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Molting in arthropods is a complex process governed by regulatory mechanisms that have evolved and adapted over millennia to allow these animals to grow, despite being confined by a hardened exoskeleton. We isolated the molt-regulating Y-organs (YO) from the common shore crab Carcinus maenas at molt stages B, C1-3, C4, and D0 to assess how changes in protein abundances might underline the unique physiology of each of these stages. We found that changes in protein abundance were most notable in the postmolt stages (B and C1-3), where an increase in energy metabolism and the reactive oxygen species stress (ROS) response proteins was observed. An increase in triosephosphate isomerase and transketolase suggest that the postmolt YO is participating in triglycerides storage and is also actively recycling excess ribose sugars manufactured during the YO’s previously activated state. We also propose as mechanism through which ROS-induced release of cyclophilin A may contribute to YO atrophy during postmolt through the remodeling of structural proteins such as collagen. We support the standing observation of YO atrophy during postmolt by drawing attention to hemolymph protein abundances, especially those of cryptocyanin isoforms, which dropped precipitously in intermolt (C4) and remained at low abundances into early premolt (D0). Finally, though our evidence is preliminary, we propose that future investigations into the YO proteome address the significance of the protein glutamate dehydrogenase. Glutamate dehydrogenase, a key enzyme involved in the formation of glutamate, represents a potential nutrient-sensing checkpoint that might be involved in YO activation. Historically, most attention has gone to the acute molt stages, where signaling mechanisms involved in the activation of the YO have been the focus. Here, we present data suggesting that other regulatory mechanism may be governing the atrophy the postmolt YO. A better understanding of crustacean physiology has the potential to benefit ecosystems and economies worldwide.

While most mammals shed their hair and skin either continuously or seasonally, northern elephant seals (Mirounga angustirostris) undergo an annual catastrophic molt, in which they shed their entire fur and underlying skin layer in the span of just three weeks. Due to the energetic and thermoregulatory constraints of molting and the large distances between their coastal rookeries and foraging grounds, elephant seals must remain on land and fast for the duration of their molt. Previous studies of molting northern elephant seals have examined endocrine and metabolic adjustments to fasting, but not the molecular processes underlying molting. We examined changes in the skin and underlying blubber tissues using histological, endocrine, and proteomic analyses during molting to provide a more in-depth understanding of the cellular mechanisms enabling rapid skin shedding and regeneration in this marine mammal. Shotgun proteome sequencing by LC-MS/MS identified 47,671 peptides and 573 protein groups in skin and outer blubber that were associated with lipid metabolism, protein processing in the endoplasmic reticulum, and collagen regulation. Label-free quantification and differential protein expression analyses identified 23 and 21 proteins that were differentially expressed during molting in the skin and outer blubber, respectively. Proteins downregulated over molting included those associated with inflammation, angiogenesis, and cellular proliferation, whereas proteins upregulated over molting included those associated with cytoskeletal remodeling, collagen synthesis, and lipid metabolism. This suggests that rapid skin regeneration involves intensive protein synthesis and increased vascularization that may be supported by fatty acid substrates from underlying blubber tissue. These data provide insights into cellular and molecular mechanisms that govern unusually rapid skin regeneration in mammals, which may further understanding of disorders affecting the skin and hair of humans and other mammals.

The Tardigrada are hydrophilous, segmented, molting micrometazoans that occupy a diversity of niches in freshwater, marine, and terrestrial habitats. A sister group of the arthropods, this phylum of bilaterally symmetrical lobopods, most less than 1 mm in length, have a hemocoel, a complete digestive tract, a dorsal gonad with one or two gonoducts, and a dorsal lobed brain with a ventral nerve cord and five ganglia. About 1000 species have been described based on the morphology of sclerified structures, especially the claws and buccal-pharyngeal apparatus. Reproduction occurs through fertilized or unfertilized eggs, with individuals being either gonochoric, unisexual, or hermaphroditic, and eggs are deposited either freely or within the shed exuvium. Parthenogenesis, very frequent in limnic and terrestrial tardigrades, allows them to colonize new territories by passive dispersal of a single individual. Quiescence (cryptobiosis: anhydrobiosis, anoxybiosis, cryobiosis, and osmobiosis) and diapause (encystment and resting eggs) occur during the tardigrade life history. Ecological parameters and global distribution patterns are poorly known or understood. Methods for collection, microscopy, and culturing have been developed.

The present research examined respiratory responses of blue crabs to long term (4, 13, and 21 days) hypercapnic hypoxia in freshwater at 23 C. Hypoxic conditions (50-60 & 75-85 mmHg O2) were induced by allowing the crabs to consume their oxygen supply, resulting in a hypercapnic induced decrease in pH that remained through the exposure. Postbranchial hemolymph responses to hypoxia/hypercapnia in freshwater demonstrate decreases in PO2, increases in PCO2, and decreases in pH. Lactate levels decreased over time, but hemocyanin concentration was highly variable with no trends. PH, lactate, and hemocyanin observations also demonstrated high variability and a variety of different responses in individual crabs. There was no evidence of improving oxygen transport abilities. Despite varying responses high mortality rates were observed. The high mortality rate suggests blue crabs are not able to survive the multiple stress of hypoxia/hypercapnia along with the stress of living in freshwater. The mortality rates observed are much greater than previous blue crab hypoxic studies in saltwater. Elevated mortality may result from a failure of oxygen transport, acid-base balance or ion regulation.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / A presente pesquisa teve como objetivo avaliar os efeitos do uso de raÃÃes de muda contendo diferentes nÃveis de torta de mamona nÃo destoxificada (TM) na induÃÃo da muda forÃada em poedeiras comerciais. Foram utilizadas 120 poedeiras da linhagem Lohman LSL, com 81 semanas de idade, distribuÃdas em delineamento inteiramente casualizado com quatro tratamentos e cinco repetiÃÃes de seis aves cada. Os tratamentos consistiram na induÃÃo da muda forÃada pelo mÃtodo do jejum e, os demais, utilizando raÃÃes de muda, composta pela mistura de raÃÃo postura com torta de mamona nos nÃveis de 20, 30 e 40%. O mÃtodo do jejum foi aplicado por 11 dias e, para os demais tratamentos, estabeleceu-se a suspensÃo da raÃÃo de muda quando as aves atingiram 23% de perda do peso inicial ou atà 21 dias de alimentaÃÃo. DiferenÃas significativas foram observadas entre os mÃtodos de induÃÃo da muda forÃada para os parÃmetros medidos durante a induÃÃo da muda e no desempenho das aves apÃs a muda. Entretanto, a qualidade dos ovos nÃo variou significativamente entre os mÃtodos avaliados. No geral, o uso de raÃÃo de muda contendo 40% de TM possibilitou a obtenÃÃo de resultados semelhante na produÃÃo e qualidade dos ovos aos obtidos com o mÃtodo do jejum, mas com menor alteraÃÃo dos parÃmetros sanguÃneos. O uso de raÃÃo contendo 20% ou 30% de TM promoveu, no perÃodo apÃs a muda, desempenho significativamente menor que os demais mÃtodos. O uso de raÃÃo de muda composta por raÃÃo de postura contendo torta de mamona nÃo destoxificada no nÃvel de 40% se mostrou uma alternativa viÃvel ao uso do mÃtodo do jejum. / This study aimed to evaluate the effects of changing diets containing different levels of non-detoxified castor bean meal (TM) in the induction of molt and compare them with the method of fasting. Used 120 Lohman LSL strain hens at 81 weeks of age were assigned to completely randomized design with four treatments and five replicates of six birds each. Treatments were the induction of molt by the method of fasting as a treatment, and others using diets change, being composed of approach ration containing castor bean at levels of 20, 30 and 40% (T2, T3 and T4 ). The method was applied to fast for 11 days and the other treatments, was established to suspend the declaration of change when the birds reached 23% loss of initial weight or up to 21 days supply. Blood samples were collected, one bird per replicate / treatment at the end of induction of changes were analyzed. Significant differences were observed between the methods of molt induction to the parameters measured during the induction of changes in performance of birds after molting. However, the quality of the eggs did not vary significantly among the evaluated methods. In general, the use of feed containing 40% of changes of TM enabled to obtain similar results in the production and egg quality to those obtained with the method of fasting, but with less alteration of blood parameters. The use of feed containing 20% or 30% of TM promoted in the period after the changes, performed significantly lower than other methods. The ration of changes made by laying diet containing not detoxified castor bean meal in the level of 40% proved to be a viable alternative to using the method of fasting.

MAGALHÃES. Kelliani de Sousa. Uso da torta de mamona não destoxificada na indução da muda forçada em poedeiras comerciais. 2011. 44 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Zootecnia, Fortaleza-CE, 2011 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-08-02T15:24:33Z No. of bitstreams: 1 2009_dis_ksmmucida.pdf: 372555 bytes, checksum: b75840a3b3a55679fa42d1927f40fdb9 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-08-02T15:24:47Z (GMT) No. of bitstreams: 1 2009_dis_ksmmucida.pdf: 372555 bytes, checksum: b75840a3b3a55679fa42d1927f40fdb9 (MD5) / Made available in DSpace on 2016-08-02T15:24:47Z (GMT). No. of bitstreams: 1 2009_dis_ksmmucida.pdf: 372555 bytes, checksum: b75840a3b3a55679fa42d1927f40fdb9 (MD5) Previous issue date: 2011 / This study aimed to evaluate the effects of changing diets containing different levels of non-detoxified castor bean meal (TM) in the induction of molt and compare them with the method of fasting. Used 120 Lohman LSL strain hens at 81 weeks of age were assigned to completely randomized design with four treatments and five replicates of six birds each. Treatments were the induction of molt by the method of fasting as a treatment, and others using diets change, being composed of approach ration containing castor bean at levels of 20, 30 and 40% (T2, T3 and T4 ). The method was applied to fast for 11 days and the other treatments, was established to suspend the declaration of change when the birds reached 23% loss of initial weight or up to 21 days supply. Blood samples were collected, one bird per replicate / treatment at the end of induction of changes were analyzed. Significant differences were observed between the methods of molt induction to the parameters measured during the induction of changes in performance of birds after molting. However, the quality of the eggs did not vary significantly among the evaluated methods. In general, the use of feed containing 40% of changes of TM enabled to obtain similar results in the production and egg quality to those obtained with the method of fasting, but with less alteration of blood parameters. The use of feed containing 20% or 30% of TM promoted in the period after the changes, performed significantly lower than other methods. The ration of changes made by laying diet containing not detoxified castor bean meal in the level of 40% proved to be a viable alternative to using the method of fasting. / A presente pesquisa teve como objetivo avaliar os efeitos do uso de rações de muda contendo diferentes níveis de torta de mamona não destoxificada (TM) na indução da muda forçada em poedeiras comerciais. Foram utilizadas 120 poedeiras da linhagem Lohman LSL, com 81 semanas de idade, distribuídas em delineamento inteiramente casualizado com quatro tratamentos e cinco repetições de seis aves cada. Os tratamentos consistiram na indução da muda forçada pelo método do jejum e, os demais, utilizando rações de muda, composta pela mistura de ração postura com torta de mamona nos níveis de 20, 30 e 40%. O método do jejum foi aplicado por 11 dias e, para os demais tratamentos, estabeleceu-se a suspensão da ração de muda quando as aves atingiram 23% de perda do peso inicial ou até 21 dias de alimentação. Diferenças significativas foram observadas entre os métodos de indução da muda forçada para os parâmetros medidos durante a indução da muda e no desempenho das aves após a muda. Entretanto, a qualidade dos ovos não variou significativamente entre os métodos avaliados. No geral, o uso de ração de muda contendo 40% de TM possibilitou a obtenção de resultados semelhante na produção e qualidade dos ovos aos obtidos com o método do jejum, mas com menor alteração dos parâmetros sanguíneos. O uso de ração contendo 20% ou 30% de TM promoveu, no período após a muda, desempenho significativamente menor que os demais métodos. O uso de ração de muda composta por ração de postura contendo torta de mamona não destoxificada no nível de 40% se mostrou uma alternativa viável ao uso do método do jejum.

The theoretical part of diploma thesis is aimed at breeding, growth and molting events in crayfish. Thesis is also aimed at life cycle and reproduction of three indigenous crayfish species (Astacus astacus, Austropotamobius torrentium, Pontastacus leptodactylus) and two introduced crayfish species (Faxonius limosus, Pacifastacus leniusculus). The results show apparent linear dependence between female size (POCL), abdomen surface (1M: y = -1061,2 + 65,4*x; r2 = 0,9829; p10-5; 2M: y = -1160,4 + 68,7*x; r2 = 0,8732; p < 10-5) and fertility (1M: y = -194,4 + 12,8*x; r2 = 0,4104 p = 0,0042; y = -330.9 + 15,9*x; r2 = 0.4372; p < 10-4) in once (1M) and twice (2M) molted females. As the length of carapax increase, the area of the abdomen increase and fertility increase in both groups of females. Furthermore, the differences in fertility, carapax length, abdomen surface, and weights in once and twice molted females of signal crayfish. In group of once molted females fertility (t-test, t = -3.12, p = 0.003), carapax length (t-test, t = -3.17, p = 0.003) = -3.24; p = 0.002) and the weight (t-test, t = -3.35; p = 0.002) was higher than in twice molted group of female. Consequentially evaluation of egg size of females. The size of the eggs did not differ between groups of females (1M and 2M) (t-test, t = -0.975, p = 0.335). The mean egg size for both groups of females was 2.71 ? 0.18 mm. Evaluation of post-ovulation conditions of the ovary, including visible signs of females that resorbed unovulated oocytes. Evaluating how much unovulated oocytes were found in the ovaries. Only in 46.4% of the total number of females were ovaries completely empty. In once and twice molted females were found in average 2.2 ? 3.8 and 1.9 ? 2.6. unovulated oocytes, respectively. In once molted, three females didn't lay eggs, two females lost the clutch of eggs. In twice molted females, six females did not lay the eggs and four females lost the clutch.

Digestion of proteins in the midgut of lepidopteran larvae relies on different types of peptidases, among the trypsins and chymotrypsins. In this work four chymotrypsinlike peptidases (MsCTLP1–4) were identified from the larval midgut of M. sexta, which are distantly related to another chymotrypsin (MsCT), a previously described peptidase present in the larval midgut of M. sexta. MsCTLP1–4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. Examination of MsCTLP expression in different tissues showed that most of the peptidases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. Expression analysis of MsCTLPs at different physiological states revealed that the mRNA amounts did not differ considerably in feeding and starving larvae except for MsCTLP2, whose mRNA dropped significantly upon starvation. During molting, however, the mRNA amounts of all MsCTLPs dropped significantly. Immunological determination of MsCTLP1 amounts showed that the mature peptidase was only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or molting larvae, suggesting that MsCTLP1 secretion is suspended during starvation or molt. Differential regulation of transcript levels as well as their partial expression in Malpighian tubules might point to a role, which is distinct from digestion for at least some MsCTLPs. In line with this assumption, MsCTLP1 was shown to interact with the chitin synthase 2 (MsCHS2), necessary for chitin synthesis in the course of peritrophic matrix formation in the midgut of M. sexta. The occurrence of this interaction in vivo is supported by colocalization and co-immunoprecipitation. The data suggest that chitin synthesis is controlled by an intestinal proteolytic signaling cascade linking chitin synthase activity to the nutritional state of the larvae. As MsCTLP1 appears to be involved in such signaling cascades, other midgut peptidases could have other targets and may therefore regulate different activities. To gain more insight into the functions of CTLPs, the gene family encoding these peptidases in the genome of the red flour beetle, T. castaneum, was analyzed. Using an extended search pattern, 14 TcCTLP genes were identified that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. Analysis of the expression patterns of seven TcCTLP genes at various developmental stages revealed that some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A). Others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP- 6C/D/E). TcCTLP genes were expressed predominantly in the midgut where they presumably function in digestion. However, TcCTLP-5C and TcCTLP-6C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, western blots were performed using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6C suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, systemic RNAi experiments were performed. While injections of dsRNAs to TcCTLP-5A/B and TcCTLP-6A/D/E into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. Recombinant expressed TcCTLP-5C2 was moreover activated by trypsin and was able to hydrolyze AAPF, hence making TcCTLP-5C the first described chymotrypsin-like peptidase ever to be involved in molting.

Molting in crustaceans is a highly complex physiological process involving negative regulation by two paired endocrine glands, the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). The XO/SG complex is responsible for making molt-inhibiting hormone (MIH) which negatively regulates synthesis of the molting hormones, ecdysteroids, by the YO. Analysis of gene expression in the XOs and YOs has led to the development of a proposed molecular signaling pathway which regulates ecdysteroidogenesis and subsequent molting in crustaceans. In this study, changes in protein abundance in the YO were characterized over the course of a molt cycle (intermolt, early premolt, mid premolt, and late premolt) induced by multiple leg autotomy (MLA) in the blackback land crab, Gecarcinus lateralis. In all, 457 distinct protein spots were detected in the molting gland using two-dimensional gel electrophoresis, of which 230 (50%) changed significantly in abundance over the course of the molt cycle (one-way permutation ANOVA, p≤0.05). Changes in protein abundance were most notable between the intermolt and the three premolt stages, indicative of a biological ‘on-off’ switch in the Y-organ. Several hemolymph species proteins, including hemocyanin, cryptocyanin, and transglutaminase, were identified which characterized physiological changes associated with molting beyond the Y-organ. An abundance of cytoskeletal proteins were identified which correspond with glandular hypertrophy and are indicative of vesicular-mediated exocytosis, possibly of ecdysteroids. Further, several proteins involved in the immune, proteostasis, and oxidative stress response are characteristic of supporting the dynamic and demanding cellular changes associated with ecdysteroidogenesis and the transition of the Y-organ from the basal to the highly active state. Many proteins involved in energetic pathways including glycolysis, the citric acid cycle, amino acid metabolism, and one-carbon metabolism changed in abundance in response to both the higher energy demands and the requirement for precursors of macromolecular synthesis of the YO over the molt cycle. Taken together, these changes in diverse physiological pathways represent the complexity involved with regulation of the Y-organ, even with just the single proposed physiological purpose of ecdysteroidogenesis.