A Morphometric Approach to Understand Prokaryoplankton: A Study in the Sicily Channel (Central Mediterranean Sea).

A new understanding of plankton ecology has been obtained by studying the phenotypic traits of free-living prokaryotes in the Sicily Channel (Central Mediterranean Sea), an area characterised by oligotrophic conditions. During three cruises carried out in July 2012, January 2013 and July 2013, the volume and morphology of prokaryotic cells were assessed microscopically using image analysis in relation to environmental conditions. The study found significant differences in cell morphologies among cruises. The largest cell volumes were observed in the July 2012 cruise (0.170 ± 0.156 µm3), and the smallest in the January 2013 cruise (0.060 ± 0.052 µm3). Cell volume was negatively limited by nutrients and positively by salinity. Seven cellular morphotypes were observed among which cocci, rods and coccobacilli were the most abundant. Cocci, although they prevailed numerically, always showed the smallest volumes. Elongated shapes were positively related to temperature. Relationships between cell morphologies and environmental drivers indicated a bottom-up control of the prokaryotic community. The morphology/morphometry-based approach is a useful tool for studying the prokaryotic community in microbial ecology and should be widely applied to marine microbial populations in nature.

New Evaluation of Postovulatory Follicle Degeneration at High-Temperature Regimes Refines Criteria for the Identification of Spawning Cohorts in the European Anchovy (Engraulis encrasicolus).

Accurate stock assessment estimates of fish resources are essential in fishery management. Wide fluctuations in abundance arising from variations in reproductive success are characteristic of many marine fish populations, including multiple spawner species. The proportion of females spawning per day is crucial in the application of egg production methods for spawning biomass evaluation and, usually, is assessed by postovulatory follicle (POF) method. Describing each degeneration stage of POF based upon its histological features allows for obtaining an aging key for postovulatory follicles. The commercially valuable European anchovy (Engraulis encrasicolus) was selected as a case study, which breeds during the summer in temperate waters (24 °C-25 °C). A collection of ovary slides, sampled in the central Mediterranean Sea during the spawning peak, provided a 24 h cycle coverage. These observations allowed us to evaluate the duration of each POF stage at water temperature experienced by anchovy in the study area. Present results demonstrated the POF degeneration progress at a faster rate than reported by previous investigations, carried out in cooler oceanic waters. Furthermore, the present study displayed the presence of two anchovy spawning cohorts sampled along a 24-h cycle. Therefore, this study not only provides useful insight for more accurate POF degeneration evaluation in temperate waters, but also suggests that current estimates should be complemented with validation studies according to different temperature regimes.

Growth-related trophic changes of Thunnus thynnus as evidenced by stable nitrogen isotopic values in the first dorsal spine.

The bluefin tuna, Thunnus thynnus, is a highly migratory and long-living fish at the top of the pelagic food web. As top predator, it plays a key role in the stability of marine food webs by exerting top-down control on its prey. The diet composition of bluefin tuna varies in relation to its growth, seasons and migratory patterns, making it difficult to evaluate spatial and temporal effects. This latter aspect is further complicated to be determined during the first months of life, when T. thynnus specimens have a rapid growth rate leading to changes in the trophic status. In this study, the potential collagen-related effects on δ15N and δ13C values were evaluated on the whole spine of adult tuna specimens collected in the central Mediterranean Sea. Obtained results showed non-significant differences between extracted and non-extracted collagen samples for δ15N in whole spine, allowing adopting the isotopic analysis both for annuli in the spine section of adults and for younger specimens, whose spine size does not permit the collagen extraction. Specifically, isotopic analysis of whole spine of the young of the year specimens, showed a rapid change in δ15N values with length, following an exponential model. For older specimens, δ15N values were higher and varied around a plateau, likely due to a higher specificity in the choice of prey and/or to change in the geographical location. Such variability was also mirrored in annuli of spines sections of adult tunas. As far as δ13C values are concerned, a strong collagen-related effect was evidenced, likely highlighting the influence of lipids. Consequently, δ13C analysis may be used only on adult specimens where collagen extraction is possible. This research also showed how isotopic analysis of both whole sample and sequence of annuli in the cross-section of dorsal spine might produce isotopic profiles useful to detect specific trophic dynamics along the bluefin tuna growth.

First annulus formation in the European anchovy; a two-stage approach for robust validation.

The age determination in fast-growing short-living species, such as European anchovy (Engraulis encrasicolus), has been widely recognized as a difficult task and bias introduced by readers leads to bias in reconstructing the population age structure. In this context, it is worth to note that age structure of fish population represents key information in fishery ecology and for stock assessment models. The uncertainty in estimating the age of the European anchovy by otolith reading is linked to the number of false-growth increments (checks) laid down before the annulus formation. While direct validation methods (e.g. mark-recapture, rearing, radiochemical dating) are difficult to implement specially for this short living species, the use of different indirect methods, supported by a coherent statistical approach, represents a robust and easier validation tool. A statistical modeling approach has been here adopted to assess the coherence of two well-known methods, namely Edge Analysis and Marginal Increment Analysis, in order to validate the first annulus formation in European anchovy. Both methodologies in two different yearly cycles converged toward the same result, thus confirming the annulus identification for the first year class. In addition, the completion dates of the checks and the first annulus were computed in order to gain a better insight into otolith growth dynamic. According to the species spawning period, the completion date of the first annulus falls in the summer period, while the first and second checks completion dates were mostly found in summer and winter respectively. General additive models using marginal increments as dependent variable showed a significant effect of the month, highlighting the presence of only one clear minimum in July/August, as well as specific relationships with condition factor and gonadosomatic index. Modeling the otolith edge morphology, the probability to find a hyaline band displayed in both years a similar shape, characterized by a minimum in July/August and higher values between November and January. The obtained results evidenced temporally coherent patterns providing a better insight in the otolith growth dynamic as well as a more robust validation of the first annulus formation in the European anchovy.

Condition of pteropod shells near a volcanic CO2 vent region.

Natural gradients of pH in the ocean are useful analogues for studying the projected impacts of Ocean Acidification (OA) on marine ecosystems. Here we document the in situ impact of submarine CO2 volcanic emissions (CO2 vents) on live shelled-pteropods (planktonic gastropods) species Creseis conica in the Gulf of Naples (Tyrrhenian Sea, Mediterranean). Since the currents inside the Gulf will likely drive those pelagic calcifying organisms into and out of the CO2 vent zones, we assume that pteropods will be occasionally exposed to the vents during their life cycle. Shell degradation and biomass were investigated in the stations located within and nearby the CO2 vent emission in relation to the variability of sea water carbonate chemistry. A relative decrease in shell biomass (22%), increase in incidence of shell fractures (38%) and extent of dissolution were observed in Creseis conica collected in the Gulf of Naples compared to those from the Northern Tyrrhenian Sea (control stations). These results suggest that discontinuous but recurrent exposure to highly variable carbonate chemistry could consistently affect the characteristic of the pteropod shells.

Micro-anatomical structure of the first spine of the dorsal fin of Atlantic bluefin tuna, Thunnus thynnus (Osteichthyes: Scombridae).

The first spine of the first dorsal fin (FS) of the Atlantic bluefin tuna (ABFT), Thunnus thynnus, is customarily used in age determination research because its transverse sections display well-defined growth marks. In this paper the FS structure was studied to explain its known dramatic age- and season-related morphological modifications, which are evidently caused by bone remodeling. Cross sections of samples from six adult ABFT were in part decalcified to be stained with histological, histochemical and immunohistochemical methods, and in part embedded in methyl-methacrylate to be either observed under a linear polarized light or microradiographed. FS showed an external compact bone zone and an inner trabecular bone zone. The compact bone zone consisted of an outer non-osteonic primary bone layer (C1) and an inner osteonic bone layer (C2). C1 was in turn characterized by alternate translucent and opaque bands. Evidence of spine bone remodeling was shown by the presence of osteoclasts and osteoblasts as well as by tartrate-resistant acid phosphatase (TRAP) positive bands at the boundary between old and newly formed bone. The examination of plain, i.e. not-fixed and not-decalcified, FS from 28 ABFT showed that the average thickness of C1 remained fairly constant during fish growth, whereas C2 increased significantly, indicating that the periosteal primary bone apposition is counterbalanced by the parallel bone remodeling occurring inside the compact bone zone. The present study revealed the structure of the ABFT FS and the pattern of its bone remodeling. Both of them underlay phenomena, never examined in detail before, such as the appearance followed by the progressive disappearance of growth bands.

Liver melanomacrophage centres and CYP1A expression as response biomarkers to environmental pollution in European anchovy (Engraulis encrasicolus) from the western Mediterranean Sea.

The goal of the present study was to verify the suitability of using melanomacrophage centres (MMCs) as response biomarkers of marine pollution in European anchovy, which are short-lived, migratory, small pelagic fish. This suitability was verified by analysing the MMC density and cytochrome P450 monooxygenase 1A (CYP1A) expression in livers of anchovies from four areas of southern Italy. Age 2 anchovies sampled from three areas exposed to pollutants of industrial/agricultural origin (Gulf of Gela, Mazara del Vallo and Gulf of Naples) showed liver areas occupied by MMCs and numbers of MMCs that were significantly higher than those in the anchovies from Pozzallo, which is a marine area not subjected to any source of pollution. Anti-CYP1A immunoreactivity was observed in the hepatocytes of all specimens sampled from the Gulf of Gela. These findings suggest the utility of liver MMCs as biomarkers of exposure to pollutants in this small pelagic fish.

Spatial dynamics and mixing of bluefin tuna in the Atlantic Ocean and Mediterranean Sea revealed using next-generation sequencing.

The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated with shared fisheries management. In an effort to resolve the species' stock dynamics, a genomewide search for spatially informative single nucleotide polymorphisms (SNPs) was undertaken, by way of sequencing reduced representation libraries. An allele frequency approach to SNP discovery was used, combining the data of 555 larvae and young-of-the-year (LYOY) into pools representing major geographical areas and mapping against a newly assembled genomic reference. From a set of 184,895 candidate loci, 384 were selected for validation using 167 LYOY. A highly discriminatory genotyping panel of 95 SNPs was ultimately developed by selecting loci with the most pronounced differences between western Atlantic and Mediterranean Sea LYOY. The panel was evaluated by genotyping a different set of LYOY (n = 326), and from these, 77.8% and 82.1% were correctly assigned to western Atlantic and Mediterranean Sea origins, respectively. The panel revealed temporally persistent differentiation among LYOY from the western Atlantic and Mediterranean Sea (FST  = 0.008, p = .034). The composition of six mixed feeding aggregations in the Atlantic Ocean and Mediterranean Sea was characterized using genotypes from medium (n = 184) and large (n = 48) adults, applying population assignment and mixture analyses. The results provide evidence of persistent population structuring across broad geographic areas and extensive mixing in the Atlantic Ocean, particularly in the mid-Atlantic Bight and Gulf of St. Lawrence. The genomic reference and genotyping tools presented here constitute novel resources useful for future research and conservation efforts.

Spatio-temporal dynamics of a planktonic system and chlorophyll distribution in a 2D spatial domain: matching model and data.

Field data on chlorophyll distribution are investigated in a two-dimensional spatial domain of the Mediterranean Sea by using for phytoplankton abundances an advection-diffusion-reaction model, which includes real values for physical and biological variables. The study exploits indeed hydrological and nutrients data acquired in situ, and includes intraspecific competition for limiting factors, i.e. light intensity and phosphate concentration. As a result, the model allows to analyze how both the velocity field of marine currents and the two components of turbulent diffusivity affect the spatial distributions of phytoplankton abundances in the Modified Atlantic Water, the upper layer of the water column of the Mediterranean Sea. Specifically, the spatio-temporal dynamics of four phytoplankton populations, responsible for about 80% of the total chlorophyll a, are reproduced. Results for phytoplankton abundances obtained by the model are converted in chlorophyll a concentrations and compared with field data collected in twelve marine sites along the Cape Passero (Sicily)- Misurata (Libya) transect. Statistical checks indicate a good agreement between theoretical and experimental distributions of chlorophyll concentration. The study can be extended to predict the spatio-temporal behaviour of the primary production, and to prevent the consequent decline of some fish species in the Mediterranean Sea.

Comparative Study of Reproductive Development in Wild and Captive-Reared Greater Amberjack Seriola dumerili (Risso, 1810).

The greater amberjack Seriola dumerili is a large teleost fish with rapid growth and excellent flesh quality, whose domestication represents an ambitious challenge for aquaculture. The occurrence of reproductive dysfunctions in greater amberjack reared in captivity was investigated by comparing reproductive development of wild and captive-reared individuals. Wild and captive-reared breeders were sampled in the Mediterranean Sea during three different phases of the reproductive cycle: early gametogenesis (EARLY, late April-early May), advanced gametogenesis (ADVANCED, late May-early June) and spawning (SPAWNING, late June-July). Fish reproductive state was evaluated using the gonado-somatic index (GSI), histological analysis of the gonads and determination of sex steroid levels in the plasma, and correlated with leptin expression in the liver and gonad biochemical composition. The GSI and sex steroid levels were lower in captive-reared than in wild fish. During the ADVANCED period, when the wild greater amberjack breeders were already in spawning condition, ovaries of captive-reared breeders showed extensive atresia of late vitellogenic oocytes and spermatogenic activity ceased in the testes of half of the examined males. During the SPAWNING period, all captive-reared fish had regressed gonads, while wild breeders still displayed reproductive activity. Liver leptin expression and gonad proximate composition of wild and captive greater amberjack were similar. However, the gonads of captive-reared fish showed different total polar lipid contents, as well as specific lipid classes and fatty acid profiles with respect to wild individuals. This study underlines the need for an improvement in rearing technology for this species, which should include minimum handling during the reproductive season and the formulation of a specific diet to overcome the observed gonadal decrements of phospholipids, DHA (22:6n-3) and ARA (20:4n-6), compared to wild breeders.

Insights on the drivers of genetic divergence in the European anchovy.

Anchovies represent the largest world's marine fish catches and the current threats on their populations impose a sustainable exploitment based on sound scientific information. In the European anchovy (Engraulis encrasicolus), the existence of several populations has been proposed but a global view is missing. Using a multidisciplinary approach, here we assessed the divergence among different ecotypes and its possible causes. SNPs have revealed two functionally distinct ecotypes overlapping in the Central Mediterranean, with one ecotype confined near the river estuaries. The same SNPs outliers also segregated two distinct populations in the near Atlantic, despite their large spatial distance. In addition, while most studies suggested that adaptation to low salinity is key to divergence, here we show that the offshore ecotype has higher environmental tolerance and an opportunistic feeding behaviour, as assessed by the study of environmental conditions, anchovy diet and trophic levels, and passive egg dispersal. These results provide insights into the anchovy evolutionary history, stressing the importance of behaviour in shaping ecotypes.

Yolked Oocyte Dynamics Support Agreement between Determinate- and Indeterminate-Method Estimates of Annual Fecundity for a Northeastern United States Population of American Shad.

Reports of American shad fecundity identify two important themes regarding egg production in fishes. First, geographic variation occurs and is biologically meaningful. Shad annual fecundity decreases with increasing latitude, but predicted lifetime fecundity does not, because of a counter-gradient of survival probability, all of which can explain the adaptive significance of natal homing. Second, the appropriate method of measuring fecundity depends on the pattern of oocyte development. Historically, the relatively simple determinate-fecundity method was used; however, a recent study in a Virginia river indicates that this method may be biased, requiring the more complicated indeterminate method. We address both themes with collections from the 2015 shad spawning run in the Connecticut River, USA. Criteria for using a determinate method were satisfied for this northern population: 1) a size gap evident in the oocyte size frequency distribution, indicating group-synchronous development of yolked oocytes; 2) a decline, early in spawning, in the standing stock of yolked oocytes; and 3) low levels of atresia at the end of spawning. The determinate-method estimate of American shad annual (2015) fecundity (303,000 ± 73,400; mean ± sd) overlapped historic estimates for this and a neighboring river. The indeterminate-method estimate of annual (2015) fecundity (311,500 ± 4,500 sd) was not significantly different from the determinate-method estimate (Student's t-test, P > 0.05). In contrast, indeterminate-method estimates of annual fecundity for a Virginia population were twice as high as that measured by the determinate method in the past. This can all be explained by fundamentally different patterns of oogenesis (i.e., group synchrony versus asynchrony with respect to yolk development) at different latitudes. American shad, which is distributed within its native range from the Canadian maritimes to Florida, USA (50-30°N), may be particularly well suited to evaluate intra-specific variation in oocyte development, a relatively unexplored life history trait.

Molecular Identification of Atlantic Bluefin Tuna (Thunnus thynnus, Scombridae) Larvae and Development of a DNA Character-Based Identification Key for Mediterranean Scombrids.

The Atlantic bluefin tuna, Thunnus thynnus, is a commercially important species that has been severely over-exploited in the recent past. Although the eastern Atlantic and Mediterranean stock is now showing signs of recovery, its current status remains very uncertain and as a consequence their recovery is dependent upon severe management informed by rigorous scientific research. Monitoring of early life history stages can inform decision makers about the health of the species based upon recruitment and survival rates. Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions. Herein we used a molecular approach employing mitochondrial and nuclear genes (CO1 and ITS1, respectively) to identify larvae (n = 188) collected from three spawning areas in the Mediterranean Sea by different institutions working with a regional fisheries management organization. Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key) and an extensive comparison of the results is presented. During this process various inaccuracies in related publications and online databases were uncovered. Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. While less than half of larvae provided were bluefin tuna, other dominant taxa were bullet tuna (Auxis rochei), albacore (Thunnus alalunga) and little tunny (Euthynnus alletteratus). We advocate an expansion of expertise for a new generation of morphology-based taxonomists, increased dialogue between morphology-based and molecular taxonomists and increased scrutiny of public sequence databases.

Interannual Changes in Biomass Affect the Spatial Aggregations of Anchovy and Sardine as Evidenced by Geostatistical and Spatial Indicators.

Geostatistical techniques were applied and a series of spatial indicators were calculated (occupation, aggregation, location, dispersion, spatial autocorrelation and overlap) to characterize the spatial distributions of European anchovy and sardine during summer. Two ecosystems were compared for this purpose, both located in the Mediterranean Sea: the Strait of Sicily (upwelling area) and the North Aegean Sea (continental shelf area, influenced by freshwater). Although the biomass of anchovy and sardine presented high interannual variability in both areas, the location of the centres of gravity and the main spatial patches of their populations were very similar between years. The size of the patches representing the dominant part of the abundance (80%) was mostly ecosystem- and species-specific. Occupation (area of presence) appears to be shaped by the extent of suitable habitats in each ecosystem whereas aggregation patterns (how the populations are distributed within the area of presence) were species-specific and related to levels of population biomass. In the upwelling area, both species showed consistently higher occupation values compared to the continental shelf area. Certain characteristics of the spatial distribution of sardine (e.g. spreading area, overlapping with anchovy) differed substantially between the two ecosystems. Principal component analysis of geostatistical and spatial indicators revealed that biomass was significantly related to a suite of, rather than single, spatial indicators. At the spatial scale of our study, strong correlations emerged between biomass and the first principal component axis with highly positive loadings for occupation, aggregation and patchiness, independently of species and ecosystem. Overlapping between anchovy and sardine increased with the increase of sardine biomass but decreased with the increase of anchovy. This contrasting pattern was attributed to the location of the respective major patches combined with the specific occupation patterns of the two species. The potential use of spatial indices as auxiliary stock monitoring indicators is discussed.

Habitat selection response of small pelagic fish in different environments. Two examples from the oligotrophic Mediterranean Sea.

A number of scientific papers in the last few years singled out the influence of environmental conditions on the spatial distribution of fish species, highlighting the need for the fisheries scientific community to investigate, besides biomass estimates, also the habitat selection of commercially important fish species. The Mediterranean Sea, although generally oligotrophic, is characterized by high habitat variability and represents an ideal study area to investigate the adaptive behavior of small pelagics under different environmental conditions. In this study the habitat selection of European anchovy Engraulis encrasicolus and European sardine Sardina pilchardus is analyzed in two areas of the Mediterranean Sea that largely differentiate in terms of environmental regimes: the Strait of Sicily and the North Aegean Sea. A number of environmental parameters were used to investigate factors influencing anchovy and sardine habitat selection. Acoustic surveys data, collected during the summer period 2002-2010, were used for this purpose. The quotient analysis was used to identify the association between high density values and environmental variables; it was applied to the entire dataset in each area in order to identify similarities or differences in the "mean" spatial behavioral pattern for each species. Principal component analysis was applied to selected environmental variables in order to identify those environmental regimes which drive each of the two ecosystems. The analysis revealed the effect of food availability along with bottom depth selection on the spatial distribution of both species. Furthermore PCA results highlighted that observed selectivity for shallower waters is mainly associated to specific environmental processes that locally increase productivity. The common trends in habitat selection of the two species, as observed in the two regions although they present marked differences in hydrodynamics, seem to be driven by the oligotrophic character of the study areas, highlighting the role of areas where the local environmental regimes meet 'the ocean triad hypothesis'.

Dynamics of two picophytoplankton groups in mediterranean sea: analysis of the deep chlorophyll maximum by a stochastic advection-reaction-diffusion model.

A stochastic advection-reaction-diffusion model with terms of multiplicative white Gaussian noise, valid for weakly mixed waters, is studied to obtain the vertical stationary spatial distributions of two groups of picophytoplankton, i.e., picoeukaryotes and Prochlorococcus, which account about for 60% of total chlorophyll on average in Mediterranean Sea. By numerically solving the equations of the model, we analyze the one-dimensional spatio-temporal dynamics of the total picophytoplankton biomass and nutrient concentration along the water column at different depths. In particular, we integrate the equations over a time interval long enough, obtaining the steady spatial distributions for the cell concentrations of the two picophytoplankton groups. The results are converted into chlorophyll a and divinil chlorophyll a concentrations and compared with experimental data collected in two different sites of the Sicily Channel (southern Mediterranean Sea). The comparison shows that real distributions are well reproduced by theoretical profiles. Specifically, position, shape and magnitude of the theoretical deep chlorophyll maximum exhibit a good agreement with the experimental values.