Cycles saisonniers, reproduction et qualite des ovocytes chez Pecten maximus en rade de Brest

This work is part of the program, "recruitment determinism in scallops" initiated in 1992 by the "Contrat de Baie". The aim of this research was to explain interannual abundance fluctuations observed for Pecten maximus, in the Bay of Brest. To this end an analytical approach to the life cycle of the species was undertaken to determine its critical phases. A phase may be considered as "critical" if the mortality rate at its level varies from one year to the other. Using data from 1989 to 1994, a precise time-table of the "physiological year" of the scallop in the Bay of Brest was determined. Fecundity and gamete production kinetics were estimated from the annual variation in the weight indices of the gonad. In the laboratory, egg quality was estimated following stimulated spawning of newly caught individuals. The digestive activity was estimated by amylase activity measurements during a three year period. Spawning lasts 6 months in the Bay of Brest from March to October. Spawning, muscle and digestive gland growths, are intimately linked to phytoplancton blooms. Gonad production shows a maximum in April-May, and two relative minima in Match and November. These results, which provide detailed understanding of the adult's annual cycle in the Bay of Brest, will be used to interpret the relationship between the annual cycle and larval and post larval abundance and survival. Already, these results are being used to improve spat production in hatcheries.

Aspects of the biology of Lutraria lutraria (L.) (bivalvia : Mactracea)

The life history of a population of Lutraria lutraria in a depth of 7m at Hunterston, Ayrshire is discussed. Much of the present population Is thought to have settled in 1967. The functional morphology of Lutraria is described and related to its life as a large, deep-burrowing bivalve. Lutraria spawned in late spring and continued to do so through the summer in 1979 and 1980. Animals became spent in August and September. Unsuccessful attempts were made to induce spawning in the laboratory. Artificial fertilization was successful but development did not proceed beyond the ciliated gastrula stage. Larvae of Lutraria were not identified in plankton samples and young stages were not encountered in sieved sediment samples. The biochemical cycle of the total animal and five component parts (gonad and visceral mass, digestive gland, adductor muscle, siphon and 'other' tissue) is investigated. A marked increase in weight, reflected in an increase in weight of the component parts, was recorded in Autumn 1979. This is thought to be related to an exceptional increase in the phytoplankton at this time. Although a relationship between the biochemical cycle and reproductive cycle remains uncertain, definite seasonal changes were recorded in the respiration rate of Lutraria. At 10°C, the maximum rate of a standard 20g animal was 0.1283m1s 02/g. dry wt./hr. in May 1980 and the minimum rate was 0.O59mls 02/g. dry wt./hr. in October 1980. The effect of temperature on respiration rate was also investigated. Significant differences were recorded for five experimental temperatures (10°C, 15°C, 20°C, 25°C and 30 °C) in August and October but only between two temperatures (10 C and 30 C) in April. There was a decrease in respiration rate at 30 C in August and October, but an increase in April. Respiration rate is affected by a reduction in oxygen tension. A variety of responses were recorded with a small degree of regulation shown. Individuals of Lutraria were able to survive 48 hours under anaerobic conditions. In fully oxygenated conditions heart rate ranged from 4-15 beats per minute with an average of 8 beats per minute. Heart beat was markedly affected by changes in temperature and oxygen tension, increasing to a maximum 22 beats per minute at 25 C, and decreasing to a minimum 2 beats per minute in anaerobic conditions. Heart rate is reduced (12 beats per minute to 5 beats per minute) on exposure to air. Lutraria exhibits an intermittent pattern of pumping activity. Under normal conditions 35% of the time is spent pumping and this Increases as oxygen is reduced (3.00mls 02/litre) to 65% of the time spent pumping. 15. Under normal conditions the respiratory flow varies between 0.382 litres per hour and 1.023 litres per hxir. Adult Lutraria maintain their ability to burrow, albeit slowly.

Efeitos do cobre no metabolismo energético do bivalve marinho Mesodesma mactroides

Apesar de ser um micronutriente essencial aos organismos, o cobre (Cu) é tóxico quando presente em elevadas concentrações na água. O mecanismo pelo qual este metal exerce sua toxicidade em invertebrados marinhos ainda não está bem estabelecido. Dentre os diversos efeitos relatados, observa-se uma redução do consumo de oxigênio corporal e tecidual no marisco Mesodesma mactroides exposto (96 h) ao Cu (150 µg L-1 ) em água do mar (salinidade 30). Portanto, o objetivo do presente estudo foi avaliar os efeitos desta exposição ao Cu no metabolismo energético em teciduais do marisco M. mactroides. Os conteúdos de ATP e coenzimas (NAD+ e NADH) nas brânquias, glândula digestiva e músculo pedal não foram alterados pela exposição ao Cu, indicando que estes tecidos mantiveram suas capacidades de produção aeróbica de energia. Porém, foi observada uma redução no conteúdo hemolinfático de ATP. Quanto ao conteúdo de proteínas, houve um aumento na glândula digestiva, que pode estar associado à maior oxidação de proteínas já relatada para esse tecido após exposição ao Cu. Os conteúdos de lipídios, glicogênio e glicose permaneceram inalterados em todos os tecidos analisados, exceto no músculo pedal, onde foi observada uma redução no conteúdo de glicose. Por isso, os conteúdos de piruvato e lactato também foram analisados no músculo pedal e na hemolinfa. Em ambos tecidos, foi observado um aumento do conteúdo de lactato, sem alteração no conteúdo de piruvato. Portanto, os resultados do presente estudo sugerem que os tecidos de M. mactroides utilizam a anaerobiose para obtenção de energia durante a exposição ao Cu, conforme demonstrado no músculo pedal e hemolinfa. Apesar disso, a hemolinfa não é capaz de manter o nível de ATP nas condições experimentais testadas. 

Validation d’un procédé automatisé d’extraction sur phase solide de glandes digestives de moules pour l’identification et la quantification des dinophysistoxines en LC/ESI/SM2 par piégeage d’ions quadripolaire

This report presents a new extraction method of the dinophysistoxins (DTXs), confirmed by quantification using high-performance liquid chromatography coupled to mass spectrometry with an ion trap and electro spray interface (HPLC/ESI/MS2). The method originality consists on the adaptation of DTXs basic extraction procedure (liquid/ liquid) to a solid phase extraction (SPE) via a robotic station: ASPEC XLi The parameters of the automatization procedure were optimized to obtain the best DTXs recovery rate. These improvements were loaded with digestive gland mussel homogenat realized on a silica cartridge SPE, activated in hexane/chloroform (50:50), washed with hexane/chloroform (50:50) and extracted by an elution gradient (chloroform methanol (65:35) and methanol (100%)). This method was validated according to two normative referentials (linearity, detection quantification limits and accuracy…) : - The Guide of the Pharmacy industry: Analytical Validation, report of the commission SFSTP 1992 (French Corporation of the Sciences and Technical Pharmaceutical), - - The Procedure of validation of an alternative method in compare to a reference method. (AFNOR, 1998. NF V 03-110). Comparison with the classical liquid/liquid extraction and the automated method present clear advantages. In an analytical method the extraction is generally considered to be the most labor-intensive and error-prone step. This new procedure allowed us to increase throughput, to improve the reproducibility and to reduce the error risks due to the individual manual treatments.

Multibiomarker assessment of cadmium-based quantum dots effects in the marine mussel Mytilus galloprovincialis

Tese de Doutoramento, Ciências do Mar, da Terra e do Ambiente, Ramo: Ciências e Tecnologias do Ambiente, Especialização em Ecotoxicologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

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.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

Three native freshwater crayfish Cherax species are farmed in Australia namely; Redclaw (Cherax quadricarinatus), Marron (C. tenuimanus), and Yabby (C. destructor). Lack of appropriate data on specific nutrient requirements for each of these species, however, has constrained development of specific formulated diets and hence current use of over-formulated feeds or expensive marine shrimp feeds, limit their profitability. A number of studies have investigated nutritional requirements in redclaw that have focused on replacing expensive fish meal in formulated feeds with non-protein, less expensive substitutes including plant based ingredients. Confirmation that freshwater crayfish possess endogenous cellulase genes, suggests their potential ability to utilize complex carbohydrates like cellulose as nutrient sources in their diet. To date, studies have been limited to only C. quadricarinatus and C. destructor and no studies have compared the relative ability of each species to utilize soluble cellulose in their diets. Individual feeding trials of late-juveniles of each species were conducted separately in an automated recirculating culture system over 12 week cycles. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch. Water temperature, conductivity and pH were maintained at constant and optimum levels for each species. Animals were fed at 3% of their body weight twice daily and wet body weight was recorded bi-weekly. At the end of experiment, all animals were harvested, measured and midgut gland extracts assayed for alpha-amylase, total protease and cellulase activity levels. After the trial period, redclaw fed with RD showed significantly higher (p<0.05) specific growth rate (SGR) compare with animals fed the TD while SGR of marron and yabby fed the two diets were not significantly different (p<0.05). Cellulase expression levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD. Amylase and protease activity in all three species were significantly higher in the animals fed with RD (Table 1). These results indicate that test animals of all species can utilize starch better than dietary soluble cellulose in their diet and inclusion of 20% soluble cellulose in diets does not appear to have any significant negative effect on their growth rate but survival was impacted in C. quadricarinatus while not in C. tenuimanus or C. destructor.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

The current study evaluated the effect of soluble dietary cellulose on growth, survival and digestive enzyme activity in three endemic, Australian freshwater crayfish species (redclaw: Cherax quadricarinatus, marron: C. tenuimanus, yabby: C. destructor). Separate individual feeding trials were conducted for late-stage juveniles from each species in an automated recirculating freshwater, culture system. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch, over a 12 week period. Redclaw fed with RD showed significantly higher (p<0.05) specific growth rates (SGR) compared with animals fed the TD, while SGR of marron and yabby fed the two diets were not significantly different. Expressed cellulase activity levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD (p<0.05). Amylase and protease activity in all three species were significantly higher in the animals fed with RD (p<0.05). These results indicate that test animals of all three species appear to utilize starch more efficiently than soluble dietary cellulose in their diet. The inclusion of 20% soluble cellulose in diets did not appear, however, to have a significant negative effect on growth rates.

Use Of Micro-Stereology And Quantitative Cyto-Chemistry To Determine The Effects Of Crude Oil-Derived Aromatic-Hydrocarbons On Lysosomal Structure And Function In A Marine Bivalve Mollusk Mytilus-Edulis

The marine bivalve mollusc,Mytilus edulis (blue mussel), is a noted accumulator of many environmental pollutants and is increasingly used for the chemical and biological assessment of environmental impact. The toxic effects of crude oil-derived aromatic hydrocarbons (30 μg/l total hydrocarbons) on the lysosomal-vacuolar system of the digestive cells have been investigated in cryostat sections of hexane-frozen digestive glands. Exposure to aromatic hydrocarbons reduced the cytochemically determined latency of lysosomal β-N-acetylhexosaminidase; lysosomal volume density and surface density increased while the numerical density decreased. Experimental exposure resulted in the formation of very large lysosomes which are believed to be largely autophagic in function and these results indicate a significant structural and functional disturbance of digestive cell lysosomes in response to hydrocarbons.

Feeding activity of Callinectes ornatus Ordway, 1863 and Callinectes danae Smith, 1869 (Crustacea, Brachyura, Portunidae) in Ubatuba, SP, Brazil

The feeding activity along the day cycle and the time consumed for extracellular digestion were evaluated in the portunids C. ornatus and C. danae. Swimming crabs were obtained from trawling in Ubatuba bay, São Paulo, Brazil, during both the rainy and dry seasons. In each season, daily scheduled samples were taken at dawn (±6 h), noon (±12 h), dusk (±18 h) and midnight (±24 h). All individuals were dissected and the degree of stomach replenishment was recorded. In order to estimate the time elapsed for extracellular digestion, crabs were fed, and groups were dissected at 30 min intervals to check the conditions of their stomachs. In general, both species show a higher feeding activity during periods of lower light intensity, as evidenced by an increased percentage of full stomachs in dusk and midnight samples. The obtained results support higher feeding activity at night in these species and indicate short time for extracellular digestion, not exceeding 8 h. Nevertheless, full stomachs were recorded in all sampling schedules. In this case, it should be considered that elimination of certain food items such as fish bones, mollusk shells and carapace fragments of crustaceans could take more time than other items. Additionally, some crab species could require a cycle of cell replacement in the midgut gland epithelium until they can take their next meal.

Enzymes present in the thoracic gland extracts from workers and males of Apis mellifera (Hymenoptera: Apidae)

This research deals with the analysis of the enzymes present in thoracic gland extracts from newly emerged, nurse workers, forager workers, newly emerged males, and mature males of A. mellifera L. (Hymenoptera, Apoidea, Apidae). The enzymes found in larger quantities in the thoracic gland occurred in all classes of workers and are digestive. Acid phosphatase and Naphtol-AS-BI-phosphohydrolase act in protein synthesis, leucine arylamidase hydrolyses proteins and a-glucosidase actuate in the nectar processing into honey. Naphtol-AS-BI-phosphohydrolase was found in larger quantities only in workers, this suggests action in protein synthesis by the thoracic gland, b-galactosidase is in larger amounts in the newly emerged bees (workers and males) this aids in the provision of other substances to be used as an energy source when glucose or sucrose are absent. Differences between enzymatic profiles from workers and males are usually related to their colony tasks, or related to their physiological necessities per individual in specific life stages.

The hypopharyngeal gland of leaf-cutting ants (Atta sexdens rubropilosa) (Hymenoptera: Formicidae)

The social behavior of ants is controlled by means of dynamic interactions between the environment and the individuals, mainly through pheromones. Among the exocrine glands is the hypopharyngeal gland (HG), located laterally over the pharyngeal plate. The present work aimed to describe and compare the HG in the different castes of the leaf-cutting ant Atta sexdens rubropilosa Forel 1908. The HG of different castes showed similar morphology varying only in relation to the secretory cell number. The secretory intracellular reservoir presented positive reaction to Xylidine Ponceau and P.A.S, indicating the presence of protein and polysaccharides in the secretion. Therefore, we suggest that the function of this gland would be the production of digestive enzymes and/or some sort of mucus, which, together with the secretions produced by the salivary glands of the thorax, would be related to the production of saliva.

Pyloric Brunner’s gland hamartoma with atypical hyperplasia

Brunner’s gland hamartoma (BGH) is an extremely rare benign digestive tumor, generally located in the duodenal bulb. We report the case of a 51-year-old asymptomatic man with a large pedunculated BGH arising from the pylorus. It was successfully removed en bloc by endoscopic resection.

Liver fluke-associated and sporadic cholangiocarcinoma: an immunohistochemical study of bile duct, peribiliary gland and tumour cell phenotypes

Aim: To compare cell phenotypes displayed by cholangiocarcinomas and adjacent bile duct lesions in patients from an area endemic in liver-fluke infestation and those with sporadic cholangiocarcinoma. Methods: 65 fluke-associated and 47 sporadic cholangiocarcinomas and 6 normal livers were studied. Serial paraffin-wax sections were stained immunohistochemically with monoclonal antibodies characterising a Brunner or pyloric gland metaplasia cell phenotype (antigens D10 and 1F6), intestinal goblet cells (antigen 17NM), gastric foveolar apomucin (MUC5AC), a gastrointestinal epithelium cytokeratin (CK20) and the p53 protein. Results: 60% of the 112 cholangiocarcinomas expressed antigen D10, 68% MUC5AC, 33% antigen 17NM and 20% CK20; 37% showed overexpression of p53. When present together in a cholangiocarcinoma, cancer cells expressing D10 were distinct from those displaying 17NM or MUC5AC. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinomas displayed 17NM and p53 expression. Most cases of hyperplastic and dysplastic biliary epithelium expressed D10 strongly. Pyloric gland metaplasia and peribiliary glands displayed D10 and 1F6, with peribiliary gland hyperplasia more evident in the livers with fluke-associated cholangiocarcinoma; goblet cells in intestinal metaplasia stained for 17NM. No notable association of expression between any two antigens (including p53) was found in the cancers. Conclusions: Most cases of dysplastic biliary epithelium and cholangiocarcinoma display a Brunner or pyloric gland cell phenotype and a gastric foveolar cell phenotype. The expression of D10 in hyperplastic and dysplastic epithelium and in cholangiocarcinoma is consistent with a dysplasia-carcinoma sequence. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinoma display an intestinal goblet cell phenotype and overexpress p53, indicating differences in the aetiopathology of the cancers in the two groups of patients.