Factors driving the seasonal dynamics of Pseudo-nitzschia species and domoic acid at mussel farming in the SW Mediterranean Sea

ABSTRACT

The seasonal variations in Pseudo-nitzschia species and domoic acid (DA) concentration were investigated, at three shellfish farms in SW coastal Mediterranean. In parallel, the toxicity of mussels was tested. Two distinct groups of species were enumerated according to morphology and size (Pseudo-nizschia delicatissima and P. seriata groups). DA was detected over a nine-week period from July to October 2012 in the Lagoon, with a maximum concentration recorded in July (12.71?ng?DA?L^?1). DA was positively correlated with the presence of P. seriata-group and P. delicatissima-group and was mostly occurred during P limitation period in seawater. No DA was found in mussels that were collected during the period of DA absence in seawater. Our results suggest that temperature, salinity, inorganic and organic nutrients were significant for the seasonal dynamics of P. seriata and P. delicatissima groups, but that the P limitation was the most driving factor for DA production in these areas. The relative influence of environmental factors should be further studied to better understand the recent surfacing of massive blooms of toxigenic Pseudo-nitzschia in SW Mediterranean coast.     

Domoic acid in benthic flatfish on the continental shelf of Monterey Bay,California, USA

Within Monterey Bay, California, USA, the food web transfer of domoic acid (DA), a neurotoxin produced by diatoms of the genus Pseudo-nitzschia, has led to major mortality events of marine mammals and birds. Less visible, and less well known, is whether invertebrates and fish associated with the benthos are also affected by blooms of DA-producing Pseudo-nitzschia spp. This study examines the presence of DA in benthic flatfish offshore of Davenport, California, (37°0′36″N, 122°13′12″W) and within Monterey Bay, California (36°45′0″N, 122°1′48″W), including species that feed primarily in the sediment (benthic-feeding) and species that feed primarily in the water column (benthopelagic-feeding). Flatfish caught between 10 December 2002 and 17 November 2003 at depths of 30–180 m had concentrations of DA in the viscera ranging from 3 to 26 μg DA g⁻¹ of viscera. Although the DA values reported are relatively low, benthic-feeding flatfish were frequently contaminated with DA, especially as compared with the frequency of contamination of flatfish species that feed in the water column. Furthermore, on days in which both benthic-feeding and benthopelagic-feeding flatfish were collected, the former had significantly higher concentrations of DA in the viscera. Curlfin turbot, Pleuronicthys decurrens, the flatfish with both the highest level and frequency of DA contamination, are reported to feed exclusively on polychaetes, suggesting that these invertebrates may be an important vector of the toxin in benthic communities and may pose a risk to other benthic-feeding organisms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

环境DNA(eDNA)宏条形码技术对枝角类浮游动物物种鉴定及其生物量监测研究

环境DNA(eDNA)宏条形码(Metabarcoding)技术越来越多地被应用于环境中物种定性识别,但如何定量监测物种在环境中的丰度尚未得到解决。本研究以太湖流域常见的5种浮游动物拟同形溞、大型溞、蚤状溞、多刺裸腹溞、老年低额溞为研究对象,建立了一种基于eDNA宏条形码技术的物种定量方法,并与实时荧光定量PCR(qPCR)相比较,研究了eDNA宏条形码技术多物种定量的准确性。结果表明,PCR引物对eDNA宏条形码的物种检测和定量影响显著。313 bp COI313引物对浮游动物物种覆盖度高,但是物种间DNA扩增的偏好性大,不适用于eDNA宏条形码定量检测。基于COI序列重新设计的短COI116引物能够同时检测出所有5个物种。荧光定量PCR(qPCR)物种拷贝数与物种相对占比呈正相关。eDNA宏条形码所检出每个物种的序列数与qPCR定量拷贝数高度一致。综上,eDNA宏条形码技术可实现对浮游动物物种的半定量检测,在生物多样性监测和生物完整性评价有显著的应用价值。     

Measuring copepod naupliar abundance in a subtropical bay using quantitative PCR

Copepod nauplii are important in plankton food web dynamics, but limited information is available about their ecology due to methodological challenges. Reported here is a new molecular method that was developed, optimized, and tested in laboratory and field samples that uses quantitative PCR (qPCR) to identify and estimate the abundance of nauplii of the planktonic copepod, Parvocalanus crassirostris. The overall approach included collection of bulk zooplankton samples in the field, size fractionation to create artificial cohorts of relatively few developmental stages, obtaining DNA copy number for each size fraction by qPCR amplification of a target gene region, and estimation of the number of animals in each fraction through application of known DNA copy number across developmental stage. Method validation studies found that our qPCR-based approach has comparable accuracy to microscope-based counts of early developmental stages. Naupliar abundance estimates obtained using the two methods on cultured populations were similar; the regression of qPCR estimates on microscope-based counts resulted in a nearly 1:1 ratio (slope = 1.09). The qPCR-based method is superior to traditional identification and quantification methods for nauplii due to its higher taxonomic resolution, sensitive detection over a range of DNA quantities, and relatively high throughput sample processing.     

Fate of domoic acid ingested by the copepod <Emphasis Type="Italic">Acartia clausi</Emphasis>

Two important issues in the studies of harmful algae include ecological role of the toxic compounds and their fate through the food web. The aims of this study were to determine whether the production of domoic acid is a strategy evolved to avoid predation and the role of copepods in the fate of this toxic compound through the food web. The copepod Acartia clausi was fed with single and mixed cultures of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia delicatissima. Ingestion rate as a function of diatom abundance was the same for the toxic and non-toxic Pseudo-nitzschia species, indicating no selective feeding behaviour against P. multiseries. The toxins ingested by the copepods did not affect mortality, feeding behaviour, egg production and egg hatching of the copepods. Copepods assimilated the 4.8% of the total domoic acid ingested. Although the amount of toxins daily detoxificated by the copepods was 63.6%, the copepods accumulated domoic acid in their tissues. We conclude that domoic acid is not toxic for copepods and, probably for this reason, this toxin does not act as feeding deterrent for copepods. However, even though the production of domoic acid has apparently not evolved to deter predation, copepods may play an important role on the fate of this toxic compound through the marine food web.     

Simple molecular method to distinguish the identity of Calanus species (Copepoda: Calanoida) at any developmental stage

Diagnostic morphological characteristics of copepods of the genus Calanus are restricted largely to minor variations in secondary sex characteristics. This presents a persistent problem in the identification of individuals to species level, especially for immature stages. We have developed a simple molecular technique to distinguish between the North Atlantic Calanus species (C. helgolandicus, C. finmarchicus, C. glacialis and C. hyperboreus) at any life stage. Using the polymerase chain-reaction (PCR), the mitochodrial large subunit (16S) ribosomal RNA (rRNA) gene was amplified from individual copepods preserved in ethanol. Subsequent digestion of the amplified products with the restriction enzymes DdeI and VspI, followed by electrophoretic separation in 2% agarose (Metaphor, FMC Ltd), produced a characteristic pattern for each species. The versatility of the method is demonstrated by the unambiguous identification to species of any life stage, from egg to adult, and of individual body parts. Received: 11 May 1998 / Accepted: 5 August 1998     

Optical–digital system applied to the identification of five phytoplankton species

An optical–digital system was used to identify five phytoplankton species of the genus Ceratium from Todos Santos Bay off the California Peninsula, Mexico. The system recognized species with at least 90% accuracy, regardless of their size, rotation, or location within the field of view. An optical filter was constructed for each species for correlation with each of six test images. The test images were prepared from phytoplankton samples containing one or more of the five species. The correlations demostrate that each optical filter recognized its corresponding species. To evaluate the effectiveness of the correlation technique, we used images of 100 different samples. The species of the Ceratium genus analyzed are important indicators, since some are responsible for red-tide blooms in areas surrounding Todos Santos Bay, Baja California, Mexico. In the near future, based on the method proposed, we plan to implement an automated plankton-identification system to determine marine eco-biological properties. Received: 9 June 1997 / Accepted: 3 June 1998     

Chemical composition of the slippery epicuticular wax blooms on Macaranga (Euphorbiaceae) ant-plants

Summary. The stems of many Macaranga ant-plants (Euphorbiaceae) are covered by epicuticular wax crystals rendering the surface very slippery for most insects. These wax blooms act as selective barriers protecting the symbiotic ant partners, which are specialized “wax-runners”, against the competition of other ants. Glaucous stems occur almost exclusively among the ant-plants of the genus Macaranga (). We analyzed the cuticular lipids of 16 Macaranga species by GC-MS and investigated the wax crystal morphology using SEM. Presence of crystalline wax blooms was strongly correlated with high concentrations (52%–88%) of triterpenoids. In contrast epicuticular waxes of glossy Macaranga surfaces contained only 0% to 36% of these dominant components. Therefore we conclude that triterpenoids are responsible for the formation of the thread-like Macaranga wax crystals. In all Macaranga ant-plants investigated, the principal components were epitaraxerol and taraxerone accompanied by smaller portions of taraxerol, β-amyrin and friedelin. Only in the case of the non-myrmecophytic M. tanarius did β-amyrin predominate. Moreover, we found that only in M. tanarius, the dense wax crystal lacework is torn into large mosaic-like pieces in the course of secondary stem diameter growth. Both chemical and macroscopic differences may contribute to a reduced slipperiness of M. tanarius stems and appear to be functionally important. The distribution of wax crystals and their composition amongst different sections of the genus suggests that glaucousness is a polyphyletic character within Macaranga. Received 7 October 1999; accepted 3 December 1999     

<Emphasis Type="Italic">Cochlodinium polykrikoides</Emphasis> blooms and clonal isolates from the northwest Atlantic coast cause rapid mortality in larvae of multiple bivalve species

Globally, many commercial bivalve populations have declined in recent decades. In addition to overharvesting and habitat loss, the increasing frequency and intensity of harmful algal blooms (HABs) are likely to contribute to bivalve losses, particularly in cases where blooms negatively impact larval stages. This paper reports on the lethal effects of clonal cultures and blooms of Cochlodinium polykrikoides from the US Atlantic coast on the larvae of three species of commercially and ecologically valuable bivalves: the Eastern oyster (Crassostrea virginica), the bay scallop (Argopecten irradians), and the Northern quahog (hard clam; Mercenaria mercenaria). Both cultures and blooms of C. polykrikoides were highly toxic to all three species of bivalve larvae causing 80–100% mortality during 24- to 72-h exposures at concentrations of 1–2 × 10³ cells ml⁻¹. Toxicity was dependent on cell densities, growth stage of C. polykrikoides (i.e. cultures in exponential stage growth were more toxic than later stages), exposure time of larvae to cells (i.e. longer exposure caused higher mortality), the age of larvae (i.e. younger larvae were more sensitive), and the relative abundance of C. polykrikoides (i.e. the presence of other microalgae decreased toxicity). Free radical-scavenging enzymes (peroxidase and catalase) and the removal of C. polykrikoides cells (i.e. culture filtrate) significantly increased larval survival suggesting toxicity is maximized by contact with live cells and may involve labile toxins bound by these compounds including e.g. reactive oxygen species. The toxicity of C. polykrikoides to bivalve larvae was generally more severe than other HAB species (e.g. Karenia brevis, Karlodinium veneficum, Alexandrium tamarense, Prorocentrum minimum). Since the bivalves in this study spawn in the months when C. polykrikoides blooms on the east coast of North America, these results suggest that these blooms may have detrimental effects on efforts to restore these already diminished populations.     

Morphology and development of benthic and pelagic life stages of North Sea jellyfish (Scyphozoa, Cnidaria) with special emphasis on the identification of ephyra stages

Jellyfish blooms or invasions could be detected in an early phase of development if the youngest medusa stages (ephyrae) and their early growth stages (post-ephyrae) were identifiable in plankton samples but a useful identification key for ephyrae in early growth stages is lacking for most species. In the present study, the identification characteristics of adult North Sea scyphomedusae (Aurelia aurita, Cyanea capillata, Cyanea lamarckii, Chrysaora hysoscella, Rhizostoma octopus) collected around the island of Helgoland (German Bight) in July?CAugust 2003 and 2004 are described. Planula larvae were measured and reared to polyps in the laboratory. The process of ephyrae development asexually produced by the polyps (strobilation) was photo-documented. Photographs of the ephyrae growth stages were combined with drawings of features useful for the species identification. The provided identification key allows discrimination among post-ephyrae from plankton samples, probably leading to conclusions on the development of jellyfish blooms and their causes.     

Development of clade-specific Symbiodinium primers for quantitative PCR (qPCR) and their application to detecting clade D symbionts in Caribbean corals

We developed quantitative PCR (qPCR) assays to distinguish each of the four clades (A–D) of dinoflagellate endosymbionts (genus Symbiodinium) commonly found in Caribbean corals. We applied these primer sets, which target portions of the multi-copy ribosomal DNA (rDNA) gene family, to assess the presence/absence of symbionts in clade D (as indicated by the detection of clade D DNA). We detected these symbionts in five of six Caribbean host species/genera (21% of samples analyzed, N = 10 of 47 colonies), from which clade D had rarely or never been observed. This suggests that Symbiodinium in clade D are present in a higher diversity of coral species than previously thought. This qPCR-based approach can improve our understanding of the total microbial diversity associated with corals, particularly in hosts thought to be relatively specific, and has many other potential applications for studies of coral reef ecology and conservation.     

Do corals select zooxanthellae by alternative discharge?

Loss of zooxanthellae (dinoflagellate Symbiodinium) from corals will sometimes lead to mass mortality of corals. To detect and quantify Symbiodinium released from corals, we developed a zooxanthellae “trap” and a quantitative PCR (qPCR) system with Symbiodinium clades A–F-specific primer sets. The trap was attached to a branch or the surface of several wild stony corals, and the water samples within the traps, including released Symbiodinium, were subjected to qPCR. All tested corals released clade C Symbiodinium at estimates of ~5,900 cells h⁻¹ cm⁻² of coral surface. Although all tested Pocillopora eydouxi harboured both clades C and D, some of these colonies released only clade C or released a lesser amount of clade D than that in the tissues. Our Symbiodinium quantification system revealed that wild hermatypic corals constantly release Symbiodinium to the environment. Our result suggests that some corals may discharge certain clades of Symbiodinium alternatively.     

Distribution of common dolphins (Delphinus spp.) in the western Atlantic Ocean: a critical re-examination

Due to indications that misidentification (largely confusion among dolphins of the genera Delphinus and Stenella) in the past had led to erroneous assumptions of distribution of the two species of common dolphins (Delphinus delphis and D. capensis) in the western Atlantic Ocean, we conducted a critical re-examination of records of the genus Delphinus from this region. We compiled 460 ‘plottable’ records, required support for confirmation of genus and species identifications, and found many records lacking (and some clearly misidentified). When we plotted only the valid records (n = 364), we found evidence of populations in only three areas, and apparent absence throughout much of the tropical/subtropical regions. Off the east coast of the US and Canada, D. delphis is found from the Georgia/South Carolina border (32°N) north to about 47–50°N off Newfoundland. Since the 1960s, they have apparently been absent from Florida waters. There is no evidence that dolphins of the genus occur in the Gulf of Mexico. Reports of common dolphins from most of the Caribbean Basin are also rejected, and the only place in that region where they are confirmed to occur is off central-eastern Venezuela (a coastal D. capensis population). Off eastern South America, common dolphins appear to be restricted to south of 20°S. There is a coastal long-beaked population found in the South Brazil Bight, and one or more short-beaked populations south and offshore of this (ranging south to at least northern Argentina). The results are very different from commonly-accepted patterns of distribution for the genus in the Atlantic. Most areas of distribution coincide with moderate to strong upwelling and common dolphins appear to avoid warm, tropical waters. This study shows that great care must be taken in identification of similar-appearing long-beaked delphinids, and that uncritical acceptance of records at face value can lead to incorrect assumptions about the ranges of the species involved.     

Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)

Quantification of feeding rates and selectivity of zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, methodological limitations have made many of these studies difficult. Recently, molecular based methods have demonstrated that DNA from prey species can be used to identify zooplankton gut contents, and further, quantitative gut content estimates by quantitative PCR (qPCR) assays targeted to the 18S rRNA gene have been used to estimate feeding rates in appendicularians and copepods. However, while standard single primer based qPCR assays were quantitative for the filter feeding appendicularian Oikopleura dioica, feeding rates were consistently underestimated in the copepod Calanus finmarchicus. In this study, we test the hypothesis that prey DNA is rapidly digested after ingestion by copepods and describe a qPCR-based assay, differential length amplification qPCR (dla-qPCR), to account for DNA digestion. The assay utilizes multiple primer sets that amplify different sized fragments of the prey 18S rRNA gene and, based on the differential amplification of these fragments, the degree of digestion is estimated and corrected for. Application of this approach to C. finmarchicus fed Rhodomonas marina significantly improved quantitative feeding estimates compared to standard qPCR. The development of dla-qPCR represents a significant advancement towards a quantitative method for assessing in situ copepod feeding rates without involving cultivation-based manipulation.     

Phaeocystis blooms and eutrophication of the continental coastal zones of the North Sea

It is suggested that novel nuisance algal blooms can result from major shifts in N/P or NH₄ ⁺/NO₃ ^- ratios. Inland hydraulic engineering caused a shift from P-limitation (before 1977) towards N-limitation (after 1977) in the Marsdiep area (Dutch coastal waters). Following this shift the colonial flagellate Phaeocystis sp. became more abundant and started to bloom during the nutrient-controlled period (later spring to autumn). Competition experiments showed that the N/P ratio can influence the species composition of marine phytoplankton. In addition, the natural distribution of some species like Rhodomonas sp. and Emiliania huxleyi may be affected by the frequency of nutrient pulses in the system. Phaeocystis was a poor competitor under P-limitation and a good competitor under N-limitation. Colony formation was absent under P- and NH₄ ⁺-limitation. Colonies were formed under NO₃ ^--limitation. These preliminary results suggest that colony-forming Phaeocystic blooms may be restricted (besides light-controlled environments) to those N-controlled environments where nitrate is consumed by Phaeocystis. The distribution of Phaeocystis along the European continental coast is evaluated on the basis of its ability to compete for nutrients and to form colonies when nitrate is the major N-source.     

Modelling the distribution and ecology of <Emphasis Type="Italic">Trichosolen</Emphasis> blooms on coral reefs worldwide

Worldwide blooms of the green alga Trichosolen have been reported on damaged coral reefs following catastrophic events. However, the global distribution of Trichosolen and the factors triggering such blooms remained elusive because of a paucity of occurrence records. This study presents a presence-only niche modelling approach to map the potential distribution and delineate bloom risk areas as well as to identify environmental response optima for non-blooming occurrences and blooms. The modelled suitability map revealed a pantropical to subtropical distribution, while high suitability values delineated bloom risk areas including important tropical reef systems where Trichosolen has not yet been reported from. While both blooms and non-bloom occurrences show a strong preference for high temperatures, blooms responded better to broader nutrient ranges than non-blooms, suggesting the importance of sudden nutrient inputs during catastrophic events in the formation of blooms.     

Phaeocystis pouchetii blooms in the East Frisian coastal waters (German Bight,North Sea)

In 1982 and 1983, blooms of the planktonic alga Phaeocystis pouchetii were studied in the East Frisian coastal waters off Norderney, FRG. Morphological variability of the colonies, population development, effects on inorganic nutrient and oxygen budget, and the role in the seasonal plankton bloom sequence were investigated. in both years two separate Ph. pouchetii blooms occurred in late spring and early summer that were formed by the globosa-type exclusively. The pouchetii-type was only present during the interval between the first and second blooms in 1982. Growth conditions for Ph. pouchetii seem to be most favourable after a breakdown of a diatom bloom when the silicate reserves have been exhausted, phosphate concentrations are relatively low, but a rich supply of dissolved inorganic nitrogen is available. Contrary to diatom blooms, Ph. pouchetii blooms result in a high production of particulate organic carbon (POC). Another characteristic is the release of great amounts of dissolved organic substances, which cause extensive sea foam formation during the peaks and the subsequent breakdown of the blooms. Due to increased assimilation during mass development and intense disintegration after the breakdown, variations of the oxygen content of the water are significant. Presumably the intensity of blooms has increased in the past decade as indicated by sea foam formation which has actually been excessive in some years.     

The development of a multi-species algal ecodynamic model for urban surface water systems and its application

An ecodynamic model that can simulate four phytoplankton species has been developed to deal with the unique characteristics of urban river systems which has manmade river profile, flow controlled by gates, severe eutrophication status, and fragile aquatic ecosystem. The ecodynamic model was developed referencing two typical models: the water quality simulation model WASP and ecological model CAEDYM. The model can simulate 11 state variables: dissolved oxygen, carbonaceous biochemical oxygen demand, ammonia nitrogen, nitrate nitrogen, organic nitrogen, inorganic phosphorus, organic phosphorus and four phytoplankton species with zooplankton as a boundary condition. The ecodynamic model was applied to Sihai section of the Beijing urban river system, where serious algal blooms broke out in recent years. The dominant phytoplankton species are Cyanophyta, Chlorophyta, Bacillariophyta, and Cryptophyta. Site-specific data on geometry, meteorology, pollution sources, and existing ecosystem parameters were collected and used for model calibration and verification The model results mimic observed trends of water quality and phytoplankton species succession and can be used for forecasting algal blooms as well as assessment of river management measures.     

Arctic-Antarctic disjunctions in the benthic seaweeds Acrosiphonia arcta (Chlorophyta) and Desmarestia viridis/willii (Phaeophyta) are of recent origin

Two examples of the most extreme biogeographic disjunctions in benthic marine algae are found in Acrosiphonia arcta (Chlorophyta) and Desmarestia viridis/willii (Phaeophyta). Both species are members of the Arctic and Antarctic boreal and subboreal marine floras. Although both genera have temperate species, neither genus has subtropical or tropical representative. Comparisons of the fast-evolving ribosomal DNA internal transcribed spacer regions among isolates in each of the two species collected from both hemispheres showed an unexpected near sequence identity suggesting that these biogeographic disjunctions are of recent origin, possibly as recent as the last Pleistocene glacial maximum (18000 yr ago). Paleoclimatic explanations that rely on a much earlier transequatorial passage of cold-adapted species through a narrowed and cooler tropical belt during the Oligocene/Miocene (38 to 7 Ma ago) are unlikely. We hypothesize that despite the separated evolutionary histories of the northern and southern hemisphere coldwater marine floras, deep-water dispersal of microthalli has occurred and probably occurs on a regular basis.