Finding copepod footprints: a protocol for molecular identification of diapausing eggs in lake sediments

Even though calanoid copepods produce diapausing eggs that stay alive in lake sediments, these eggs have rarely been used paleolimnologically, as they lack diagnostic morphological features. In this study, we developed a method to identify copepod diapausing eggs in Japan as a clue toward reconstructing past plankton populations. We first determined a 28S ribosomal DNA (rDNA) (i.e., nc28S) regional sequence library (240 bp) of various calanoid copepod species using ethanol-fixed plankton samples collected from across the Japanese archipelago. Then we applied the UltraSHOT method to extract DNA from an individual diapausing egg. Finally, the nc28S region of diapausing eggs collected from various lakes was sequenced and compared with the regional library for species identification. In total, 21 haplotypes of the nc28S region were recovered from planktonic samples of 11 Japanese freshwater calanoid copepod species. Despite the short length of this region, no identical haplotypes were shared among the species analyzed, including the Acanthodiaptomus pacificus complex treated as a species. Even different lineages of A. pacificus could be separated. These results indicate that the nc28S region can be used as a barcode in Japan. A total of 112 diapausing eggs collected from various lakes and ponds was processed, and the nc28S region of each was successfully sequenced. All of these egg sequences matched one or the other of the nc28S haplotypes in the regional library mentioned above. The set of protocols we applied (i.e., preparing a comprehensive regional sequence library and sequencing egg DNA) is thus useful for involving copepod diapausing eggs in paleolimnological studies in lakes. The nc28S region treated in this study has a strong potential to uncover the paleodiversity of copepods, at least in Japan.     

Spatial and temporal abundance patterns for the late stage copepodites of Metridia lucens (Copepoda: Calanoida) in the US northeast continental shelf ecosystem

The annual cycle of abundance for the calanoid copepod Metridialucens and its interannual variability are described from 17611 samples collected on 193 broad-scale plankton surveys ofthe US northeast continental shelf region from 1977 to 1999.The copepod’s seasonal distribution is illustrated withbi-monthly geographic abundance plots. Abundance of M. lucensis compared with various environmental variables, abundanceof the copepod Calanus finmarchicus, density of potential predatorsand the North Atlantic Oscillation (NAO) index. Metridia lucensabundance increased throughout the ecosystem during spring warmingand usually reached peak levels in late spring. The highestmean densities were measured in the Gulf of Maine (GOM), wherethe copepod is usually found year round and exhibits a weak,decreasing west to east abundance gradient. The less dense populationslocated on Georges Bank and within the Middle Atlantic Bightare characterized by year-round onshore to offshore gradientsthat strengthen during the summer months when the copepod concentratesoffshore where there is a cool layer of subthermocline water,the region often referred to as the cold pool. Interannual variabilityin abundance showed two persistent periods: above average valuesfrom 1979 to 1982, followed by very low levels from 1983 toat least 1987. Metridia lucens abundance in the 1990s has usuallybeen at or above time series means. None of the environmentalvariables measured during the surveys could be correlated tothis variability. The copepod’s abundance in the GOM wasnot related to variation in the NAO index or to changes in predatorabundance found in the region. The seasonal abundance cycleand interannual variability of M. lucens in the GOM were foundto be very similar to those found for the dominant copepod,Calanus finmarchicus. Reasons for this coupling are discussed.     

Copepod production estimated by combining in situ data and specific temperature-dependent somatic growth models

Although growth in adult copepods is frequently assumed to be similar to juvenile growth, some evidence have pointed out that under in situ conditions, it can be lower, with, as a consequence, underestimation of secondary production. In addition, under field conditions, juvenile growth in copepods is close to maximum rates estimated at food-saturated conditions. Based on previous assumptions, this study aimed to test the applicability of a new approach for copepod production estimate, derived from temperature-dependent growth models and in situ data, such as seawater temperature and copepod biomass. For this purpose, site-specific copepod juvenile growth models, defined for Acartia tonsa and A. clausi populations from a Southern European estuary (Canal de Mira, Ria de Aveiro, Portugal), were used and copepod biomass was taken from zooplankton samples collected during 2 years at six sampling stations. By comparing the obtained results with published data, the feasibility of the approach for copepod secondary production estimates and its applicability in worldwide marine ecosystems was confirmed. Future studies should combine the estimates of adult and juvenile production in order to evaluate its relative contribution and to obtain a more precise estimate of secondary production.     

Intrashoot distributions of leaf dwelling harpacticoid copepods on the seagrass Zostera marina L.: implications for sampling design

The distribution of leaf dwelling harpacticoid copepods within seagrass (Zostera marina L.) shoots was investigated on four dates in 1986 and 1987. Copepods were found to be non-uniformly distributed on shoots, with higher abundances observed on older leaves. Patterns of abundance within shoots could not be explained by the surface area of individual leaves except on the sampling date with the highest copepod densities. It is suggested that harpacticoid copepod distributions on seagrass shoots are primarily determined by the pattern of epiphytic biomass. However, at high population densities, habitable surface area may be the limiting factor. Increased habitat complexity at high epiphyte loads does not seem to be the cause of the copepod distributions observed in this study. An accurate method for estimating seagrass copepod abundance per unit sediment area using intrashoot distributions is described and compared to existing methodology.     

Salinity tolerance of the copepod Apocyclops dengizicus (Lepeschkin, 1900), a key food chain organism in the Salton Sea,California

The copepod Apocyclops dengizicus is a key item in the food chain of the Salton Sea where the salinity is currently 45 g 1^–1. The salinity of the Salton Sea may reach 90 g 1 ^–1 within the next 20 years. This study examined the salinity tolerance of this copepod.Large copepodite and adult A. dengizicus were introduced into various salinities with and without acclimation. The 96 h LC₅₀ without acclimation was 101 g 1^–1. Mortality (at 96 h) without acclimation was low at salinities of 90 g 1 ^–1 or less.Copepod cultures were maintained, with successful reproduction of at least one new generation, at salinities of from 0.5 to 68 g 1 ^–1 for at least 120 days. Copepods maintained at higher salinities, up to 79 g 1 ^–1, remained alive up to 90 days, but a new generation was not produced. In laboratory studies of larval production and survivorship, few nauplii were released at salinities of 68 g 1 ^–1 or higher, and none survived to the copepodite stage.     

Assessment of the importance of fish predation versus copepod predation on life history traits of Daphnia hyalina

Daphnia hyalina is a cladoceran present throughthe whole year except for late summer in Maranhão,a meso-eutrophic reservoir in central Portugal. Apartfrom the influence of food, both vertebrate andinvertebrate predation pressures seem to have aneffect on D. hyalina population dynamics.Enclosure experiments were designed to assess therelative importance of both types of predation. Afterthe summer crash, D. hyalina reached highernumbers in the fishless enclosures than in the lakedespite of high predation pressure upon juveniles byAcanthocyclops robustus. Fish predation upon thelargest individuals, especially large egg bearingfemales, was responsible for the lower fertility ofthe open water population when compared with theenclosure population. In the enclosures an increase intail spine length was observed. The longer tail spineprobably offered protection from copepod predation,allowing at least some of the juveniles to coexistwith their potential predator and reach the adultstage, less susceptible to copepod predation.     

DMSP-consuming bacteria associated with the calanoid copepod Acartia tonsa (Dana)

DMSP-consuming bacteria (DCB) were recovered from the body and fecal pellets of the copepod Acartia tonsa (Dana). The most probable number of DCB associated with starved A. tonsa was 9.2x10(2) cells copepod(-1). The abundance of DCB recovered from the copepod body increased to 1.6-2.8x10(4) after the copepod fed on DMSP-containing alga. DCB abundance associated with fecal pellets averaged 1.2x10(4) cells pellet(-1). In enrichment cultures, the DCB grew with a doubling time of 1.1-2.9 days, and consumed DMSP at a rate of 4.5-7.5 fmol cell(-1) day(-1). The apparent DMSP-to-DMS conversion efficiency was 25-41% for DCB from copepod body, and 99% for DCB from fecal pellets. Our study demonstrated that copepods and their fecal pellets may harbour dense populations of DCB, and that the copepod-bacteria coupling represents a novel mechanism for DMSP consumption in the water column.     

Copepod communities related to water masses in the southwest East China Sea

The East China Sea is characterized by a complex hydrographic regime and high biological productivity and diversity. This environmental setting in particular challenged a case study on the use of mesozooplankton community parameters as indicators of water masses. In order to reveal spatial patterns of zooplankton communities during summer, a large scale oceanic transect study was conducted. Two transects were taken in the southwest East China Sea region, covering for the first time the China shelf, slope, and the estuaries of the Yangtze river and of the Minjiang river, the northern Taiwan Strait, and the Kuroshio Current region. A total of 77 copepod species were quantified. Copepod abundance was significantly higher in the estuary of the Yangtze River runoff mixture waters and lowest at the Kuroshio Current Region. The calanoid Parvocalanus crassirostris was the most frequently occurring and abundant species retrieved from 27 samples of a total of 39 samples. The use of multivariate cluster analysis separated the Mainland China Shelf from the northern Taiwan Strait and the Kuroshio Current Region at the first hierarchical level. The use of an indicator value method (IndVal) associated with each cluster of stations revealed characteristic species assemblages. Two hierarchical levels defined 4 assemblages within geographical sectors representing copepod assemblages of the Kuroshio Current Region, of the northern Taiwan Strait and the southern China Shelf near the estuary of the Minjiang River and northern stations near the estuary of the Yangtze River. Overall, there was a strong correspondence between the distribution of certain copepod species and water masses. Differences between the Mainland China shelf, the northern Taiwan Strait and the Kuroshio Current Region were characterized by differences in species composition and abundance. Water mass boundaries in the study area were exclusively indicated by distinct differences in species composition, emphasizing a correlation between copepod communities and water masses of the southwest East China Sea in summer.     

First record of Pseudomyicola spinosus in Argopecten ventricosus in Baja California, Mexico

This is the first record of the copepod Pseudomyicola spinosus in the scallop Argopecten ventricosus in northwestern Mexico, and describes: (1) the known annual prevalence and intensity of this copepod on scallops from culture sites (Gulf of California) and natural populations (Pacific coast), (2) the histopathological effects caused on the soft tissues of scallops, and (3) the relationship between prevalence and intensity records and environmental parameters. The copepod was present throughout the period of investigation, showing similar prevalence and ratio of copepod to scallop patterns in both cultured scallops and wild specimens from natural habitats. Highest prevalence and ratio values were detected in summer-autumn at both sites, probably because scallops showed a weak condition from the combined effects of spawning, reabsorption of residual gametes, and high temperature. The condition index of A. ventricosus showed a significant correlation with the presence of the copepod in Magdalena Bay (-0.67). P. spinosus was observed in the gills of scallops, producing alterations or rupture of filaments, and in the stomach, causing detachment and loss of the epithelium. No relationship between copepod infestation with temperature, salinity, chlorophyll, and seston were found during the investigation. Although P. spinosus was present year-round at both sites, no association between infestation and scallop mortalities was detected.     

Gyrodinium undulans Hulburt, a marine dinoflagellate feeding on the bloom-forming diatomOdontella aurita, and on copepod and rotifer eggs

The marine dinoflagellateGyrodinium undulans was discovered as a feeder on the planktonic diatomOdontella aurita. Every year, during winter and early spring, a certain percentage of cells of this bloom-forming diatom, in the Wadden Sea along the North Sea coast, was regularly found affected by the flagellate. Supplied with the food diatomO. aurita the dinoflagellate could be maintained successfully in clonal culture. The vegetative life cylce was studied, mainly by light microscopy on live material, with special regard to the mode of food uptake. Food is taken up by a so-called phagopod, emerging from the antapex of the flagellate. Only fluid or tiny prey material could be transported through the phagopod. Larger organelles like the chloroplasts ofOdontella are not ingested and are left behind in the diatom cell. Thereafter, the detached dinoflagellate reproduces by cell division, occasionally followed by a second division. As yet, stages of sexual reproduction and possible formation of resting cysts could not be recognized, neither from wild material nor from laboratory cultures. Palmelloid stages (sometimes with a delicate wall) occurring in ageing cultures may at least partly function as temporary resting stages. The winter speciesG. undulans strongly resemblesSyltodinium listii, a summer species feeding on copepod and rotifer eggs. Surprisingly, in a few cases this prey material was accepted byG. undulans as well, at least under culture conditions. When fed with copepod eggs, the dinoflagellate developed into a large trophont, giving rise thereafter by repeated binary fission to 4, 8 or 16 flagellates, as a result of a single feeding act. A re-examination of both species under simultaneous culture conditions is planned.     

Phylogenetic implications of the Crustacean nauplius

The plesiomorphic mode of crustacean development is widely accepted to be via a larva called the nauplius. Extant taxa like the Cephalocarida, Branchiopoda, Ostracoda, Mystacocarida, Copepoda, Cirripedia, Ascothoracida, Facetotecta, Euphausiacea and Penaeidea hatch from an egg as a free-living nauplius. Other crustaceans show an embryonic phase of development suggestive of a naupliar organization. Several features of the nauplius larva have been proposed as diagnostic characters for the Crustacea: a median (nauplius) eye; at least three pairs of head appendages (antennules, antennae, mandibles); a posteriorly directed fold (the labrum) extending over the mouth and a cephalic (nauplius) shield. The relationship between trilobite protaspis with at least four appendages and the crustacean nauplius remains unclear, but reports of a copepod orthonauplius with four appendages are rejected. Swimming is suggested to represent the underived mode of locomotion for the crustacean nauplius, and that naupliar swimming directly results in naupliar feeding which also is underived.     

Intrusions of the Kuroshio Current in the northern South China Sea affect copepod assemblages of the Luzon Strait

We analyse the influence of the Kuroshio Current on copepod assemblages in the northern South China Sea. The assumption was tested whether predominant current regimes bring marine zooplankton and Copepoda from subtropical and tropical waters to the south of Taiwan. A total of 101 copepod species were identified from 26 families and 48 genera that include Calanoida, Cyclopoida, Harpacticoida and Poecilostomatoida. High copepod abundances in the study area are shown to be caused by both, a year-round Kuroshio Current intrusion and the SW monsoon, prevailing in the South China Sea during summer. Calanus sinicus did not appear in the samples, indicating that there was no cold water mass intrusion in the area during sampling. Both, the intrusion of the Kuroshio Branch Current to the Luzon Strait and the South China Sea circulation may play a more important role in shaping copepod assemblages in the region than hitherto expected. The abundance of copepods was higher above the 50 m isoline than at deeper strata. Species number and the Shannon-Wiener diversity index were higher with increasing depth. Copepod assemblage structure changed with different sampling depth and different sampling areas. Copepod abundance and species richness were higher in the northern South China Sea than in the Kuroshio Current area, and higher at lower latitudes than at higher latitudes. Some indicator species are characteristic for the Kuroshio Current and indicate with others that the study area accomodated water masses from the northern South China Sea as well as from the Kuroshio Current.     

Diapausing fertilized adults

Summary A lake population of the cyclopoid copepod Cyclops strenuus Fischer had a 1-year life cycle including a diapause from June to January. Dormant stages consisted of fertilized females, not previously described as a diapause stage, and a small fraction of cop V. The longevity of the sperm in a diapausing female was at least 6–7 months. Pre-fertilization is assumed to be an adaptive mechanism to assure fertilization when male abundance is low. This life cycle pattern also gives a shorter time-lag between diapause break and offspring development, and also the low total adult population exerts a lower predation pressure on their offspring. The flexibility of life cycle patterns of copepods is emphasized.     

Comparison of the predation rate of freshwater cyclopoid copepod species on larvae of the mosquito Culex pipiens

The predation rates of six copepod species: Acanthocyclops robustus G.O. Sars, Eucyclops neumani Pesta, Macrocyclops albidus Jurine, Mesocyclops longisetus Thibaud, Metacyclops grandis Kiefer and Metacyclops mendocinus Wierzejski (Copepoda: Cyclopidae) on mosquito larvae, Culex pipiens Linnaeus (Diptera: Culicidae) were assessed. Experiments consisted of 24-h incubations of one copepod and 10 larvae without extra food at 16 degrees C and 26 degrees C. Nine replicates were considered for each species and temperature. Predation rates (larvae per copepod per day) were: M. mendocinus (1.8), M. grandis (3.1), E. neumani (3.8), A. robustus (3.8), Ma. albidus (6.1) and Me. longisetus (7.0). There was a significant effect of both species and temperature on predation: all species experienced higher predation at 26 degrees C than at 16 degrees C, except for A. robustus whose predation rate was similar at both temperatures. These observations are consistent with previous results that point to Macrocyclops and Mesocyclops genera as important larval predators and suggest the need for field trials to evaluate the response of Ma. albidus and Me. longisetus under natural conditions in Uruguay.     

Nematode species abundance patterns and their use in the detection of environmental perturbations

If the concepts of biological indices and biomonitoring at the multi-species level are to prove viable, then meiofauna and marine nematodes in particular should be an ideal group with which to test the hypothesis. Many attempts to assess the structure of species assemblages, such as the use of diversity indices, nematode: copepod ratios and the graphical method of log normal plots, have been shown to be theoretically unsound and/or impractical, especially for routine use by extension workers. A method of assessing shifts in dominance patterns which involves all the proportional species abundances is suggested as a better means of comparing diversity. A modified method of rapidly assessing Simpson's dominance-weighted diversity index is also advocated as being of practical use. In combination, they should enable the diversity aspect of the multi-species approach to biomonitoring to be rigorously and exhaustively evaluated.     

Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp. and Temora sp. from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.     

Lake construction has facilitated calanoid copepod invasions in New Zealand

Aim We tested the hypothesis that construction of lakes and ponds has facilitated both inter‐ and intracontinental invasions of calanoid copepod species. Location North Island, New Zealand. Methods We sampled both natural and constructed lakes, ponds and reservoirs for calanoid copepods in the North Island, New Zealand. Species records were supplemented by examining historically collected samples and literature review. Distributions of non‐indigenous calanoid copepod species were compared between constructed and natural waters. Species distributions of native species were compared with the basement terranes (microplates) of the North Island to determine if they possess ‘natural ranges’, and to assess whether construction of new water bodies had altered these distributions. Results Ten calanoid copepod species have been recorded. At least four, and possibly five, of these species are non‐indigenous and were restricted to constructed water bodies. Occurrences in constructed water bodies were not restricted to dammed valleys, but also included ponds constructed on farms, ornamental ponds, disused quarries and retired mines. Four Boeckella species had distributions in natural waters closely related to the North Island basement terranes, and therefore possess ‘natural ranges’ on the island. One species, Boeckella propinqua, was found in natural lakes over a small geographical range only, but has spread with construction of new water bodies to now be widely distributed over the island. Main conclusions Construction of lakes and ponds has facilitated the invasion of calanoid copepod species at both inter‐ and intracontinental scales. Our findings suggest that resident native calanoid copepod species may reduce the risk of invasion to natural water bodies, as similar‐sized species are commonly unable to co‐occur. Spread of the non‐indigenous representatives from constructed into natural waters is inevitable, with established populations providing local propagule supplies for regular introductions.     

Epizootiology of Amblyospora connecticus (Microsporida) in field populations of the saltmarsh mosquito, Aedes cantator, and the cyclopoid copepod, Acanthocyclops vernalis

The natural ecology of a heterosporous microsporidium, Amblyospora connecticus was investigated at three different salt marsh habitats during 1986-1989. The parasite has a well-defined seasonal transmission cycle that occurs regularly each year and intimately involves the primary mosquito host, Aedes cantator, and the intermediate copepod host, Acanthocyclops vernalis. In the spring, the microsporidium is horizontally transmitted from the copepod, where it appears to overwinter, to the mosquito via the ingestion of haploid spores produced in the copepod. Mosquitoes develop a benign infection, and females transmit the microsporidium transovarially to their progeny via infected eggs. Oviposition occurs during the summer and infected eggs hatch synchronously in the fall causing widespread epizootics. Infected larvae die, and the cycle is completed when meiospores are released into the pool and subsequently are eaten by A. vernalis, which reappears in the fall and early winter. Amblyospora connecticus thereby persists by surviving in one of two living hosts throughout most of its life cycle rather than in the extra-corporeal environment. This represents an important survival strategy for A. connecticus as results show the salt marsh habitat to be a relatively unstable environment that is subject to periodic flooding and drying. The adaptive significance of utilizing an intermediate host in the life cycle is discussed as it directly facilitates transmission and enhances survival of the microsporidium.     

Development of a microplate-based fluorescence immunoassay using quantum dot streptavidin conjugates for enumeration of putative marine bacteria, Alteromonas sp., associated with a benthic harpacticoid copepod

Attached bacteria inhabit the surfaces of many marine animals--a process that may play important roles in the survival and transport through aquatic systems. However, efficient detection of these bacteria has been problematic, especially small aquatic animals such as benthic harpacticoid copepod. Quantum dots (QD) have recently emerged as a significant tool in immunofluorescence detection because of their unique properties compared to other fluorescent probes. In the present study, a polyclonal antibody was raised against the Gram-negative marine bacterium, Alteromonas sp. A microplate-based immunofluorescence bioassay using QD strepavidin conjugates was developed for quantifying putative Alteromonas sp. cells located on the surfaces of a marine harpacticoid copepod, Microarthridion littorale. The number of attached Alteromonas sp. was estimated to be 10(2)+/-8 CFU using this method. The QD approach, coupled to a microplate assay can potentially provide an efficient and accurate method for rapidly detecting multiple bacteria species attached to small invertebrate animals because of their unique excitation and emission characteristics.     

海南岛西北沿岸海域浮游桡足类的分布及群落特征

为了解昌江沿岸海域生态系统的现状, 探讨海域环境因素对浮游动物的生存环境造成的影响。本文根据2008年11月至2009年7月在海南西部昌江沿岸水域21个测站、4个季度月调查所获的浮游桡足类样品数据, 对该海域浮游桡足类群落结构、分布、季节变化及影响因素进行了分析。本调查共鉴定出桡足类44种, 隶属4目17科24属, 其中秋季25种, 冬季23种, 春季22种, 夏季23种。本次调查共发现优势种6种, 分别是微刺哲水蚤(Canthocalanus pauper)、亚强次真哲水蚤(Subeucalanus subcrassus)、锥形宽水蚤(Temora turbinata)、刺尾纺锤水蚤(Acartia spinicauda)、椭形长足水蚤(Calanopia elliptica)和精致真刺水蚤(Euchaeta concinna), 优势种以近岸暖水种居多。浮游桡足类丰度季节变化明显: 冬季最高, 达409 ind./m³; 秋季次之, 为144 ind./m³, 春季为55 ind./m³, 夏季最低仅为17 ind./m³。其丰度的平面分布显示: 秋、冬季节分别在海区中部和南部形成明显密集区, 春、夏季节则大致呈现由外海向近岸逐渐递减的趋势。浮游桡足类的多样性指数(H')表现为夏季>春季>秋季>冬季, 春、夏季的均匀度指数(J')明显高于秋、冬季。本调查反映出该海区的桡足类群落具有热带—亚热带区系特征, 种类组成季节更替明显, 桡足类种群受海域水温和硅藻的影响明显, 受盐度影响不明显。