The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus.

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools.

The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis.

The daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as "gating." Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term "gating" suggests that it is separate from the developmental process of metamorphosis. Here, we follow the progression through the final stages of Drosophila adult development with single-animal resolution and show that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. Here, we show that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.

Prediction of neuropeptide precursors and differential expression of adipokinetic hormone/corazonin-related peptide, hugin and corazonin in the brain of malaria vector Nyssorhynchus albimanus during a Plasmodium berghei infection.

Insect neuropeptides, play a central role in the control of many physiological processes. Based on an analysis of Nyssorhynchus albimanus brain transcriptome a neuropeptide precursor database of the mosquito was described. Also, we observed that adipokinetic hormone/corazonin-related peptide (ACP), hugin and corazonin encoding genes were differentially expressed during Plasmodium infection. Transcriptomic data from Ny. albimanus brain identified 29 pre-propeptides deduced from the sequences that allowed the prediction of at least 60 neuropeptides. The predicted peptides include isoforms of allatostatin C, orcokinin, corazonin, adipokinetic hormone (AKH), SIFamide, capa, hugin, pigment-dispersing factor, adipokinetic hormone/corazonin-related peptide (ACP), tachykinin-related peptide, trissin, neuropeptide F, diuretic hormone 31, bursicon, crustacean cardioactive peptide (CCAP), allatotropin, allatostatin A, ecdysis triggering hormone (ETH), diuretic hormone 44 (Dh44), insulin-like peptides (ILPs) and eclosion hormone (EH). The analysis of the genome of An. albimanus and the generated transcriptome, provided evidence for the identification of myosuppressin neuropeptide precursor. A quantitative analysis documented increased expression of precursors encoding ACP peptide, hugin and corazonin in the mosquito brain after Plasmodium berghei infection. This work represents an initial effort to characterize the neuropeptide precursors repertoire of Ny. albimanus and provides information for understanding neuroregulation of the mosquito response during Plasmodium infection.

Completion of metamorphosis after adult emergence in Ceratitis capitata (Diptera: Tephritidae).

The ecdysis of the imago is a crucial step in the development of holometabolous insects. However, no information on several aspects of Ceratitis capitata imago emergence and subsequent body maturation is available. We analysed behavioural events and evaluated the oxygen consumption and the dynamics of carbohydrate and lipid reserves during puparium extrication and in newly emerged imago until full wing expansion. A system for recording images with the corresponding software for image analysis was built for this purpose. After extrication, C. capitata showed two early postemergence phases: walking (6.56 ± 4.01 min, 6.2% of the wing spreading period, WSP) and the phase without locomotor motion (98.75 ± 26.04 min; 93.8% WSP). Three main events were recognized during the last phase. The first involved an initial expansion of the wings (11.12 ± 4.32 min). The second event was the progressive tanning of the body cuticle in general and the wing veins in particular, and the last entailed the definitive expansion of the wings to attain the characteristic arrow-shaped wing aspect. Our studies here complement previous descriptions of the tanning process of newly emerged medfly adults (Pérez et al., 2018). As a consequence of the results presented here, we consider that the initial events of the newly emerged adult could be interpreted as the last steps of metamorphosis.

Moulting cycle stages in Macrobrachium rosenbergii by setogenesis method / Estágios de muda em Macrobrachium rosenbergii pelo método de setogênese

This study had the objective of evaluating the duration of the moulting cycle stages of Macrobrachium rosenbergii, using the method of setogenesis. The experiment was conduced in the Prawn Farming Laboratory of the Federal University in Paraiba, in the town of Bananeiras, Paraiba, Brazil. Fifteen prawns (5.7 ± 0.3 g) were distributed in 3 aquariums (60 x 30 x 40 cm), maintained in natural photoperiod (12 x 12 hours), at a controlled temperature (28 °C), and feed was offered 3 times a day (8 a.m., 12 and 16 p.m.), throughout the experimental period. For identification, the animals were marked with colored silicone rings on the ocular peduncle and with colored plastic discs fixed on the carapace. From the setogenesis method, the prawns were classified into stage A, B, C, D0, D1, D2 or D3 of the moulting cycle. Setogenesis was observed stereomicroscopically every day, at the same time, 7 a.m., from the first ecdysis to the next ecdysis. The duration of the moulting cycle was 27.7 ± 3.2 days, and the intermoult stage (C) was the longest (8.0 ± 2.38 days). Stage A was the shortest (1 day), and the pre-moult stage (stages D) lasted 12.98+1.65 days. We concluded that juvenile of M. rosenbergii have a diecdysis type moulting cycle with shorter intermoult than pre-moult periods.

O presente estudo teve como objetivo avaliar a duração dos estágios de muda do Macrobrachium rosenbergii, utilizando o método da setogênese. O experimento foi realizado no Laboratório de Carcinicultura da Universidade Federal da Paraíba, em Bananeiras, Paraíba, Brazil. Quinze camarões (5,7 ± 0,3 g) foram distribuídos em 3 aquários (60 x 30 x 40 cm), mantidos em fotoperíodo natural, temperatura controlada (28 °C) e ração ofertada 3 vezes ao dia (8 a.m., 12 e 16 p.m.), durante todo o período experimental. Para identificação, os animais foram marcados com anéis coloridos de silicone no pedúnculo ocular e com discos plásticos coloridos fixados à carapaça. Os camarões foram classificados em estágio A, B, C, D0, D1, D2 ou D3 do ciclo de muda, através do método da setogênese. A setogênese foi observada estereomicroscopicamente todos os dias, às 7 a.m., no mesmo horário, a partir da primeira ecdise até a ecdise seguinte. Como resultado, a duração do ciclo de muda foi 27,7 ± 3,2 dias, com o estágio de intermuda (C) sendo o mais duradouro (8,00 ± 2,38 dias). O estágio A foi o mais breve (1 dia), e a fase de pré-muda (os estágios D) durou 12,98 ± 1,65 dias. Conclui-se que juvenis de M. rosenbergii tem o ciclo de muda do tipo diecdise com o período de intermuda menor que o de pré-muda.

Moulting cycle stages in Macrobrachium rosenbergii by setogenesis method / Estágios de muda em Macrobrachium rosenbergii pelo método de setogênese

This study had the objective of evaluating the duration of the moulting cycle stages of Macrobrachium rosenbergii, using the method of setogenesis. The experiment was conduced in the Prawn Farming Laboratory of the Federal University in Paraiba, in the town of Bananeiras, Paraiba, Brazil. Fifteen prawns (5.7 ± 0.3 g) were distributed in 3 aquariums (60 x 30 x 40 cm), maintained in natural photoperiod (12 x 12 hours), at a controlled temperature (28 °C), and feed was offered 3 times a day (8 a.m., 12 and 16 p.m.), throughout the experimental period. For identification, the animals were marked with colored silicone rings on the ocular peduncle and with colored plastic discs fixed on the carapace. From the setogenesis method, the prawns were classified into stage A, B, C, D0, D1, D2 or D3 of the moulting cycle. Setogenesis was observed stereomicroscopically every day, at the same time, 7 a.m., from the first ecdysis to the next ecdysis. The duration of the moulting cycle was 27.7 ± 3.2 days, and the intermoult stage (C) was the longest (8.0 ± 2.38 days). Stage A was the shortest (1 day), and the pre-moult stage (stages D) lasted 12.98+1.65 days. We concluded that juvenile of M. rosenbergii have a diecdysis type moulting cycle with shorter intermoult than pre-moult periods.(AU)

O presente estudo teve como objetivo avaliar a duração dos estágios de muda do Macrobrachium rosenbergii, utilizando o método da setogênese. O experimento foi realizado no Laboratório de Carcinicultura da Universidade Federal da Paraíba, em Bananeiras, Paraíba, Brazil. Quinze camarões (5,7 ± 0,3 g) foram distribuídos em 3 aquários (60 x 30 x 40 cm), mantidos em fotoperíodo natural, temperatura controlada (28 °C) e ração ofertada 3 vezes ao dia (8 a.m., 12 e 16 p.m.), durante todo o período experimental. Para identificação, os animais foram marcados com anéis coloridos de silicone no pedúnculo ocular e com discos plásticos coloridos fixados à carapaça. Os camarões foram classificados em estágio A, B, C, D0, D1, D2 ou D3 do ciclo de muda, através do método da setogênese. A setogênese foi observada estereomicroscopicamente todos os dias, às 7 a.m., no mesmo horário, a partir da primeira ecdise até a ecdise seguinte. Como resultado, a duração do ciclo de muda foi 27,7 ± 3,2 dias, com o estágio de intermuda (C) sendo o mais duradouro (8,00 ± 2,38 dias). O estágio A foi o mais breve (1 dia), e a fase de pré-muda (os estágios D) durou 12,98 ± 1,65 dias. Conclui-se que juvenis de M. rosenbergii tem o ciclo de muda do tipo diecdise com o período de intermuda menor que o de pré-muda.(AU)

Orcokinins regulate the expression of neuropeptide precursor genes related to ecdysis in the hemimetabolous insect Rhodnius prolixus.

Ecdysis is a vital process for insects, during which they shed the old cuticle in order to emerge as the following developmental stage. Given its relevance for survival and reproduction, ecdysis is tightly regulated by peptidic hormones that conform an interrelated neuromodulatory network. This network was studied in species that undergo a complete metamorphosis, but not in hemimetabola. In a recent work, we demonstrated that orcokinin neuropeptides are essential for ecdysis to occur in the kissing bug Rhodnius prolixus. Here we performed gene silencing, quantitative PCR and in vitro treatments in order to study the interrelationships between RhoprOKs and hormones such as ecdysis triggering hormone, corazonin, eclosion hormone, crustacean cardioactive peptide and ecdysone. Our results suggest that RhoprOKs directly or indirectly regulate the expression of other genes. Whereas RhoprOKA is centrally involved in the regulation of gene expression, RhoprOKB is implicated in processes related to midgut physiology. Therefore, we propose that the different transcripts encoded in RhoprOK gene could integrate signaling cues, in order to coordinate the nutritional state with development and ecdysis. Given the emerging data that point to OKs as important factors for survival and reproduction, they could be candidates in the search for new insect management strategies based on neuroendocrine targets.

Stereotyped responses of Drosophila peptidergic neuronal ensemble depend on downstream neuromodulators.

Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. Here, we focus on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and we show that the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, depends fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. Our findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors.

Ecdysis period of Rhodnius prolixus head investigated using phase contrast synchrotron microtomography.

Microtomography using synchrotron sources is a useful tool in biological imaging research since the phase coherence of synchrotron beams can be exploited to obtain images with high contrast resolution. This work is part of a series of works using phase contrast synchrotron microtomography in the study of Rhodnius prolixus head, the insect vector of Chagas' disease, responsible for about 12,000 deaths per year. The control of insect vector is the most efficient method to prevent this disease and studies have shown that the use of triflumuron, a chitin synthesis inhibitor, disrupted chitin synthesis during larval development and it's an alternative method against insect pests. The aim of this work was to investigate the biological effects of treatments with triflumuron in the ecdysis period (the moulting of the R. prolixus cuticle) using the new imaging beamline IMX at LNLS (Brazilian Synchrotron Light Laboratory). Nymphs of R. prolixus were taken from the Laboratory of Biochemistry and Physiology of Insects, Oswaldo Cruz Foundation, Brazil. Doses of 0.05mg of triflumuron were applied directly to the abdomen on half of the insects immediately after feeding. The insects were sacrificed 25days after feeding (intermoulting period) and fixed with glutaraldehyde. The results obtained using phase contrast synchrotron microtomography in R. prolixus showed amazing images of the effects of triflumuron on insects in the ecdysis period, and the formation of the new cuticle on those which were not treated with triflumuron. Both formation and malformation of this insect's cuticle have never been seen before with this technique.

Effects of chitin synthase double-stranded RNA on molting and oogenesis in the Chagas disease vector Rhodnius prolixus.

In this study, we provided the demonstration of the presence of a single CHS gene in the Rhodnius prolixus (a blood-sucking insect) genome that is expressed in adults (integument and ovary) and in the integument of nymphs during development. This CHS gene appears to be essential for epidermal integrity and egg formation in R. prolixus. Because injection of CHS dsRNA was effective in reducing CHS transcript levels, phenotypic alterations in the normal course of ecdysis occurred. In addition, two phenotypes with severe cuticle deformations were observed, which were associated with loss of mobility and lifetime. The CHS dsRNA treatment in adult females affected oogenesis, reducing the size of the ovary and presenting a greater number of atresic oocytes and a smaller number of chorionated oocytes compared with the control. The overall effect was reduced oviposition. The injection of CHS dsRNA modified the natural course of egg development, producing deformed eggs that were dark in color and unable to hatch, distinct from the viable eggs laid by control females. The ovaries, which were examined under fluorescence microscopy using a probe for chitin detection, showed a reduced deposition on pre-vitellogenic and vitellogenic oocytes compared with control. Taken together, these data suggest that the CHS gene is fundamentally important for ecdysis, oogenesis and egg hatching in R. prolixus and also demonstrated that the CHS gene is a good target for controlling Chagas disease vectors.

Growth, Shedding and Food Intake in Captive Eunectes murinus (Linnaeus, 1758) (Serpentes: Boidae) / Crecimiento, Muda y Consumo de Alimento en Cautiverio de la Eunectes murinus (Linnaeus, 1758) (Serpentes: Boidae)

The anaconda Eunectes murinus (Linnaeus, 1758) inhabits large hydrographic basins in tropical America and figures among the world's largest snakes, attaining a length of 12 m. This study analyzed the growth of three female anaconda siblings, with records from their birth in captivity up to around 14 months of age. The snakes were kept in a controlled environment with constant temperature and data related to biometry, feeding and skin shedding were recorded. At the end of these 445 days, the siblings had grown on average 2.6 times their initial length and increased their initial weight by 3830.lOg, incorporating about 43.5% of total food ingested to their body mass. They showed a total of 0.69 skin sheddings per month in that period, and did not exhibit significant differences in shedding intervals, nor in body growth (weight and length), when compared among themselves. Food was refused at times, coinciding with the days that preceded the ecdyses. Sheddings do not seem to be explained by feeding or growth, which suggests a relation to other endogenous factors. A more detailed study of this species is needed to better understand its growth to the adult phase and its hormonal levels during growth.

La anaconda Eunectes murinus (Linnaeus, 1758) se encuentra en una ampia área hidrográfica de América tropical y figura entre las más grandes serpientes del mundo, alcanzando una longitud de 12 metros. Este estudio analizó el crecimiento de tres anacondas hembras hermanas, con registros de su nacimiento en cautiverio hasta alrededor de 14 meses de edad. Las serpientes fueron mantenidas en un medio controlado con temperatura constante y se obtuvieron registros relacionados con su biometría, alimentación y mudas de piel. Al final de 445 días, las hermanas crecieron en promedio 2,6 veces su longitud inicial e incrementaron su peso inicial en 3810,1 g, incorporando a su masa corporal alrededor de 43,5% del alimento ingerido. Ellas tuvieron un total de 0,69 mudas de piel por mes en ese periodo y no hubo diferencias significativas en los intervalos de mudas, ni en el crecimiento corporal (peso y longitud), cuando comparadas entre ellas. La comida, a veces, fue rechazada coincidiendo con los días que precedían al cambio de piel. Las mudas de piel parecen no tener relación con la alimentación o crecimiento, lo cual sugiere que puede deberse a otros factores endógenos. Es necesario un estudio más detallado de esta especie para comprender mejor su crecimiento en la fase adulta y sus niveles hormonales durante el crecimiento.

Lesions (ulcers and/or erosions) and desquamations location in the gastric mucosa from asymptomatic Quarter Horse foals: endoscopic survey / Localização de lesões (úlceras e/ou erosões) e descamações da mucosa gástrica em potros assintomáticos da raça Quarto de Milha: estudo endoscópico

Gastric ulcer accounts for an important cause of abdominal discomfort in young horses. Concerning either the presence or absence of clinical signs and their variations, the lesions location in the gastric mucosa and complications resulting from ulcerative lesions, four clinical syndromes have been described in foals: 1) Asymptomatic or silent ulcers; 2) Symptomatic or active ulcers; 3) Perforated ulcers; and 4) Gastric or duodenal obstruction. With the aim of studying the distribution of lesions (ulcers and/or erosions) and desquamations from the non-glandular epithelium in the gastric mucosa of young horses and a possible relationship between both alterations (lesion/desquamation), sixty Quarter Horse foals without signs of gastric disease underwent gastroscopy. Foals were divided by age in four groups of 15 animals as follows: 1 to 30 days, 31 to 60 days, 61 to 90 days and 91 to 120 days. Lesions were most prevalent in the stratified squamous epithelial mucosa mainly adjacent to the margo plicatus along the greater curvature followed by squamous mucosa next to the cardia along the lesser curvature, glandular and non-glandular fundus and antrum. Regions of the fundus and margo plicatus were similarly affected by desquamations. There was no association between lesions and desquamations occurrence.(AU)

A úlcera gástrica figura como uma importante causa de desconforto abdominal em eqüinos jovens. De acordo com a localização das lesões na mucosa gástrica, a presença ou ausência de sinais clínicos e possíveis complicações resultantes de sua ocorrência, quatro síndromes clínicas são freqüentemente descritas em potros: 1) Úlceras assintomáticas ou silenciosas; 2) Úlceras sintomáticas ou ativas; 3) Úlceras perfuradas; e 4) Obstruções gástrica ou duodenal. Com o objetivo de se verificar a distribuição de lesões gástricas (úlceras e/ou erosões) e descamações do epitélio aglandular no estômago de eqüinos jovens assim como uma possível relação entre as alterações mencionadas (lesão/descamação) sessenta potros da raça Quarto de Milha não-portadores de sinais clínicos compatíveis com úlceras gástricas foram submetidos à gastroscopia. Os potros foram divididos em quatro faixas etárias de 15 animais cada uma, sendo: 1 a 30 dias, 31 a 60 dias, 61 a 90 dias e 91 a 120 dias de idade. As lesões ocorreram em ordem decrescente de freqüência nas regiões aglandular próximo ao margo plicatus ao longo da curvatura maior, aglandular próximo à cárdia ao longo da curvatura menor, fundo glandular e aglandular e antro. As descamações do epitélio aglandular ocorreram de forma similar nas regiões de fundo e margo plicatus. Não houve associação entre a ocorrência de lesões e descamações.(AU)