Seasonal foods, gonadal maturation, and length-weight relationships for nine fishes commonly captured by shrimp trawl on the Northwest Gulf of Mexico Continental Shelf

Recent emphasis on ecosystem approaches to fisheries management renews interest in, and the need for, trophic information about fish communities. A program was started in 1980 at the National Marine Fisheries Service Galveston Laboratory to develop a trophic database for continental shelf fishes. Collections were made during 1982-1983 that were processed but never published, yet the data remain valid today for historical purposes and for delimiting food web components within ecosystem assessments. I examined spring, summer, and fall foods in offshore populations of nine common species of trawl-susceptible fishes, with particular reference to predation on commercial penaeid shrimps (Farfantepenaeus and Litopenaeus). Diets were evaluated with the Index of Relative Importance (IRI) which combines the occurrence, number, and weight of each food item. Bank sea bass (Centropristis ocyurus) and bighead searobin (Prionotus tribulus) primarily consumed crabs, more so by larger than smaller fish. Inshore lizardfish (Synodus foetens) was almost entirely piscivorous. Ocellated flounder (Ancylopsetta ommata) consumed fishes, crabs, and stomatopods. Dwarf sand perch (Diplectrum bivittatum), blackwing searobin (Prionotus rubio), rock sea bass (Centropristis philadelphica), southern kingfish (Menticirrhus americanus), and red snapper (Lutjanus campechanus) fed mainly on shrimps. Most fish diets varied with respect to size (age), time of day, area sampled, depth, or season. Rimapenaeus and Sicyonia were the most frequently identified shrimp genera - only five Farfantepenaeus and no Litopenaeus were identified in almost 4,300 fish stomachs. I also examined gonadal development and documented fish length-weight relationships. Ripe gonads were most frequently found during summer in dwarf sand perch, during fall in ocellated flounder and bighead searobin, and during spring for other species, except no ripe red snapper or bank sea bass were collected. Rock sea bass was found to be a protogynous hermaphrodite, while dwarf sand perch is a synchronous hermaphrodite. Only ocellated flounder and southern kingfish exhibited sex-related differences in length-weight relationships. (PDF contains 40 pages.)

Characterization of the benthos, marine debris and bottom fish at Gray’s Reef National Marine Sanctuary

Executive Summary: Baseline characterization of resources is an essential part of marine protected area (MPA) management and is critical to inform adaptive management. Gray’s Reef National Marine Sanctuary (GRNMS) currently lacks adequate characterization of several key resources as identified in the 2006 Final Management Plan. The objectives of this characterization were to fulfill this need by characterizing the bottom fish, benthic features, marine debris, and the relationships among them for the different bottom types within the sanctuary: ledges, sparse live bottom, rippled sand, and flat sand. Particular attention was given to characterizing the different ledge types, their fish communities, and the marine debris associated with them given the importance of this bottom type to the sanctuary. The characterization has been divided into four sections. Section 1 provides a brief overview of the project, its relevance to sanctuary needs, methods of site selection, and general field procedures. Section 2 provides the survey methods, results, discussion, and recommendations for monitoring specific to the benthic characterization. Section 3 describes the characterization of marine debris. Section 4 is specific to the characterization of bottom fish. Field surveys were conducted during August 2004, May 2005, and August 2005. A total of 179 surveys were completed over ledge bottom (n=92), sparse live bottom (n=51), flat sand (n=20), and rippled sand (n=16). There were three components to each field survey: fish counting, benthic assessment, and quantification of marine debris. All components occurred within a 25 x 4 m belt transect. Two divers performed the transect at each survey site. One diver was responsible for identification of fish species, size, and abundance using a visual survey. The second diver was responsible for characterization of benthic features using five randomly placed 1 m2 quadrats, measuring ledge height and other benthic structures, and quantifying marine debris within the entire transect. GRNMS is composed of four main bottom types: flat sand, rippled sand, sparsely colonized live bottom, and densely colonized live bottom (ledges). Independent evaluation of the thematic accuracy of the GRNMS benthic map produced by Kendall et al. (2005) revealed high overall accuracy (93%). Most discrepancies between map and diver classification occurred during August 2004 and likely can be attributed to several factors, including actual map or diver errors, and changes in the bottom type due to physical forces. The four bottom types have distinct physical and biological characteristics. Flat and rippled sand bottom types were composed primarily of sand substrate and secondarily shell rubble. Flat sand and rippled sand bottom types were characterized by low percent cover (0-2%) of benthic organisms at all sites. Although the sand bottom types were largely devoid of epifauna, numerous burrows indicate the presence of infaunal organisms. Sparse live bottom and ledges were colonized by macroalgae and numerous invertebrates, including coral, gorgonians, sponges, and “other” benthic species (such as tunicates, anemones, and bryozoans). Ledges and sparse live bottom were similar in terms of diversity (H’) given the level of classification used here. However, percent cover of benthic species, with the exception of gorgonians, was significantly greater on ledge than on sparse live bottom. Percent biotic cover at sparse live bottom ranged from 0.7-26.3%, but was greater than 10% at only 7 out of 51 sites. Colonization on sparse live bottom is likely inhibited by shifting sands, as most sites were covered in a layer of sediment up to several centimeters thick. On ledge bottom type, percent cover ranged from 0.42-100%, with the highest percent cover at ledges in the central and south-central region of GRNMS. Biotic cover on ledges is influenced by local ledge characteristics. Cluster analysis of ledge dimensions (total height, undercut height, undercut width) resulted in three main categories of ledges, which were classified as short, medium, and tall. Median total percent cover was 97.6%, 75.1%, and 17.7% on tall, medium, and short ledges, respectively. Total percent cover and cover of macroalgae, sponges, and other organisms was significantly lower on short ledges compared to medium and tall ledges, but did not vary significantly between medium and tall ledges. Like sparse live bottom, short ledges may be susceptible to burial by sand, however the results indicate that ledge height may only be important to a certain threshold. There are likely other factors not considered here that also influence spatial distribution and community structure (e.g., small scale complexity, ocean currents, differential settlement patterns, and biological interactions). GRNMS is a popular site for recreational fishing and boating, and there has been increased concern about the accumulation of debris in the sanctuary and potential effects on sanctuary resources. Understanding the types, abundance, and distribution of debris is essential to improving debris removal and education efforts. Approximately two-thirds of all observed debris items found during the field surveys were fishing gear, and about half of the fishing related debris was monofilament fishing line. Other fishing related debris included leaders and spear gun parts, and non-gear debris included cans, bottles, and rope. The spatial distribution of debris was concentrated in the center of the sanctuary and was most frequently associated with ledges rather than at other bottom types. Several factors may contribute to this observation. Ledges are often targeted by fishermen due to the association of recreationally important fish species with this bottom type. In addition, ledges are structurally complex and are often densely colonized by biota, providing numerous places for debris to become stuck or entangled. Analysis of observed boat locations indicated that higher boat activity, which is an indication of fishing, occurs in the center of the sanctuary. On ledges, the presence and abundance of debris was significantly related to observed boat density and physiographic features including ledge height, ledge area, and percent cover. While it is likely that most fishing related debris originates from boats inside the sanctuary, preliminary investigation of ocean current data indicate that currents may influence the distribution and local retention of more mobile items. Fish communities at GRNMS are closely linked to benthic habitats. A list of species encountered, probability of occurrence, abundance, and biomass by habitat is provided. Species richness, diversity, composition, abundance, and biomass of fish all showed striking differences depending on bottom type with ledges showing the highest values of nearly all metrics. Species membership was distinctly separated by bottom type as well, although very short, sparsely colonized ledges often had a similar community composition to that of sparse live bottom. Analysis of fish communities at ledges alone indicated that species richness and total abundance of fish were positively related to total percent cover of sessile invertebrates and ledge height. Either ledge attribute was sufficient to result in high abundance or species richness of fish. Fish diversity (H`) was negatively correlated with undercut height due to schools of fish species that utilize ledge undercuts such as Pareques species. Concurrent analysis of ledge types and fish communities indicated that there are five distinct combinations of ledge type and species assemblage. These include, 1) short ledges with little or no undercut that lacked many of the undercut associated species except Urophycis earlii ; 2) tall, heavily colonized, deeply undercut ledges typically with Archosargus probatocephalus, Mycteroperca sp., and Pareques sp.; 3) tall, heavily colonized but less undercut with high occurrence of Lagodon rhomboides and Balistes capriscus; 4) short, heavily colonized ledges typically with Centropristis ocyurus, Halichoeres caudalis, and Stenotomus sp.; and 5) tall, heavily colonized, less undercut typically with Archosargus probatocephalus, Caranx crysos and Seriola sp.. Higher levels of boating activity and presumably fishing pressure did not appear to influence species composition or abundance at the community level although individual species appeared affected. These results indicate that merely knowing the basic characteristics of a ledge such as total height, undercut width, and percent cover of sessile invertebrates would allow good prediction of not only species richness and abundance of fish but also which particular fish species assemblages are likely to occur there. Comparisons with prior studies indicate some major changes in the fish community at GRNMS over the last two decades although the causes of the changes are unknown. Species of interest to recreational fishermen including Centropristis striata, Mycteroperca microlepis, and Mycteroperca phenax were examined in relation to bottom features, areas of assumed high versus low fishing pressure, and spatial dispersion. Both Mycteroperca species were found more frequently when undercut height of ledges was taller. They often were found together in small mixed species groups at ledges in the north central and southwest central regions of the sanctuary. Both had lower mode size and proportion of fish above the fishery size limit in heavily fished areas of the sanctuary (i.e. high boat density) despite the presence of better habitat in that region. Black sea bass, C. striata, occurred at 98% of the ledges surveyed and appeared to be evenly distributed throughout the sanctuary. Abundance was best explained by a positive relationship with percent cover of sessile biota but was also negatively related to presence of either Mycteroperca species. This may be due to predation by the Mycteroperca species or avoidance of sites where they are present by C. striata. Suggestions for monitoring bottom features, marine debris, and bottom fish at GRNMS are provided at the end of each chapter. The present assessment has established quantitative baseline characteristics of many of the key resources and use issues at GRNMS. The methods can be used as a model for future assessments to track the trajectory of GRNMS resources. Belt transects are ideally suited to providing efficient and quantitative assessment of bottom features, debris, and fish at GRNMS. The limited visibility, sensitivity of sessile biota, and linear nature of ledge habitats greatly diminish the utility of other sampling techniques. Ledges should receive the bulk of future characterization effort due to their importance to the sanctuary and high variability in physical structure, benthic composition, and fish assemblages. (PDF contains 107 pages.)

Age determination methods for northwest Atlantic species

The successful application of techniques to enhance detection of age marks in biological specimens is of vital importance in fisheries research. This manual documents age determination techniques used by staff at the Woods Hole Laboratory, National Marine Fisheries Service. General information on procedures for preparing anatomical structures is described, together with criteria used to interpret growth patterns and assign ages. Annotated photographs of age structures are provided to illustrate criteria. Detailed procedures are given for the following species: Atlantic herring (Clupea harengus), haddock (Melanogrammus aeglefinus), Atlantic cod (Gadus morhua), pollock (Pollachius virens), silver hake (Merluccius bilinearis), red hake (Urophycis chuss), black sea bass (Centropristis striata), weakfish (Cynoscion regalis), Atlantic mackerel (Scomber scombrus), butterfish (Peprilus triacanthus), redfish (Sebastes fasciatus), summer flounder (Paralichthys dentatus), winter flounder (Pseudopleuronectes americanus), witch flounder (Glyptocephalus cynoglossus), American plaice (Hippoglossoides platessoides), yellowtail flounder (Limanda ferruginea), surf clam (Spisula solidissima), and ocean quahog (Arctica islandica). (PDF file contains 142 pages.)

Effects of stress on the immune system of fish

The effects of stress on the immune system of various fish species including dab Limanda limanda, flounder Platichthys flesus, sea bass Dicentrarchus labrax and gobies Zosterisessor ophiocephalus, were investigated from laboratory and field experiments, using various assays to measure immunocompetence, correlated with histological and ultrastructural observations. Modulation of the immune system was demonstrated at tissue, cellular and biochemical levels following exposure to various stressors. The spleen somatic index was depressed in dab stressed in the laboratory and gobies collected from polluted sites in the Venice Lagoon. Differential blood cell counts consistently showed an increase in phagocytes and decrease in thrombocytes in fish exposed to various stressors. Phagocytic activity from spleen and kidney adherent cells was stimulated in dab stressed by transportation but depressed in fish exposed to chemical pollutants. Respiratory burst activity in phagocytic cells was also stimulated in stressed dab but depressed in sea bass exposed to cadmium. The results are discussed in relation to current concepts on stress in fish and the regulation of the immune system.

Application of Entropy and Fractal Dimension Analyses to the Pattern Recognition of Contaminated Fish Responses in Aquaculture

The objective of the work was to develop a non-invasive methodology for image acquisition, processing and nonlinear trajectory analysis of the collective fish response to a stochastic event. Object detection and motion estimation were performed by an optical flow algorithm in order to detect moving fish and simultaneously eliminate background, noise and artifacts. The Entropy and the Fractal Dimension (FD) of the trajectory followed by the centroids of the groups of fish were calculated using Shannon and permutation Entropy and the Katz, Higuchi and Katz-Castiglioni's FD algorithms respectively. The methodology was tested on three case groups of European sea bass (Dicentrarchus labrax), two of which were similar (C1 control and C2 tagged fish) and very different from the third (C3, tagged fish submerged in methylmercury contaminated water). The results indicate that Shannon entropy and Katz-Castiglioni were the most sensitive algorithms and proved to be promising tools for the non-invasive identification and quantification of differences in fish responses. In conclusion, we believe that this methodology has the potential to be embedded in online/real time architecture for contaminant monitoring programs in the aquaculture industry.

Analysis of permanent magnets as elasmobranch bycatch reduction devices in hook-and-line and longline trials

Previous studies indicate that elasmobranch fishes (sharks, skates and rays) detect the Earth’s geomagnetic field by indirect magnetoreception through electromagnetic induction, using their ampullae of Lorenzini. Applying this concept, we evaluated the capture of elasmobranchs in the presence of permanent magnets in hook-and-line and inshore longline fishing experiments. Hooks with neodymium-iron-boron magnets significantly reduced the capture of elasmobranchs overall in comparison with control and procedural control hooks in the hook-and-line experiment. Catches of Atlantic sharpnose shark (Rhizoprionodon terraenovae) and smooth dogfish (Mustelus canis) were signif icantly reduced with magnetic hook-and-line treatments, whereas catches of spiny dogfish (Squalus acanthias) and clearnose skate (Raja eglanteria) were not. Longline hooks with barium-ferrite magnets significantly reduced total elasmobranch capture when compared with control hooks. In the longline study, capture of blacktip sharks (Carcharhinus limbatus) and southern stingrays (Dasyatis americana) was reduced on magnetic hooks, whereas capture of sandbar shark (Carcharhinus plumbeus) was not affected. Teleosts, such as red drum (Sciaenops ocellatus), Atlantic croaker (Micropogonias undulatus), oyster toadfish (Opsanus tau), black sea bass (Centropristis striata), and the bluefish (Pomatomas saltatrix), showed no hook preference in either hook-and-line or longline studies. These results indicate that permanent magnets, although eliciting species-specific capture trends, warrant further investigation in commercial longline and recreational fisheries, where bycatch mortality is a leading contributor to declines in elasmobranch populations.

日本花鲈细胞色素b基因部分序列的研究

首次报道日本花鲈线粒体DNA细胞色素b基因片段的PCR扩增及其序列测 定。得到410bp的碱基序列，其A、T、G、C含量分别为99bp(24．15哟、113bp(27．56旧、 72bp(17．56嗡、126bp(30．73哟，与其他鱼类相同基因片段碱基序列含量相似。

Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus)

Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

SNAP-23蛋白在鲈鱼巨噬细胞白细胞介素1β分泌过程中的功能

SNARE蛋白家族是所有真核细胞胞吐及分泌作用中的关键因子，由其介导的运输囊泡膜与靶膜的锚靠、融合为胞内蛋白的运出提供了一条重要途径。体外试验表明，Syntaxin6-Syntaxin7-Vti1b，SNAP-23-Syntaxin4等SNARE核心蛋白之间精确的相互作用是哺乳动物巨噬细胞肿瘤坏死因子α (TNF-α)运输和分泌的必备条件，在机体非特异性免疫应答反应过程中起重要作用。 本研究受上述启示，旨在揭示SNARE蛋白在海洋鱼类免疫细胞内重要细胞因子白细胞介素1β (IL-1β)的分泌过程中的作用。参照Percoll密度梯度离心技术，从鲈鱼头肾组织分离纯化巨噬细胞进行稳定培养；利用RT-PCR方法克隆出鲈鱼t-SNARE蛋白SNAP-23和Syntaxin3的部分cDNA序列，再结合先前克隆的VAMP2和已知的鲈鱼IL-1β，TNF-α和IL-8的基因序列，设计特异性引物。利用Real-time PCR技术在mRNA水平上精确测定鲈鱼巨噬细胞中上述6种基因在革兰氏阴性菌脂多糖(LPS)分子刺激下的表达变化，发现SNAP-23基因与三种细胞因子基因的表达正相关；通过免疫印迹检测SNAP-23蛋白表达变化，利用酶联免疫吸附试验(ELISA)检测IL-1β的分泌水平，在蛋白水平上验证了SNAP-23表达与IL-1β分泌的正相关性；利用5`RACE和3`RACE技术克隆出鲈鱼SNAP-23全长基因，结合定点突变策略和靶向PCR克隆手段，构建鲈鱼SNAP-23野生型融合质粒pEGFP-SNAP-23wt，Cys缺失突变融合质粒pEGFP-SNAP-23ΔCys和模拟E型肉毒神经毒素(BoNT/E)切割突变融合质粒pEGFP-SNAP-23ΔBoNT/E，以及鲈鱼IL-1β野生型融合表达质粒IL-1β-pEGFP和IL-1β-pEYFP。所有融合蛋白均在鲈鱼巨噬细胞内成功表达，结合ELISA实验结果发现，SNAP-23野生型的表达对IL-1β的分泌有促进作用，而Cys缺失突变体的表达则抑制IL-1β向胞外分泌。首次证实了鱼类巨噬细胞内SNAP-23蛋白在IL-1β分泌过程中的重要作用。此外通过与GFP共表达，定位了IL-1β分子在巨噬细胞内的分布，发现新合成的IL-1β分子很可能像TNFα一样经“内质网－胞质－伪足－胞外” 的分泌路径运出胞外。

评价水产动物生长性能的生理生化指标和分子生物学指标

以奥利亚罗非鱼(Oreochromis aureus)为实验对象，设计了3种不同的摄食类型，分别是鲜活饵料组、饥饿3周后饱食投喂组和人工饲料组。鲜活饵料组投喂冰冻赤子爱胜蚓，利用蚯蚓体内丰富的营养成分和活性物质，以期获得奥利亚罗非鱼良好的生长状况；饥饿后饱食组是指饥饿3周后，以人工饲料饱食投喂2周，用于研究饥饿与补偿生长获得快速生长时血液理化指标的变化情况；人工饲料组作为对照组。纯淡水条件下养殖，水温25±2℃。测定了奥利亚罗非鱼在3种摄食类型饲喂下某些血液生理生化指标变化的情况，并将指标变化情况与增重率做相关性分析，试图找出能够反映奥利亚罗非鱼生长性能的血液生理生化指标。 研究结果表明，奥利亚罗非鱼在饥饿3周后获得了补偿生长，补偿生长时的增重率和特定生长率显著高于人工饲料组(P<0.05)，高于鲜活饵料组，但差别不显著；相关性分析研究表明血清总蛋白、胆固醇、四碘甲状腺原氨酸（T4）与增重率极显著相关(P<0.01)，血红蛋白显著相关(P<0.05)，红细胞、白细胞、碱性磷酸酶高度相关(相关系数为0.580、0.551和0.557)，因此，建议血清总蛋白、胆固醇和血红蛋白可作为能够反映罗非鱼生长性能的新指标。 根据序列设计引物，PCR反应条件：变性温度：95 ℃，3 min；退火温度：57℃，20 sec；延伸温度：72℃，5 min，共36个循环，从牙鲆、黑鲪和鲈鱼中克隆出胰岛素样生长因子（IGF-Ⅰ）部分序列，首次证实了IGF-Ⅰ在3种海水鱼中的存在。 利用蛋氨酸与ZnSO4•7H2O，在pH 5.5、80℃下，反应1小时，采用蛋氨酸与硫酸锌2：1的配料比，合成出了产物蛋氨酸螯合锌，蛋氨酸螯合锌外观白色，粉状，室温下微溶于水，不溶于乙醇，并用原子吸收光谱法测定其含锌量为15%，螯合率为88.2%。

Efeitos genotóxicos e bioquímicos de xenobióticos em animais aquáticos

No presente trabalho, foi estudado um largo espectro de efeitos genotóxicos e bioquímicos na tainha-garrento (Liza aurata). Nos Capítulos II e III são descritos os efeitos de exposição de curta duração ao fenantreno, um hidrocarboneto aromático policíclico (HAP). A exposição de curta duração (16 horas) demonstrou a capacidade deste composto induzir a actividade da enzima de fase I da biotransformação, etoxiresorufina O-desetilase (EROD), provocar decréscimos de integridade no ADN hepático e aumento de anomalias nucleares eritrocíticas (ANE). Em termos de respostas de stresse, os níveis plasmáticos de cortisol e glucose aumentaram face à exposição a este HAP. A exposição ao fenantreno induziu o decréscimo da glutationa peroxidase (GPx) nas guelras, enquanto que no fígado a actividade da GPx aumentou. No rim, a actividade da glutationa S-transferase (GST) foi inibida. Nas guelras, verificou-se um aumento da catalase. O fenantreno demonstrou igualmente a capacidade de induzir um aumento dos níveis de glutationa nas guelras e fígado. Estas respostas demonstraram a sensibilidade de L. aurata, a este HAP, realçando a especificidade das respostas em termos de órgãos. Apesar dos aumentos das defesas antioxidantes, o potencial tóxico deste composto foi demonstrado pelo aumento da peroxidação lipídica nos três órgãos. Nos capítulos seguintes, são descritas as respostas de L. aurata capturada na Ria de Aveiro, em locais com diferentes perfis de contaminação, inicialmente no Outono de 2005 (Capítulos III a IX) e posteriormente analisando respostas sazonais (Capítulos X e XI). A análise de respostas de stresse (cortisol, glucose e lactato) revelou que L. aurata capturada em Vagos (local contaminado por HAPs) apresentava níveis baixos de cortisol, enquanto que no Laranjo (local contaminado por mercúrio) apresentavam elevados níveis de glucose e lactato. Relativamente às hormonas do eixo hipotálamo – hipófise – tiróide (HHT), foram observados elevados níveis plasmáticos da hormona estimuladora da tiróide (TSH) nos organismos capturados no Laranjo, baixos níveis de tiroxina (T4) nos organismos da Barra (local sujeito a tráfego naval) e baixos níveis de triiodotironina (T3) no Rio Novo do Príncipe (próximo de um antigo efluente de pasta de papel), Laranjo e Vagos. A avaliação das defesas antioxidantes, dano oxidativo e genotóxico nas guelras, rim e fígado revelou diferenças significativas nas respostas dos órgãos. L. aurata capturada na Barra apresentou dano oxidativo nas guelras (Capítulo V). No rim foi detectada uma diminuição da integridade do ADN no Rio Novo do Príncipe e Vagos (Capítulo VI), enquanto que no fígado foi observado dano lipídico na Gafanha e Vagos (Capítulo VIII). O dano não esteve sempre associado a um decréscimo das defesas. As análises da água e do sedimento da Ria de Aveiro (Outono de 2005) revelaram elevadas concentrações de metais (Cd, Hg, Cu e Zn),principalmente, no Laranjo e Rio Novo do Príncipe. L. aurata capturada nestes locais apresentou os níveis mais elevados de metalotioninas hepáticas (Capítulo VII) que parecem responsáveis pela inexistência de danos no fígado (Capítulo VIII). O dano oxidativo no ADN, avaliado através da quantificação dos níveis plasmáticos de 8-hidroxi-2-desoxiguanosina (8-OHdG) e o dano clastogénico/aneugénico, avaliado através da quantificação da frequência de ANE, foram estudados, no Outono de 2005, em duas espécies de peixes (L. aurata e Dicentrarchus labrax - robalo) (Capítulo IX). Os resultados revelaram grande sensibilidade de D. labrax em termos de dano oxidativo no ADN na Gafanha, Rio Novo do Príncipe e Vagos, enquanto que L. aurata apresentou dano oxidativo apenas no Laranjo. O aumento da frequência de ANE apenas foi detectado em L. aurata, em Vagos, não se tendo detectado correlação entre estes dois parâmetros. O estudo sazonal (Maio de 2006 a Março de 2007) do dano oxidativo no ADN e frequência de ANE em L. aurata (Capítulo X) demonstrou a variação destes parâmetros com a estação do ano, apesar de não se ter verificado correlação com os parâmetros hidrológicos determinados. No entanto, no local de referência não se verificaram diferenças sazonais, o que sugere que estes biomarcadores reflectem variações de biodisponibilidade de contaminantes. A análise global dos resultados das diferentes estações do ano revelou que L. aurata capturada no Rio Novo do Príncipe e em Vagos apresentou maior susceptibilidade a dano oxidativo no ADN. No entanto, apenas L. aurata capturada em Vagos apresentou frequência de ANE superior à do local de referência. Os dados do estudo sazonal revelaram uma correlação entre dano oxidativo e ANE, sugerindo o stresse oxidativo como um possível mecanismo envolvido na formação de anomalias. A integridade do ADN das guelras, rim, fígado e sangue de L. aurata foi igualmente estudada ao longo de um ano (Capítulo XI), tendo-se verificado uma grande variabilidade ao longo deste período. Não foi demonstrada sensibilidade a um perfil de contaminação específico, tendo-se verificando variabilidade sazonal no local de referência. Globalmente, os resultados demonstraram a importância da utilização de uma bateria de biomarcadores na monitorização ambiental e a especificidade da resposta dos diferentes órgãos de L. aurata.

Mercury in feral fish: distribution, accumulation and toxicity

O principal objectivo desta dissertação foi estudar a acumulação de mercúrio em vários tecidos de peixes marinhos, a sua relação com factores biológicos e as respectivas respostas bioquímicas. O trabalho realizado permitiu obter novos conhecimentos sobre a acumulação de mercúrio em peixes, possibilitando avaliar a influência da biodisponibilidade do elemento e as suas possíveis implicações no ambiente. O trabalho foi desenvolvido na Ria de Aveiro (Portugal), uma zona costeira onde existe um gradiente ambiental de mercúrio, o que oferece a oportunidade de estudar a sua acumulação e os seus efeitos tóxicos em condições realísticas. As amostragens foram efectuadas em dois locais considerados críticos em termos de contaminação por mercúrio – Largo do Laranjo (L1 e L2) e num local afastado da principal fonte de poluição, usado como termo de comparação (Referência; R); L1 e L2 corresponderam a locais moderadamente e altamente contaminados, respectivamente. Foram escolhidos juvenis de duas espécies ecologicamente diferentes e representativas da comunidade piscícola local, a tainha garrento (Liza aurata) e o robalo (Dicentrarchus labrax). Em cada local foram recolhidas amostras de água e de sedimento para determinação de mercúrio. Foram quantificadas as concentrações de mercúrio total (T-Hg) e orgânico (O-Hg) em vários tecidos dos peixes, escolhidos tendo em conta a sua função relativamente à toxicocinética e toxicodinâmica de metais. As respostas antioxidantes (Catalase- CAT, glutationa peroxidase- GPx, glutationa reductase- GR, glutationa –S-transferase- GST e conteúdo em glutationa total- GSHt), o dano peroxidativo (LPO) e o conteúdo em metalotioninas (MTs) foram também avaliados. A acumulação de T-Hg foi semelhante para as duas espécies de peixes estudadas, embora D. labrax tenha apresentado concentrações tendencialmente maiores. Ambas as espécies demonstraram capacidade de reflectir o grau de contaminação ambiental existente, indicando claramente que a acumulação depende da concentração ambiental. A acumulação revelou-se específica de cada tecido. O padrão da acumulação em L. aurata foi rim > fígado > músculo > cérebro > guelras > sangue e em D. labrax foi fígado > rim > músculo > cérebro ≈ guelras > sangue. Relativamente à acumulação de OHg, verificou-se que D. labrax exibiu concentrações mais elevadas que L. aurata. Todos os tecidos foram capazes de reflectir diferenças entre R e L2. Os níveis de O-Hg no fígado, músculo e nos conteúdos intestinais foram diferentes entre espécies, sendo mais elevados para D. labrax. As guelras e o intestino foram os tecidos onde se obtiveram os valores mais baixos de O-Hg e observaram-se valores idênticos para as duas espécies. Com excepção das guelras, as concentrações de O-Hg variaram em função do valor observado nos conteúdos intestinais, indicando que a alimentação é a via dominante da acumulação. As concentrações de O-Hg nos conteúdos intestinais revelaram ser uma informação relevante para prever a acumulação de O-Hg nos tecidos, pois verificou-se uma razão praticamente constante entre o teor de mercúrio no fígado, no músculo e nos conteúdos intestinais. A percentagem de O-Hg no músculo e no fígado variou de acordo com o grau de contaminação ambiental e com o tipo de assimilação preferencial do elemento (alimentação vs. água), sugerindo que o fígado exerce um papel protector em relação à acumulação de mercúrio nos outros órgãos. Ambas as espécies de peixes demonstraram ser boas sentinelas da contaminação ambiental com mercúrio (T-Hg e O-Hg), sendo o cérebro e o músculo os tecidos que melhor reflectiram o grau de acumulação com o elemento. A análise conjunta dos dados de bioacumulação e de respostas ao stress oxidativo permitiram estabelecer uma relação entre as concentrações de mercúrio nas guelras, fígado, rim e cérebro e a sua toxicidade. As respostas do cérebro aos efeitos tóxicos do mercúrio revelaram ser específicas de cada espécie. Enquanto que para o cérebro de L. aurata se verificou um decréscimo de todos os parâmetros antioxidantes estudados nos locais contaminados, sem haver evidência de qualquer mecanismo compensatório, no D. labrax observaram-se respostas ambivalentes, que indicam por um lado a activação de mecanismos adaptativos e, por outro, o decréscimo das respostas antioxidantes, ou seja, sinais de toxicidade. Embora em ambas as espécies de peixe fosse evidente uma condição pró-oxidante, o cérebro parece possuir mecanismos compensatórios eficientes, uma vez que não se verificou peroxidação lipídica. As respostas antioxidantes do cérebro de D. labrax foram comparadas em diferentes períodos do ano - quente vs. frio. O período quente mostrou ser mais crítico, uma vez que no período frio não se verificaram diferenças nas respostas entre locais, ou seja, a capacidade antioxidante do cérebro parece ser influenciada pelos factores ambientais. As guelras revelaram susceptibilidade à contaminação por mercúrio, uma vez que se verificou uma tendência para o decréscimo da actividade de CAT em L2 e ausência de indução em L1. O fígado e o rim demonstraram mecanismos adaptativos face ao grau de contaminação moderada (L1), evidenciados pelo aumento de CAT. O rim também demonstrou adaptabilidade face ao grau elevado de contaminação (L2), uma vez que se verificou um aumento GST. Embora o grau de susceptibilidade tenha sido diferente entre os órgãos, não se verificou peroxidação lipídica em nenhum. A determinação do conteúdo em MTs em D. labrax e em L. aurata revelou que este parâmetro depende não só da espécie, mas também do tecido em causa. Assim, em D. labrax foi observado um decréscimo de MTs no cérebro, bem como a incapacidade de síntese de MTs no sangue, guelras, fígado, rim e músculo. Em L. aurata observou-se um aumento do conteúdo em MTs no fígado e no músculo. Estes resultados indicam que a aplicabilidade das MTs como biomarcador de exposição ao mercúrio parece ser incerta, revelando limitações na capacidade de reflectir os níveis de exposição ao metal e por consequência o grau de acumulação. Este trabalho comprova a necessidade de se integrarem estudos de bioacumulação com biomarcadores de efeitos, de modo a reduzir os riscos de interpretações erróneas, uma vez que as respostas nem sempre ocorrem para os níveis mais altos de contaminação ambiental com mercúrio.

Evolution of the extracellular matrix in deuterostomes and the influence of calcitropic hormones

Tese de dout., Biologia (Biologia Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50–55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the ‘‘classical’’ role of pituitary function regulation.

An integrated and comparative approach to genetic selection of the aquaculture species Dicentrarchus labrax

The European sea bass, Dicentrarchus labrax, is one of the most important marine species cultivated in Southern Europe and has not benefited from selective breeding. One of the major goals in the sea bass (D. labrax) aquaculture industry is to understand and control the complexity of growth associated traits. The aim of the methodology developed for the studies reported in the thesis was not only to establish genetic and genomic resources for sea bass, but to also develop a conceptual strategy to efficiently create knowledge in a research environment that can easily be transferred to the aquaculture industry. The strategy involved; i) establishing an annotated sea bass transcriptome and then using it to, ii) identify new genetic markers for target QTL regions so that, iii) new QTL analysis could be performed and marker based resolution of the DNA regions of interest increased, and then iv) to merge the linkage map and the physical map in order to map the QTL confidence intervals to the sea bass genome and identify genes underlying the targeted traits. Finally to test if genes in the QTL regions that are candidates for divergent growth phenotypes have modified patterns of transcription that reflects the modified whole organism physiology SuperSAGE-SOLiD4 gene expression was used with sea bass with high growth heterogeneity. The SuperSAGE contributed to significantly increase the transcriptome information for sea bass muscle, brain and liver and also led to the identification of putative candidate genes lying in the genomic region of growth related QTL. Lastly all differentially expressed transcripts in brain, liver and muscle of the European sea bass with divergent specific growth rates were mapped to gene pathways and networks and the regulatory pathways most affected identified and established the tissue specific changes underlying the divergent SGR. Owing to the importance of European sea bass to Mediterranean aquaculture and the developed genomics resources from the present thesis and from other studies it should be possible to implement genetic selection programs using marker assisted selection.