Differential expression profiling of components associated with exoskeletal hardening in crustaceans

Background: Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Glycoproteins have been implicated in the calcification process of many matrices. Sclerotization, on the other hand, is catalysed by phenoloxidases, which also play a role in melanization and the immunological response in arthropods. Custom cDNA microarrays from Portunus pelagicus were used to identify genes possibly associated with the activation pathways involved in these processes. Results: Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display molt cycle-related differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the post-molt stage, during which calcification takes place, implicating both in the regulation of calcification. Genes presumed to be involved in the phenoloxidase activation pathway that facilitates sclerotization also displayed molt cycle-related differential expression profiles. Members of the serine protease superfamily, trypsin-like and chymotrypsin-like, were up-regulated in the intermolt stage when compared to post-molt, while trypsin-like was also up-regulated in pre-molt compared to ecdysis. Additionally, up-regulation in pre- and intermolt stages was observed by transcripts encoding other phenoloxidase activators including the putative antibacterial protein carcinin-like, and clotting protein precursor-like. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and intermolt. Conclusion: Cuticle hardening in crustaceans is a complex process that is precisely timed to occur in the post-molt stage of the molt cycle. We have identified differential expression patterns of several genes that are believed to be involved in biomineralization and sclerotization and propose possible regulatory mechanisms for these processes based on their expression profiles, such as the potential involvement of C-type lectin receptors and mannose binding protein in the regulation of calcification.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR Could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts Occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. (C) 2009 Published by Elsevier Inc.

Relação entre padrão comportamental, estágios do ciclo de muda e atividade de enzimas digestivas proteolíticas em juvenis do camarão marinho Litopenaeus vannamei (Crustacea: Penaeidae)

Shrimp farming in Brazil is a consolidated activity, having brought economical and social gains to several states with the largest production concentrated in the northeast. This fact is also reflected in higher feed intake, necessitating a more efficient feed management. Currently, management techniques already foresee food loss due to molting. In this sense, studies relating shrimp s digestive physiology, molting physiology and behavioral response of shrimp feed can optimize the feed management. Thus, our study aimed to evaluate the behavioral response of the marine shrimp L. vannamei (Crustacea: Penaeidae) in accordance with the stages of moulting cycle and feeding schedules based on higher or lower activity of proteolytic digestive enzymes; also, to investigate the influence of feeding schedule on hepatosomatic index and non-specific and specific protease activity (trypsin). Experiments were carried out at the Laboratory of Shrimp Behavioral Studies at UFRN in partnership with the Laboratory of Enzimology UFPE. Juveniles of L. vannamei weighting 5.25 g (+ 0.25 g) were kept in aquaria at a density of 33 shrimp m -2. In the first experiment, shrimp were fed in the light phase or in the dark phase for 8 days; in the ninth day, the animals were observed for 15 minutes every hour during the 12 hours of each phase of the photoperiod. We recorded the frequency of inactivity, exploration, food intake, burrowing, swimming and crawling behavior. At the end of the 12th observation session, the shrimp were sacrified and classified by the method of setogenesis in the molt cycle stages A, B, C, D0, D1, D2 or D3. We found that the shrimp in A stage show high levels of inactivity. Moreover, the frequency of food intake was very low. The shrimp in D3 stage also had low food intake and high inactivity associated with elevated frequencies of burrowing. In the second experiment, shrimp were kept in physiological acclimation to experimental conditions for 28 days, distributed in 12 treatments in the light phase and 12 treatments in the dark phase. In the end, the animals were sacrified and dissected to assess non-specific and specific protease activity (trypsin) activity. In general, these parameters did not vary among animals fed in the light phase and those fed in the dark phase. However, significant differences were found in the activity of specific and nonspecific proteases in relation to food treatment. In the light phase, the major proteolytic activities converged to 10 hours after the start of the light phase, while the lowest activities converged to 6 hours after the beginning of this phase. In the dark phase, the highest enzyme activity converged to 12 hours after the onset of phase, while the lowest activities converged to 3 hours after the onset of phase. In the third experiment, we sought to evaluate the behavioral responses of shrimp in relation to dietary treatments based on higher or lower activity of proteolytic enzymes, considering the results of the second experiment. The behavioral categories observed were the same as the ones in the first experiment, with observations of 30 minutes (15min before and 15min after food supply). We found variation in behavioral responses as a function of the treatments, with greater intake of food in shrimp fed during the period of greatest activity of proteolytic enzymes, in the light phase. Thus we see that periodic events associated with the shrimp s physiology interfere in their behavioral responses, revealing situations that are more adjustable to the provision of food, and consequently optimizing feeding management

Influência da densidade de estocagem sobre o crescimento, ciclo de muda e o comportamento em juvenis do camarão marinho Litopenaeus vannamei

One of the factors that may interfere with the cultivation of Litopenaeus vannamei is the population density. This study aimed to assess the effect of density on growth, mortality, physical integrity and behavior of shrimp. The study was divided into two stages. At first, the shrimp were placed in tanks at densities of 50, 75 and 100 shrimp m-2. The animals were monitored in relation to the degree of proventricular filling, the stage of the molt cycle and physical integrity three times a week and in relation to the weight and length once a week. Mortality, growth and proventricular filling were not influenced by the density; frequency of records in specific stages of the molt cycle varied according to the density. The lower proportion of broken appendages and higher frequency of necrotic lesions occurred in lower density. The second stage of the research, conducted in aquaria, was divided into two parts. The first described social or feeding behavioral categories: slow displacement by contact, slow displacement by approximation, abrupt displacement by contact, abrupt displacement by approximation, reactivity, cannibalism, occupying the tray, get feed in the tray and get feed outside the tray. In the second part, these and other behavioral categories, described in the literature, were recorded in densities of 50, 75 and 100 shrimp m-2. Mortality was more frequent in higher density. The frequency of most behaviors mentioned above was very low, not differing between densities or being too low to determine differences between them. The behavioral profile of animals in different densities was, in general, very similar, with no difference in exploration, digging and cleaning between the densities. Even so, inactivity, feeding, crawling, burrowing, swimming, and proximity between animals were influenced by the density. These results suggest that some behaviors suffer greater interference from population density. However, the density may not have a broader influence on the animals when other factors, such as physico-chemical parameters of water and feed offering, are adequate

Physiological maturity and relationships of growth and reproduction in the red mangrove crab Goniopsis cruentata (Latreille) (Brachyura, Grapsidae) on the coast of São Paulo, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ciclo reprodutivo do caranguejo violinista Uca rapax (Smith) (Crustacea, Brachyura, Ocypodidae) habitante de um estuário degradado em Paraty, Rio de Janeiro, Brasil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epibiosis in decapod crustaceans by stalked barnacle Octolasmis lowei (Cirripedia: Poecilasmatidae)

Stalked barnacles Octolasmis lowei Darwin, 1851 are frequently found attached to decapod crustaceans. Their epibiotic association depends on many factors, which are mainly related to characteristics of the host's biology. This study evaluated the infestation and distribution of stalked barnacles in the branchial chambers of crabs, and analyzed the data with respect to the host's sex, maturity stage, molt cycle and size. The crab species Arenaeus cribrarius Lamarck, 1818, Callinectes danae Smith, 1869, Callinectes ornatus Ordway, 1863, Hepatus pudibundus Herbst, 1785, Libinia ferreirae Brito Capello, 1871, and Persephona punctata Linnaeus, 1758 were sampled and found to be infested by O. lowei. No juvenile crabs were infested. The prevalence of infestation by O. lowei was significantly different among C. danae, C. ornatus, and H. pudibundus males and females. All infested hosts were in the intermolt period. The mean size of infested crabs was larger than that observed for non-infested individuals. Internally, stalked barnacles were concentrated on the central gills or walls and floor of branchial chambers, suggesting that these gills provide more favorable conditions for the settlement and development of these epibionts. These results highlight the relationship between epibiont infestation and host biology, as well as the role of decapod crustaceans as a suitable substrate for the development of stalked barnacle O. lowei. © 2013 Sociedade Brasileira de Zoologia All rights reserved.

Cryptocyanin, a crustacean molting protein: Evolutionary link with arthropod hemocyanins and insect hexamerins

Cryptocyanin, a copper-free hexameric protein in crab (Cancer magister) hemolymph, has been characterized and the amino acid sequence has been deduced from its cDNA. It is markedly similar in sequence, size, and structure to hemocyanin, the copper-containing oxygen-transport protein found in many arthropods. Cryptocyanin does not bind oxygen, however, and lacks three of the six highly conserved copper-binding histidine residues of hemocyanin. Cryptocyanin has no phenoloxidase activity, although a phenoloxidase is present in the hemolymph. The concentration of cryptocyanin in the hemolymph is closely coordinated with the molt cycle and reaches levels higher than hemocyanin during premolt. Cryptocyanin resembles insect hexamerins in the lack of copper, molt cycle patterns of biosynthesis, and potential contributions to the new exoskeleton. Phylogenetic analysis of sequence similarities between cryptocyanin and other members of the hemocyanin gene family shows that cryptocyanin is closely associated with crustacean hemocyanins and suggests that cryptocyanin arose as a result of a hemocyanin gene duplication. The presence of both hemocyanin and cryptocyanin in one animal provides an example of how insect hexamerins might have evolved from hemocyanin. Our results suggest that multiple members of the hemocyanin gene family—hemocyanin, cryptocyanin, phenoloxidase, and hexamerins—may participate in two vital functions of molting animals, oxygen binding and molting. Cryptocyanin may provide important molecular data to further investigate evolutionary relationships among all molting animals.

Biological polarized light reflectors in stomatopod crustaceans

Body parts that can reflect highly polarized light have been found in several species of stomatopod crustaceans (mantis shrimps). These polarized light reflectors can be grossly divided into two major types. The first type, usually red or pink in color to the human visual system, is located within an animal’s cuticle. Reflectors of the second type, showing iridescent blue, are located beneath the exoskeleton and thus are unaffected by the molt cycle. We used reflection spectropolarimetry and transmission electron microscopy (TEM) to study the reflective properties and the structures that reflect highly polarized light in stomatopods. For the first type of reflector, the degree of polarization usually changes dramatically, from less than 20% to over 70%, with a change in viewing angle. TEM examination indicates that the polarization reflection is generated by multilayer thin-film interference. The second type of reflector, the blue colored ones, reflects highly polarized light to all viewing angles. However, these reflectors show a slight chromatic change with different viewing angles. TEM sections have revealed that streams of oval-shaped vesicles might be responsible for the production of the polarized light reflection. In all the reflectors we have examined so far, the reflected light is always maximally polarized at around 500 nm, which is close to the wavelength best transmitted by sea water. This suggests that the polarized light reflectors found in stomatopods are well adapted to the underwater environment. We also found that most reflectors produce polarized light with a horizontal e-vector. How these polarized light reflectors are used in stomatopod signaling remains unknown.

Influence of different fat sources on the performance, egg quality, and lipid profile of egg yolks of commercial layers in the second laying cycle

This study was designed to evaluate the effects of different fat sources on the performance, egg quality, and lipid profile of the egg yolks of layers in their second production cycle. The fat sources were cottonseed oil, soybean oil, lard, sunflower oil, or canola oil. Experimental diets were fed to postmolt ISA Brown layers at 70 wk of age and the experimental period was 74 to 86 wk of age. The different fat sources did not influence performance or eggshell quality, but lipid profile of the egg yolk changed as a function of dietary fat sources. In general, the best changes, such as lower level of saturated fatty acids, higher levels of alpha-linolenic acid and DHA, and lower linoleic acid levels, were promoted by the addition of canola oil, but it did not promote enrichment of the eggs with polyunsaturated fatty acids.

Reproductive biology and moulting cycle of the crab Callinectes ornatus (Decapoda, Portunidae) from the Ubatuba region, Sao Paulo, Brazil

To investigate the reproductive biology of Callinectes ornatus gonadal and moulting cycles were studied over two consecutive years in the Ubatuba region. Six stages of gonadal development were detected in females and four in males. Observations on the number of ovigerous females, on the moulting stages of males and females, and on the percentage of individuals exhibiting mature gonads reveal a continuous reproductive cycle, the stages of which progress at different rates. These data support the hypothesis that C. ornatus shows staggered spawning, with possibly more than one annual reproductive period. A pause in growth before the terminal moult may occur in this species.