Scientific Background

Spondyliosoma cantharus, Diplodus vulgaris and Diplodus sargus are important sparid species found in the NE Atlantic, from Great Britain to Senegal, including Madeira, Azores and Canaries Archipelagos, and also in the Mediterranean [1]. These coastal fish live on rocky and sandy substrates and in seagrass meadows, at depths ranging between 50 and 160 m [2]. Adults spawn offshore in deep waters, during winter and spring, with reproductive strategies varying between species [3,4,5]. During spring/summer, and after 1-2 months of pelagic life, the larvae are transported by oceanographic currents towards inshore shallow nursery areas, such as coastal lagoons, estuaries and artificial reefs, where they settle and remain for several months [6]. After reaching 4.5 to 5.5 cm in length, the juveniles disperse outside the nursery area to join the adult population [7,8,9,10]. Sparids are a valuable resource in the south of Portugal [11], being a target for gill nets and longlines used by artisanal fisheries [9]. Landings of sea breams in general and for most Sparidae species have shown a steady decline over the past ten years. However, no regulations are currently imposed on this fishery, other than minimum landing sizes and catch quotas [12]. Despite its fishery value and ecological importance, the existing studies regarding the population structure, fish movement patterns and connectivity are scarce in SW Portugal [13,14,15]. Recent studies show the existence of spatially distinct elemental tags in otoliths of D. vulgaris juveniles (age 0+) collected in several Portuguese estuaries [16]. However the attempt to assess the contribution of these nurseries to the adult recruitment failed, probably because it did not take into account some unidentified nursery areas [17]. Otolith signatures of young adults (age 2+) suggest that D. vulgaris captured on the Portuguese SW coast is resident in the regional fishing areas during the adult stage. However, it could not be conclusively determined whether or not recruitment to these grounds was derived from different spawning and/or nursery areas [14]. For the above-mentioned species, it is unclear, at present, whether young juveniles that recruit to different inshore nursery areas are derived from different spawning sources and which inshore nursery areas are important sources of recruits to the adult spawning populations and fishery grounds in different coastal regions [14, 15]. Although genetic techniques are useful for studying fish population structure, they are often inconclusive if gene flow is sufficient to maintain genetic homogeneity across populations that are widely separated and potentially characterised by different local recruitment, growth and mortality rates [19]. This may often be the case in meta-population complexes, where sub-populations may have similar genetics but for management purposes should be treated independently. Resolving meta-population structures is a problem in fisheries management because of the difficulty of quantifying connectivity rates or interdependency of replenishment among populations in different areas. Otolith chemistry and shape are non-genetic methods to investigate population structure for fish, since it provides one alternative solution for addressing such questions of connectivity and separation between spawning and juvenile sources and adult stocks [20, 21, 22, 23]. The purpose of this work will be to assess the utility of chemical signatures and shape analysis of otoliths to provide information on the population structure, movement patterns and connectivity of some sparid fish (Diplodus vulgaris, Diplodus sargus and Spondyliosoma cantharus) along the Portuguese southwest coast.

 

Objectives

 

Methodology

Biological Sampling: juveniles (age 0+) will be collected in spring 2016 using beach seine and beam trawl in the estuaries and coastal lagoons known as nurseries grounds for sea breams [8] (Mondego, Tejo, Sado, Mira, Ria Formosa, Alvor, Arade, Guadiana, Odiel and Guadalquivir). Additional samples obtained by fish traps from artificial reefs off Algarve will be included [10]. Adults (age 2+) will be collected in the SW fisheries grounds (Mira, Sesimbra, Sines, Sagres, Portimão, Olhão and Vila Real Santo António) [9] in spring 2018 through the artisanal fleets using trammel nets, gillnets and longlines. Information about the location of capture will be requested from fishermen. If available, some adults specimens from Madeira, Azores, Canaries and Mediteranean will be included as outgroups. Individuals from the same age (cohort) will be used. The ages will be later confirmed by absence of annual ring (0+) or by counting the annual growth increments (2+) [14,15]. A sub-sample of thirty individuals (0+ and 2+) will be selected from each nursery and fishery ground for further analyses. A few specimens from 2017 (juveniles or adults) will be used to assess the temporal homogeneity of the otolith (edge) signatures. Fish will be measured, weighed and frozen for later analyses. Otoliths will be carefully extracted, differentiated to left and right sagitta and stored. A portion of muscle will be extracted and preserved in ethanol for RNA/DNA analysis.

 

Age Determination: The annual growth increments will be counted using, preferentially, non-destructive methodologies. Otoliths will be immersed in a clearing mixture and annuli counted using a stereoscopic microscope under reflected light against a dark background at 25X magnification. If needed, otoliths will be mounted in epoxy resin and slight sections will be made using a low speed saw. The age will be estimated by counting the translucent increments, taking into consideration the assumed date of birth (1st January) and the date of capture. Three readers will age each otolith independently. Only fish with 100% concordance between age readings will be used [14,15].

 

Otolith Microchemistry: Otolith fingerprint analysis will focus on the core region of the otoliths (natal origin) from both young juveniles (0+: 2014) and adults (2+: 2016) using laser ablation inductively coupled plasma mass spectrometry (ICP-MS-LA). Sagittae will be cleaned of organic tissues, rinsed with Milli-Q water, air dried and mounted in epoxy. Transverse sections will be taken through the primordium with a diamond saw. Otolith sections will be grounded with silicon carbide papers and further polished with diamond pastes until the core became visible. The polished sections will be fixed on microscope slides with epoxy resin, sonicated in Milli-Q water, further rinsed and dried in a laminar flow cabinet. Data will be collected for a range of isotopes already known to be informative for these species (88Sr, 138Ba, 55Mn, 25Mg, 7Li, 65Cu, Pb208 and 66Zn) along with 43Ca which is used as the internal standard [14,15]. The laser spot diameter will be of 200 µm which corresponds to the otolith section in age 0+ juveniles [17]. Calibration will be achieved with the National Institute of Standards (NIST) and accuracy assessed using a standard reference otolith material (FEBS-1). The core isotopic composition (16O/18O and 13C/12C) of the otoliths will be also performed by Isotope Ratio Mass Spectrometry (IRMS) using a high-resolution computer-controlled micromill sampling device. Edge analysis (ICP-MS-LA) of a few juveniles or adults collected in two consecutive years will be also assessed (temporal stability of the fingerprint). Whole otolith analysis of adults will be made through ICP-MS-SB (solution based) and IRMS using standard protocols [14,15]. 

 

Otolith Shape Analysis: Otoliths will be photographed for shape analysis of the outline using fourier descriptors and shape indices (circularity, aspect ratio, roundness and solidity). Morphometric measurements and elliptical fourier coefficients will be obtained using ImageJ V.1.4.3 and Shape V.1.3 softwares, respectively. Data will be analysed multivariate statistical procedures after removing the possible confounding effect related with different fish sizes [21,22,23].

Fish Condition Indices: The use of general well being proxies for juveniles (0+), such as biochemical (RNA:DNA and TAG:ST) morphometric (Fulton’s Condition, K) and growth (Otolith Marginal Increment Width index, OG) will allow the habitat quality of the nurseries to be assessed. K will be calculated by the formula K=100 (W/L3) where W is the eviscerated body mass (mg) and L is the standard length (mm). RNA:DNA concentrations for each individual will be obtained using ethidium bromide through the fluorimetric method. ATG/ST (triacylglycerols:sterols) ratio will be quantified following an extraction of the lipids using standard protocols. OG is the mean otolith daily increments width during the last 10 days prior to capture [18]. 

 

Statistics: Data will be checked for normality and homogeneity of variances prior to statistical analysis. If needed, data will be transformed as appropriate to conform to distributional assumptions. Univariate and multivariate tests will be made in accordance with the specific objective of each study. One-Way ANOVA, followed by the Tukey pos-hoc test, if needed, will be used to discriminate mean differences against several groups (e.g. variables: µg element g-1 of calcium; Factor: location). MANOVA will be used to test for spatial and temporal differences in otolith elemental signatures. Locations and year will be treated as fixed factors. For the MANOVA, we will report the approximate F-ratio statistic for the most robust test of multivariate statistics (Pillai’s trace). Post-hoc multivariate pairwise comparisons between locations will be performed using the Hotelling T-square test. Linear or Quadratic Discriminant Function Analyses (LDFA or QDFA) will be use to visualize spatial differences and to examine the re-classification accuracy success of fishes to this original location. Cross validations will be performed by using jackknife (‘‘leave one out’’) procedures [19]. Additionally, Maximum Likelihood Analysis (MLA) of the juvenile core otolith chemistry data (natal baseline) will be used to determine the contributions of the nurseries areas to the adults stocks using the HISEA programme. For each cohort, the simulation mode with 1000 simulations will be used to estimate the variability of the estimator (i.e. baseline data). Bootstrapping (1000 re-samplings) of the baseline and mixed sample data will be used to estimate the mean and standard deviations of the proportions of adults originating from each nursery area.

 

Consortium and Supervision team

The 3-year PhD proposal has been designed to provide an integrated and multidisciplinary doctoral training across three leading European marine research institutes (CIIMAR, GMIT and UAlg). The three institutes will provide complementary expertise, equipment and facilities to the candidate. Furthermore partners have within national and European ongoing research projects that will support field trips, laboratorial and analytical costs. All the supervisors (Alberto Correia, Deirdre Brophy and Jorge Gonçalves from CIIMAR, GMIT and UAlg, respectively) are independent senior researchers holding a PhD in Biological Sciences. They worked in the last 10-15 years in Marine Biology and Fisheries Sciences with a broad experience in the use of otolith chemical signatures to study fish population structure, to track migratory patterns, to assess connectivity issues and to discriminate fish stock, including sea breams. They have very good track record in high rated journals, participate regularly in international conferences and have a large post-graduate teaching experience. All have/had several national and international funded projects in the Marine Sciences area, including as principal investigator.

 

References