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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., Introduction Copepod nauplii are important members of the marine planktonic community and are often the most abundant component of the microzooplankton (Calbet et al. 2001; Lucic et al. 2003; Turner [...]

Evolutionary diversification of the broadly distributed copepod sibling species complex Eurytemora affinis has been documented in the northern hemisphere. However, the fine scale geographic distribution, levels of genetic subdivision, evolutionary, and demographic histories of European populations have been less explored. To gain information on genetic subdivision and to evaluate heterogeneity among European populations, we analyzed samples from 8 locations from 58° to 45°N and 0° to 23°E, using 549 base pairs of the mitochondrial cytochrome oxidase subunit I (COI) gene. We discovered three distinct lineages of E. affinis in Western Europe, namely the East Atlantic lineage, the North Sea/English Channel (NSEC) lineage, and the Baltic lineage. These geographically separated lineages showed sequences divergence of 1.7-2.1%, dating back 1.9 million years (CI: 0.9-3.0 My) with no indication of isolation by distance. Genetic divergence in Europe was much lower than among North American lineages. Interestingly, genetic structure varied distinctively among the three lineages: the East Atlantic lineage was divided between the Gironde and the Loire populations, the NSEC lineage comprised one single population unit spanning the Seine, Scheldt and Elbe rivers and the third lineage was restricted to the Baltic Proper (Sweden). We revealed high haplotype diversity in the East Atlantic and the Baltic lineages, whereas in the NSEC lineage haplotype diversity was comparatively low. All three lineages showed signs of at least one demographic expansion event during Pleistocene glaciations that marked their genetic structure. These results provide a preliminary overview of the genetic structure of E. affinis in Europe., Author(s): Gesche Winkler [sup.1] , Sami Souissi [sup.2] [sup.3] [sup.4] , Céline Poux [sup.2] [sup.5] , Vincent Castric [sup.2] [sup.5] Author Affiliations: (1) grid.265702.4, 000000012185197X, Institut des Sciences de la [...]

The nature of epistatic interactions between genes encoding interacting proteins in hybrid organisms can have important implications for the evolution of postzygotic reproductive isolation and speciation. At this point very little is known about the fitness differences caused by specific closely interacting but evolutionarily divergent proteins in hybrids between populations or species. The intertidal copepod Tigriopus californicus provides an excellent model in which to study such interactions because the species range includes numerous genetically divergent populations that are still capable of being crossed in the laboratory. Here, the effect on fitness due to the interactions of three complex III proteins of the electron transport system in [F.sub.2] hybrid copepods resulting from crosses of a pair of divergent populations is examined. Significant deviations from Mendelian inheritance are observed for each of the three genes in [F.sub.2] hybrid adults but not in nauplii (larvae). The two-way interactions between these genes also have a significant impact upon the viability of these hybrid copepods. Dominance appears to play an important role in mediating the interactions between these loci as deviations are caused by heterozygote/homozygote deleterious interactions. These results suggest that the fitness consequences of the interactions of these three complex III-associated genes could influence reproductive isolation in this system.

A common feature of interspecific animal and plant hybrids is the uniparental silencing of ribosomal RNA gene transcription, or nucleolar dominance. A leading explanation for the genetic basis of nucleolar dominance in animal hybrids is the enhancer-imbalance model. The model proposes that limiting transcription factors are titrated by a greater number of enhancer-bearing subrepeat elements in the intergenic spacer (IGS) of the dominant cluster of genes. The importance of subrepeats for nucleolar dominance has repeatedly been supported in competition assays between Xenopus laevis and X. borealis minigene constructs injected into oocytes. However, a more general test of the importance of IGS subrepeats for nuclear dominance in vivo has not been conducted. In this report, rRNA gene expression was examined in interpopulation hybrids of the marine copepod Tigriopus californicus. This species offers a rare opportunity to test the role of IGS subrepeats in nucleolar dominance because the internal subrepeat structure, found in the IGS of virtually all animal and plant species, is absent in T. californicus. Our results clearly establish that nucleolar dominance occurs in [F.sub.1] and [F.sub.2] interpopulation hybrids of this species. In the [F.sub.2] generation, nucleolar dominance appears to break down in some hybrids in a fashion that is inconsistent with a transcription factor titration model. These results are significant because they indicate that nucleolar dominance can be established and maintained without enhancer-bearing repeat elements in the IGS. This challenges the generality of the enhancer-imbalance model for nucleolar dominance and suggests that dominance of rRNA transcription in animals may be determined by epigenetic factors as has been established in plants.

We investigated the phylogenetic relationships among different size groups of the pelagic microcopepod Oncaea venusta Philippi, 1843, by comparing the patterns of genetic variation of specimens collected at five locations of the Indo-West Pacific Ocean. Phylogenetic analyses were based on sequence data obtained from two DNA markers: A 310 bp fragment of the mitochondrial cytochrome b (cyt b) gene and a 480 bp fragment of the nuclear internal transcribed spacer 1 (ITS1). The cyt b sequences showed a much higher level of variation than those from ITS1, but the conclusion from both genes was concordant. Four genetic clades could be differentiated. A small- and a large-size group were unambiguously assigned to two distinct clades or lineages. Unexpectedly, the medium-sized individuals could be divided into another two different genetic clades. All four lineages were supported by high bootstrap values. The high levels of sequence divergence under sympatric conditions indicated that at least the two main groups, the large and the small one, may be assigned to different species. For the medium-size group additional morphological studies and more sensitive nuclear markers are required to clarify their taxonomic status., Introduction The application of DNA analytical methods combined with a rising interest in biodiversity has led to the identification of numerous new lineages in morphologically similar taxa in the marine [...]

Portions of the mitochondrial genome (ca. 4 kb), encoding three protein-coding (COI, ND4L, ND6) and two ribosomal RNA (srRNA, lrRNA) genes, were sequenced for all six currently recognized species, plus one form, of the pelagic calanoid copepod genus Neocalanus. In Neocalanus gracilis, the ND6 gene was not found in the sequenced portion of the mitochondrial genome. Unambiguously aligned sequences were subjected to Bayesian, maximum-likelihood, maximum-parsimony, and neighbor-joining analyses using Eucalanus bungii as an outgroup. The resultant tree topologies from these four methods were congruent, robust, and all nodes were supported by high bootstrap values and posterior probabilities of 92-100%. Two tropical and subtropical species (N. gracilis and N. robustior) occupied the most basal position, and a subantarctic (N. tonsus) and three subarctic Pacific species (N. cristatus, N. plumchrus, and N. flemingeri) diverged subsequently. Transequatorial dispersal of the ancestral population during glaciations is suggested for this pattern of speciation, in which sister clades exhibited antitropical distributions. Although the area of ocean is much broader in the subantarctic than the subarctic Pacific, a higher number of species occur in the subarctic Pacific (three) than the subantarctic (one). The possibility that marginal seas, such as Japan Sea and Okhotsk Sea, function as natal areas for the divergence of species is discussed., Introduction The pelagic calanoid copepod genus Neocalanus currently includes six species plus one form, (the long form of N. flemingeri) which is known to occur in the western North Pacific [...]

The functional response of the aspects of reproductive success of a southwestern Baltic population of Acartia tonsa (Copepoda: Calanoida) was quantified in the laboratory using wide ranges in temperatures and salinities. Specifically, daily egg production (EP, # femal[e.sup.-1] [day.sup.-1]) was determined for 4 or 5 days at 18 different temperatures between 5 and 34[degrees]C and the time course and success of hatching were evaluated at 10 different temperatures between 5 and 23[degrees]C. The effect of salinity (0 to 34 psu) on egg hatching success was also examined. The highest mean rates of EP were observed between 22 and 23[degrees]C (46.8-50.9 eggs femal[e.sup.-1] [day.sup.-1]). When studied at 18 psu, hatching success of eggs increased with increasing temperature and was highest (92.2%) at 23[degrees]C. No hatching was observed for eggs incubated at low temperatures ([less than or equal to] 12[degrees]C) that were produced by females acclimated to temperatures [less than or equal to] 10[degrees]C indicating a possible thermal threshold between 10.0 and 13.0[degrees]C below which only the production of diapause (or low quality) eggs exists in this population. When tested at 18[degrees]C, the hatching success of eggs incubated at 15 different salinities increased asymptotically with increasing salinity and was maximal (81.4-84.5%) between 17 and 25 psu. The high reproductive success observed over wide ranges in temperatures and salinities in this Baltic population demonstrates one of the mechanisms responsible for the cosmopolitan distribution of this species within productive, estuarine and marine habitats., Calanoid copepods play a key role in the cycling of nutrients and energy in marine ecosystems by forming a trophodynamic link between primary (phytoplankton) and tertiary (e.g., planktivorous fish) production [...]

We examined patterns of genetic isolation-by-distance in two sister species of freshwater copepods, Eudiaptomus graciloides and Eudiaptomus gracilis, using the polymorphism of microsatellite markers. We assumed an enhanced dispersal potential of the species possessing diapausing eggs (E. graciloides) compared with E. gracilis with no diapause. On a longitudinal gradient spanning from northern Germany to Russia, we sampled 17 E. graciloides populations and 16 E. gracilis populations in order to investigate the scale dependence of isolation-by-distance from 0.4 to 1,340 km distance. In the 100-1,000 km range, no isolation could be detected in either Eudiaptomus species, suggesting either similar moderate gene flow over large geographic distances or persistence of historic patterns after postglacial recolonization. Our hypothesis was partly supported at the small-scale level (100 km range) where differences in population structure among species existed. We found isolation-by-distance in E. graciloides, the species with resting eggs, whereas E. gracilis exhibited a pattern of persistent founder effects. Further, in E. graciloides we found significant isolation- by-distance in the 1,340 km range with significantly reduced gene flow at distances >1,000 kin. In E. gracilis, isolation-by-distance at distances >l,000 km could not be detected with our data because this species was not found in the Russian lakes. Our results suggest that short- and long-distance dispersal in Eudiaptomus are due to different processes, with diapausing eggs only being advantageous for short-distance dispersal. We also argue that different spatial scales must be sampled to understand the geographic partitioning of genetic variance at marker loci.

Molecular systematic analyses of marine taxa are crucial for recording ocean biodiversity, so too are elucidation of the history of population divergence and the dynamics of speciation. In this paper we present the joined phylogeography of the calanoid copepod Calanus helgolandicus (Claus 1863) from the North East (NE) Atlantic and the Adriatic Sea and the closely related C. euxinus (Hulsemann 1991) from the Black Sea based on sequences of a mitochondrial Cytochrome Oxidase subunit I (COI) fragment. Coalescent-based Bayesian methods and minimum spanning networks are used to reconstruct the history of population divergence. Our results reveal that copepod populations from all three basins share a great number of haplotypes and demonstrate a close genetic affinity of C. euxinus with C. helgolandicus. The data do not support significant genetic structuring among samples within seas. Coalescent analyses suggest divergences between NE Atlantic, Mediterranean, and Black Sea populations dating back to the middle Pleistocene, with the NE Atlantic-Mediterranean divergence being the earliest and the Mediterranean-Black Sea divergence the most recent. These middle Pleistocene dates are much older than the estimated dates of colonisation of the Mediterranean and Black Seas based on paleoclimatic scenarios. Our results do not rule out that the assumed colonisations took place but they indicate that the populations colonising the Mediterranean and the Black Sea were already, and have since remained, diverged. The chaetognath Sagitta setosa, which has a comparable distribution pattern and feeds upon the copepods, provides a unique opportunity to compare phylogeographic patterns and distinguish among alternative hypotheses. The dates produced in this paper are in agreement with those estimated elsewhere for S. setosa. We propose that a great deal of the genetic make-up of marine planktonic populations comprises divergences that date back to long before the last glacial maximum. We consider questions on the taxonomic status of C. euxinus to remain open. However, its high genetic affinity to the C. helgolandicus calls for further investigation., Introduction The study of marine biodiversity, at the species level as well as the genetic diversity within species, is requisite for understanding the patterns and processes of marine speciation, ecology, [...]

The activity of the enzymes aminoacyl-tRNA synthetases (AARS) in adult females and males and copepodites stage V of Calanus helgolandicus was studied at the L4 time-series station in the English Channel from June 2002 to December 2003. AARS activity was explored as an index of somatic growth in the laboratory as well as in the field by comparison with other methods of measuring growth: (1) the direct method (Heinle in Chesapeake Sci 7:59-74, 1966), (2) the weight increment (WI) method, (3) the Hirst and Bunker (HB) equation (Limnol Oceanogr 48(5):1988-2010, 2003) and (4) the egg production (EPR) method. AARS activity showed a significant correlation with the direct measurement of growth in the laboratory ([R.sup.2]=0.55). However, the correlation was lower for growth assessed either with the WI or the HB approaches in the field ([R.sup.2]=0.05-0.17). Female AARS activity showed a positive correlation with specific EPR during the reproductive season ([R.sup.2]=0.40) but no relationship was found during the non-reproductive period.

Molecular systematic analyses of marine taxa are crucial for recording ocean biodiversity, so too are elucidation of the history of population divergence and the dynamics of speciation. In this paper we present the joined phylogeography of the calanoid copepod Calanus helgolandicus (Claus 1863) from the North East (NE) Atlantic and the Adriatic Sea and the closely related C. euxinus (Hulsemann 1991) from the Black Sea based on sequences of a mitochondrial Cytochrome Oxidase subunit I (COI) fragment. Coalescent-based Bayesian methods and minimum spanning networks are used to reconstruct the history of population divergence. Our results reveal that copepod populations from all three basins share a great number of haplotypes and demonstrate a close genetic affinity of C. euxinus with C. helgolandicus. The data do not support significant genetic structuring among samples within seas. Coalescent analyses suggest divergences between NE Atlantic, Mediterranean, and Black Sea populations dating back to the middle Pleistocene, with the NE Atlantic--Mediterranean divergence being the earliest and the Mediterranean--Black Sea divergence the most recent. These middle Pleistocene dates are much older than the estimated dates of colonisation of the Mediterranean and Black Seas based on paleoclimatic scenarios. Our results do not rule out that the assumed colonisations took place but they indicate that the populations colonising the Mediterranean and the Black Sea were already, and have since remained, diverged. The chaetognath Sagitta setosa, which has a comparable distribution pattern and feeds upon the copepods, provides a unique opportunity to compare phylogeographic patterns and distinguish among alternative hypotheses. The dates produced in this paper are in agreement with those estimated elsewhere for S. setosa. We propose that a great deal of the genetic make-up of marine planktonic populations comprises divergences that date back to long before the last glacial maximum. We consider questions on the taxonomic status of C. euxinus to remain open. However, its high genetic affinity to the C. helgolandicus calls for further investigation.

Eucalanus inermis is an abundant species in the eastern tropical and subtropical South Pacific, including the oceanic and coastal waters off Chile and Peru. Its annual life cycle was studied through a time-series sampling (weekly intervals) during 2002, at a fixed coastal station at an upwelling site (Mejillones Bay, 23[degrees]S) off northern Chile. The more-or-less continuous occurrence and abundance of naupliar and copepodid stages indicated that the species reproduces during most of the year, with two peaks: one during the austral spring and the other during the summer. Thereafter, an abrupt decline in the population was observed during winter. The abundances of E. inermis copepodids and nauplii were positively correlated with sea surface temperature, suggesting temperature-dependent development and growth during the spring-summer period. Three cohorts could be distinguished during one annual cycle, with generation times > 30 days. The estimate of mean weight-specific daily growth (0.12 [day.sup.-1]) is lower than that of other species in the area, but it is consistent with a slower development rate. The distribution of this copepod is associated with the equatorial subsurface waters characterized by low-oxygen content ( 200 m depth), probably in a lethargic mode, within the oxygen minimum zone in the adjacent oceanic area.

Replicate hybrid populations were initiated with first generation backcross hybrids between two genetically distinct populations from California such as Royal Palms (RP) and San Diego (SD). Computer simulations showed that selection must be invoked to explain the shift in allele frequencies, thus suggesting that hybrid inferiority in early generations gave way to hybrid superiority in later generations.

We investigated the impact of copepods on the seston community in a mesocosm set-up, and assessed how the changes in food quantity, quality and size affected the condition of the grazers, by measuring the RNA:DNA ratios in different developmental stages of Calanus finmarchicus. Manipulated copepod densities did not affect the particulate carbon concentration in the mesocosms. On the other hand, chlorophyll a content increased with higher copepod densities, and increasing densities had a positive effect on seston food quality in the mesocosms, measured as C:N ratios and [omega]3: [omega]6 fatty acid ratios. These food quality indicators were significantly correlated to the nutritional status of C. finmarchicus. In contrast to our expectations, these results suggest a lower copepod growth potential on higher quality food. However, in concordance with earlier studies, we found that when copepods were in high densities the large particles (> 1000 [micro][m.sub.3]) decreased and that the smaller particles (< 1000 [micro][m.sub.3]) increased in number. These patterns were closely linked to the condition of C. finmarchicus, which were of better condition (RNA:DNA ratios) with increasing biovolumes of large particles, and, conversely, lower RNA:DNA ratios with increasing biovolumes of smaller particles. Consequentially, the selective grazing by copepods stimulated increased biovolumes of smaller plankton, and this increase was responsible for the increased food quality, in terms of C:N and [omega]3: [omega]6 ratios. Thus, we conclude that the decreasing growth potentials of C. finmarchicus were a result of a decrease of favourably sized food particles, induced by copepod grazing.

A study was conducted to examine the recombination in interpopulation hybrids of the Copepod Tigriopus californicus. Findings suggest that recombinant hybrids were found to have significant faster development time for both males and females.

Patterns of reproductive isolation in copepod populations of Acanthocyclops vernalis, for which morphology, chromosome number, DNA content and rDNA sequences are known, are examined to clarify the pattern of biological species within this group and crosses are performed. A model is presented in which alterations due to Robertonian fusions, translocations and/or loss of chromosomal fragments generate heritable variations, only some of which lead to reproductive isolation.

The two distinguished sympatric and circumglobal sister species, Eucalanus hyalinus and Eucalanus spinifer were examined to study the population genetic structure throughout their global biogeographic ranges. The observed species-specific differences proved to be an important determining factor for the various patterns of their dispersal, which can be further used to determine the population genetic structure of open-ocean planktons.

Epistatic interactions between cytochrome c (CYC) alleles and mitochondrial DNA (mtDNA) variation are largely influenced by environmental temperature in interpopulation hybrids of the copepod Tigriopus californicus. The fitness of particular hybrid genotypes depends on additional non-mtDNA encoded genes that interact with CYC.

The intracellular accumulation of free amino acids is a common response of diverse organisms to increasing salinities in the extracellular environment. In crustaceans, proline is most often the dominant free amino acid accumulated in response to hyperosmotic stress. In the euryhaline copepod Tigriopus californicus the synthesis of proline from its metabolic precursors is tightly regulated by environmental salinities. Previous work has suggested that the enzyme ?1-pyrroline-5-carboxylase synthase (P5CS) lies at the key regulatory point in this pathway. We have characterized the gene encoding this enzyme in T. californicus and monitored its expression under hyperosmotic stress. The abundance of the transcript of this gene does not increase in response to hyperosmotic stress. Two additional enzymes in the proline synthesis pathway have also been examined: Neither glutamate dehydrogenase nor ?1-pyrroline-5-carboxylase reductase shows increased transcript abundance or enzyme activity under hyperosmotic stress. This work suggests that if P5CS is indeed the key enzyme in proline regulation under hyperosmotic stress in T. californicus, control must be mediated by either translational control or post-translational modification of this enzyme.