Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system

Background Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology. Results Analyses of the bacterial community identified in water from BFT and “clear seawater” (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments. Conclusion This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system.

Molecular characterization of a crustinlike antimicrobial peptide in the giant tiger shrimp, Penaeus monodon, and its expression profile in response to various immunostimulants and challenge with WSSV

A crustinlike antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustinlike AMP from other shrimp crustins. The differential expression of the crustinlike AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two bglucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two grampositive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded preand postchallenge white spot syndrome virus (WSSV) by semiquantitative RTPCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustinlike AMP was found to be constitutively expressed in the animal and a significant downregulation could be noted postchallenge WSSV. Remarkable downregulation of the gene was observed in the immunostimulant fed animals prechallenge followed by a significant upregulation postchallenge WSSV. Tissuewise expression of crustinlike AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustinlike AMP and confer significant protection to P. monodon against WSSV infection

Nursery performance of the pink shrimp Farfantepenaeus paulensis (Crustacea: Decapoda: Penaeidae) postlarvae in different salinities

Nursery performance, development, and RNA:DNA ratio were investigated in Farfantepenaeus paulensis (Pérez-Farfante, 1967) postlarvae acclimated from a salinity of 30‰ to higher (35‰) or lower (16, 22 and 29‰) salinities and reared for 20 days. Overall, higher final weight, yield and growth rate were observed at a salinity of 29‰. RNA:DNA ratio indicated reduced growth potential at a salinity of 35‰. Low salinities resulted in more developed individuals. Thus, early postlarval F. paulensis should not be stocked in salinities higher than that of the original hatchery, otherwise in lower salinities postlarvae should be older and/or have an extended nursery phase. Results may assist in the development of nursery rearing protocols for F. paulensis, an alternative species for aquaculture in subtropical areas.

ASSOCIATION OF A MYOSIN MOTOR WITH MEMBRANE-BOUNDED PIGMENT GRANULES IN FRESHWATER SHRIMP CHROMATOPHORES: EVIDENCE FROM THE NITELLA ACTIN-CABLE ASSAY

We have adapted an actin-mosin motility assay to examine the interactions in vitro between actin cables isolated from the giant internodal cells of the freshwater alga, Nitella, and pigment granules extracted from red ovarian chromatophores of the freshwater palaemonid shrimp, Macrobrachium olfersi. The chromatophore pigment mass consists of large (0.5-1.0-mu m diameter) membrane-bounded granules, and small (140-nm diameter), a membranous granules, both structurally continuous with the abundant smooth endoplasmic reticulum. Our previous immunocytochemical studies show a myosin motor to be stably associated with the pigment mass; however, to which granule type or membrane the myosin motor is attached is unclear. Here, we show that sodium vanadate, a myosin ATPase inhibitor, markedly increases the affinity of isolated, large, membrane-bounded granules for Nitella actin cables to which they become permanently attached. This interaction does not occur in granule preparations containing ATP with uninhibited, active myosin without vanadate. We propose that a stable state of elevated affinity is established between the granule-located myosin motor and the Nitella actin cables, resulting from a vanadate-inhibited acto-myosin-ADP complex. This finding provides further evidence for a myosin motor positioned on the surface of the membrane-bounded pigment granules in shrimp ovarian chromatophores.

INTERPOPULATION COMPARISON OF REPRODUCTION OF THE ATLANTIC SHRIMP HIPPOLYTE OBLIQUIMANUS (CARIDEA: HIPPOLYTIDAE)

The genus Hippolyte is represented by typically small shrimps with intriguing mechanisms of reproduction. In order to study possible variability in reproductive aspects among different populations, we conducted an exhaustive comparative study of H. obliquimanus from South (Brazil) and Central American (Costa Rica) waters. The study focuses on fecundity and reproductive output. Mean size of ovigerous females was significantly larger, and both mean reproductive output and mean fecundity were significantly higher in specimens from Costa Rica then in those collected in Brazil. Embryo volume was significantly smaller in the Costa Rican population, and in both populations embryos doubled their volume during embryogenesis. We discuss and compare our findings with the information available regarding H. obliquimanus and other hippolytid shrimp. The reproductive traits of both populations of H. obliquimanus show some important differences which may reflect adaptations to local environmental conditions, demonstrating a high plasticity of reproductive features of the species in Brazilian and Costa Rican waters.

PLANKTON-CAUGHT ZOEAL STAGES AND MEGALOPA OF THE LOBSTER SHRIMP AXIUS SERRATUS (DECAPODA: AXIIDAE) FROM THE BAY OF FUNDY, CANADA, WITH A SUMMARY OF AXIIDEAN AND GEBIIDEAN LITERATURE ON LARVAL DESCRIPTIONS

Wild-caught larvae, attributed to the lobster shrimp Arius serratus, consisting of two zoeal stages and a decapodid (megalopa), are described in detail. Parentage of larvae was ascertained based on geographic distribution of axiideans and gebiideans (= former thalassinideans) within the study area and close morphological resemblance to other congeneric larval stages. Larvae of A. serratus represent the first described 'thalassinidean' larvae from Canadian Atlantic waters and the first for Axiidae within the northwest Atlantic. Among axiidean larvae, those of A. serratus most closely resemble larvae of A. stirhynchus from the eastern Atlantic. Distinct features include the spination of the pleon that set A. serratus zoeae apart from those of most other 'thalassinideans' but that, in combination with a telson very similar to Homarus americanus, contributes to the general resemblance of A. serratus larvae to those of the American lobster. The primary distinction between these taxa is the presence of a chela on the third pereiopod in the latter that is not present in the former. In view of these appendages being prone to loss or damage, other characters that separate these taxa are listed and discussed. Given the uncertain status of some taxa within Axiidae and limited detailed information of larvae with certain parentage, difficulties in delineating the family based on larvae persist, as they do for cladistic analyses using adult morphology and molecular approaches.

Bacteria in shrimp pond sediments: their role in mineralizing nutrients and some suggested sampling strategies

Strategies for sampling sediment bacteria were examined in intensive shrimp, Penaeus monodon (Fabricius), ponds in tropical Australia. Stratified sampling of bacteria at the end of the production season showed that the pond centre, containing flocculated sludge, had significantly higher bacterial counts (15.5 X 10(9) g(-1) dw) than the pond periphery (8.1 X 10(9) g(-1) dw), where the action of aerators had swept the pond floor. The variation in bacterial counts between these two zones within a pond was higher than that between sites within each zone or between ponds. Therefore, sampling effort should be focused within these zones: for example, sampling two ponds at six locations within each of the two zones resulted in a coefficient of variation of approximate to 5%. Bacterial numbers in the sediment were highly correlated with sediment grain size, probably because eroded soil particles and organic waste both accumulated in the centre of the pond. Despite high inputs of organic matter added to the ponds, principally as pelleted feeds, the mean bacterial numbers and nutrient concentrations (i.e. organic carbon, nitrogen and phosphorus) in the sediment were similar to those found in mangrove sediments. This suggests that bacteria are rapidly remineralizing particulates into soluble compounds. Bacterial numbers were highly correlated with organic carbon and total kjeldahl nitrogen in the sediment, suggesting that these were limiting factors to bacterial growth.

Hippolyte obliquimanus (Decapoda : Caridea : Hippolytidae): a gonochoric or hermaphroditic shrimp species?

Several studies have reported the occurrence of hermaphroditism in caridean shrimps of the family Hippolytidae. Here we provide the first observations of population traits from the small Western Atlantic shrimp, Hippolyte obliquimanus, to determine its sexual system using descriptive and experimental approaches. Specimens were collected at 2-month intervals from March 2005 to May 2006 in Ubatuba Bay on the northern coast of the state of Sao Paulo, Brazil. The sex of individuals was checked and morphometric dimensions (carapace length, maximum pleura of second abdominal segment width, appendix masculina length, maximum propodus width, and dactyl length of the third pereopod) were also analyzed. The gonads were dissected and examined for signs of abnormalities. The possibility of sex change was experimentally evaluated under laboratory conditions. A total of 674 specimens were collected: 211 males, 339 non-ovigerous females, and 124 ovigerous females. The carapace length ranged from 0.55 to 3.20 mm, with females being significantly larger than males. Hippolyte obliquimanus showed sexual dimorphism in the third pereopod, absence of cincinnuli in the first pleopod, and no reduction of the appendix masculina in the largest males. All males examined had only testes, and all females had only ovaries. There was no sex change observed in the experiments. Together, these data indicate a population with females reaching larger sizes than males, males with a well-developed appendix masculina, and no transitional individuals. The results presented allow characterizing H. obliquimanus as a gonochoric species.

Signal Transduction, Plasma Membrane Calcium Movements, and Pigment Translocation in Freshwater Shrimp Chromatophores

Crustacean color change results from the differential translocation of chromatophore pigments, regulated by neurosecretory peptides like red pigment concentrating hormone (RPCH) that, in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi, triggers pigment aggregation via increased cytosolic cGMP and Ca(2+) of both smooth endoplasmatic reticulum (SER) and extracellular origin. However, Ca(2+) movements during RPCH signaling and the mechanisms that regulate intracellular [Ca(2+)] are enigmatic. We investigate Ca(2+) transporters in the chromatophore plasma membrane and Ca(2+) movements that occur during RPCH signal transduction. Inhibition of the plasma membrane Ca(2+)-ATPase by La(3+) and indirect inhibition of the Na(+)/Ca(2+) exchanger by ouabain induce pigment aggregation, revealing a role for both in Ca(2+) extrusion. Ca(2+) channel blockade by La(3+) or Cd(2+) strongly inhibits slow-phase RPCH-triggered aggregation during which pigments disperse spontaneously. L-type Ca(2+) channel blockade by gabapentin markedly reduces rapid-phase translocation velocity; N- or P/Q-type blockade by omega-conotoxin MVIIC strongly inhibits RPCH-triggered aggregation and reduces velocity, effects revealing RPCH-signaled influx of extracellular Ca(2+). Plasma membrane depolarization, induced by increasing external K(+) from 5 to 50 mM, produces Ca(2+)-dependent pigment aggregation, whereas removal of K(+) from the perfusate causes pigment hyperdispersion, disclosing a clear correlation between membrane depolarization and pigment aggregation; K(+) channel blockade by Ba(2+) also partially inhibits RPCH action. We suggest that, during RPCH signal transduction, Ca(2+) released from the SER, together with K(+) channel closure, causes chromatophore membrane depolarization, leading to the opening of predominantly N- and/or P/Q-type voltage-gated Ca(2+) channels, and a Ca(2+)/cGMP cascade, resulting in pigment aggregation. J. Exp. Zool. 313A:605-617, 2010. (C) 2010 Wiley-Liss, Inc.

Revision of the larval morphology (Zoea I) of the family Hippolytidae (Decapoda, Caridea), with a description of the first stage of the shrimp Hippolyte obliquimanus Dana, 1852

The aim of this study was to summarize the available data on larval morphology of the first zoea of the family Hippolytidae and describe the first zoeal stage of H. obliquimanus from two geographically distinct populations, Brazilian and Caribbean in order to discuss inter- and intraspecific variability. Ovigerous females of Hippolyte obliquimanus were collected at Cahuita (Limon, Costa Rica) and at Ubatuba (Sao Paulo, Brazil). We compiled the published descriptions of all available hippolytid Zoea I (66 spp., 21%), and all zoeae share several characteristics. However, such morphological features cannot be used to distinguish the first zoeae of Hippolytidae from other caridean larvae. Historically, the presence of an exopodal seta at the maxillule and the absence of the anal spine/papilla have been considered as characteristic for the Zoea I of the genus Hippolyte. The results of our revision, however, did not support these conclusions: although H. obliquimanus showed an exopodal seta at the maxillule, four congeners did not bear such structure; moreover, H. obliquimanus as well as one other congener have an anal spine/papilla. All morphological characters observed in the first zoeal stage of H. obliquimanus are shared with others species of the family Hippolytidae. Intraspecific variability in Hippolyte obliquimanus was detected in one morphological aspect: the first zoea had four denticles on the ventral margin of the carapace in the Brazilian population, while specimens from the Costa Rican population had three.

Maternal investment in egg production: environmental and population-specific effects on offspring performance in the snapping shrimp Alpheus nuttingi (Schmitt, 1924) (Decapoda, Alpheidae)

Among marine invertebrates, the overall biomass invested in egg production varies widely within populations, which can result from the interaction of endogenous and exogenous factors. Species that have constant reproduction throughout the year can be good models to study the influence of environmental factors on reproductive processes. We conducted a seasonal comparison of egg production in the intertidal snapping shrimp Alpheus nuttingi, which shows a continuous reproductive pattern, to examine the hypothesis that differences in egg production are driven by environmental conditions and population features. This population showed an uncommon strategy, characterized by females that produce eggs of varying sizes within their clutches, with reduced egg volume when the number of eggs is higher (Spring-Summer). In these seasons, higher temperatures and greater food availability may allow the production of more eggs compared to the Autumn-Winter seasons. Compared to other alpheid shrimps, this population produces small eggs, but in larger numbers. Despite the higher fecundity, the reproductive output is relatively low, this production being supported by the large size of females from the southern Atlantic region. Our findings showed that the egg production of A. nuttingi was greatly influenced by environmental factors. Therefore, this shrimp, and probably other decapods that possess continuous reproduction, adopt different reproductive strategies during the year. (C) Koninklijke Brill NV, Leiden, 2010.

Sexual ratio, reproductive period and seasonal variation of the gonochoric shrimp Hippolyte obliquimanus (Caridea: Hippolytidae)

Hippolyte obliquimanus is a small, gonochoric shrimp found in algal substrates along the western Atlantic coast of Brazil, particularly in association with seaweed of the genus Sargassum. We studied population features (sexual ratio, reproductive period and temporal distribution) of H. obliquimanus in southeastern Brazil, including its relationships with the seasonality of banks of this alga. Specimens were collected at two-monthly intervals from March 2005 to January 2006, in Ubatuba Bay. The sex of individuals was checked, and the carapace length measured. In total, 668 individuals were collected: 211 males (0.70-2.50 mm carapace length), 341 non-ovigerous females (0.55-2.90 mm), and 116 ovigerous females (1.55-3.20 mm). Hippolyte obliquimanus showed seasonal-continuous reproduction and variable continuous recruitment. The highest number of animals (75%) was collected in fall-winter. The percentages of ovigerous females/total females (fall-winter: 27%; spring-summer: 26%) and the sexual ratio (fall-winter: 31%; spring-summer: 32%) were practically equal in both periods. The sexual ratio showed a predominance of females in almost all size classes, and we detected a new sex ratio pattern for this species. The seasonal variation in the number of individuals can be related to its migration to deeper areas, due to the decrease in the abundance of Sargassum sp. in shallower waters in spring-summer.

Cyclic Guanosine Monophosphate Signaling Cascade Mediates Pigment Aggregation in Freshwater Shrimp Chromatophores

The cell signaling cascades that mediate pigment movements in crustacean chromatophores are not yet well established, although Ca(2+) and cyclic nucleotide second messengers are involved. Here, we examine the participation of cyclic guanosine monophosphate (cGMP) in pigment aggregation triggered by red pigment concentrating hormone (RPCH) in the red ovarian chromatophores of freshwater shrimp. In Ca(2+)-containing (5.5 mmol l(-1)) saline, 10 mu mol l(-1) dibutyryl cGMP alone produced complete pigment aggregation with the same time course (approximate to 20 min) and peak velocity (approximate to 17 mu m/min) as 10(-8) mol l(-1) RPCH; however, in Ca(2+)-free saline (9 X 10(-11) mol l(-1) Ca(2+)), db-cGMP was without effect. The soluble guanylyl cyclase (GC-S) activators sodium nitroprusside (SNP, 0.5 mu mol l(-1)) and 3-morpholinosydnonimine (SIN-1, 100 mu mol l(-1)) induced moderate aggregation by themselves (approximate to 35%-40%) but did not affect RPCH-triggered aggregation. The GC-S inhibitors zinc protoporphyrin IX (ZnPP-XI, 30 mu mol l(-1)) and 6-anilino-5,8-quinolinedione (LY83583, 10 mu mol l(-1)) partially inhibited RPCH-triggered aggregation by approximate to 35%. Escherichia coli heat-stable enterotoxin (STa, 1 mu mol l(-1)), a membrane-receptor guanylyl cyclase stimulator, did not induce or affect RPCH-triggered aggregation. We propose that the binding of RPCH to an unknown membrane-receptor type activates a Ca(2+)-dependent signaling cascade coupled via cytosolic guanylyl cyclase and cGMP to protein kinase G-phosphorylated proteins that regulate aggregation-associated, cytoskeletal molecular motor activity. This is a further example of a cGMP signaling cascade mediating the effect of a crustacean X-organ neurosecretory peptide.