Prediction of apparent protein digestibility of ingredients and diets by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for juvenile Pacific white shrimp Litopenaeus vannamei

Aquafeed production faces global issues related to availability of feed ingredients. Feed manufacturers require greater flexibility in order to develop nutritional and cost-effective formulations that take into account nutrient content and availability of ingredients. The search for appropriate ingredients requires detailed screening of their potential nutritional value and variability at the industrial level. In vitro digestion of feedstuffs by enzymes extracted from the target species has been correlated with apparent protein digestibility (APD) in fish and shrimp species. The present study verified the relationship between APD and in vitro degree of protein hydrolysis (DH) with Litopenaeus vannamei hepatopancreas enzymes in several different ingredients (n = 26): blood meals, casein, corn gluten meal, crab meal, distiller`s dried grains with solubles, feather meal, fish meals, gelatin, krill meals, poultry by-product meal, soybean meals, squid meals and wheat gluten. The relationship between APD and DH was further verified in diets formulated with these ingredients at 30% inclusion into a reference diet. APD was determined in vivo (30.1 +/- 0.5 degrees C, 32.2 +/- 0.4%.) with juvenile L vannamei (9 to 12 g) after placement of test ingredients into a reference diet (35 g kg(-1) CP: 8.03 g kg(-1) lipid; 2.01 kcal g(-1)) with chromic oxide as the inert marker. In vitro DH was assessed in ingredients and diets with standardized hepatopancreas enzymes extracted from pond-reared shrimp. The DH of ingredients was determined under different assay conditions to check for the most suitable in vitro protocol for APD prediction: different batches of enzyme extracts (HPf5 or HPf6), temperatures (25 or 30 degrees C) and enzyme activity (azocasein): crude protein ratios (4 U: 80 mg CP or 4 U: 40 mg CP). DH was not affected by ingredient proximate composition. APD was significantly correlated to DH in regressions considering either ingredients or diets. The relationships between APD and DH of the ingredients could be suitably adjusted to a Rational Function (y = (a + bx)/(1 + cx + dx2), n = 26. Best in vitro APD predictions were obtained at 25 degrees C, 4 U: 80 mg CP both for ingredients (R(2) = 0.86: P = 0.001) and test diets (R(2) = 0.96; P = 0.007). The regression model including all 26 ingredients generated higher prediction residuals (i.e., predicted APD - determined APD) for corn gluten meal, feather meal. poultry by-product meal and krill flour. The remaining test ingredients presented mean prediction residuals of 3.5 points. A model including only ingredients with APD>80% showed higher prediction precision (R(2) = 0.98: P = 0.000004; n = 20) with average residual of 1.8 points. Predictive models including only ingredients from the same origin (e.g., marine-based, R(2) = 0.98; P = 0.033) also displayed low residuals. Since in vitro techniques have been usually validated through regressions against in vivo APD, the DH predictive capacity may depend on the consistency of the in vivo methodology. Regressions between APD and DH suggested a close relationship between peptide bond breakage by hepatopancreas digestive proteases and the apparent nitrogen assimilation in shrimp, and this may be a useful tool to provide rapid nutritional information. (C) 2009 Elsevier B.V. All rights reserved.

Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 degrees C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2-9.0) and 55 degrees C. CaCl(2) did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 degrees C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P < 0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.

CLEANER SHRIMP (CARIDEA: PALAEMONIDAE) ASSOCIATED WITH SCYPHOZOAN JELLYFISH

Shrimp are found in association with many hosts (sponges, anthozoans, mollusks, echinoids, ascidians and fish). Shrimp are rarely observed in association with jellyfish. The interaction between the cleaner shrimp Periclimenes paivai and jellyfish is poorly known, with only one published report for Brazilian waters. We add new information about this association, based on field and laboratory observations. Shrimp were collected from three locations on the coast Of Sao Paulo State (Cananeia, Santos and Sao Sebastiao) and from two species of scyphozoan rnedusae: Chrysaora lactea and Lychnorhiza lucerna. We classify the association as facultative commensalism, since the shrimp gain protection and probably feed on mucus produced by the jelly fish. The maximum carapace length of the shrimp collected was 9.2 min. Medusa bel I diameter ranged between about 10 and 19 cm for C. lactea and between 4.5 and 25 cm for L. lucerna. We also provide a list of records of Periclimenes in association with different scyphornedUsae, and additional information on the size and coloration of P. paivai.

Ecological distribution of the shrimp Litopenaeus schmitti (Burkenroad, 1936) (Decapoda, Penaeoidea) in Ubatuba Bay, Sao Paulo, Brazil

The penaeidean Litopenaeus schmitti, popularly known as white shrimp, is a species of great economic importance, being a target of fishing fleets in the southeast region of Brazil. It is distributed through the western Atlantic, from Cuba to Brazil, until Rio Grande do Sul. Adults are found from shallow depths up to 30 m and have been found to depths of 47 m in the state of Rio de Janeiro, while juveniles are located in bays and estuaries. The studied species is seasonally distributed in the region of Ubatuba. The objectives of this study are to analyze the abundance and ecological distribution of L. schmitti and to assess if and when juveniles use the Indaia estuary during their life cycle. Furthermore, the hypothesis was tested that the main period of recruitment in the bay coincides with the period of closure of fisheries defined by the Instruction of IBAMA. To that end, samples were taken monthly from July 2005 through June 2007, both in Ubatuba Bay and in the estuary formed by the Rio Indaia. At each sampling station, salinity, temperature (bottom), depth, organic matter content (%), and grain size of sediments were measured. We found that the largest catches in the estuary were in late spring and early summer. In Ubatuba Bay, peak catches occurred during winter and early spring, whereas in the second year, already in May, there was a high peak capture. The variation in the number of individuals was correlated with some environmental factors both in the estuary and in the inlet (p < 0.05). In the estuary, abundance was positively correlated with temperature (p = 0.008) and organic matter (p = 0.025) and negatively with depth (p = 0.009). Regarding the Ubatuba Bay, only temperature (p = 0.034) and depth (p = 0.013) were significantly associated with the distribution of the shrimp, both being negative relations. The shrimp L. schmitti uses both the estuarine as well the shore environment, particularly the Ubatuba estuary and its adjacent bay, to complete its life cycle. The proposed period of fisheries closure (between March to May in the state of Sao Paulo) for this and other shrimps coincides with individuals capable of reproduction entering the inlet and thus are being protected.

Taxonomic revision of doubtful Brazilian freshwater shrimp species of genus Macrobrachium (Decapoda, Palaemonidae)

The freshwater prawns of the genus Macrobrachium Spence Bate, 1868 are widely distributed in rivers of tropical and subtropical regions and represent an interesting group with controversial taxonomy. The morphological characters traditionally used to separate species have shown a high intraspecific variation. Doubts about the status of M. birai Lobao, Melo & Fernandes, 1986, M. holthuisi Genofre & Lobao, 1978 and M. petronioi Melo, Lobao & Fernandes, 1986 have been arisen due to the high resemblance of the former two species with M. olfersi (Wiegmann, 1836), and the latter one with M. potiuna (Muller, 1880). Therefore, we performed a detailed morphological analysis of these species, including new characters not usually used in the species recognition. The present results here with molecular data lead us to conclude that M. birai and M. holthuisi are junior synonyms of M. olfersi, and M. petronioi is a junior synonym of M. potiuna. Considering these synonymies, 17 valid species are now reported for the Brazilian territory.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg2+, Na+, K+ and NH4 (+) and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, . (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP ( (M) = 0.09 +/- A 0.01 mmol L-1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ ( (0.5) = 0.91 +/- A 0.04 mmol L-1) in decapodid III than in other stages; NH4 (+) had no modulatory effect. The affinity for Na+ ( (0.5) = 13.2 +/- A 0.6 mmol L-1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4 (+) obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval The NH4 (+)-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Milk hygienic practices and occurrence of Staphylococcus aureus and Escherichia coli O157:H7 in small-scale dairy farms in Sao Paulo, Brazil

The objective of this study was to assess the relationship between somatic cell counts (SCC), the use of different milking practices, and the occurrence of Staphylococcus aureus and Escherichia coli O157:H7 in 42 small-scale dairy farms located in the state of Sao Paulo, Brazil. S. aureus and E. coli O157:H7 were isolated in the milk from dairy cows with low (< 200,000 cells/ml) and high SCC (>200,000 cells/ml), although no effect of SCC (p > 0.05) was observed on the incidence of the bacteria in raw milk. The use of disposable gloves during milking reduced S. aureus counts in milk (p < 0.05), but did not affect the occurrence of E. coli O157:H7. The other milking practices evaluated (closed milking system, use of pre- and post-dipping, mastitis diagnosis by strip cup test, and disinfection of teat cups) did not affect (p < 0.05) the occurrence of S. aureus or E. coli O157:H7 in raw milk. Results indicate the need for effective educational programs addressed to prevent the contamination of milk with S. aureus and E. coli O157:H7 in Brazilian small-scale dairy farms.

Reproductive biology of the ghost shrimp Lepidophthalmus bocourti (A. Milne-Edwards, 1870) (Decapoda: Axiidea: Callianassidae): A tropical species with a seasonal reproduction

Based on the hypothesis that reproduction is a continuous process in tropical habitats, we analysed reproductive periodicity and egg production in the callianassid ghost shrimp Lepidophthalmus bocourti, one of the most common species in mangrove systems along the Pacific coast of Central America. During one year (May 2008 to April 2009), individuals of L. bocourti (N = 499) were collected nearshore Gulf of Nicoya, Pacific coast of Costa Rica. Observations were made on presence or absence of incubated embryos, and gonad activity of females was analysed as gonadosomatic index (GSI). Our results revealed that L. bocourti has a marked seasonal breeding period, which contradicts previous reports regarding coastal marine decapods from the tropics. Ovigerous females were found only from June to August, while high GSI values were obtained from March to July. The increase of GSI and appearance of ovigerous females were associated with a concomitant decrease of salinity, but not with temperature. We assume that reproduction of L. bocourti is adapted to local changes of environmental conditions, and that a decrease in salinity during rainy season may serve as a triggering factor for ovarian development. Compared to other ghost shrimps, L. bocourti produced on average more (2002 +/- 1365) and smaller (0.87 +/- 0.109 mm) eggs, which seems to suggest that this species does not have an abbreviated larval development as reported for other species of genus. The deviation from the generalization of constant reproduction in the tropics for shallow water marine invertebrates and its probable cause are adequately discussed.

Characterization of Staphylococcus aureus isolates in milk and the milking environment from small-scale dairy farms of Sao Paulo, Brazil, using pulsed-field gel electrophoresis

This research aimed to evaluate the occurrence of Staphylococcus aureus isolates in milk and in the milking environment of 10 small-scale farms (<400 L/d) located in the regions of Franca and Ribeirao Preto, state of Sao Paulo, Brazil. Two-hundred twenty samples of milk were collected from individual cows, along with 120 samples from bulk tank milk, 389 samples from milking equipment and utensils (teat cups, buckets, and sieves), and 120 samples from milkers' hands. Fifty-six Staph. aureus strains were isolated from 849 analyzed samples (6.6%): 12 (5.5%) from milk samples of individual cows, 26 (21.7%) from samples of bulk tank milk, 14 (3.6%) from samples collected from equipment and utensils, and 4 (3.3%) from samples from milkers' hands. Pulsed-field gel electrophoresis typing of the 56 Staph. aureus isolates by SmaI restriction enzyme resulted in 31 profiles (pulsotypes) arranged in 12 major clusters. Results of this study indicate a low incidence, but wide distribution of Staph. aureus strains isolated from raw milk collected from individual cows and surfaces of milkers' hands and milking equipment in the small-scale dairy farms evaluated. However, the high percentage of bulk milk samples found with Staph. aureus is of public health concern because raw, unprocessed milk is regularly consumed by the Brazilian population.

Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca2+ cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca2+/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca2+ cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca2+ (int), activating the Ca2+/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

A novel spermatozoan ultrastructure in the shrimp Hippolyte obliquimanus Dana, 1852 (Decapoda: Caridea: Hippolytidae)

The aim of this study was to describe and illustrate the morphology of the spermatozoon of the Western Atlantic shrimp, Hippolyte obliquimanus. Individuals were sampled from Itagua Beach (Ubatuba, southern Brazil). The male reproductive system was dissected and morphological analysis was undertaken using a stereomicroscope, a light microscope, and transmission electron and scanning electron microscopes. When viewed from the nuclear or acrosomal poles, each spermatozoon has many translucent radiating arms (about 20) from a denser cell body, while laterally the cell body and arms resemble a "cnidarian medusa", with all the arms projecting away from the bell-like cell body. This sperm morphology is distinct from the "thumbtack"-shaped spermatozoa observed in the majority of carideans but has similarities to the spermatozoa of Rhynchocinetes spp. The morphology of sperm of several species of the genus Hippolyte resembles the spermatozoon of H. obliquimanus with the presence of posterior nuclear arms, but it is necessary to study other Hippolyte species to place these arms in the context of the genus.

Assessment of trophic transfer of benzo(a)pyrene genotoxicity from the post-larval pink shrimp F. brasiliensis to the juvenile Florida pompano T. carolinus

In the present study, the polycyclic aromatic hydrocarbon (PAH) genotoxicity was investigated in a one-step predator-prey relationship with the trophic-related marine species. Florida pompanos were fed for 5 and 10 days with pink shrimp post larvae previously exposed to benzo(a)pyrene (BaP) concentrations. Parent BaP body burden was measured in samples of Farfantepenaeus brasiliensis. BaP metabolites were determined in bile samples of Trachinotus carolinus and DNA damage was assessed through the comet and erythrocyte nuclear abnormalities (ENAs) assays in fish erythrocytes. BaP body burden increased significantly with the PAH concentration in pink shrimp PLs as well as the fish bile BaP metabolites. Both, comet and ENAs assays indicated significant increase on erythrocyte DNA damage of Florida pompanos fed with BaP-exposed pink shrimp on both feeding periods. The trophic route of BaP genotoxicity is discussed as well as the PAH biotransformation as the inducing mechanism for the DNA damages observed.

EROD activity and genotoxicity in the seabob shrimp Xiphopenaeus kroyeri exposed to benzo[a]pyrene (BaP) concentrations

Seabob shrimp Xiphopenaeus kroyeri is a marine species that lives in shallow waters of coastal environments, often impacted by polycyclic aromatic hydrocarbons (PAH) pollution. In the present study, seabob shrimp were exposed for 96 h to benzo[a]pyrene (BaP) at the nominal concentrations of 100, 200, 400 and 800 microg.L-1. Animals of the control groups were exposed either to clean water or to the BaP-carrier (DMSO). At the end of the exposures, muscle tissues were sampled for BaP uptake assessment and hepatopancreas and hemolymph for EROD enzyme activity and hemocytes DNA damage, respectively. EROD activity and DNA damage increased significantly as a function of BaP exposure concentrations. Significant correlations between BaP uptake and both EROD activity and DNA damage suggest that they can be used as suitable tools for integrated levels of study on the biomarkers of PAH exposure.

Ontogenetic and evolutionary change of external morphology of the neotropical shrimp potimirim (Holthuis, 1954) explained by a molecular phylogeny of the genus

A phylogenetic analysis of a fragment of the mitochondrial gene 16S was used to test the monophyletic status of Potimirim. Existing doubts on the taxonomic status of brasiliana (once P glabra) and P potimirim (once P mexicana) were clarified. Potimirim mexicana and P potimirim are distinct species according to molecular data and appendix masculina morphology. A new species (Potimirim sp. 1) from Puerto Rico was revealed with molecular data, and it is evolutionarily related to P potimirim and P mexicana according to our analysis. We found out three distinct species under the name P glabra. Then, we recommend the application of the name P glabra for the populations of the Pacific slope of Central America and revalidation of P brasiliana for the Brazilian ones. The need for a new name to those "P glabra" of the Caribbean is highlighted, and it was provisionally referred as Potimirim sp. 2. The ontogenetic (juveniles to adults) development of the appendix masculina of P brasiliana was observed and compared to the other species of Potimirim (adults). In the light of our phylogenetic hypothesis, we postulate a pattern of character addition for the evolution of the appendix masculina of Potimirim. This hypothesis is plausible for two key reasons. First. Potimirim is a monophyletic group according to our hypothesis. Second, the shape of appendix masculina found in adults of P. americana is similar and comparable to those found in the earliest juvenile stages of P brasiliana, a derived species according to our phylogeny (P americana, ((P mexicana, Potimirim sp. 1. P potimirim), (P glabra, (brasiliana, Potimirim sp. 2)))). As so, the basal P americana retain the ancestral morphological state of the appendix masculina when compared to the other species of Potimirim. In our interpretation the ontogeny of the appendix masculina recapitulated the proposed phylogeny, giving further support to it.

A longitudinal study of Neospora caninum infection on three dairy farms in Brazil

Neospora caninum is an apicomplexan protozoan parasite that is one of the most important infectious causes of abortion in both dairy and beef cattle in many countries. The objectives of this longitudinal study were to determine the prevalence, rates of vertical and horizontal transmission of N. caninum and hazard for culling of N. caninum-seropositive animals in three Brazilian dairy herds. Blood samples from all animals were collected nine times at each of the three farms over a two-year period. Serum was tested for antibodies against N. caninum using the indirect fluorescent antibody test with a cutoff value of 1:100. The percentage of N. caninum-positive samples at each sampling time ranged at Farm 1 from 3.32% to 11.71%, at Farm 11 from 3.90% to 22.06% and at Farm III from 3.90% to 22.06%. The number of positive serological reactions varied in relation to the number of repeated samples taken from individual animals at each farm. In all herds, there was a high degree (P < 0.05) of association between the N. caninum serological status of dams and daughters. The seropositive conversion rate was estimated as 0.37%, 3.00% and 6.94% per 100 cow-years at Farms I, II and III, respectively. The seronegative conversion rate was estimated as 31.58% and 11.11% per 100 cow-years at Farms 1 and III, respectively. In all herds, there was no difference (P > 0.05) in the culling rate between the cattle that were seropositive cattle and seronegative for N. caninum infection. The results from this study confirm the importance of vertical transmission in the epidemiology of N. caninum. Although a few positive seroconversions indicated horizontal transmission, it does not appear to be the major route of infection for N. caninum. (C) 2012 Elsevier B.V. All rights reserved.