Macrozooplankton predation impact on anchovy (Engraulis encrasicolus) eggs mortality at the Bay of Biscay shelf break spawning centre

A real-time PCR based method involving a species-specific probe was applied to detect Engraulis encrasicolus eggs predation by the macrozooplank- ton community during the 2011 spawning season. Three locations along the shelf break presenting contrasting but high prey densities were sampled. A total of 840 individuals from 38 taxa of potential macrozooplankton predators were assayed for E. encrasicolus DNA presence and 27 presented at least one positive signal. Carnivorous copepods were responsible for the most predation events (66%) followed by euphausiids (16%), chaetognaths (5%), and myctophid fish (4%). Macrozooplankton predation on anchovy eggs followed a type-I functional response with daily mortalities ,4% of available prey abundance suggesting a negligible impact on the species recruitment at the shelf break spawning centre.This research was financially supported by the projects ECOGENBAY (MICINN CTM2009-13570-C02-02), funded by the Ministry of Science and Research of the Government of Spain, and BIOMAN, funded by the Department of Economic Development and Competitiveness of the Basque Government and by the European Commission

A real-time PCR assay to estimate invertebrate and fish predation on anchovy eggs in the Bay of Biscay

In order to investigate the role of predation on eggs and larvae in the recruitment of anchovy (Engraulis encrasicolus), sardine (Sardina pilchardus), sprat (Sprattus sprattus) and 52 macrozooplankton taxa were assayed for anchovy remains in the gut during the 2010 spawning season using a molecular method. This real-time PCR based assay was capable of detecting 0.005 ng of anchovy DNA (roughly 1/100 of a single egg assay) in a reliable way and allowed detecting predation events up to 6 h after ingestion by small zooplankton taxa. A total of 1069 macrozooplankton individuals, 237 sardines and 213 sprats were tested. Both fish species and 32 macrozooplankton taxa showed remains of anchovy DNA within their stomach contents. The two main findings are (1) that the previously neglected macrozooplankton impact in anchovy eggs/larvae mortality is in the same order of magnitude of that due to planktivorous fishes and that, (2) the predation pressure was notably different in the two main spawning centers of Bay of Biscay anchovy. While relatively low mortality rates were recorded at the shelf-break spawning center, a higher predation pressure from both fish and macrozooplankton was exerted at the shelf one.This research was financially supported by the projects ECOGENBAY (MICINN CTM2009-13570-C02-02), funded by the Ministry of Science and Research of the Government of Spain, and BIOMAN, funded by the Department of Economic Development and Competitiveness of the Basque Government and by the European Commissio

Unraveling the environmental and anthropogenic drivers of bacterial community changes in the Estuary of Bilbao and its tributaries

In this study, 16S rRNA gene sequencing was used to characterize the changes in taxonomic composition and environmental factors significantly influencing bacterial community structure across an annual cycle in the Estuary of Bilbao as well as its tributaries. In spite of this estuary being small and characterized by a short residence time, the environmental factors most highly correlated with the bacterial community mirrored those reported to govern larger estuaries, specifically salinity and temperature. Additionally, bacterial community changes in the estuary appeared to vary with precipitation. For example, an increase in freshwater bacteria (Comamonadaceae and Sphingobacteriaceae) was observed in high precipitation periods compared to the predominately marine-like bacteria (Rhodobacterales and Oceanospirillales) that were found in low precipitation periods. Notably, we observed a significantly higher relative abundance of Comamonadaceae than previously described in other estuaries. Furthermore, anthropic factors could have an impact on this particular estuary's bacterial community structure. For example, ecosystem changes related to the channelization of the estuary likely induced a low dissolved oxygen (DO) concentration, high temperature, and high chlorophyll concentration period in the inner euhaline water in summer (samples with salinity >30 ppt). Those samples were characterized by a high abundance of facultative anaerobes. For instance, OTUs classified as Cryomorphaceae and Candidatus Aquiluna rubra were negatively associated with DO concentration, while Oleiphilaceae was positively associated with DO concentration. Additionally, microorganisms related to biological treatment of wastewater (e.g Bdellovibrio and Zoogloea) were detected in the samples immediately downstream of the Bilbao Wastewater Treatment Plant (WWTP). There are several human activities planned in the region surrounding the Estuary of Bilbao (e.g. sediment draining, architectural changes, etc.) which will likely affect this ecosystem. Therefore, the addition of bacterial community profiling and diversity analysis into the estuary's ongoing monitoring program would provide a more comprehensive view of the ecological status of the Estuary of Bilbao.The authors received funding from Geonomic resources group (Grant IT558-10) to AE, and supported by SIEBM XVIII congress (Gijon). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Insights on the Origin of Invasive Copepods colonizing Basque Estuaries; a DNA Barcoding Approach

Background: The introduction of NIS to estuaries and coastal embayment is of great concern. Commercial ships’ ballast water discharge and the northwards progression of species due to the ongoing climate change arise as the main factors explaining the rising occurrence of NIS species in Northern Atlantic waters. In this regard, regular monitoring of the plankton communities is paramount as to be able to respond properly to this potential issue. Results: While monitoring the invasive copepod Acartia tonsa populations in the estuaries of Bilbao and Urdaibai (Basque country, Spain), we report here the Asian copepod Pseudodiaptomus marinus for the first time in the Iberian Peninsula waters. Individuals from both species were collected from July to October, 2013 for DNA sequencing of mitochondrial cytochrome c oxidase subunit I gene (MT-CO1). Phylogenetic analysis of MT-CO1 confirmed P. marinus identity. Conclusions: Phylogeographic distribution of A. tonsa haplotypes in Europe along with the Bilbao port traffic patterns suggested a secondary invasion from an European source to Basque estuaries. The successful establishment of the A. tonsa population and the appearance of Pseudodiaptomus marinus confirm the need for regular plankton monitoring of estuarine and port waters. This applies also to nearby systems as these populations could represent a source of future dispersal.DA’s work was supported by the ZabaldUz Program (PhD fellowship). SGIker technical and human support (UPV/EHU) is gratefully acknowledged. Thanks to T. Matellanes (Bilbao Port Authority) for providing maritime traffic data. Special thanks to the anonymous reviewers that greatly improved the different manuscript versions. The sampling program received funds from the University of the Basque Country (UFI11/37) and the Basque Government (GIC10/168)

Single nucleotide polymorphism discovery in albacore and Atlantic bluefin tuna provides insights into worldwide population structure.

The optimal management of the commercially important, but mostly over-exploited, pelagic tunas, albacore (Thunnus alalunga Bonn., 1788) and Atlantic bluefin tuna (BFT; Thunnus thynnus L., 1758), requires a better understanding of population structure than has been provided by previous molecular methods. Despite numerous studies of both species, their population structures remain controversial. This study reports the development of single nucleotide polymorphisms (SNPs) in albacore and BFT and the application of these SNPs to survey genetic variability across the geographic ranges of these tunas. A total of 616 SNPs were discovered in 35 albacore tuna by comparing sequences of 54 nuclear DNA fragments. A panel of 53 SNPs yielded FST values ranging from 0.0 to 0.050 between samples after genotyping 460 albacore collected throughout the distribution of this species. No significant heterogeneity was detected within oceans, but between-ocean comparisons (Atlantic, Pacific and Indian oceans along with Mediterranean Sea) were significant. Additionally, a 17-SNP panel was developed in Atlantic BFT by cross-species amplification in 107 fish. This limited number of SNPs discriminated between samples from the two major spawning areas of Atlantic BFT (FST = 0.116). The SNP markers developed in this study can be used to genotype large numbers of fish without the need for standardizing alleles among laboratories.This work was supported by ATM2010Hegaluze (351BI20090047), ATM2009Hegalabur (351BI20090034) and ATM2008Bonorte (ACM2008BONORTE) projects funded by the Basque Government, and the ACEITUNA (CTM2011-27505) project funded by the Spanish Ministerio de Economía y Competitividad

18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species

The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 9 150 bp) technology. To critically evaluate the method's performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.The sampling was financially supported by the projects ECOGENBAY (MICINN CTM2009-13570-C02-02), funded by the Government of Spain, and the project "Bay of Biscay anchovy biomass estimates for year 2010", funded by the Basque Government and by the European Commission within the frame of the National Sampling Programme (DCF No. 665/2008 14 July 2008). The General Secretariat of Sea also collaborated providing the R/V Emma Bardan. AA and DA work was supported by a contract with the Euskampus Foundation (Euskampus Fundazioa) and a doctoral fellowship of the Basque Government (UPV/EHU "ZabaldUz" program), respectively. This study was funded by a Basque Government grant to "Grupo de Investigacion consolidado del sistema universitario vasco, IT558-10"

Multiple SNP Markers Reveal Fine-Scale Population and Deep Phylogeographic Structure in European Anchovy (Engraulis encrasicolus L.)

10 p.Geographic surveys of allozymes, microsatellites, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have detected several genetic subdivisions among European anchovy populations. However, these studies have been limited in their power to detect some aspects of population structure by the use of a single or a few molecular markers, or by limited geographic sampling. We use a multi-marker approach, 47 nDNA and 15 mtDNA single nucleotide polymorphisms (SNPs), to analyze 626 European anchovies from the whole range of the species to resolve shallow and deep levels of population structure. Nuclear SNPs define 10 genetic entities within two larger genetically distinctive groups associated with oceanic variables and different life-history traits. MtDNA SNPs define two deep phylogroups that reflect ancient dispersals and colonizations. These markers define two ecological groups. One major group of Iberian-Atlantic populations is associated with upwelling areas on narrow continental shelves and includes populations spawning and overwintering in coastal areas. A second major group includes northern populations in the North East (NE) Atlantic (including the Bay of Biscay) and the Mediterranean and is associated with wide continental shelves with local larval retention currents. This group tends to spawn and overwinter in oceanic areas. These two groups encompass ten populations that differ from previously defined management stocks in the Alboran Sea, Iberian-Atlantic and Bay of Biscay regions. In addition, a new North Sea-English Channel stock is defined. SNPs indicate that some populations in the Bay of Biscay are genetically closer to North Western (NW) Mediterranean populations than to other populations in the NE Atlantic, likely due to colonizations of the Bay of Biscay and NW Mediterranean by migrants from a common ancestral population. Northern NE Atlantic populations were subsequently established by migrants from the Bay of Biscay. Populations along the Iberian-Atlantic coast appear to have been founded by secondary waves of migrants from a southern refuge.This work was supported by a research grant to IZ from the Education, Universities and Investigation Department of the Basque Government, by the European Commission (FACTS, FP7-KBBE-2009-3, grant agreement 244966) and two funded projects from the Ministry of Science and Innovation of Spain (RTA2006-00068-C02-02) and the Agriculture and Fisheries Department of the Basque Government (ECOANCHOA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript