The potential of Asparagopsis armata to control the bacterial load associated to live feed to improve Seabream (Sparus aurata) larvae performance

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Allozyme and mtDNA variation of white seabream Diplodus sargus populations in a transition area between western and eastern Mediterranean basins (Siculo-Tunisian Strait)

To investigate the possible influence of the Siculo-Tunisian Strait on the genetic structure of white seabream Diplodus sargus, 13 polymorphic allozyme loci and a fragment of the cytochrome b mitochondrial DNA were analysed. Allozyme data indicated a moderate but significant differentiation between some north-eastern (Bizerta, Ghar El Melh Lagoon and Mahdia) and southern (Gabes Gulf and El Biban Lagoon) samples. This heterogeneity was also highlighted after removing PGM* and PGI-1* loci which may be under selection. These results can be explained by the chaotic genetic patchiness hypothesis. In contrast, the mtDNA data indicated genetic homogeneity among localities showing the absence of structure in white seabream populations across the Siculo-Tunisian Strait. Historical demography of this species suggests that it has undergone a recent population expansion as a consequence of a bottleneck event during the Pleistocene glaciations.

Phylogeographical history of the white seabream Diplodus sargus (Sparidae): Implications for insularity

Partial sequences of the mitochondrial control region and its comparison with previously published cytochrome b (cyt-b) and microsatellite data were used to investigate the influence of island isolation and connectivity on white seabream genetic structure. To achieve this, a total of 188 individuals from four island localities (Castellamare and Mallorca, Mediterranean Sea; Azores and Canary Islands, Atlantic Ocean) and five coastal localities (Banyuls, Murcia and Tunisia, Mediterranean Sea; Galicia and Faro, Atlantic Ocean) were analysed. Results showed high haplotype diversity and low to moderate nucleotide diversity in all populations (except for the Canary Islands). This pattern of genetic diversity is attributed to a recent population expansion which is corroborated by other results such as cyt-b network and demographic analyses. Low differentiation among Mediterranean/Atlantic and coastal/island groups was shown by the AMOVA and FST values, although a weak phylogeographic break was detected using cyt-b data. However, we found a clear and significant island/ distance effect with regard to the Azores islands. Significant genetic differentiation has been detected between the Azores islands and all other populations. The large geographical distance between the European continental slope and the Azores islands is a barrier to gene flow within this region and historic events such as glaciation could also explain this genetic differentiation.

In two waters: contemporary evolution of lagoonal and marine white seabream (Diplodus sargus) populations

Brackish water ecosystems are often exposed to wide variations in environmental variables, including temperature and salinity, which may cause strong selective pressures on organisms modifying the genetic patterns of species. The aim of this work was to test whether there is a ‘divergence-with-gene flow’ in coastal lagoon populations of white seabream (Diplodus sargus) (Ria Formosa, S Portugal and Mar Menor, SE Spain) respect to four marine populations, by using partial sequences of cyt b mitochondrial gene and information from nine microsatellite loci. Genetic diversity was highest in both coastal lagoons (Mar Menor and Ria Formosa) considering mitochondrial and nuclear markers. Although some of FST population pairwise comparisons were not significant, analyses of molecular variance (AMOVAs) detected differences between groups (coastal lagoon and marine) close to significance. Also, only two haplotypes (Cytb-17 and Cytb-18) were detected in both coastal lagoon sampling sites and these localities (Mar Menor and Ria Formosa) showed the highest number of singletons, some of them with a high number of mutations, as has been already described for other Mar Menor populations (Pomatochistus marmoratus and Holothuria polii). Also, several tests detected significant positive and balancing selection considering mtDNA and microsatellite data. These data support the hypothesis of selection as one of the drivers of the genetic differences found between coastal lagoon and marine populations. The life strategy adopted by Diplodus sargus in coastal lagoons allows it to decrease its mortality rate and improve the heritability of its genes. Also, the increase time spent in coastal lagoons with different temperatures and salinities favours the fitness selection and the maintenance of exclusive haplotypes and genotypes in coastal lagoon inhabitants favouring the ‘divergence-with-gene-flow’.

Connectivity patterns inferred from the genetic structure of white seabream (Diplodus sargus L.)

Themarine environment seems, at first sight, to be a homogeneousmediumlacking barriers to species dispersal. Nevertheless, populations of marine species show varying levels of gene flow and population differentiation, so barriers to gene flow can often be detected. Weaimto elucidate the role of oceanographical factors ingenerating connectivity among populations shaping the phylogeographical patterns in the marine realm, which is not only a topic of considerable interest for understanding the evolution ofmarine biodiversity but also formanagement and conservation of marine life. For this proposal,we investigate the genetic structure and connectivity between continental and insular populations ofwhite seabreamin North East Atlantic (NEA) and Mediterranean Sea (MS) aswell as the influence of historical and contemporary factors in this scenario using mitochondrial (cytochrome b) and nuclear (a set of 9 microsatellite) molecular markers. Azores population appeared genetically differentiated in a single cluster using Structure analysis. This result was corroborated by Principal Component Analysis (PCA) and Monmonier algorithm which suggested a boundary to gene flow, isolating this locality. Azorean population also shows the highest significant values of FST and genetic distances for both molecular markers (microsatellites and mtDNA). We suggest that the breakdown of effective genetic exchange between Azores and the others' samples could be explained simultaneously by hydrographic (deep water) and hydrodynamic (isolating current regimes) factors acting as barriers to the free dispersal of white seabream(adults and larvae) and by historical factors which could be favoured for the survival of Azorean white seabream population at the last glaciation. Mediterranean islands show similar genetic diversity to the neighbouring continental samples and nonsignificant genetic differences. Proximity to continental coasts and the current system could promote an optimal larval dispersion among Mediterranean islands (Mallorca and Castellamare) and coasts with high gene flow.

Genetic considerations on the introduction of farmed fish in marine protected areas: The case of study of white seabream restocking in the Gulf of Castellammare (Southern Tyrrhenian Sea)

Human exploitation has drastically reduced the abundance and distribution of several marine fish and invertebrate populations through overfishing and habitat destruction. Restocking can potentially mitigate these impacts and help to reconstitute depleted stocks but genetic repercussions must be considered. In the present study, the degree of genetic similarity between white seabream (Diplodus sargus Linnaeus 1758) individuals reared for restocking purposes and the receiving population in the Gulf of Castellammare fishery reserve (Sicily, Italy) was assessed using microsatellites. We also inferred the spatial pattern of the genetic structure of D. sargus and connectivity along Sicilian coasts. The farmed population showed significant heterozygosity deficiency in 6 loci and an important reduction in the number of alleles, which could indicate an incipient inbreeding. Both the farmed population and the target one for restocking (Castellammare fishery reserve), showed high and significant values of genetic differentiation due to different allele frequencies, number of privative alleles and total number of alleles. These findings indicate a low degree of genetic similarity between both populations, therefore this restocking initiative is not advisable. The genetic connectivity pattern, highly consistent with oceanographic currents, identified two distinct metapopulations of white seabream around Sicily. Thus it is recommended to utilize broods from the same metapopulation for restocking purposes to provide a better genetic match to the wild populations.

Changes in movements of white seabream (Diplodus sargus) during the reproductive season

It has been hypothesized, based on anecdotal information and reports, that adult white seabream migrate and aggregate during spawning. Because most of the past telemetric studies on this ecologically and commercially important species were short in duration and did not cover the reproductive season, we set out to use longer-term passive acoustic telemetry to test this hypothesis. We found that white seabream expands its home range and increases the frequency of forays during the reproductive season, but found no obvious signs of spawning aggregations. Our results thus provide evidence for short-range behavioral shifts in fish space use related to reproductive activity, and support the notion that small MPAs may be effective in managing reef fish populations by effectively protecting their spawning biomass. (c) 2015 Elsevier Ltd. All rights reserved.

Feeding habits of the gilthead seabream (Sparus aurata) from the Ria Formosa (southern Portugal) as compared to the black seabream (Spondyliosoma cantharus) and the annular seabream (Diplodus annularis)

The feeding habits of Sparus aurata L., Diplodus annularis L. and Spondyliosoma cantharus L. in the Ria Formosa (southern Portugal) lagoon system were studied using three simple methods (frequency of occurrence, numeric percentage and percentage weight) and a composite index [index of relative importance (IRI)]. The Ivlev index was used to evaluate diet selectivity, while the Schoener overlap index was used to compare diets, and diet diversity was characterized by the Simpson index. The diets of the three species consist of a wide variety of food organisms, nevertheless S. aurata seems to be the most specialized. No significant dietary overlap was found, with S. aurata preferentially selecting gastropods and bivalves. while S. cantharus preferentially selected a wide variety of crustaceans and D. sargus a wider array, including crustaceans, gastropods and bivalves.

Population structure of the black seabream Spondyliosoma cantharus along the south-west Portuguese coast inferred from otolith chemistry

The chemistry of black seabream Spondyliosoma cantharus otoliths from three main fishery grounds (Olhao, Sagres and Sesimbra) located along c. 400 km of the Portuguese south and west coasts was examined. Element:Ca ratios were determined in whole otoliths and otolith cores of young adult specimens of 23 years of age. Using the data from whole otoliths, it was possible to discriminate among S. cantharus from the three fishing grounds with an average accuracy of 91%. Differences among fishing grounds were significant for all element:Ca ratios, and otoliths from Sagres had significantly higher levels of all ratios compared to the other fishing grounds. In contrast, the chemical composition of the otolith core, representative of the larval stage, showed limited variation among the fishing grounds, with an average discrimination accuracy of only 44%, although the Mg:Ca ratio of the otolith cores was also significantly higher for the Sagres samples. The data suggest that larval stages experienced a homogenous environment consistent with an offshore oceanic spawning. Juveniles appeared to display local residency on the inshore fishing grounds, areas probably characterized by greater environmental heterogeneity. Spondyliosoma cantharus population structure is consistent with distinct local population units that share a spawning ground providing recruits to different coastal fishery areas.

Residency, movements and habitat use of adult white seabream (Diplodus sargus) between natural and artificial reefs

Artificial reefs are used as management tools for coastal fisheries and ecosystems and the knowledge of habitat use and fish movements around them is necessary to understand their performance and improve their design and location. In this study wild specimens of Diplodus sargus were tagged with acoustic tags and their movements were tracked using passive acoustic telemetry. The monitored area enclosed a natural rocky reef, an adjacent artificial reef (AR) and shallower sandy bottoms. Most of the fish were close to full time residents in the monitored area. Results revealed that D. sargus use the natural reef areas on a more frequent basis than the AR. However, excursions to the adjacent AR and sandy bottoms were frequently detected, essentially during daytime. The use of acoustic telemetry allowed a better understanding of the use of artificial reef structures and its adjacent areas by wild D. sargus providing information that is helpful towards the improvement of AR design and location. (c) 2013 Elsevier Ltd. All rights reserved.

Age, growth and reproduction of the Axilary Seabream, "Pagellus acarne" (Risso, 1827), from the South coast of Portugal

Axillary seabream, Pagellus acarne, caught by longlines in the Algarve (Southern Portugal), were sampled between August 1995 and August 1996. Age was studied by counting growth increments on otoliths and the estimated von Bertalanffy parameters were Linf = 28.82 cm, K = 0.29 year-1 and t0 = -1.47 year for males and Linf = 32.30 cm, K = 0.18 year-1 and t0 = -2.56 year for females. Evidence of the annual periodicity of the deposition of increments was found by marginal increment analyses. Macroscopic analysis of the gonads and the gonad somatic index showed that reproduction occurred over an extensive period of time, from May to November. Lengths at first maturity were 18.10 and 17.60 cm for males and females, respectively. This species was characterized as being a protandric hermaphrodite.

Regulation of Gonadotropin-Releasing Hormone (GnRH)-Receptor Gene Expression in Tilapia: Effect of GnRH and Dopamine

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5–50 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1–100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Characterization of muscle-regulatory gene, MyoD, from flounder (Paralichthys olivaceus) and analysis of its expression patterns during embryogenesis

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix-loop-helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

真鲷（Pagrus major）胚胎超低温损伤机理研究

鱼类胚胎由于其自身结构特征：体积大、含水量高、多室结构等，迄今超低温保存尚未成功。超低温保存过程中所造成的冷冻损伤是制约鱼类胚胎超低温保存成功与否的关键，具体表现为渗透压影响、抗冻剂毒性、冰晶损伤等。系统研究并阐明鱼类胚胎冷冻损伤机理，是成功建立鱼类胚胎超低温保存技术的基础。本论文主要针对胚胎对渗透压的耐受性、抗冻剂对胚胎的渗透性、降温速率对胚胎内外冰晶形成温度的影响等冷冻损伤机理进行了系统研究，主要研究结果如下： 1.通过检测胚胎在不同浓度人工海水（0%、25%、50%、75%、1×、2×、3×、4×，渗透压范围0~3740 mOsm/kg）中的孵化率，确定了真鲷不同发育时期胚胎对渗透压的耐受范围，以及心跳期胚胎浸泡不同时间对渗透压的耐受范围。结果显示：①真鲷2-4细胞期、原肠期、10-14体节期胚胎、心跳期和出膜前期胚胎孵化率>50%时渗透压的范围依次为：919~1391 mOsm/kg、919~1391 mOsm/kg、462 ~1391 mOsm/kg、232~1878 mOsm/kg和692~1391 mOsm/kg，表明心跳期胚胎对渗透压变化的耐受范围最广；②在不同浓度人工海水中分别浸泡10 min、30 min、1 h、5 h和10 h后，真鲷胚胎孵化率无显著变化的渗透压范围分别为0~2804 mOsm/kg、0~1878 mOsm/kg、232~1391 mOsm/kg、232~1391 mOsm/kg和919~1391 mOsm/kg；结果表明心跳期胚胎对渗透压的耐受范围随浸泡时间的延长而减小。 2.采用毛细管电泳技术检测胚胎内部DMSO的浓度，并且分析了胚胎孵化率和胚胎内部DMSO的浓度随浸泡时间变化与外部抗冻剂的关系。结果表明胚胎孵化率随胚胎外部抗冻剂溶液浓度和浸泡时间的增加而降低；胚胎内部DMSO浓度随胚胎外部抗冻剂溶液浓度和浸泡时间的增加而增加。对胚胎孵化率（y1）随抗冻剂溶液浓度（x）的变化进行一元三次多项式回归，当浸泡时间分别为10 min、30 min和60 min时，回归方程依次为：y1 = -2832.7x3 + 575.01x2 - 37.011x + 99.641（R2 = 0.9722）；y1 = 30288x3 - 16322x2 + 2077.3x + 27.603（R2 = 0.9876）；y1 = 16052x3 - 5985.2x2 - 32.696x + 119.6（R2 = 0.9124）。对胚胎内部DMSO浓度（y2）随抗冻剂溶液浓度（x）的变化进行回归，当浸泡时间分别为10 min、30 min和60 min时，回归方程依次为：y2 = 0.2584e6.7294x（R2 = 0.9876）；y2 = 0.2521e10.964x（R2 = 0.9644）；y2 = 0.4054e10.95x（R2 = 0.8954）。 3. 利用低温显微镜观察了不同降温速率（20、40、60、80、100、120℃/min）对胚胎内外冰晶形成温度的影响。胚胎外部冰晶形成温度（TEIF）随降温速率的增加显著下降，在降温速率大于80℃/min之后，TEIF随降温速率增加而降低的幅度减小；胚胎内部冰晶形成温度（TIIF）在降温速率小于80℃/min 时随降温速率的升高而降低，在降温速率大于80℃/min 时随降温速率的升高而升高；胚胎内外冰晶形成温度差值（TEIF - TIIF）在降温速率小于80℃/min时随降温速率的升高而增大，在降温速率大于80℃/min时随降温速率的升高而减小。 4. 在低温显微镜下观察了真鲷胚胎低温保存中有复活胚胎记录的保存方法在冷冻解冻过程中的冰晶形成过程，结果表明：①在冷冻过程中，玻璃化法冷冻的胚胎的内部冰晶形成温度（-53.70，-64.33℃）显著低于程序降温法（-17.51，-21.40℃）；而且在玻璃化法冷冻的胚胎内部冰晶形成温度高于外部冰晶后形成（-70.30℃），程序降温法中则相反，胚胎内部冰晶形成温度显著低于外部冰晶形成温度（-4.93，-5.00℃）；玻璃化法中，40%PG冷冻的胚胎外部溶液出现玻璃化现象，其他组均未出现；②在解冻过程中，各组均出现重结晶现象；解冻后，玻璃化法的胚胎完整率（62.82%）远高于程序降温法（9.21%）。

Impacts of ocean acidification in a warming Mediterranean Sea: An overview

Mediterranean Sea fisheries supply significant local and international markets, based largely on small pelagic fish, artisanal fisheries and aquaculture of finfish (mainly seabass and seabream) and shellfish (mussels and oysters). Fisheries and aquaculture contribute to the economy of countries bordering this sea and provide food and employment to coastal communities employing ca 600,000 people. Increasing temperatures and heat wave frequency are causing stress and mortality in marine organisms and ocean acidification is expected to worsen these effects, especially for bivalves and coralligenous systems. Recruitment and seed production present possible bottlenecks for shellfish aquaculture in the future since early life stages are vulnerable to acidification and warming. Although adult finfish seem able to withstand the projected increases in seawater CO2, degradation of seabed habitats and increases in harmful blooms of algae and jellyfish might adversely affect fish stocks. Ocean acidification should therefore be factored into fisheries and aquaculture management plans. Rising CO2 levels are expected to reduce coastal biodiversity, altering ecosystem functioning and possibly impacting tourism being the Mediterranean the world’s most visited region. We recommend that ocean acidification is monitored in key areas of the Mediterranean Sea, with regular assessments of the likely socio-economic impacts to build adaptive strategies for the Mediterranean countries concerned.