Trying to resolve the taxonomic confusion of Paracalanus parvus species complex (Copepoda, Calanoida) in the Mediterranean and Black Seas through a combined analysis of morphology, molecular taxonomy and DNA metabarcoding

Paracalanus parvus is reported as the most abundant representative of the genus and one of the main components of the coastal zooplankton in the Mediterranean and Black Seas. However, the subtle taxonomic differences between P. parvus and the congeneric species P. indicus and P. quasimodo, in combination with the ample morphological variation found in Mediterranean specimens, render problematic the correct identification. A recent molecular study by Cornils and Held (2014) provided evidence of cryptic speciation in the P. parvus complex and indicated that P. parvus s.s. does not have global distribution, but may be restricted to the northeastern Atlantic. In order to clarify the taxonomic status and distribution of this species complex in the Mediterranean and Black Seas, a study was conducted on Paracalanus specimens collected from different locations across the aforementioned marine basins and sequenced for portion of the COI mitochondrial gene. An accurate taxonomic analysis was also carried out to correlate morphological characteristics with the molecular species' assignation. The phylogenetic analysis of the specimens together with the publicly available sequences of P. parvus complex revealed the presence of four molecular operational taxonomic units (MOTUs) in the Mediterranean, which differed in abundance and geographic distribution. The combination of morphological and molecular data revealed great inconsistencies between morphospecies and MOTUs. Moreover, several bulk zooplankton samples were analyzed through DNA metabarcoding in the frame of the “MetaCopepod” project to provide more extensive information on the spatiotemporal distribution and abundance of the target specie

The “MetaCopepod” project: Designing an integrated DNA metabarcoding and image analysis approach to study and monitor the diversity of zooplanktonic copepods and cladocerans in the Mediterranean Sea

The timely and accurate analysis of marine zooplankton diversity is a challenge in ecological and monitoring studies. Morphology-based identification of taxa, which requires taxonomy experts, is time consuming and cannot provide accurate resolution at species level in several cases (e.g. immature stages, cryptic species, broken specimens). The “MetaCopepod” project is aimed at overcoming these limitations by developing a high-throughput and cost effective methodology that integrates DNA metabarcoding and image analysis. Utilizing the accuracy of DNA metabarcoding in species recognition and the quantitative results of image analysis, zooplankton diversity (mainly of copepods and cladocerans) is assessed both qualitatively (species' composition) and quantitatively (abundance, biomass and size-distribution). To achieve this goal, bulk zooplankton samples are first scanned and analyzed with ZooImage and then massively sequenced for a selected fragment of the mitochondrial 16S rRNA gene. Through a bioinformatic pipeline, sequences are compared to a reference genetic database, constructed within the project, and identified at species- level. The methodology was calibrated by using both mock and taxonomically identified samples and demonstrated on samples collected monthly from monitoring stations across the Mediterranean Sea. It is currently optimized for higher integration and accuracy and is expected to become a powerful tool for monitoring zooplankton in the long term and for early warning of bioinvasions and other ecosystem change

Comparison of mesozooplankton faecal pellet characteristics from the Southern North Sea and the Mediterranean Sea during spring bloom conditions

peer reviewedMesozooplankton faecal pellets participate in the carbon and nutrient cycles, in the nutrition of marine organisms, and in the transport of toxins, pollutants, and sediments. The importance of this role depends on their nature and production, which are highly variable parameters that, in turn, depend on many factors. For example, a phytoplankton bloom may significantly change the nature of mesozooplankton faecal pellets because it affects their shape, content, density, and volume. The aim of the present study is to compare the eutrophic Southern North Sea and the oligotrophic Bay of Calvi in the Meditenanean Sea with respect to the seasonal variability of faecal pellet characteristics (shape, size, sinking speed, density) during bloom conditions

Impact of a Dinophysis acuminata Bloom on the Copepod Acartia clausi: First Indications

Faecal pellet production and content along with egg production of the dominant copepod species Acartia clausi were studied in the Thermaikos Gulf (NW Aegean Sea) during a pre-bloom and a bloom of the toxic dinoflagellate Dinophysis acuminata. Both faecal pellet production (6.8–8.6 ind−1 d−1) and egg production (15.8–47.6 ind−1 d−1) appeared unrelated to the D. acuminata bloom. Less than 11% of the copepod faecal pellets contained one or two D. acuminata cells, almost intact, whereas the other material in the pellets was broken into small pieces or amorphous shapes. The toxin outflux seemed to be insignificant when compared to the mean toxin concentration from the whole D. acuminata population. Finally, the potential grazing impact of A. clausi on D. acuminata during the study period was low

Faecal pellet production of copepoda collected in water depth up to 30 meters in the eastern Mediterranean Sea in Oktober 2008 during SES_GR2

Mesozooplankton is collected by vertical tows within the Black sea water body mass layer in the NE Aegean, using a WP-2 200 µm net equipped with a large non-filtering cod-end (10 l). Macrozooplankton organisms are removed using a 2000 µm net. A few unsorted animals (approximately 100) are placed inside several glass beaker of 250 ml filled with GF/F or 0.2 µm Nucleopore filtered seawater and with a 100 µm net placed 1 cm above the beaker bottom. Beakers are then placed in an incubator at natural light and maintaining the in situ temperature. After 1 hour pellets are separated from animals and placed in separated flasks and preserved with formalin. Pellets are counted and measured using an inverted microscope. Animals are scanned and counted using an image analysis system. Carbon- Specific faecal pellet production is calculated from a) faecal pellet production, b) individual carbon: Animals are scanned and their body area is measured using an image analysis system. Body volume is then calculated as an ellipsoid using the major and minor axis of an ellipse of same area as the body. Individual carbon is calculated from a carbon- total body volume of organisms (relationship obtained for the Mediterranean Sea by Alcaraz et al. (2003) divided by the total number of individuals scanned and c) faecal pellet carbon: Faecal pellet length and width is measured using an inverted microscope. Faecal pellet volume is calculated from length and width assuming cylindrical shape. Conversion of faecal pellet volume to carbon is done using values obtained in the Mediterranean from: a) faecal pellet density 1,29 g cm**3 (or pg µm**3) from Komar et al. (1981); b) faecal pellet DW/WW=0,23 from Elder and Fowler (1977) and c) faecal pellet C%DW=25,5 Marty et al. (1994)

Faecal pellet production of copepoda collected in water depth up to 100 meters in the eastern Mediterranean Sea in March and April 2008 during SES_GR1

The SES_UNLUATA_GR1-Mesozooplankton faecal pellet production rates dataset is based on samples taken during March and April 2008 in the Northern Libyan Sea, Southern Aegean Sea and in the North-Eastern Aegean Sea. Mesozooplankton is collected by vertical tows within the 0-100 m layer or within the Black sea water body mass layer in the case of the NE Aegean, using a WP-2 200 µm net equipped with a large non-filtering cod-end (10 l). Macrozooplankton organisms are removed using a 2000 µm net. A few unsorted animals (approximately 100) are placed inside several glass beaker of 250 ml filled with GF/F or 0.2 µm Nucleopore filtered seawater and with a 100 µm net placed 1 cm above the beaker bottom. Beakers are then placed in an incubator at natural light and maintaining the in situ temperature. After 1 hour pellets are separated from animals and placed in separated flasks and preserved with formalin. Pellets and are counted and measured using an inverted microscope. Animals are scanned and counted using an image analysis system. Carbon- Specific faecal pellet production is calculated from a) faecal pellet production, b) individual carbon: Animals are scanned and their body area is measured using an image analysis system. Body volume is then calculated as an ellipsoid using the major and minor axis of an ellipse of same area as the body. Individual carbon is calculated from a carbon- total body volume of organisms (relationship obtained for the Mediterranean Sea by Alcaraz et al. (2003) divided by the total number of individuals scanned and c) faecal pellet carbon: Faecal pellet length and width is measured using an inverted microscope. Faecal pellet volume is calculated from length and width assuming cylindrical shape. Conversion of faecal pellet volume to carbon is done using values obtained in the Mediterranean from: a) faecal pellet density 1,29 g cm**3 (or pg µm**3) from Komar et al. (1981); b) faecal pellet DW/WW=0,23 from Elder and Fowler (1977) and c) faecal pellet C%DW=25,5 Marty et al. (1994)

Faecal pellet production of copepoda collected in water depth up to 20 meters in the eastern Mediterranean Sea in March and April 2008 during SES_GR1

The SES_GR1-Mesozooplankton faecal pellet production rates dataset is based on samples taken during April 2008 in the North-Eastern Aegean Sea. Mesozooplankton is collected by vertical tows within the Black sea water body mass layer in the NE Aegean, using a WP-2 200 µm net equipped with a large non-filtering cod-end (10 l). Macrozooplankton organisms are removed using a 2000 µm net. A few unsorted animals (approximately 100) are placed inside several glass beaker of 250 ml filled with GF/F or 0.2 µm Nucleopore filtered seawater and with a 100 µm net placed 1 cm above the beaker bottom. Beakers are then placed in an incubator at natural light and maintaining the in situ temperature. After 1 hour pellets are separated from animals and placed in separated flasks and preserved with formalin. Pellets are counted and measured using an inverted microscope. Animals are scanned and counted using an image analysis system. Carbon- Specific faecal pellet production is calculated from a) faecal pellet production, b) individual carbon: Animals are scanned and their body area is measured using an image analysis system. Body volume is then calculated as an ellipsoid using the major and minor axis of an ellipse of same area as the body. Individual carbon is calculated from a carbon- total body volume of organisms (relationship obtained for the Mediterranean Sea by Alcaraz et al. (2003) divided by the total number of individuals scanned and c) faecal pellet carbon: Faecal pellet length and width is measured using an inverted microscope. Faecal pellet volume is calculated from length and width assuming cylindrical shape. Conversion of faecal pellet volume to carbon is done using values obtained in the Mediterranean from: a) faecal pellet density 1,29 g cm**3 (or pg µm**3) from Komar et al. (1981); b) faecal pellet DW/WW=0,23 from Elder and Fowler (1977) and c) faecal pellet C%DW=25,5 Marty et al. (1994)

Seawater carbonate chemistry and egg production, hatching and metabolic rates of a Mediterranean copepod species (Acartia clausi) in a laboratory experiment

This study includes the first information on the combined effect of low pH and raised temperature on egg production rate (EP), hatching success (HS), excretion and respiration of the Mediterranean copepod Acartia clausi. Adult individuals of A. clausi and fresh surface seawater were collected at a coastal station in Saronikos Gulf during April 2012. Four different conditions were applied: two different pH levels (present: 8.09 and future: 7.83) at two temperature values (present: 16°C and present+4 °C= 20°C). EP and HS success decreased significantly over the duration of exposure at future pH at both temperature conditions. However, the analysis of the combined effect of pH, T, chlorophyll a and the duration of the experiments on EP and HS revealed that ocean acidification had no discernible effect, whereas warming; food and the duration of exposure were more significant for the reproductive output of A. clausi. Temperature appeared to have a positive effect on respiration and excretion. Acidification had no clear effect on respiration, but a negative effect on the A. clausi excretion was observed. Acidification and warming resulted in the increase of the excretion rate and the increase was higher than that observed by warming only. Our findings showed that a direct effect of ocean acidification on copepod's vital rates was not obvious, except maybe in the case of excretion. Therefore, the combination of acidification with the ambient oligotrophic conditions and the warming could result in species being less able to allocate resources for coping with multiple stressors