Use of biomarkers in zooplankton for assessment of the ‘health status’ of marine and brackish environments: a short overview

The biomarker approach has been used for 25 years to study the environmental quality of marine, brackish and freshwater ecosystems. Biomarkers may indicate health status and can be applied to organisms of all zoological phyla by destructive or non destructive methods. For 5 years we have been using this approach in zooplankton to detect ecotoxicological alterations at low levels of the food chain due to contaminants. Here we review our approach to validate and apply biomarker techniques in zooplankton. We discuss advantages, limitations, some results and future research. We indicate that biomarkers in zooplankton can be used as new indices of trophic status and ecological integrity of Italian marine coastal and lagoon environments, to be included among the tools specified by Italian law D.Lgs. 152/2006.     

Geographic variation and behavioral evolution in marine plankton: the case of<Emphasis Type="Italic"> Mastigias</Emphasis> (Scyphozoa,Rhizostomeae)

Although complex behavior in marine zooplankton has been considered strong evidence of adaptation, ethological studies of marine zooplankton generally have not employed either the comparative approach or evolutionary perspective necessary to distinguish adaptation from any alternative. Consequently, the potential for intra-specific variation in the behavior of marine zooplankton has received insufficient attention and conclusions of adaptation remain poorly substantiated. Intra-specific comparison of patterns of migration and behavior for seven populations of golden jellyfish, Mastigias (Scyphozoa: Rhizostomeae), inhabiting isolated marine lakes and semi-enclosed lagoon coves in Palau document population specific differences in patterns of horizontal migration, vertical migration, pulse rate, swimming speed, and turning behavior. Evidence was found for symplesiomorphic behaviors, canalization, exaptation, adaptation, and probably once-deleterious traits. Behavioral evolution likely proceeded via, at least, relaxation of selection, trade-offs with morphology, and natural selection effected by predation. Behavioral patterns also may change with ontogeny. Geographic variation in the behavior of marine plankton therefore can be substantial and patterns of evolution complex. Behavioral evolution can rapidly generate coastal biodiversity. Thus, geographic variation in marine plankton is of potential interest to ethologists, evolutionary biologists, biogeographers, and conservation biologists.Electronic Supplementary Supplementary material is available in the online version of this article at Communicated by G.F. Humphrey, Sydney     

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.     

Interacting trophic forcing and the population dynamics of herring

Small pelagic fish occupy a central position in marine ecosystems worldwide, largely by determining the energy transfer from lower trophic levels to predators at the top of the food web, including humans. Population dynamics of small pelagic fish may therefore be regulated neither strictly bottom-up nor top-down, but rather through multiple external and internal drivers. While in many studies single drivers have been identified, potential synergies of multiple factors, as well as their relative importance in regulating population dynamics of small pelagic fish, is a largely unresolved issue. Using a statistical, age-structured modeling approach, we demonstrate the relative importance and influence of bottom-up (e.g., climate, zooplankton availability) and top-down (i.e., fishing and predation) factors on the population dynamics of Bothnian Sea herring (Clupea harengus) throughout its life cycle. Our results indicate significant bottom-up effects of zooplankton and interspecific competition from sprat (Sprattus sprattus), particularly on younger age classes of herring. Although top-down forcing through fishing and predation by grey seals (Halichoerus grypus) and Atlantic cod (Gadus morhua) also was evident, these factors were less important than resource availability and interspecific competition. Understanding key ecological processes and interactions is fundamental to ecosystem-based management practices necessary to promote sustainable exploitation of small pelagic fish.     

Habitat specialization and the exploitation of allochthonous carbon by zooplankton

The significance of spatial subsidies depends on consumer resource interactions in the recipient habitat. Lakes are subsidized by terrestrial carbon sources, but the pathways of allochthonous carbon through lake food webs are complex and not well understood. Zooplankton vertically partition resources within stratified lakes in response to life history trade-offs that are governed by predators, the quantity and quality of food, and abiotic conditions (e.g., UV, temperature, and viscosity). We measured habitat specialization of zooplankton in an oligotrophic lake where allochthonous and autochthonous resources varied with depth. During stratification, the quantity and quality of zooplankton food was highest in the hypolimnion. We used a yearlong time series of the delta13C of zooplankton and particulate organic matter (POM) to determine which zooplankton species exploited hypolimnetic rather than epilimnetic resources. Because the delta13C of POM decreased with depth, we used the delta13C of zooplankton to detect inter- and intraspecific variation in habitat selection. We incubated Daphnia pulex at discrete depths in the water column to confirm that the delta13C of zooplankton can indicate habitat specialization. Zooplankton that specialized in the epilimnion relied more on allochthonous carbon sources than those that specialized in the hypolimnion. Therefore, the fate of allochthonous carbon subsidies to lakes depends on spatially explicit consumer-resource interactions.     

The variations of exergies and structural exergies along eutrophication gradients in Chinese and Italian lakes

Taking 29 Chinese lakes and 29 Italian lakes as two separate case studies, the paper presented the variations of exergies and structural exergies along eutrophication gradients in Chinese and Italian lakes. The exergies (Ex) and structural exergies (Exst) were calculated based on phytoplankton biomass (BA) and zooplankton biomass (BZ). A trophic state index (TSI) scaling from 0 to 100 was developed to classify trophic status for Chinese and Italian lakes based on three indicators, chlorophyll-a concentration (Chl-a, in mg/m³), total phosphorus (TP in mg/m³) and transparency in Secchi disk depth (SD in m). The relationships between TSI and Ex, Exst, total biomass, BZ/BA ratios were analyzed. The following results were obtained: (1) with the increase of TSI in Chinese and Italian lakes, there is an increasing trend for Ex, and a decreasing trend for Exst, generally. The obvious negative correlations exist between TSI and Exst, at the significant level of 0.01 for Italian lakes, and 0.05 for Chinese lakes. The obvious positive correlations exist between TSI and Ex, at the significant level of 0.01 for Chinese lakes, and for Italian lakes in Spring, Autumn and the all-year. (2) The structural exergy is more dependent on the ratio of phytoplankton biomass to zooplankton biomass (BZ/BA) than the exergy, and the exergy is more dependent on total biomass than the structural exergy. (3) The phytoplankton biomass (BA) and zooplankton biomass (BZ) are increased with the increasing TSI in Chinese and Italian lakes, and phytoplankton biomass (BA) increases more rapidly then zooplankton biomass (BZ) does. This results in the definite decrease of BZ/BA ratio with the increasing trophic status index. Such changes of BA, BZ and BZ/BA ratio could explain successfully the variations of exergies and structural exergies along eutrophication gradients in Chinese and Italian lakes. From the two separate case studies of Chinese lakes and Italian lakes, it could be concluded that exergy and structural exergy are feasible to serve as the system-level ecological indicators to give appropriate information on the trophic status of different lakes.     

Flexible foraging movements of leatherback turtles across the North Atlantic Ocean

Some marine species have been shown to target foraging at particular hotspots of high prey abundance. However, we show here that in the year after a nesting season, female leatherback turtles (Dermochelys coriacea) in the Atlantic generally spend relatively little time in fixed hotspots, especially those with a surface signature revealed in satellite imagery, but rather tend to have a pattern of near continuous traveling. Associated with this traveling, distinct changes in dive behavior indicate that turtles constantly fine tune their foraging behavior and diel activity patterns in association with local conditions. Switches between nocturnal vs. diurnal activity are rare in the animal kingdom but may be essential for survival on a diet of gelatinous zooplankton where patches of high prey availability are rare. These results indicate that in their first year after nesting, leatherback turtles do not fit the general model of migration where responses to resources are suppressed during transit. However, their behavior may be different in their sabbatical years away from nesting beaches. Our results highlight the importance of whole-ocean fishing gear regulations to minimize turtle bycatch.     

Using ecosystem-services assessments to determine trade-offs in ecosystem-based management of marine mammals

The goal of ecosystem-based management (EBM) is to support a sustainable and holistic multisectored management approach, and is recognized in a number of international policy frameworks. However, it remains unknown how these goals should be linked to assessments and management plans for marine fauna, such as mammals and fish stocks. It appears particularly challenging to carry out trade-off analyses of various ocean uses without a framework that integrates knowledge of environmental, social, and economic benefits derived from nonstationary marine fauna. We argue this gap can be filled by applying a version of the ecosystem-service approach at the population level of marine fauna. To advance this idea, we used marine mammals as a case study to demonstrate what indicators could operationalize relevant assessments and deliver an evidence base for the presence of ecosystem services and disservices derived from marine mammals. We found indicators covering common ecosystem service categories feasible to apply; examples of indicator data are already available in the literature for several populations. We encourage further exploration of this approach for application to marina fauna and biodiversity management, with the caveat that conceptual tensions related to the use of the ecosystem service concept itself needs to be addressed to ensure acceptance by relevant stakeholders.     

Density-gradient centrifugation as an aid to sorting planktonic organisms. I. Gradient materials

Fish eggs and larvae can be separated from invertebrate zooplankton by isopycnic centrifugation in gradients of sucrose or silica. Preserved samples of invertebrate zooplankton, fish eggs, and fish larvae, representing a typical assortment of marine plankton, were layered over linear gradients of 25 to 60% w/w (weight/weight) sucrose or 0 to 15% w/w silica (as Ludox AM) in 100 c³ swinging buckets, and centrifuged for 1 h at 1000 rpm (revs per minute). In sucrose gradients, the invertebrate zooplankton were confined to the two ends of the gradient, while 85% of the fish eggs were recovered from an intermediate zone (27.5 to 55% w/w). In Ludox AM, the fish eggs banded in a narrow region between 2 and 3% w/w, while fish larvae banded at the bottom of the gradient between 10 and 14% w/w. Of the 6 dominant classes of zooplankton, only Salpa overlapped appreciably with the fish eggs and none overlapped with the fish larvae. Of the gradient materials tested, Ludox AM offers the most advantages; sucrose may also be useful for subfractionation. Gradients of sodium bromide and dextran have been found to be totally unsuitable.Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903, USA.     

Predator-induced morphological defenses in marine zooplankton: a larval case study

While there are numerous reports of predator-induced morphological defenses for freshwater zooplankton, freshwater larvae, and benthic marine animals, a literature search revealed no reports of predator-induced morphological defenses for marine zooplankton. Rarity of predator-induced morphological defenses in marine zooplankton would imply a difference in predation risks compared to those experienced by freshwater organisms and benthic marine adults, whereas the presence of such plasticity in defenses would imply that risks are modified by developmental responses. This study reports a predator-induced change in defenses and vulnerability of a marine planktonic larva. Specifically, when reared in the presence of zoea larvae of Cancer spp., veliger larvae of the intertidal snail Littorina scutulata developed significantly smaller shell apertures and rounder shells than did cohort veligers reared in the absence of predator cues. Pairwise predation trials demonstrated that veligers reared with caged zoeas throughout development had greater survival than predator-naive veligers during short-term exposure to zoeas. The development of predator-induced morphological defenses by some marine larvae introduces a range of testable hypotheses on developmental plasticity that reduces vulnerability of planktonic larvae and other marine zooplankton to predators.     

The effects of zooplankton swimming behavior on prey-capture kinematics of red drum larvae, <Emphasis Type="Italic">Sciaenops ocellatus</Emphasis>

Most marine fishes undergo a pelagic larval phase, the early life history stage that is often associated with a high rate of mortality due to starvation and predation. We present the first study that examines the effects of prey swimming behavior on prey-capture kinematics in marine fish larvae. Using a digital high-speed video camera, we recorded the swimming velocity of zooplankton prey (Artemia franciscana, Brachionus rotundiformis, a ciliate species, and two species of copepods) and the feeding behavior of red drum (Sciaenops ocellatus) larvae. From the video recordings we measured: (1) zooplankton swimming velocity in the absence of a red drum larva; (2) zooplankton swimming velocity in the presence of a red drum larva; and (3) the excursion and timing of key kinematic events during prey capture in red drum larvae. Two-way ANOVA revealed that: (1) swimming velocity varied among zooplankton prey; and (2) all zooplankton prey, except rotifers and ciliates, increased their swimming velocity in the presence of a red drum larva. The kinematics of prey capture differed between two developmental stages in S. ocellatus larvae. Hyoid-stage larvae (3–14 days old) fed on slow swimming B. rotundiformis (rotifers) while hyoid-opercular stage larvae (15 days and older) ate fast moving A. franciscana. Hyoid-opercular stage red drum larvae had a larger gape, hyoid depression and lower jaw angle, and a longer gape cycle duration relative to their hyoid-stage conspecifics. Interestingly, the feeding repertoire within either stage of red drum development was not affected by prey type. Knowledge of the direct relationship between fish larvae and their prey aids in our understanding of optimal foraging strategies and of the sources of mortality in marine fish larvae.     

Use of environmental DNA in assessment of fish functional and phylogenetic diversity

Assessing the impact of global changes and protection effectiveness is a key step in monitoring marine fishes. Most traditional census methods are demanding or destructive. Nondisturbing and nonlethal approaches based on video and environmental DNA are alternatives to underwater visual census or fishing. However, their ability to detect multiple biodiversity factors beyond traditional taxonomic diversity is still unknown. For bony fishes and elasmobranchs, we compared the performance of eDNA metabarcoding and long-term remote video to assess species’ phylogenetic and functional diversity. We used 10 eDNA samples from 30 L of water each and 25 hr of underwater videos over 4 days on Malpelo Island (pacific coast of Colombia), a remote marine protected area. Metabarcoding of eDNA detected 66% more molecular operational taxonomic units (MOTUs) than species on video. We found 66 and 43 functional entities with a single eDNA marker and videos, respectively, and higher functional richness for eDNA than videos. Despite gaps in genetic reference databases, eDNA also detected a higher fish phylogenetic diversity than videos; accumulation curves showed how 1 eDNA transect detected as much phylogenetic diversity as 25 hr of video. Environmental DNA metabarcoding can be used to affordably, efficiently, and accurately census biodiversity factors in marine systems. Although taxonomic assignments are still limited by species coverage in genetic reference databases, use of MOTUs highlights the potential of eDNA metabarcoding once reference databases have expanded.     

Effects of ocean acidification, temperature and nutrient regimes on the appendicularian Oikopleura dioica: a mesocosm study

Increasing pCO₂ is hypothesized to induce shifts in plankton communities toward smaller cells, reduced carbon export rates and increased roles of gelatinous zooplankton. Appendicularians, among the most numerous pan-global “gelatinous” zooplankton, continuously produce filter-feeding houses, shortcutting marine food webs by ingesting submicron particles, and their discarded houses contribute significantly to carbon fluxes. We present a first mesocosm-scale study on the effects of temperature, pCO₂ and bloom structures on the appendicularian, Oikopleura dioica. There were effects of temperature and nutrients on phytoplankton communities. No shifts in functional phytoplankton groups, nor changes in particle sizes/morphotypes, known to impact appendicularian feeding, were observed under manipulated pCO₂ conditions. However, appendicularian abundance was positively correlated with increased pCO₂, temperature and nutrient levels, consistent with hypotheses concerning gelatinous zooplankton in future oceans. This suggests appendicularians will play more important roles in marine pelagic communities and vertical carbon transport under projected ocean acidification and elevated temperature scenarios.     

Research On Zooplankton in the Gulf of Rapallo

Zooplankton samples, collected in autumn 1996 from two stations in the Gulf of Rapallo, Ligurian Sea (Rapallo Harbour and Prelo Bay, which is a more open site with lower human impacts) were analysed. at both stations, the community was dominated by copepods (mainly juveniles and adults of different species of Acartia and Oithona) and meroplankton (mainly polychaete larvae). Total zooplankton abundance in the harbour waters was significantly higher than in the nearby bay. the occurrence of Acartia grani in Rapallo Harbour and Pteriacartia josephinae in Prelo Bay is noteworthy, as they are reported for the first time in the Ligurian Sea, and A. grani has not previously been recorded to the Italian coastline. A three-day benthic chamber experiment aimed to quantify nutrient fluxes between the sediment and water column was performed in the Rapallo Harbour: the profound effects of confinement on the composition and abundance of zooplankton within the incubation chamber are discussed.     

Dynamic model of nutrification in Huntington Bay,New York

A dynamic simulation model of nutrification with respect to phytoplankton and zooplankton levels in Huntington Bay is developed. The validity of relationships used by previous researchers in freshwater environments is tested under marine conditions.Nutrients are modelled, and it is shown that nitrogen is the limiting nutrient in the bay. Phosphorus is not found to be limiting.     

Marine food-web analysis: An experimental study of demersal zooplankton using isotopically labelled prey species

Short-term incubations in seawater containing H¹⁴CO₃ ^- or ³H₂O in place of the naturally predominant isotopes can yield highly radioactive preparations of living phytoplankton or zooplankton. Subsequent in situ incubation of these labelled organisms with the community from which they were taken results in the rapid transfer of radioisotope to those species which prey upon them. This technique has been employed to map a portion of a marine food web involving demersal zooplankton; experiments were conducted in summer and autumn on a coral reef and in a subtropical estuary. Similar results were obtained from these initial experiments at each study site during both seasons. Prey supplied as zooplankton (124 to 410 m nominal diameter), which consisted mainly of Oithona oculata, was fed upon by zooplankton size classes ranging from 410 to 850 m and containing amphipods, ostracods, cumaceans and polychaetes. In experiments employing labelled phytoplankton as prey a wide size spectrum was used (10 to 106 m) in order to include representative samples of most of the available planktonic autotrophs as estimated by primary production measurements. In two separate experiments, only 7 out of 63 samples evidenced grazing of phytoplankton by demersal zooplankters. In contrast, labelled diatom auxospores, employed in one experiment as they constituted the most numerically abundant species in the water column, were found to be grazed upon in nearly half the samples examined.     

Network-based exploration and visualisation of ecological data

Networks – structured graphs consisting of sets of nodes connected by edges – provide a rich framework for data visualisation and exploratory analyses. Although rarely used for the visualisation of ecological data, networks are well suited to this purpose, including data that one might not normally think of as a network. We present a simple method for transforming a data matrix into network format, and show how this can be used as the basis for interactive exploratory analyses of ecological data.The method is demonstrated using a database of marine zooplankton samples acquired in the Southern Ocean. The network analyses revealed zooplankton community structures that are in good agreement with previously published results. Variations in community structure were observed to be related to the temporal and spatial pattern of sampling, as well as to physical environmental factors such as sea ice cover. The analyses also revealed a number of errors in the data, including taxon identification errors and instrument failures.The method allows the analyst to generate networks from different combinations of variables in the data set, and to examine the effects of varying parameters such as the scales of spatial, temporal, and taxonomic aggregation. This flexibility allows the analyst to rapidly gain a number of perspectives on the data and provides a powerful mechanism for exploration.     

From science to policy—a geostatistical approach to identifying potential areas for cetacean conservation: a case study of bottlenose dolphins in the Pelagos sanctuary (Mediterranean Sea)

Cetaceans are top-level predators that serve as sentinels of the health and status of lower trophic levels in the marine ecosystem. For this reason they attract significant attention in marine conservation planning and often have been used to promote designation of reserve areas in many countries (e.g., Ligurian Sea, Moray Firth, Hawaiian Islands, The Gully, Wadden Sea, Banks Peninsula, and Golfo San José). Many policies are designed to protect cetaceans. For example, the Habitat Directive requires member states to select, designate, and protect sites that support certain natural habitats or species, such as the bottlenose dolphin, as Special Areas of Conservation (SACs) that aim to create a network of protected areas across the European Union known as Natura 2000. The boundaries of protected areas for cetacean species must be defined for management purposes. In recent years, many techniques have been developed to define the distribution of cetaceans in relation to habitat preferences. Although these models can provide an understanding of the ecological processes that determine species distribution, their application requires prior knowledge of the variables that should be included in the model, the interactions among these variables, and their effects on species distribution. Thus, the lack of available data in understudied areas precludes the application of these types of models. As an alternative, we describe a geostatistical approach to identifying areas that potentially should be designated as marine protected areas for cetaceans. We illustrate the application of the kriging algorithm to the bottlenose dolphin population that resides in the northwestern Mediterranean Sea. The data derived from a 7-year survey were used. The encounter rate is the only variable required for this method, making it very easy to apply. The resulting georeferenced and high resolution map includes areas most visited by bottlenose dolphins, which are called core areas. Core areas are helpful for establishing the boundaries of marine reserves for the protection of the species. The approach described herein is accurate, precise, unbiased, replicable to all highly mobile species and easy to understand by both researchers and policy makers.     

Feeding in Arctic darkness: mid-winter diet of the pelagic amphipods Themisto abyssorum and T. libellula

The pelagic amphipods Themisto abyssorum and Themisto libellula represent important links between the herbivore zooplankton community and higher trophic levels of the Arctic marine food webs. Large double structured eyes of both of these hyperiid species are assumed to be used for visual prey detection. However, no information is available on the feeding strategies of these visually searching predators for the period of the polar night, a time of year with no or very low levels of daylight. Here, we report on the stomach and gut content of both Themisto species collected during a January expedition around Svalbard (78° to 81°N). Results indicate that T. abyssorum and T. libellula feed actively during the Arctic winter. The major food source of both amphipods consisted of calanoid copepods, most frequently Calanus finmarchicus.     

The measurement of respiratory electron-transport-system activity in marine zooplankton

A modification of the tetrazolium reduction method (Packard, 1971) for measurement of respiratory electron transport system activity in marine zooplankton is described. A major modification is the addition of Triton X-100 to the reaction mixture to solubilize formazan produced, thus eliminating an organic extraction step and increasing the sensitivity and versatility of the method. The kinetic parameters of the assay are presented and the apparent effects of Triton X-100 are discussed. The correlation between electron-transport-system (ETS) activity and in vivo respiration (ETS/R) for Calanus pacificus has been found to be 2.02±0.29 (standard deviation). The method is presented as providing a reliable estimate of the zooplankton component of oxygen consumption in the sea.