molecules to be expressed and the environmental spread of the transgenic bacteria into wild insect populations. The results of experimental models of paratransgenesis developed with triatomines, mosquitoes, sandflies and tsetse flies are analysed. Finally, the regulatory and safety rules to be satisfied for the successful environmental release of the genetically engineered organisms produced in paratransgenesis are considered.

New Law of Brazilian Biodiversity: Legal Aspects and Impact in the Field of Biotechnology.

Access to genetic resources (GR) and/or traditional knowledge associated with genetic resources (ATK) has been regulated in Brazil since 2001. The law 13,123 / 2015 determined a significant change in the theme, mainly on the rules of distribution of benefits obtained for conservation and sustainable use of biodiversity, the access to technology and technology transfer, the exploitation of products or reproductive material from the GR or ATK and consignment to the outside of part or all the living or dead organism shipped for GR. The implementation of international treaties on GR and ATK for research, biotechnological development and bioprospecting have been causing difficulties for Brazilian researchers, mainly due to the lack of information and dissemination available for compliance with the legislation. In this work, the members of the Committee for Access to Genetic Resources and Associated Traditional Knowledge of the Federal Fluminense University (UFFGEN) - Brazil, and collaborators performed a critical reflection on the new law, helping Brazilian researchers with information necessary to understand the changes made by the new legislation, especially in the field of Biotechnology associated with Brazilian Biodiversity.

Exploring the Insecticide and Acaricide Potential of Development Regulators obtained from Restinga vegetation from Brazil.

As a part of our continuing search for insect and arthropod development regulators from Brazilian restinga vegetation of the Rio de Janeiro State, crude extracts, purified fractions and essential oils were submitted to screening tests seeking for biological activities on the development of the insects Rhodnius prolixus, Dysdercus peruvianus, Oncopeltus fasciatus and Rhipicephalus (Boophilus) microplus. Up to now, 102 secondary metabolites have been detected in the fractions, among them monoterpenes, sesquiterpenes and two triterpenes which were obtained from the species, Eugenia sulcata, Pilocarpus spicatus, Manilkara subsericea, Myrciaria floribunda and Zanthoxylum caribaeum. These secondary plant metabolites are considered of interest for the use of studies related to arthropod endocrinology, vector-parasite interaction system, and population control of vector insect and agricultural pest. The observed biological activities were surprisingly high, involving increased mortality, molting and metamorphosis inhibition, paralysis, corporeal deformities, apparition of permanent nymphs, of adultoids and juvenoids, partial or total inhibition of oviposition and egg hatching. These compounds are now being studied further to determine if they may or may not be useful in controlling insect populations and/or interfere with the life cycle and vector transmission of parasites to animal and human populations.

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection.

Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite Trypanosoma cruzi, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total T. cruzi parasite counts in R. prolixus were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

Laboratory evaluation of Clusia fluminensis extracts and their isolated compounds against Dysdercus peruvianus and Oncopeltus fasciatus

ABSTRACT The effects of the hexanic extracts of the fruits and flowers of Clusia fluminensis Planch. & Triana, Clusiaceae, as well as their main constituents, the triterpene lanosterol and the benzophenone clusianone, were evaluated on hemipterans Dysdercus peruvianus and Oncopeltus fasciatus. The topical treatments of insects with the hexanic extracts significantly affected the survival of O. fasciatus, but not that of D. peruvianus. Concomitantly, extracts delayed the development of both hemipterans. Moreover, isolated lanosterol significantly reduced both the survival and development of O. fasciatus and D. peruvianus, while clusianone only reduce the survival of D. peruvianus and marginally inhibited the development of both insects. The results show the specific activity of lanosterol and clusianone against the two evaluated insect species and indicate the potential of compounds derived from C. fluminensis for the development of specific biopesticides for the control of agricultural pests. Subsequent work will examine the mode of action of lanosterol and clusianone isolates from C. fluminensis.

Biocontrol evaluation of extracts and a major component, clusianone, from Clusia fluminensisPlanch. & Triana againstAedes aegypti

Studies evaluated the effects of hexanic extracts from the fruits and flowers ofClusia fluminensis and the main component of the flower extract, a purified benzophenone (clusianone), against Aedes aegypti. The treatment of larvae with the crude fruit or flower extracts from C. fluminensis did not affect the survival ofAe. aegypti (50 mg/L), however, the flower extracts significantly delayed development of Ae. aegypti. In contrast, the clusianone (50 mg/L) isolate from the flower extract, representing 54.85% of this sample composition, showed a highly significant inhibition of survival, killing 93.3% of the larvae and completely blocking development of Ae. aegypti. The results showed, for the first time, high activity of clusianone against Ae. aegypti that both killed and inhibited mosquito development. Therefore, clusianone has potential for development as a biopesticide for controlling insect vectors of tropical diseases. Future work will elucidate the mode of action of clusianone isolated from C. fluminensis.

Biocontrol evaluation of extracts and a major component, clusianone, from Clusia fluminensis Planch. & Triana against Aedes aegypti.

Studies evaluated the effects of hexanic extracts from the fruits and flowers of Clusia fluminensis and the main component of the flower extract, a purified benzophenone (clusianone), against Aedes aegypti. The treatment of larvae with the crude fruit or flower extracts from C. fluminensis did not affect the survival ofAe. aegypti (50 mg/L), however, the flower extracts significantly delayed development of Ae. aegypti. In contrast, the clusianone (50 mg/L) isolate from the flower extract, representing 54.85% of this sample composition, showed a highly significant inhibition of survival, killing 93.3% of the larvae and completely blocking development of Ae. aegypti. The results showed, for the first time, high activity of clusianone against Ae. aegypti that both killed and inhibited mosquito development. Therefore, clusianone has potential for development as a biopesticide for controlling insect vectors of tropical diseases. Future work will elucidate the mode of action of clusianone isolated from C. fluminensis.

Effects of essential oil from leaves of Eugenia sulcata on the development of agricultural pest insects

Essential oils are known for their insect control potential, which is mainly attributed to the presence of terpenes that interfere with hormonal and physiological processes of arthropods. The aim of the present study was to evaluate the effects of essential oil from the leaves of Eugenia sulcata Spring ex Mart., Myrtaceae, on the development of two species of agricultural pest insects, Dysdercus peruvianus and Oncopeltus fasciatus. Results showed that the essential oil induced mortality, and reduced numbers of adults. Topical treatment of Oncopeltus fasciatus using pure essential oil caused significant mortality rates (96.67%), while Dysdercus peruvianus had a higher tolerance, with 80% mortality at the end of the experiments. Results suggest that essential oil from the leaves of Eugenia sulcata may be used in agriculture for insect pest control.

Laboratory evaluation of the effects of essential oil of Myrciaria floribunda leaves on the development of Dysdercus peruvianus and Oncopeltus fasciatus

Myrciaria floribunda (H. West ex Willd.) O. Berg, Myrtaceae, is popularly known as "camboim-amarelo" and was collected at Restinga de Jurubatiba (RJ, Brazil). Leaves from this species were submitted to hydrodistillation to extract its essential oil. Monoterpenes were the main compounds found (53.9%), and 1,8-cineole was the major constituent (38.4%). Studies were carried out to evaluate the effects of this essential oil on the development of two species of agricultural pests (Oncopeltus fasciatus and Dysdercus peruvianus). The essential oil was considered effective against D. peruvianus and O. fasciatus, causing mortality in both insects. The LD50 values (µg/insect) observed were 112.44 µg/insect (O. fasciatus) and 309.64 µg/insect (D. peruvianus) after one day of treatment, and 72.18 µg/insect (O. fasciatus) and 94.42 µg/insect (D. peruvianus) after 22 days of treatment. The present study reports for the first time the bioinsecticidal activity of essential oil of Myrciaria floribunda leaves, and provides important data regarding the use of essential oils in complementary programs for pest control.

Trypanosoma cruzi TcSMUG L-surface mucins promote development and infectivity in the triatomine vector Rhodnius prolixus.

BACKGROUND: TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. CONCLUSION AND SIGNIFICANCE: Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.

Physalin B inhibits Trypanosoma cruzi infection in the gut of Rhodnius prolixus by affecting the immune system and microbiota.

Physalin B is a natural secosteroidal, extracted from the Solanaceae plant, Physalis angulata, and it presents immune-modulator effects on the bloodsucking bug, Rhodnius prolixus. In this work, R. prolixus was treated with physalin B at a concentration of 1 mg/ml of blood meal (oral application), or 20 ng/insect (applied topically) or 57 ng/cm(2) of filter paper (contact treatment), and infected with Trypanosoma cruzi Dm28c clone (2×10(6) epimastigotes/insect). The three types of applications significantly decreased the number of T. cruzi Dm28c in the gut comparing with the non-treated infected insects (controls). All groups of infected insects treated with physalin B had higher numbers of bacterial microbiota in the gut than the non-treated controls infected with T. cruzi. We observed that the infected physalin B insects with topical and contact treatments had a lower antibacterial activity in the gut when compared with control infected insects. Furthermore, infected insects with the physalin B oral treatment produced higher levels of nitrite and nitrate in the gut than control infected insects. These results demonstrate that physalin B decreases the T. cruzi transmission by inhibiting the parasite development in the insect vector R. prolixus. Herein the importance of physalin B modulation on the immune system and microbiota population in terms of parasite development and transmission are discussed.

Trypanosoma cruzi immune response modulation decreases microbiota in Rhodnius prolixus gut and is crucial for parasite survival and development.

Trypanosoma cruzi in order to complete its development in the digestive tract of Rhodnius prolixus needs to overcome the immune reactions and microbiota trypanolytic activity of the gut. We demonstrate that in R. prolixus following infection with epimastigotes of Trypanosoma cruzi clone Dm28c and, in comparison with uninfected control insects, the midgut contained (i) fewer bacteria, (ii) higher parasite numbers, and (iii) reduced nitrite and nitrate production and increased phenoloxidase and antibacterial activities. In addition, in insects pre-treated with antibiotic and then infected with Dm28c, there were also reduced bacteria numbers and a higher parasite load compared with insects solely infected with parasites. Furthermore, and in contrast to insects infected with Dm28c, infection with T. cruzi Y strain resulted in a slight decreased numbers of gut bacteria but not sufficient to mediate a successful parasite infection. We conclude that infection of R. prolixus with the T. cruzi Dm28c clone modifies the host gut immune responses to decrease the microbiota population and these changes are crucial for the parasite development in the insect gut.

Phase contrast X-ray synchrotron imaging for assessing external and internal morphology of Rhodnius prolixus.

PhC-SR-µCT is a nondestructive technique that allows the microanatomical investigations and 3D images reconstructions. This technique is performed in blood sucker, Rhodnius prolixus--one of the most important insect vectors of Trypanosoma cruzi, ethiologic agent of Chagas' disease in Latin America--was able to provide excellent information about the microanatomy of the thorax and head allowing a new tool for further studies of development and physiology of triatomine by a non-invasive method of observation.

Involvement of sulfated glycosaminoglycans on the development and attachment of Trypanosoma cruzi to the luminal midgut surface in the vector, Rhodnius prolixus.

In the present study, we investigated the involvement of sulfated glycosaminoglycans in both the in vivo development and adhesion of T. cruzi epimastigotes to the luminal surface of the digestive tract of the insect vector, Rhodnius prolixus. Pre-incubation of T. cruzi, Dm 28c epimastigotes with heparin, chondroitin 4-sulfate, chondroitin 6-sulfate or protamine chloridrate inhibited in vitro attachment of parasites to the insect midgut. Enzymatic removal of heparan sulfate moieties by heparinase I or of chondroitin sulfate moieties by chondroitinase AC from the insect posterior midgut abolished epimastigote attachment in vitro. These treatments also reduced the labelling of anionic sites exposed at the luminal surface of the perimicrovillar membranes in the triatomine midgut epithelial cells. Inclusion of chondroitin 4-sulfate or chondroitin 6-sulfate and to a lesser extent, heparin, in the T. cruzi-infected bloodmeal inhibited the establishment of parasites in R. prolixus. These observations indicate that sulfated glycosaminoglycans are one of the determinants for both adhesion of the T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine and the parasite infection in the insect vector, R. prolixus.

Effect of fruit and leaves of Meliaceae plants (Azadirachta indica and Melia azedarach) on the development of Lutzomyia longipalpis larvae (Diptera: Psychodidae: Phlebotominae) under experimental conditions.

This no-choice, laboratory study focuses on the feeding of dried, ground, homogeneous powdered, unprocessed fruit and leaves of Azadirachta indica and Melia azedarach to Lutzomyia longipalpis larvae to determine the effects on their mortality and metamorphosis. A. indica and M. azedarach fruit and leaves significantly increased larval mortality in comparison to larvae fed the untreated, standard diet. A. indica fruit and leaves blocked the molting of the larvae to the fourth instar, resulting in them remaining as third instars until the end of the experiment. M. azedarach fruit also blocked the molting of larvae, which remained permanently in the fourth instar. Feeding M. azedarach leaves resulted in greater molt inhibition. All insects in this group stopped their development as second-instar larvae. No antifeedant effect was detected for any experimental treatment. The results indicate that nontoxic, unprocessed materials obtained from A. indica and M. azedarach are potent development inhibitors of L. longipalpis larvae.

Synthesis and mobilization of glycogen and trehalose in adult male Rhodnius prolixus.

The vector of Chagas' disease, Rhodnius prolixus, feeds exclusively on blood. The blood meals are slowly digested, and these insects wait some weeks before the next meal. During the life of an insect, energy-requiring processes such as moulting, adult gonadal and reproductive growth, vitellogenesis, muscular activity, and fasting, lead to increased metabolism. Carbohydrates are a major source of energy and their mobilization is important. We determined the amounts of glycogen, trehalose, and glucose present in the fat body and/or hemolymph of adult males of R. prolixus and recorded the processes of accumulation and mobilization of these carbohydrates. We also tested our hypothesis that these processes are under endocrine control. The amount of glycogen in the fat body progressively increased until the fourth day after feeding (from 9.3+/-2.2 to 77. 3+/-7.5 microg/fat body), then declined to values around 36.3+/-4.9 microg/fat body on the fifteenth day after the blood meal. Glycogen synthesis was eliminated in decapitated insects and head-transplanted insects synthesized glycogen. The amount of trehalose in the fat body increased until the sixth day after feeding (from 16. 6+/-1.7 to 40. 6+/-5.3 nmol/fat body), decreased abruptly, and stabilized between days 7 and 15 at values ranging around 15-19 nmol/fat body. Decapitated insects did not synthesize trehalose after feeding, and this effect was reversed in head-transplanted insects. The concentration of trehalose in the hemolymph increased after the blood meal until the third day (from 0.07+/-0.01 to 0.75+/-0.05 mM) and at the fourth day it decreased until the ninth day (0.21+/-0.01 mM), when it increased again until the fourteenth day (0.79+/-0.06 mM) after the blood meal, and then declined again. In decapitated insects, trehalose concentrations did not increase soon after the blood meal and at the third day it was very low, but on the fourteenth day it was close to the control values. The concentration of glucose in the hemolymph of untreated insects remained low and constant (0.18+/-0.01 mM) during the 15 days after feeding, but in decapitated insects it progressively increased until the fifteenth day (2.00+/-0.10 mM). We recorded the highest trehalase activity in midgut, which was maximal at the eighth day after feeding (2,830+/-320 nmol of glucose/organ/h). We infer that in Rhodnius prolixus, the metabolism of glycogen, glucose, and trehalose are controlled by factors from the brain, according to physiological demands at different days after the blood meal.

Cytochemical characterization of microvillar and perimicrovillar membranes in the posterior midgut epithelium of Rhodnius prolixus.

Perimicrovillar membranes (PMM) are structures present on the surface of midgut epithelial cells of the hematophagous insect, Rhodnius prolixus. They cover the microvilli and are especially evident 10 days after blood meal, providing the compartmentalization of the enzymatic processes in the intestinal microenvironment. Using an enzyme cytochemical approach, Mg2+-ATPase and ouabain-sensitive Na+K+-ATPase activities were observed in the plasma (or microvillar) membrane (MM) of midgut cells and in the PMM. In contrast, alkaline phosphatase was only detected in MM. Using cationized ferritin and colloidal iron hydroxide particles, anionic sites were found only on the luminal surface of the PMM. Using fluorescein isothiocyanate (FITC)-labeled lectins, residues of alpha-d-galactose, mannose, N-acetyl-neuraminic acid, N-acetyl-d-galactosamine and N-acetyl-galactosamine-alpha-1,3-galactose were detected on the apical surface of posterior midgut epithelial cells. On the other hand, using FITC-labeled neoglycoproteins (NGP) it was possible to detect the presence of carbohydrate binding molecules (CBM) recognizing N-acetyl-d-galactosamine, alpha-d-mannose, alpha-l-fucose and alpha-d-glucose in the posterior midgut epithelium. The use of digitonin showed the presence of sterols in the MM and PMM. These results have led the authors to suggest that for some components the PMM resembles the MM lining the midgut cells of R. prolixus, composing a system which covers the microvilli and stretches to the luminal space.

Effects of the essential oil obtained from Pilocarpus spicatus Saint-Hilaire (Rutaceae) on the development of Rhodnius prolixus nymphae

Bioassays against fifth-instar nymphae of Rhodnius prolixus were conducted with essential oil of Pilocarpus spicatus extracted by hydrodistillation. The main results may be summarized as follows: (i) high levels of toxicity and paralysis together with discrete moulting inhibition were caused by topical application of either 0.5 µL or 1.0 µL per insect of the crude essential oil; (ii) partial fagoinhibition, high moulting inhibition, prolonged intermoulting period and high number of paralyzed insects, but no toxicity were observed after oral treatment using either 5 µL or 10 µL of Pilocarpus spilcatus essential oil per mL of ingested blood meal. The importance of these results in relation to the relevant biological events in R. prolixus is herein discussed.

Bioensaios contra ninfas de 5º estádio de Rhodnius prolixus foram conduzidos utilizando-se óleo essencial de Pilocarpus spicatus extraído por hidrodestilação. Os principais resultados podem ser resumidos como se segue: (i) altos níveis de toxicidade e paralisia associados à discreta inibição da muda foram induzidos pela aplicação tópica de 0,5 µL ou 1,0 µL do óleo essencial por inseto; (ii) fagoinibição parcial, altos níveis de inibição da muda, período intermuda prolongado e alto número de insetos paralisados mas ausência de toxicidade foram observados após tratamento oral com 5,0 µL ou 10 µL de óleo essencial de P. spicatus por mL de sangue ingerido. A importância destes resultados em relação a eventos biológicos relevantes em R. prolixus é aqui discutida.