Ultraviolet irradiation alters the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions in copepod myocytes.

The efficient production of energy via oxidative phosphorylation is essential to the growth, survival, and reproduction of eukaryotes. The behavior (position of, and communication between, mitochondria) and morphology of mitochondria play key roles in efficient energy production and are influenced by oxidative stressors such as ultraviolet (UV) radiation. We tested the hypothesis that mitochondria change their behavior and morphology to meet energetic demands of responding to changes in oxidative stress. Specifically, we predicted that UV irradiation would increase the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions to influence whole-animal metabolic rate. Using transmission electron microscopy, we found that both three and six hours of UV-A/B irradiation (0.5 W/m2) increased the proportion of inter-mitochondrial junctions (with increasing mitochondrial aspect ratio) and the density of inner mitochondrial membrane in myocytes of Tigriopus californicus copepods. Mitochondrial density increased following both irradiation treatments, but mitochondrial size decreased under the six hour treatment. Metabolic rate was maintained under three hours of irradiation but decreased following six hours of exposure. These observations demonstrate that the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions can play formative roles in maintaining whole-animal metabolic rate, and ultimately organismal performance, under exposure to an oxidative stressor.

The discovery of Lepeophtheirus lichiae Barnard, 1948 (Copepoda: Caligidae) parasitic on leerfish, Lichia amia (Linnaeus) in the Mediterranean Sea.

Lepeophtheirus lichiae Barnard, 1948 (Copepoda: Caligidae), a very rare and poorly known sea louse, is redescribed based on a single newly collected female. The specimen was collected from the dorsal body surface of the leerfish, Lichia amia (Linnaeus) caught in north-eastern Mediterranean waters off the Turkish coast. The original and only available description of L. lichiae was incomplete and with only three illustrations: the dorsal view of the female habitus, the sternal furca, and the postantennal process. Here, we present a full re-description of female of L. lichiae and report it from the Mediterranean Sea for the first time.

The cleavage pattern of calanoid copepods-a case study.

Many crustacean groups show stereotyped cleavage patterns during early ontogeny. However, these patterns differ between the various major crustacean taxa, and a general mode is difficult to extract. Previous studies suggested that also copepods undergo an early cleavage with a more or less stereotyped pattern of blastomere divisions and fates. Yet, copepod embryology has been largely neglected. The last investigation of this kind dates back more than a century and the results are somewhat contradictory when compared with those of other researchers. To overcome these problems, we studied the early development of a so far undescribed calanoid copepod species, Skistodiaptomus sp., applying histochemical staining, confocal laser scanning microscopy, and bifocal 4D microscopy. The blastomere arrangement of the four-cell stage of this species varies to a large degree. It can either form a typical radial pattern with the four blastomeres lying in one plane or a tilted orientation of the axes connecting the sister cells of the previous division. In both cases, a stereotyped division pattern is maintained inside each quadrant during subsequent cleavages. In addition, we found two types of blastomere arrangements with a mirror symmetry. Most divisions within the quadrants follow the perpendicularity rule until the eighth cleavage. Deviations from this rule occur only in the narrow regions where the different quadrants touch and near the site of gastrulation. Gastrulation is initiated around the descendants of one individually identifiable blastomere of the 16-cell stage. This cell divides in a specific manner forming a characteristic cell arrangement, the gastrulation triangle. This gastrulation triangle initiates the internalization process of the gastrulation and it is encircled by another characteristic cell type, the crown cells. Our observations reveal several similarities to the early development of Calanus finmarchicus, another calanoid species. These relate to blastomere arrangements and divisions and the pattern of gastrulation. As Calanoida represent a basal or near basal branch of the copepod tree, this description will provide the ground for reconstruction of the cleavage pattern of the last common ancestor of Copepoda.

A new species of Clausidium Kossmann, 1874 (Copepoda: Cyclopoida) associated with ghost shrimps from the Persian Gulf, including female-male interlocking mechanisms and remarks on host specificity.

A new clausidiid copepod was found associated with the ghost shrimp Neocallichirus jousseaumei (Nobili) in the Persian Gulf, on the Iranian coast. The new species shares the armature formula of swimming legs 2 to 4 with C. persiaensis, but can be easily distinguished from its congeners by unique characteristics of the females: the prominent spine on endopodal segment 1 of the antenna, the armature of the maxilliped, and the elongated basis of the swimming legs. Distinguishing features observed in males include the distinct projections on the maxilliped and the armature of legs 1 and 4. In addition to traditional light microscopy-based descriptions, confocal laser scanning microscopy (CLSM) was used to obtain high resolution images and 3-D reconstructions of entire copepods. Structures of taxonomic importance that exhibit complex shapes (male maxilliped and female urosome) were scanned to generate 3-D prints that gave valuable insights about female/male interlocking mechanisms. The taxonomic status and host specificity of Clausidium spp. are discussed and a key to valid species is provided.

Molecular characterization and functional analysis of a salmon louse (Lepeophtheirus salmonis, Krøyer 1838) heme peroxidase with a potential role in extracellular matrixes.

Heme peroxidases are the most abundant type of peroxidase catalyzing a H2O2-dependent oxidation of a wide variety of substrates. They are involved in numerous processes like the innate immune response, hormone and prostaglandin synthesis and crosslinking of proteins within extracellular matrixes (ECM) as well as molecules within the cuticle and chorion of arthropods and nematodes. In the present study, a Lepeophtheirus salmonis heme peroxidase (LsHPX) 1 was characterized. Amino acids in the active site of heme peroxidases were conserved, and the predicted protein sequence showed the highest similarity to genes annotated as chorion peroxidases and genes suggested to be involved in cuticle hardening or adhesion. LsHPX1 exhibited a dynamic expression during ontogenesis and during the nauplius molting cycle. Transcripts were localized to muscle cells near the muscle-tendon junction, in nerve tissue especially at neuromuscular junctions, subcuticular epithelium, subepithelial cells facing the hemolymph, exocrine glands within the subepithelial tissue and in isolated cells within the testis. Knock-down of LsHPX1 in nauplius larvae decreased the swimming activity of emerging copepodids. Histological analysis of knock-down animals revealed increased spacing between myofibers and changes in subepithelial and exocrine gland tissue. Considering these results, the potential role of LsHPX1 in crosslinking molecules of salmon louse ECMs is discussed.

RNA interference mediated knockdown of the KDEL receptor and COPB2 inhibits digestion and reproduction in the parasitic copepod Lepeophtheirus salmonis.

Retrograde transport of proteins from the endoplasmic reticulum to the Golgi is an essential part of the secretory pathway that all newly synthesised secreted and membrane proteins in eukaryotic cells undergo. The aim of this study was to characterise two components of the retrograde transport pathway in the parasitic copepod Lepeophtheirus salmonis (salmon louse) on a molecular and functional level. LsKDELR and LsCOPB2 were confirmed to be the salmon louse homologues of the chosen target proteins by sequence similarity. Ontogenetic analysis by qRT-PCR revealed the highest expression levels of both genes in adult females and the earliest larval stage. LsKDELR and LsCOPB2 localisation in adult females was detected by immunofluorescence and in situ hybridisation, respectively. Both LsKDELR and LsCOPB2 were found in the ovaries, the oocytes and the gut. LsKDELR and LsCOPB2 were knocked down by RNA interference in preadult females, which was confirmed by qRT-PCR. LsCOPB2 knockdown lice had a significantly higher mortality and failed to develop normally, while both LsCOPB2 and LsKDELR knockdown caused disturbed digestion and the absence of egg strings. This shows the potential of LsKDELR and LsCOPB2 as suitable target candidates for new pest control methods.

Egg number-egg size: an important trade-off in parasite life history strategies.

Parasites produce from just a few to many eggs of variable size, but our understanding of the factors driving variation in these two life history traits at the intraspecific level is still very fragmentary. This study evaluates the importance of performing multilevel analyses on egg number and egg size, while characterising parasite life history strategies. A total of 120 ovigerous females of Octopicola superba (Copepoda: Octopicolidae) (one sample (n=30) per season) were characterised with respect to different body dimensions (total length; genital somite length) and measures of reproductive effort (fecundity; mean egg diameter; total reproductive effort; mean egg sac length). While endoparasites are suggested to follow both an r- and K-strategy simultaneously, the evidence found in this and other studies suggests that environmental conditions force ectoparasites into one of the two alternatives. The positive and negative skewness of the distributions of fecundity and mean egg diameter, respectively, suggest that O. superba is mainly a K-strategist (i.e. produces a relatively small number of large, well provisioned eggs). Significant sample differences were recorded concomitantly for all body dimensions and measures of reproductive effort, while a general linear model detected a significant influence of season*parasite total length in both egg number and size. This evidence suggests adaptive phenotypic plasticity in body dimensions and size-mediated changes in egg production. Seasonal changes in partitioning of resources between egg number and size resulted in significant differences in egg sac length but not in total reproductive effort. Evidence for a trade-off between egg number and size was found while controlling for a potential confounding effect of parasite total length. However, this trade-off became apparent only at high fecundity levels, suggesting a state of physiological exhaustion.

Circular polarization of transmitted light by sapphirinidae copepods.

UNLABELLED: Circularly polarized light, rare in the animal kingdom, has thus far been documented in only a handful of animals. Using a rotating circular polarization (CP) analyzer we detected CP in linearly polarized light transmitted through epipelagic free living Sapphirina metallina copepods. Both left and right handedness of CP was detected, generated from specific organs of the animal's body, especially on the dorsal cephalosome and prosome. Such CP transmittance may be generated by phase retardance either in the muscle fibers or in the multilayer membrane structure found underneath the cuticle. Although the role, if any, played by circularly polarized light in Sapphirinidae has yet to be clarified, in other animals it was suggested to take part in mate choice, species recognition, and other forms of communication. HIGHLIGHTS: Planktonic Sapphirinidae copepods were found to circularly polarize the light passing through them. Circular polarization may be created by unique, multilayered features of the membrane structure found under their cuticle or by organized muscle fibers.

Elevated oxidative damage is correlated with reduced fitness in interpopulation hybrids of a marine copepod.

Aerobic energy production occurs via the oxidative phosphorylation pathway (OXPHOS), which is critically dependent on interactions between the 13 mitochondrial DNA (mtDNA)-encoded and approximately 70 nuclear-encoded protein subunits. Disruptive mutations in any component of OXPHOS can result in impaired ATP production and exacerbated oxidative stress; in mammalian systems, such mutations are associated with ageing as well as numerous diseases. Recent studies have suggested that oxidative stress plays a role in fitness trade-offs in life-history evolution and functional ecology. Here, we show that outcrossing between populations with divergent mtDNA can exacerbate cellular oxidative stress in hybrid offspring. In the copepod Tigriopus californicus, we found that hybrids that showed evidence of fitness breakdown (low fecundity) also exhibited elevated levels of oxidative damage to DNA, whereas those with no clear breakdown did not show significantly elevated damage. The extent of oxidative stress in hybrids appears to be dependent on the degree of genetic divergence between their respective parental populations, but this pattern requires further testing using multiple crosses at different levels of divergence. Given previous evidence in T. californicus that hybridization disrupts nuclear/mitochondrial interactions and reduces hybrid fitness, our results suggest that such negative intergenomic epistasis may also increase the production of damaging cellular oxidants; consequently, mtDNA evolution may play a significant role in generating postzygotic isolating barriers among diverging populations.

[Cytophotometric determination of genome size in two species of Cyclops Lake Baikal (Crustacea: Copepoda: Cyclopoina) in ontogenetic development].

Genome size of Cyclops in cells at early stages of cleavage (up to the 5th division) and in somatic cells were estimated by a static digital Feulgen cytophotometry in order to study the quantitative changes in DNA content during chromatin diminution. Our realization of the cytophotometric method was approbeted on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation of the obtained and known from the literature data on the genome size and a high reproducibility, which allows to use these systems in future work. We also optimized the conditions for DNA hydrolysis of both blood smears, and for two species of Cyclops from the Moscow population, as 30 min in 5 N HCl at 24 degrees C. Here we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus estimated the extent ofchromatin diminution in Diacyclops galbinus as 95.5-96.2 %. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution. We also optimized the conditions for DNA hydrolysis of both blood smear preparations, and for two species of copepods from the Moscow population, as 30 min in 5N HCl at 24 degrees C.

Caligus sclerotinosus (Copepoda: Caligidae), a serious pest of cultured red seabream Pagrus major (Sparidae) in Korea.

Caligid copepods (Crustacea) known as sea lice are pests of cultured fish, causing serious diseases and economic losses in fish aquaculture worldwide. One species, Caligus sclerotinosus Roubal, Armitage & Rohde, 1983 (Caligidae), is considered a serious pest of the highly prized red seabream Pagrus major (Temminck and Schlegel, 1843) (Sparidae) cultured in Japan. Recently, in neighboring Korea, red seabream culture has intensified and almost replaced yellow tail culture. However, until now, there have been no reports on infection of this sea louse from red seabream in Korea. We surveyed 120 (20 fish per month) P. major from a sea ranched Tongyeong Marine Research Center aquaculture facility, Gyeongsangnamdo, Korea for six months in 2011 (June to November). We recorded severe infection by the sea louse C. sclerotinosus on the skin of P. major. Prevalence was 100%, mean intensity 7.06, maximum intensity 49, and minimum intensity 2. Adult females (624), males (219) and few chalimi (5) were observed and identified by their morphology. As an average of all our collections, less than 0.6% of individuals were chalimi. We suggest, therefore, that adults of C. sclerotinosus undergo ontogenetic host switching after their final moult. No infection of C. sclerotinosus was found on wild P. major collected from Tongyeong and Yeosu fish markets on the southern coast of Korea. Severe infection by this sea louse may cause secondary infections of the host. This copepod is already reported from Australia and Japan and hence, this is the first report from Korea. We expect this pest to have an impact on Korean red seabream fisheries equally serious to that being experienced in Japan.

Molecular phylogeny of the Calanoida (Crustacea: Copepoda).

The order Calanoida includes some of the most successful planktonic groups in both marine and freshwater environments. Due to the morphological complexity of the taxonomic characters in this group, subdivision and phylogenies have been complex and problematic. This study establishes a multi-gene molecular phylogeny of the calanoid copepods based upon small (18S) and large (28S) subunits of nuclear ribosomal RNA genes and mitochondrial encoded cytochrome b and cytochrome c oxidase subunit-I genes, including 29 families from 7 superfamilies of the order. This analysis is more comprehensive than earlier studies in terms of number of families, range of molecular markers, and breadth of taxonomic levels resolved. Patterns of divergence of ribosomal RNA genes are shown to be significantly heterogeneous among superfamilies, providing a likely explanation for disparate results of previous studies. The multi-gene phylogeny recovers a monophyletic Calanoida, as well as the superfamilies Augaptiloidea, Centropagoidea, Bathypontioidea, Eucalanoidea, Spinocalanoidea and Clausocalanoidea. The phylogeny largely agrees with previously-published morphological phylogenies, including e.g., enlargement of the Bathypontioidea to include the Fosshageniidae.

Morphological characterization and intraspecific variation of Zoothamnium intermedium Precht, 1935 (Ciliophora, Peritrichia) attached to calanoid copepods in the Chesapeake Bay, USA.

A redescription of Zoothamnium intermedium Precht, 1935, a peritrich epibiont on copepods, is provided using specimens colonizing Acartia tonsa and Eurytemora affinis in Chesapeake Bay, USA. Bell-shaped zooids of Z. intermedium ranged in size from 31.2-54.7 microm x 16.7-31.3 microm in vivo. A single contractile vacuole and a "C shaped" macronucleus lie in the upper half of the body. Colonies had up to 30 zooids, but most presented two to four zooids. The single myoneme was continuous from the zooids through the lateral branches and basal stalk, terminating 4-73.2 mm before the attachment point of the colony. The ciliature of Z. intermedium was typical of sessile peritrichs, consisting of an outer haplokinety and an inner polykinety 1 (PK1) that made approximately turns around the peristomial disk before entering the infundibulum. Within the infundibulum, PK 1 was accompanied by polykinetid 2 (PK2) and polykinetid 3 (PK3), each consisting of three kinetosomal rows: PK2 of Z. intermedium terminated adoral to the aboral end of PK1 and had a central row of kinetosomes shorter than the lateral rows. Scanning electron microscopy revealed an annular pattern of pellicular ridges with pellicular pores. Morphological features used in peritrich species identification are quantified for specimens attached to both hosts and compared with other species of Zoothamnium. Statistically significant differences found between specimens attached to A. tonsa and E. affinis may reflect phenotypic plasticity rather than infestation by multiple species of Zoothamnium.

Cryptic ecological diversification of a planktonic estuarine copepod, Acartia tonsa.

The recent discovery of cryptic species in marine holoplankton, organisms that 'drift' in oceanic currents throughout their life cycle, contrasts with their potential for long-distance passive dispersal and presumably high gene flow. These observations suggest that holoplankton species are adapting to surprisingly small-scale oceanographic features and imply either limited dispersal or strong selection gradients. Acartia tonsa is a widespread and numerically dominant estuarine copepod containing deep mitochondrial lineages within and among populations along the northwestern Atlantic coast. In this study, we intensively investigated A. tonsa populations in Chesapeake Bay with the goals of testing species status for the deep lineages and testing for their association with environmental features over space and time. Phylogenetic analyses of DNA sequences from mitochondrial cytochrome c oxidase I (mtCOI) and the nuclear ribosomal internal transcribed spacer (nITS) resolved two concordant monophyletic clades. Deep divergence between the two clades (13.7% uncorrected sequence divergence for mtCOI and 32.2% for nITS) and genealogical concordance within sympatric populations strongly suggest that the two clades represent reproductively isolated cryptic species. Based on restriction fragment length polymorphisms of mtCOI, representatives from the two clades were found consistently associated with contrasting salinity regimes (oligohaline vs. meso-polyhaline) with an overlap between 2 and 12 PSU in samples from 1995 to 2005. Finding these patterns in one of the best-known estuarine copepods reinforces the conclusion that marine biodiversity is underestimated, not only in terms of species numbers, but also with respect to niche partitioning and the potential importance of ecological divergence in marine holoplankton.

Histological approaches for high-quality imaging of zooplanktonic organisms.

The investigation of the internal organization of zooplankton communities provides important information on the plankton biology with special interest for the study of ecological processes. Zooplanktoners can play a structural function as indicators for ecosystem health or stress, but their study using histological techniques is still limited. Here we report that the internal structure of zooplanktonic organisms can be facilely observed by a histological approach that combines optimal fixation and processing with a plastic resin (glycol methacrylate) embedding, resulting in increased tissue resolution. Using copepods, organisms that can dominate zooplankton assemblages, as models, collected from a tropical ecosystem (Paraibuna river, Brazil), we showed fine histological details of their muscular, nervous and digestive systems, structure of appendages and cell features. Critical advantages of this approach are that it permits optimal preservation and adequate handling of the organisms (embedded in agar after fixation) for further histological processing and investigation. This is important because it prevents both mechanically induced artifacts and loss of these diminutive organisms during the different steps of processing. Moreover, embedding in plastic resin showed a superior imaging of copepod internal structures compared to paraffin embedding. The use of glycol methacrylate is advantageous over paraffin/paraplast embedding by avoiding heat damage, tissue retraction and allowing faster embedding procedure and better tissue resolution. The value of histological approaches in enabling high-quality imaging of the internal structure of copepods is particularly important because these organisms can be used as indicators of environmental changes.

Deleterious epistatic interactions between electron transport system protein-coding loci in the copepod Tigriopus californicus.

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 F2 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 F2 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.

Effect of Nannochloris sp. on the toxicity of four algae.

The effect of Nannochloris sp. on the toxicity of cultures of four algae was evaluated using a Microtox 500 that measures the effect on a light-producing bacterium, Vibrio fischeri. Cultures of four algae produced a toxic effect, but in the presence of Nannochloris sp., the effect was reversed, and a stimulatory effect was observed. The effect was tested for Microcystis aeroginosa, Cyclotella menengheniana, Scenedesmus dimorphis, and Lyngbya sp. using cultures obtained from the University of Texas Culture Collection of Algae.

Molecular cloning of trypsin cDNAs and trypsin gene expression in the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae).

The salmon louse, Lepeophtheirus salmonis, is a marine ectoparasitic copepod that infects salmonid fishes. We are studying the interactions between this parasite and its salmonid hosts, as it is a common cause of disease in both wild and farmed stocks of salmon. In this paper, we report on the cloning and sequencing of seven trypsin-like enzymes from a cDNA library prepared from whole body preadult female and male L. salmonis. The predicted trypsin activation peptides are 23 or 24 residues in length, considerably longer than previously reported activation peptides of other animals. Differences in the putative signal and activation peptide sequences of the trypsin isoforms suggest that these forms differ in their regulation and function. The calculated molecular weights of the trypsins range from 23.6 to 23.7 kDa. There are eight cysteine residues, which suggest the presence of four disulfide bridges. These trypsins are very similar (>or=46% aa identity) to other crustacean trypsins and insect hypodermins. Using in situ hybridization techniques trypsinogen expression could be identified in all three cell types of the midgut.