Assessing feeding of a carnivorous copepod using species-specific PCR

The polymerase chain reaction (PCR) offers a sensitive and selective way to detect trace amounts of biological remnants. Here, we show that this simple molecular technique can be applied to identify prey copepods in the fecal pellets of carnivorous zooplankton. Using variation in the mitochondrial cytochrome C oxidase subunit I (mtCOI) sequence, we developed a species-specific oligonucleotide PCR primer (COI-2026) for Calanus helgolandicus. In a touch-down PCR, Calanus DNA was amplified from pellets collected from freshly incubated individuals of the carnivorous copepod Pareuchaeta norvegica. Positive results could easily be detected by agarose gel electrophoresis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M. Kühl, Helsingør     

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.     

Summer copepod production in subtropical waters adjacent to Australia's North West Cape

Growth and secondary production of pelagic copepods near Australia's North West Cape (21° 49 S, 114° 14 E) were measured during the austral summers of 1997/1998 and 1998/1999. Plankton communities were diverse, and dominated by copepods. To estimate copepod growth rates, we incubated artificial cohorts allocated to four morphotypes, comprising naupliar and copepodite stages of small calanoid and oithonid copepods. Growth rates ranging between 0.11 and 0.83 day^–1 were low, considering the high ambient temperatures (23–28°C). Calanoid nauplii had a mean growth rate of 0.43±0.17 day^-1 (SD) and calanoid copepodites of 0.38±0.13 day^-1. Growth rates of oithonid nauplii and copepodites were marginally less (0.38±0.19 day^–1 and 0.28±0.11 day^–1 respectively). The observed growth rates were suggestive of severe food limitation. Although nauplii vastly outnumbered copepodite and adult copepods, copepodites comprised the most biomass. Copepodites also contributed most to secondary production, although adult egg production was sporadically important. The highest copepod production was recorded on the shelf break (60 mg C m^-2 day^-1). Mean secondary production over both shelf and shelf break stations was 12.6 mg C m^-2 day^-1. Annual copepod secondary production, assuming little seasonality, was estimated as ~ 3.4 g C m^-2 year^-1 (182 kJ m^-2 year^-1).Communicated by G.F. Humphrey, Sydney     

Comparison of mercury bioaccumulation between wild and mariculture food chains from a subtropical bay of Southern China

Bioaccumulation and trophic transfer of mercury (Hg) both in the natural marine ecosystem and the mariculture ecosystem were studied at Daya Bay, a subtropical bay in Southern China. Averaged Hg concentrations in sediment, phytoplankton, macrophyte, shrimp, crab, shellfish, planktivorous fish, carnivorous fish, farmed pompano, farmed snapper, compound feed and trash fish were 0.074, 0.054, 0.044, 0.098, 0.116, 0.171, 0.088, 0.121, 0.210, 0.125, 0.038 and 0.106 μg g⁻¹ dw, respectively. These Hg levels were at the low–middle ends of the global range. Positive correlation between Hg concentrations in farmed fish and fish weights/sizes was observed, whereas no clear correlation between Hg concentrations and lipid contents was found. Hg concentrations followed macrophyte < phytoplankton < sediment < planktivorous fish < shrimp < crab < carnivorous fish < shellfish, and commercial feed < trash fish < farmed fish. Hg was biomagnified along the marine food chain in the ecosystem of Daya Bay. Hg levels in the farmed fish were higher than those in the wild fish primarily because of the higher Hg level in fish feed and the smaller size of marine wild fish.

     

Species abundance estimation using point-to-plant sampling in a design-based setting

A new species abundance estimator is proposed when point-to-plant sampling is adopted in a design-based framework. The method is based on the relationship between each species abundance and the probability density function of the relative squared point-to-plant distance. Using this result, a kernel estimator for species abundance is provided and the nearest neighbor method is suggested for bandwidth selection. The proposed estimator requires no assumptions about the species point patterns nor corrections for sampling near the edges of the study region. Moreover, the estimator shows suitable statistical properties as well as good practical performance as is shown in a simulation study.     

The effects of boat propeller scarring intensity on nekton abundance in subtropical seagrass meadows

Seagrasses play a critical role in the function and structure of coastal ecosystems, and they are an important habitat for a variety of marine organisms. Damage to seagrass beds caused by boat propeller scarring is significant in many areas. This study was designed to assess the impact of varying scarring intensities on nekton density. We selected ten replicate (10 m×25 m quadrats) sites representing four distinct scarring intensities: reference (0%), low (5% or less), moderate (5–15%), and severe (>15%). Sites were sampled in 2003–2004 for nekton during four seasons (summer, fall, winter, and spring) using epibenthic sleds. There were eight taxa numerically dominant in all seasons and an additional four seasonally dominant species. We were unable to detect a significant effect of propeller scarring on nekton density at any scarring level. Additionally, regression analysis indicated no relationship between scarring intensity and nekton density. These results suggest that propeller scarring intensities of up to ca. 27% may not impact nekton densities. However, seagrass loss, higher scarring intensity, and scale may play a critical role in determining the impact of propeller scarring on nekton.     

Functional responses of copepod nauplii using a high efficiency gut fluorescence technique

To investigate copepod nauplii ingestion rates on phytoplankton, we have adapted the traditional gut fluorescence technique as it can be used with lower gut pigment concentrations. With the improved technique, laboratory experiments were performed to estimate functional responses for nauplii of Calanus helgolandicus and Centropages typicus. Nauplii were raised from eggs to copepodites and the experiments were performed with stages NIV-NV. Gut evacuation rates and ingestion rates were measured on Isochrysis galbana at different concentrations. Specific ingestion rates ranged between 0.038–0.244 μg C μg⁻¹ nauplii C d⁻¹ for C. typicus and 0.041–1.412 μg C μg⁻¹ nauplii C d⁻¹ for C. helgolandicus. Both species showed a type III functional response, reaching a saturation concentration at around 600 μgC l⁻¹ for C. typicus and 800 μgC l⁻¹ for C. helgolandicus. An erratum to this article can be found at     

Seasonal contrasts in abundance and reproductive parameters of Penilia avirostris (Cladocera, Ctenopoda) in a coastal subtropical area

We studied the population dynamics and the reproductive biology of Penilia avirostris during three consecutive years on the inner shelf off Ubatuba, Brazil. Penilia avirostris individuals and its eggs and embryos were counted, measured, and classified into stages. The species occurred throughout the studied period, in a wide temperature range (14.8–28.2°C). Cladoceran densities were usually higher (>2,000 ind m⁻³) in warm seasons, when the water column was stratified as a consequence of bottom intrusions of the cold- and nutrient-rich South Atlantic Central Water. Juveniles, non-reproducing females, and parthenogenic females were the dominant developmental stages. Males and gamogenic females were rare and only occurred when females reached peak abundances. This suggests that in tropical and subtropical coastal seas gamogenesis in P. avirostris is not as common as in temperate seas, but may play a significant role in the density-dependent control of the population preceding unfavourable periods.     

Food ingestion by the marine planktonic copepod Paracalanus in relation to abundance and size distribution of food

Juveniles and adult females were presented a food spectrum of three algae of different sizes (4.5, 12 and 20 m cell width). The increase in rate of ingestion of the medium-sized alga with an increase in copepod size was significantly greater than the increase in rate of ingestion of the small alga. It is hypothesized that the perception of chemical signals from the small alga by a copepod decreases as the copepod moults from stage to stage. The rate of ingestion of the large alga by copepod stage V (CV) and adult females was lower than the rate of ingestion of the medium-sized alga at mid- and high phytoplankton concentrations. The amount of nitrogen ingested when the medium-sized alga alone was offered was either higher than (stage C II) or not significantly different from that when the three algae were offered together (stage C IV). Ingestion rates are reduced when there is a multialgal food source. This implies that there is increased stability in the ocean because multiparticle food sources are more slowly depleted than unialgal foods. Weight-specific ingestion rates of copepods fed the three algae simultaneously increased from nauplius to stage C III and then decreased as adulthood approached. The contribution of the small alga to the total amount of nitrogen ingesied was greatest for naupliar stages while the contribution of the medium-sized cells was greater for later stages. The largest alga was readily ingested by stage C V and adult females but never contributed more than 25% of the nitrogen ingested. Eight to 12% of the nitrogen ingested by adult females was from the small alga. It is hypothesized that the algal cell size for maximum nitrogen ingestion in upwelled waters is relatively small, round or square and close to the size threshold below which adult females do not sense individual cells.     

Gpi genotypic effect on quantitative traits in the northern bay scallop,Argopecten irradians irradians

The relationships between glucose-6-phosphate isomerase (Gpi) genotype and quantitative variation related to reproduction and growth were explored over the lifespan of a single cohort of northern bay scallops, Argopecten irradians irradians (Lamarck), from September 1986 to April 1988 in the Niantic River estuary, Connecticut, USA. Analyses revealed that Gpi genotype explained an increasingly significant proportion of variation in scallop size (up to 15%) as the cohort aged. The genotype-specific effects were consistent across sampling dates and among measured traits. There was no evidence for heterosis with respect to size at this locus; however, rare genotypes contributed substantially to the relationship and showed some tendency to fall on the extremes of the phenotypic distribution. The strength and consistency of the genotypic effect on scallop size suggest that genetic variation for Gpi, or some locus in linkage disequilibrium with Gpi, may translate into biochemical and/or physiological variation and affect fitness in this species.     

使用底栖和浮游桡足类生物测试评估沉积物修复方案的效果

三丁基锡（TBT）是一种常见于船舶防污涂料中的有机锡化合物。尽管该化学物已于2008年被禁用,但在ports和港口等海上交通区域仍可检出致毒水平（浓度）的TBT。一项补救方案在新西兰的一个港口实施,以将TBT和铜的浓度降低到可接受的（浓度）值。这项研究使用分析化学和桡足类生物测试相结合的方法评估了该方案的有效性。在港口内的三个空间梯度上采集沉积物和水样,并在修复前后测定了有机锡和金属含量。利用底栖和浮游桡足类生物测试估算了沉积物和淘析样品的毒性。虽然底泥样品的急性毒性在修复后减少,但对底栖桡足类的繁殖率仍有影响。本研究所采用的方法有望评估沿海地区修复过程的效果。     

Alkaline phosphatase activity in subtropical Central North Pacific waters using a sensitive fluorometric method

This paper describes a fluorometric method for assaying low levels of the enzyme alkaline phosphatase in seawater. The assay is based on the hydrolysis of the monophosphate ester bond of 3-0-methylfluorescein phosphate. This enzyme is synthesized by many microorganisms when phosphate becomes limiting. Alkaline phosphatase activity was detected in phytoplankton from the nutrient-impoverished surface waters of the subtropical Central North Pacific Ocean. The presence of naturally occurring phosphatase suggests that phosphorus may be limiting to phytoplankton growth in these waters. The phytoplankton in water samples lacking enzyme activity at the time of collection produced phosphatase within 1 to 2 days of incubation at in situ temperatures.     

Development and environmental application of a genus-specific quantitative PCR approach for Pseudo-nitzschia species

Quantitative polymerase chain reaction (qPCR) for the identification and quantification of microbes has become a common tool for the study of harmful algal blooms (HABs). We developed a qPCR method for the diatom genus Pseudo-nitzschia. Several species of this genus form toxic blooms through the production of the neurotoxin domoic acid (DA). Outbreaks of toxicity attributed to DA along the US west coast have caused sickness and death of marine mammals and seabirds through food web contamination. The method developed here quantifies Pseudo-nitzschia spp. at low abundances in natural samples, thereby, providing a method to improve our understanding of the environmental conditions leading to blooms of these species. This has been accomplished previously by techniques for identification and quantification that are slow and laborious compared to qPCR. The approach was successfully tested and validated using eight species of Pseudo-nitzschia and 33 non-target organisms and employed to follow local bloom dynamics.     

Estimating total abundance of a large temperate-reef fish using visual strip-transects

Total abundance estimates for the large, common, reef fish Cheilodactylus spectabilis (Hutton) were obtained for a marine reserve and adjacent section of coast in north-eastern New Zealand during 1985. Visual strip-transects were used to estimate abundance and size structure in both areas. The accuracy, precision and cost efficiency of five transect sizes (500, 375, 250, 100, 75 m²) were examined over three times per day (dawn, midday and dusk), by simulating transects over mapped C. spectabilis populations. Two transect sizes showed similarly high efficiency. The smaller of the two (20x5 m) was chosen for the survey because of the general advantages attributable to small sampling units. Biases related to strip-transect size are discussed. Preliminary sampling indicated that C. spectabilis was distributed heterogeneously, and that density was habitat-related. An optimal stratified-random design was employed in both locations, to obtain total abundance and size-structure estimates. This reduced the between-habitat source of variability in density. The total number of sampling units used was governed by the time available. The resulting total abundance estimates obtained were 18 338±2 886 (95% confidence limit) for the 5 km marine reserve, compared to 3 987±1 117 for an adjacent, heavily fished 4 km section of coast. When corrected for total area and habitat area sampled, this represented a 2.3-fold difference in abundance. If sampling had been designed to detect an arbitrary 10% difference in abundance within each habitat, an infeasible 440 h of sampling would have been required. Size-frequency distributions of C. spectabilis at the reserve had a larger model size class than distributions from the adjacent area. The data suggest that reserve status is causal in these differing abundance and size structure estimates.     

Tiger shark (Galeocerdo cuvier) abundance and growth in a subtropical embayment: evidence from 7 years of standardized fishing effort

The tiger shark (Galeocerdo cuvier Peron and Lesueur 1822) is a widely distributed predator with a broad diet and the potential to affect marine community structure, yet information on local patterns of abundance for this species is lacking. Tiger shark catch data were gathered over 7 years of tag and release research fishing (1991–2000, 2002–2004) in Shark Bay, Western Australia (25°45′S, 113°44′E). Sharks were caught using drumlines deployed in six permanent zones (~3 km² in area). Fishing effort was standardized across days and months, and catch rates on hooks were expressed as the number of sharks caught h⁻¹. A total of 449 individual tiger sharks was captured; 29 were recaptured. Tiger shark catch rate showed seasonal periodicity, being higher during the warm season (Sep–May) than during the cold season (Jun–Aug), and was marked by inter-annual variability. The most striking feature of the catch data was a consistent pattern of slow, continuous variation within each year from a peak during the height of the warm season (February) to a trough in the cold season (July). Annual growth rates of recaptured individuals were generally consistent with estimates from other regions, but exceeded those for populations elsewhere for sharks >275 cm fork length (FL), perhaps because mature sharks in the study area rely heavily on large prey. The data suggest that (1) the threat of predation faced by animals consumed by tiger sharks fluctuates dramatically within and between years, and (2) efforts to monitor large shark abundance should be extensive enough to detect inter-annual variation and sufficiently intensive to account for intra-annual trends.     

High mortality in a copepod population caused by a parasitic dinoflagellate

Infection of copepods by parasitic dinoflagellates has been known for many years, but the ecological consequences of this parasitism have been largely neglected. We estimated mortality rates in the copepodParacalanus indicus Wolfenden due to parasitism by the dinoflagellateAtelodinium sp. by applying laboratory mortality rates to a field population of infected copepods in Port Phillip Bay, Australia, sampled in 1982–1985. Adult female copepods were most often infected, with an incidence of 0 to 28.5% (median 6.2%). Stage V female copepodites were less often infected, and males were never infected. The median mortality rate in females was about 7% d^–1, or about one-third of total mortality, and the maximum was 41% d^–1. The frequent occurrence of dinoflagellate parasitoids in some species of copepod implies an important, species-specific mechanism for the regulation of populations.     

Evaluation of the effect of antimicrobials in marine cultures,using the copepod Acartia tonsa as a bioindicator

Cultures of the copepod Acartia tonsa are used both in aquaculture and ecotoxicology studies. However, the cultivation of these crustaceans at high densities results in the proliferation of microorganisms that can affect the organisms of interest, leading to illness or death. Antimicrobials inhibit microbial growth and may favour the cultivated species, aiding the development of ecological studies. This study investigated the potential of antimicrobials (antibiotic + antifungal) to inhibit bacteria and fungi when applied to marine zooplankton cultures, using the copepod A. tonsa as a bioindicator of acute toxicity. Treatment with 0.025?g?L^?1 of penicillin G potassium + 0.08?g?L^?1 of streptomycin sulphate + 0.04?g?L^?1 of neomycin sulphate + 0.005?g?L^?1 of nystatin resulted in 95% bacterial inhibition (after 12?h of exposure); however, after this time, the inhibitory effect was lost. The antimicrobial combination tested in this study prevented colonisation by fungi until 168?h after exposure, without causing acute toxicity to A. tonsa. Thus, it has potential for use in marine cultures of less sensitive organisms.     

Late winter abundance and distribution of phaeodarian radiolarians,other large protozooplankton and copepod nauplii in the Weddell Sea,Antarctica

Large (>64 µm) protozooplankton (radiolarians, thecate dinoflagellates, foraminiferans, and tintinnid ciliates) and copepod nauplii were sampled at six stations located in sea-ice-covered waters, using a multinet at five depth intervals between the surface and 1000 m depth during a late winter expedition of R.V. Polarstern on a west-east transect across the Weddell Sea in 1989. Large (>400 µm) phaeodarian radiolarians contributed more than 98% of the carbon in the large protozooplankton fraction; large protozooplankton represented ca. 38% of the total protozooplankton carbon during late winter under the close pack ice in the upper 250 m. Large protozooplankton may therefore be an important food source for small particle-feeding zooplankton in the upper 250 m, and a modest food source down to 1000 m. Phaeodarian species distributions showed a distinct vertical pattern. Except for spumellarian and nassellarian radiolarians all other protozooplankton (foraminiferans, thecate dinoflagellates, tintinnids) were most abundant in the upper 250 m. The horizontal variation showed a western, central and eastern pattern most probably due to the differing regional hydrography. Feeding ecology of the dominant species of phaeodarians was examined using transmission electron microscopy of contents of feeding vacuoles. The 58 specimens were trophic generalists. The predominance of silica fragments and amorphous material in vacuoles and the similarity of vacuole contents of specimens from three depth zones suggest that phaeodarians in late winter may have fed on organic aggregates.Publication no. 469 of the Alfred-Wegener Institute for Polar and Marine Research     

The mutualism-parasitism continuum in ectomycorrhizas: a quantitative assessment using meta-analysis

Context dependency is deemed to position the outcomes of species interactions along a continuum of mutualism to parasitism. Thus, it is imperative to understand which factors determine where a particular interspecific interaction falls along the continuum. Over the past 20 years research on the ectomycorrhizal symbiosis has resulted in sufficient independent studies to now generalize about the factors and mechanisms that affect host response to ectomycorrhizas. Using meta-analysis we quantitatively evaluated the role of biotic (partner identity and colonization levels of ectomycorrhizal fungi) and abiotic (phosphorus levels) factors in determining host biomass, height, and shoot:root responses to ectomycorrhizal associations. On average, seedlings across multiple host genera increased in total biomass when inoculated with ectomycorrhizal fungi regardless of the identity of the fungal associate; host genera differed in the magnitude of response for both total biomass and shoot:root ratio. Association with different fungal genera modified only host allocation of biomass to shoots and roots. Neither level of colonization on inoculated seedlings nor the level of contamination on control seedlings relative to colonization levels by target fungi on inoculated seedlings was important in explaining variation in effect sizes for any growth response. None of our proposed factors (identity of partners, colonization level, magnitude of contamination, or duration of association) explained variation in effect sizes for shoot height, although in general seedlings were taller when inoculated with ectomycorrhizal fungi. Phosphorus additions did not influence effect sizes. Although the general trend across studies was for a positive response of hosts to ectomycorrhizal inoculation, publication bias and methodological issues effectively reduce and distort the spectrum on which we evaluate host responses to ectomycorrhizal inoculation. Our results indicate that the variation in ectomycorrhizal fungi perceived by the host may be of a discrete (presence/absence of ectomycorrhizal fungi) rather than continuous nature (variation in identity or abundance of ectomycorrhizal fungi).     

Calanoid copepod escape behavior in response to a visual predator

Calanoid copepods typically exhibit escape reactions to hydrodynamic stimuli such as those generated by the approach of a predator. During the summers of 2000, 2001 and 2004, two small calanoid species, Temora turbinata Dana, 1849 and Paracalanus parvus Claus, 1863 were exposed to a visual predatory fish, the blenny Acanthemblemaria spinosa Metzelaar, 1919, and their predator–prey interactions were recorded using both high-speed and standard videographic techniques. Copepod escape reaction components, including swimming pattern, reactive distance, turning rate, and jump kinetics, were quantified from individual predation events using motion analysis techniques. Among the observed escape reaction components, differences were noted between the species’ swimming patterns prior to attack and their response latencies. Temora turbinata was a continuous cruiser and P. parvus exhibited a hop-and-sink swimming pattern. During periods of sinking, P. parvus stopped beating its appendages, which presumably reduced any self-generated hydrodynamic signals and increased perceptual abilities to detect an approaching predator. Response latency was determined for each copepod species using a hydrodynamic stimulus produced by a 1 ms acoustic signal. Response latencies of T. turbinata were significantly longer than those of P. parvus. Despite some apparent perceptual advantages of P. parvus, the blenny successfully captured both species by modifying its attack behavior for the targeted prey.