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     

Comparison of four methods to estimate meiobenthic copepod Amonardia normani ingestion rates

Four different methods were used in the control conditions of laboratory to estimate the ingestion rate of a female meiobenthic harpacticoid copepod Amonardia normani: (1) reduction of algal biomass, (2) the quantification of total pigments in fecal pellets, (3) the gut fluorescence method, (4) the percentage of assimilation and the total egestion rate. The food used during all experiments was the diatom Nitzschia constricta in an axenic condition at the concentration of 0.13 μg Chl-a mL^?1 at stationary growth phase. All experiments were made at 20 °C and 30 salinity. All tested methods excepted the quantification of total pigments in fecal pellets resulted in similar estimatives. The gut fluorescence method indicated that during the day gut contents are smaller than during the night but the gut passage time was faster, resulting in similar ingestion rates during the day and the night. The reduction of algal biomass and the percentage of assimilation and the total egestion rate also indicated similar ingestion rates in the day and in the night. The daily ingestion rate represents 107 % of female carbon weight per day (903 ng C cop^?1day^?1).     

Ontogenetic feeding shifts in the meiobenthic harpacticoid copepod Nitocra lacustris

¹⁴C-radiolabelling experiments indicate that adult stages of the salt-marsh harpacticoid copepod Nitocra lacustris (Schmankevitsch) receive a large part of their nutrition through the ingestion and assimilation of certain diatoms. An abundance of empty diatom frustules occurs in the gutpellet contents of field-collected individuals. Naupliar stages do not ingest diatoms in the laboratory, and nauplii from the field do not contain frustules in their gut pellets. Ingestion of diatoms in the laboratory first occurs during the second or third copepodite stage. ³H-radiolabel expeiments and grazing experiments using bacterium-sized beads adhering to the diatoms indicated that both adults and nauplii ingest bacteria adhering to the outer mucus coating of the diatoms (and probably ingest the diatom mucus itself). Adults ingest bacteria (and probably mucus exopolymer) coincidently while ingesting diatoms. The nauplii ingest these components by scraping the outer surface of the diatoms. SEM observations indicate that diatoms are not punctured by the nauplii during feeding. While diatom mucus and associated bacteria play an (as yet unquantified) role in the nutrition of the adults, these components may comprise the bulk of food resources for naupliar stages.     

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.     

Effects of feeding experience in the copepod Eucalanus pileatus: a cinematographic study

High-speed microcinematography was used to examine the effects of prior experience with particular cell types on the feeding efficiency of a calanoid copepod. Female Eucalanus pileatus were fed monocultures of either the 5-m diatom Thalassiosira pseudonana or the 11-m diatom T. weissflogii during a 2-to 3-d preconditioning period. The smaller diatoms are accumulated passively by the second maxillae while the larger diatoms are detected and actively captured as individual cells. Four females from each preconditioning culture were transferred to a monoculture of the large cells and their behavior filmed at five intervals over a 24-h period to determine whether a loss of efficiency occurs when the copepods must shift capture modes. Ingestion rates for females experienced with the larger cells were approximately 2.5 times higher than those of inexperienced females. Six sequential behavioral steps in the feeding process could alter ingestion rates: (1) amount of time spent flapping the feeding appendages. (2) rate of flapping of the feeding appendages, (3) ability to detect individual cells, (4) success rate of capture attempts, (5) capture and handling time per cell and (6) rejection rate of captured cells. An increased ability to detect cells and a decreased rejection rate contributed significantly to the higher ingestion rate of experienced feeders, indicating that copepods have the ability to learn during the feeding process. Grazing rates may be seriously underestimated in experiments which do not include a preconditioning period, especially those which calculate ingestion over short time intervals. Such effects may also influence the feeding of copepods in the field when encountering changes in particle spectra through vertical migration or horizontal displacement.     

Toxin accumulation and feeding behaviour of the planktonic copepod Calanus finmarchicus exposed to the red-tide dinoflagellate Alexandrium excavatum

The planktonic copepod Calanus finmarchicus is a dominant member of the zooplankton community in the lower St. Lawrence Estuary in eastern Canada. Blooms of the toxic marine dinoflagellate Alexandrium excavatum which produces high cellular levels of paralytic shellfish poisoning (PSP) toxins, occur during the period of high C. finmarchicus production in summer in this region. To study the feeding behaviour of C. finmarchicus in the presence of Alexandrium spp., experiments were conducted in which female adult copepods collected from the St. Lawrence Estuary between May and September 1991 were exposed under controlled conditions to two toxic isolates of A. excavatum (Pr18b and Pr11f) from the estuary and to a non-toxic control (PLY 173) of a closely related species, A. tamarense isolated from the Tamar Estuary, Plymouth, U.K. Clearance rates on non-toxic A. tamarense cells averaged 5.5 ml ind^-1 h^-1 but were nearzero with either toxic isolate. When presented with a mixture of A. excavatum and the non-toxic diatom Thalassiosira weissflogii in varying proportions, C. finmarchicus fed upon the diatom but avoided the toxic dinoflagellate. Although feeding rates on A. excavatum were very low, toxin analysis by high-performance liquid chromatography with fluorescence detection (HPLC-FD) revealed that the PSP toxins were accumulated in copepods exposed to toxigenic dinoflagellates.The toxin composition in copepods was similar to that of the toxic dinoflagellate, but not necessarily identical, particularly after short-term (2-h) exposure, when relatively elevated levels of N-sulfocarbamoyl toxins were detected. The evidence suggests that C. finmarchicus ingests toxic dinoflagellate cells, either mistakenly or during exploratory bouts of feeding, and accumulates PSP toxins in its gut system and perhaps in other tissues.     

A method for the determination of copepod feeding rates during short time intervals

An experimental technique for measuring the feeding behavior of copepods on small time scales is important because grazer/micro-patch interactions in the ocean are probably short (minutes to hours). By placing copepods with empty guts into a known food concentration and monitoring the rate of increase in gut contents until defecation begins, usually 20 to 100 min, the volume of water swept clear of particles per min can be calculated. This process is easily quantified by using phytoplankton as food and measuring the gut contents by fluorometry. The utility of this technique is demonstrated in the laboratory and at sea.     

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.     

Discriminate feeding of the calanoid copepod Acartia clausi in mixtures of phytoplankton and inert particles

Adult female Acartia clausi were allowed to feed in the laboratory on each of three algae, the small green alga Dunaliella tertiolecta (5.6 m ESD), the diatoms Thalassiosira decipiens (13.4 m ESD) and T. nordenskioldii (17.7 m ESD), singly or in mixtures with polystyrene beads (15.7 m ESD). The ingestion rate on beads was much lower than that on cells, even when the supply of beads was one order of magnitude higher than that of cells. Beads offered singly were not ingested. In the experiments with T. nordenskioldii, the more cells adult females ingested, the higher the ingestion rate on beads. In addition, a linear regression equation describing the relationship between bead concentration and ratio of ingested beads to ingested cells was highly significant. These results suggest that beads are ingested by chance in feeding bouts on T. nordenskioldii. The bead interference on the ingestion rate on cells was observed in the experiments with D. tertiolecta and T. decipiens, but not with T. nordenskioldii. In a mixture of beads and T. decipiens, juvenile A. clausi discriminated beads from cells more successfully than adults. The presence of beads did not affect the ingestion rate of juveniles on cells. Starved adult females tended to ingest more beads than well fed individuals. The possible effects of body size and hunger on the discriminate feeding of copepods are discussed.     

Observations of copepod feeding and vertical distribution under natural turbulent conditions in the North Sea

We present results of simultaneous measurements of turbulent-dissipation rate, zooplankton vertical distribution and copepod gut pigments in the northern North Sea. Analysis shows that some, but not all, copepods (by species, sex and stage) exhibit significant dependence on turbulence in respect to vertical distribution and feeding rate. Oithona similis (female and copepodite stages) exhibits an avoidance of the surface layer when turbulence is strong there. For the range of turbulence (10⁻⁷ to 10⁻³m² s⁻³) and ambient chlorophyll concentration (0.5–0.8 μg l⁻¹) encountered, Calanus spp. and Metridia lucens exhibited a significant negative response in feeding-rate index with increasing turbulence. Centropages typicus and Pseudocalanus spp. also exhibited a negative response but of less significance. Received: 12 October 2000 / Accepted: 11 December 2000     

Distribution and feeding of the carnivorous copepod Paraeuchaeta norvegica in habitats of shallow prey assemblages and midnight sun

Paraeuchaeta norvegica was found to be widely distributed in the Norwegian Sea. They were least abundant in north-western areas, but otherwise no clear horizontal patterns appeared with respect to latitude, longitude or water mass. Females and males had similar vertical distributions. The highest concentrations of adults occurred at 400-500 m depth; they largely avoided the upper 50-100 m, even at night. Stages CIV and CV lived shallower in the water column than the adults, with the highest concentration between 100 and 300 m. Stages CII-CIII were most abundant at 50-100 m, while CI was distributed slightly deeper (maxima at 100-200 m). Potential prey were most abundant in the upper 100 m; i.e. shallower than P. norvegica. Numbers of fecal pellets produced by freshly collected adult females were relatively low (estimated at 0.7 pellets per individual on average for the entire sea), with maximal numbers for individuals captured in shallow waters. This suggests food limitation during summer, when food is concentrated in upper waters, and short and light nights limit nocturnal access to the shallow food resources. Pellets mainly contained copepod remains.     

Specialized feeding mechanism in the pelagic copepod genus Heterorhabdus (Calanoida: Heterorhabdidae), with special reference to the mandibular tooth and labral glands

Scanning and transmission electron microscopy and gut-content analysis revealed the fine structure of the mandibular ventralmost tooth and labral glands of the carnivorous copepods Heterorhabdus spp. on the basis of the specimens collected in the waters near Nansei Islands, Southwestern Japan, in November 1993. The ventralmost tooth is hollow with a subterminal pore and a basal opening. The distal region of the tooth is like a hypodermic needle and is strengthened by opal layers. The tip of the tooth including the subterminal pore usually protrudes from between the labrum and the paragnath, while the basal region of the ventralmost tooth and the whole parts of the other teeth are semi-enclosed in the preoral food chamber formed by the labrum and the paragnath. There is a large glandular cell in the basal region of the mandibular gnathobase, but no direct connection exists between the gnathobasal cells and the tubular lumen of the ventralmost tooth. The posterior surface of the labrum carries a lateral pair of large glandular pores, which are located close to, and appear to fit exactly the basal openings of the ventralmost teeth. Each of these glands has two massive secretory cells, and the secretory materials from these cells appear to be mixed before discharge from the pore. A specialized feeding mechanism is proposed, wherein venom or anaesthetic is injected from the labral-gland pores into the tubular lumen of the mandibular ventralmost tooth, and then relcased into the prey from its subterminal pore immediately after capture using the stout maxillae of prey macrozooplankters such as copepodids and polychaetes.     

亚硝态氮胁迫下菲律宾蛤仔实时定量P CR内参基因的筛选

为了更好地从基因角度研究亚硝态氮对贝类的生态毒性,需要筛选一个合适的内参基因作为参考,对其毒性相关基因进行定量表达分析。本研究以菲律宾蛤仔(Ruditapes philippinarum)作为研究对象,以亚硝态氮胁迫的鳃组织cDNA作为模板,利用geNorm软件和NormFinder软件对6个候选内参基因进行表达稳定性分析,并对2个结果做了综合赋权分析。结果显示,geNorm分析的基因表达稳定性结果为ActinCyPAEF1αUbi18STubu,NormFinder软件分析的结果为ActinUbiCyPAEF1α18STubu,最后经加权赋值法综合分析获得6个候选基因的表达稳定性为ActinCyPAUbiEF1α18STubu,即Actin为表达最稳定的基因。因此,Actin可以作为衡量亚硝态氮胁迫后菲律宾蛤仔鳃组织中基因表达的内参基因。     

Quantitative feeding ecology of the hydromedusan Nemopsis bachei in Chesapeake Bay

We determined feeding rates of the hydromedusan Nemopsis bachei L. Agassiz in the mesohaline region of Chesapeake Bay, USA during the spring of 1989 and 1990 from gut contents, digestion rates and abundances of medusae and zooplankton. The medusae consumed primarily copepodites of Acartia tonsa, selecting against naupliar stages. The peak abundance of N. bachei medusae was in April to May, when densities averaged more than 10 m^-3. Medusa densities were similar in both years, but were greatest (maximum of 132 medusae m^-3) along a southern transect sampled only in 1990. At peak densities, N. bachei medusae consumed 30% d^-1 of the copepodite standing stocks, but they consumed <1% d^-1 at the lower densities typical of late May or early June. The predation effects were generally greater than those reported for other hydromedusan species. But even at peak predation, N. bachei medusae could not have controlled or reduced A. tonsa copepod populations, which had a production rate of 85% d^-1 at that time. Medusa feeding rates were highest at nighttime, and were correlated with prey density in the field, but not in the laboratory.Communicated by J. Grassle, New Brunswick     

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.     

抗除草剂转基因作物实时荧光定量PC检测

建立了一种以SYBR Green Ⅰ为结合染料、快速准确检测转抗除草剂基因成分的实时荧光定量PCR方法.以转基因大豆与转基因玉米标准品为材料,通过使用特异性引物和SYBR Green Ⅰ结合染料实时荧光定量PCR技术,对转基因农作物中外源抗除草剂基因进行了定量检测,绘制了两种基因扩增的标准曲线图,根据标准曲线方程计算外源基因含量;并作了溶解曲线、检测方法检测灵敏度和精密度的分析.研究发现,两者标准曲线方程线性关系良好.R~2 值分别达到0.993 9与0.992 4.通过已知标准品进行验证,实测值与真值接近,与实际含量的相埘偏差是6.52%和7.90%.结果表明,SYBR Green Ⅰ结合染料法完全可以用于转基因农作物定量PCR检测.图5表2参11     

基于分子信标探针的荧光定量PCR方法检测转基因食品

选择了内源基因大豆植物凝集素(lectin)、玉米转化酶(invertase)和外源基因花椰菜花叶病毒35S(CaMV35S)启动子的分子信标探针,确定了探针浓度和镁离子浓度等反应条件,分别对转基因大豆和转基因玉米系列标准品进行内源基因和外源基因的荧光PCR扩增,在PCR反应过程中分别以两种荧光通道信号分别追踪同一样品DNA内源基因和外源基因的扩增动力学变化,并依此绘制了循环阈值与转基因食品百分比含量之间的标准曲线,建立了转基因大豆和转基因玉米的分子信标探针-荧光定量PCR检测方法,该方法具有特异性强、敏感性高的特点,实现了对转基因食品的定量分析.图7表1参11     

Evaluation of the copepod Tigriopus californicus as a bioassay organism for the detection of chemical feeding deterrents produced by marine phytoplankton

Marine phytoplankton have been shown to use chemical feeding deterrents to reduce or inhibit zooplankton grazing. In order to screen phytoplankton species for feeding deterrent production and to isolate and identify feeding deterrent compounds, a new, rapid, and reliable laboratory bioassay was developed. This bioassay used the laboratory-reared harpacticoid copepod Tigriopus californicus and measured inhibition of feeding by measuring the fecal pellet production rate. The bioassay was capable of detecting deterrent compounds: (1) adsorbed onto ground fish food (a normally palatable food); (2) dissolved in a mixture of seawater and live Thalassiosira pseudonana cells (a species of diatom which had no feeding deterrent activity); and (3) present in live cell cultures. Method (2) was recommended for use in bioassay-guided fractionation (isolation of chemical compounds), as it was reliable, rapid, accurate, and easy to perform with large numbers of samples. The total bioassay time was < 48 h, and data collection required only a microscope. Methanolic cell extracts of several phytoplankton species were screened for feeding deterrent activity. Extracts from the diatom Phaeodactylum tricornutum and the dinoflagellate Gonyaulax grindleyi gave feeding deterrent responses, while extracts from the diatom Thalassiosira pseudonana gave no feeding deterrent responses. Live P. tricornutum cells deterred feeding at densities of 6x10⁵ cells ml^-1. This bioassay should provide a valuable tool in screening phytoplankton for feeding deterrent compounds and determining the chemical nature of these compounds.     

A method to estimate pre-exploitation population size

Antarctic fur seals (Arctocephalus gazella) were commercially exploited on the subantarctic island of South Georgia for over 100 years and nearly driven to extinction. Since the cessation of harvesting, however, their populations have rebounded, and they are now often considered a nuisance species whose impact on the terrestrial landscape should be mitigated. Any evaluation of their current population requires the context provided by their historic, pre-exploitation abundance, lest ecologists fall prey to shifting baseline syndrome in which their perspective on current abundance is compared only with an altered state resulting from past anthropogenic disturbance. Estimating pre-exploitation abundance is critical to defining species recovery and setting recovery targets, both of which are needed for the International Union for the Conservation of Nature's recent efforts to develop a green list of recovering species. To address this issue, we reconstructed the South Georgia fur seal harvest from 1786 to 1908 from ship logbooks and other historical records and interpolated missing harvest data as necessary with a generalized linear model fit to the historical record. Using an approximate Bayesian computation framework, harvest data, and a stochastic age-structured population model, we estimated the pre-exploitation abundance of Antarctic fur seals on South Georgia was 2.5 million females (95% CI 1.5–3.5 million). This estimate is similar to recent abundance estimates, and suggests current populations, and the ecological consequences of so many fur seals on the island, may be similar to conditions prior to human harvest. Although the historic archive on the fur sealing era is unavoidably patchy, the use of archival records is essential for reconstructing the past and, correspondingly, to understanding the present. Article impact statement: Defining species recovery requires an understanding of baseline population state, which can be estimated through statistical methods.     

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