Feeding deterrence properties of apo-fucoxanthinoids from marine diatoms. II. Physiology of production of apo-fucoxanthinoids by the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana,and their feeding deterrent effects on the copepod Tigriopus californicus

The marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana have been shown to produce apo-fucoxanthinoid compounds which act as feeding deterrents against the harpacticoid copepod Tigriopus californicus. The amounts and types of apo-fucoxanthinoids produced were species specific. Th. pseudonana produced small quantities of apo-12-fucoxanthinal and apo-13-fucoxanthinone only during senescence, while P. tricornutum produced much greater quantities of these two compounds during both log and senescence phases, in addition to producing a third compound, apo-10-fucoxanthinal, only during senescence. For both species, production of apo-fucoxanthinoids increased as the cells entered senescence phase due to phosphate limitation. The amounts of apo-fucoxanthinoids necessary to reduce feeding in T. californicus by 50% ranged from 2.22 to 20.2 ppm. This range was approximately 1000 times lower than the total apo-fucoxanthinoid concentration in P. tricornutum. The amounts of apo-fucoxanthinoids necessary to cause a 50% mortality in a population of T. californicus ranged from 36.8 to 76.7 ppm. Thus, these compounds are present in concentrations which may have ecological significance in the control of bloom formation and grazing. The production of apo-fucoxanthinoids may be a phenomenon common to many diatoms, particularly as they enter senescence due to nutrient limitation.     

Mating system of the intertidal copepod Tigriopus californicus

Male Tigriopus californicus clasp immature females (copepodid stages II–V) for a period of up to a week prior to the female's terminal molt; upon maturation (stage VI) the female is inseminated and released. While females can mate anytime after their terminal molt, experiments using electrophoretically-detected genetic markers indicate that each mates only once in her lifetime. No evidence of sperm displacement was observed. Hence, male mating behavior can be interpreted as pre-copulatory mate guarding, a strategy employed to assure that a potential mate has not been previously inseminated. Males minimize the time investment required to insemiate a single female successfully by preferentially choosing to clasp more developmentally-advanced females; males clasped to stage III females will release them in order to clasp stage V females if the latter are present. Since males are capable of multiple mating, under most conditions of population sex ratio, this mating system results in low availability of unclasped, developmentally-advanced females; consequently, males must clasp successively younger (i.e. developmentally less-advanced) females in order to obtain a successful insemination.     

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.     

Genetic differentiation and reproductive incompatibility among Baja California populations of the copepod Tigriopus californicus

Populations of the harpacticoid copepod Tigriopus californicus were sampled from five sites from San Diego, California, to Playa Altamira, Baja California, Mexico. Allozyme analyses revealed that all the study populations are sharply differentiated genetically. At the extreme, two populations, Punta Baja and Playa Altamira, have no alleles in common at the seven allozyme loci studied. All pairwise interpopulation crosses successfully produced F₂ hybrids except those involving the Playa Altamira population. All crosses using Playa Altamira females failed to produce F₁ hybrids, while Playa Altamira males successfully produced F₁ progeny with females from all other sites. These F₁ offspring, however, were completely sterile (with San Diego and Punta Banda females) or only occasionally produced F₂ offspring (with Punta Morro and Punta Baja females). These results suggest that allopatric differentiation among Baja populations has resulted in exceptionally high levels of genetic divergence and nearly complete reproductive isolation of the Playa Altamira population, which should now be recognized at the semispecies (or perhaps sibling species) level.     

Feeding deterrence properties of apo-fucoxanthinoids from marine diatoms. I. Chemical structures of apo-fucoxanthinoids produced by Phaeodactylum tricornutum

Methanol extracts of freshly harvested cells of the marine diatom Phaeodactylum tricornutum were found to produce a feeding deterrent effect in the copepod Tigriopus californicus. Bioassay guided fractionation of the methanol extracts led to the isolation of four compounds possessing feeding deterrent activity. The compounds were identified as apo-10-fucoxanthinal (1), apo-12-fucoxanthinal (2), apo-12-fucoxanthinal (3), and apo-13-fucoxanthinone (4) by detailed spectroscopic analysis and comparison with authentic compounds produced semi-synthetically from fucoxanthin. Compounds 1 to 4 exhibited feeding deterrent responses in T. californicus at concentrations of less than 20 ppm.     

Survival and sex ratios of the intertidal copepod,Tigriopus californicus,following ultraviolet-B (290–320 nm) radiation exposure

The marine harpacticoid copepod, Tigriopus californicus (Baker), is a successful colonizer of supralittoral splash pools from Torch Bay, Alaska, to Baja California, Mexico. As these pools are subject to abundant amounts of direct solar radiation, it was of interest to determine the sensitivity of T. californicus to ultraviolet-B (UV-B, 290 to 320 nm) radiation. During 1980, copepods were raised under diurnal conditions in the laboratory and fed a mixture of unicellular algae and bacteria. Larval and adult stages were irradiated for 0 to 26 h on a rotating turntable under enhanced ultraviolet radiation. Following irradiation, the copepods were maintained in the culture area and checked daily for survival. All life stages of T. californicus tolerated enhanced UV-B radiation irrespective of exposure period; in contrast, survival of an irradiated planktonic copepod, Acartia clausii (Giesbrecht), was significantly lower. Additionally, there appeared to be asex ratio shift in adults that developed from the youngest naupliar stages (N₁/N₂) of T. californicus that were irradiated. Extraction and tentative identification of the pigment(s) responsible for the bright orange color of the carapace of T. californicus were performed. These pigments may function to absorb UV radiation and protect internal structures from photochemically induced damage.     

Mate choice in the face of both inbreeding and outbreeding depression in the intertidal copepod Tigriopus californicus

 In species vulnerable to both inbreeding and outbreeding depression, individuals might be expected to choose mates at intermediate levels of genetic relatedness. Previous work on the intertidal copepod Tigriopus californicus has repeatedly shown that crosses between populations result in either no effect or hybrid vigor in the first generation, and hybrid breakdown in the second generation. Previous work also shows that mating between full siblings results in inbreeding depression. The present study again found inbreeding depression, with full sibling mating causing significant fitness declines in two of the three populations assayed. In the mate choice assays, a single female was combined with two males. Despite the costs of both inbreeding and outbreeding, mate choice showed clear inbreeding avoidance but no clear pattern of outbreeding avoidance. This lack of outbreeding avoidance may be attributed either to the temporary increase in fitness in the F₁ generation or to the absence of selection for premating isolation in wholly allopatric populations with infrequent migration. If this inability to avoid unwise matings is common to other taxa, it may contribute to the problem of outbreeding depression when allopatric populations are mixed together. Received: 18 May 1999 / Accepted: 25 January 2000     

Feeding of the copepod Acartia tonsa on the diatom Nitzschia closterium and brown algae (Fucus vesiculosus) detritus

Integumental sensilla were examined in 39 species of meso- and bathypelagic shrimps and 6 species of epibenthic shrimps. A tuft organ, consisting of a group of openended, tubular setae and so probably functioning as a chemosensor, is described. A single tuft with ancillary smaller tufts occur on the dorsal median surface of the fourth abdominal segment and two pairs of tufts occur on the proximal dorsal region of the telson of all oplophorid and pandalid shrimps examined. An additional tuft is present on the fifth abdominal segment of the rare bathypelagic Physetocaris microphthalma Chace. No tuft organs were identified in Processa canaliculata Leach, suggesting that they may not occur in all families of caridean shrimps. Acanthephyrid, systellaspid and pandalid shrimps have the integument completely covered by scales. These are extremely delicate and are normally removed during the process of catching the shrimp through abrasion of the integument by the net. The scales are peculiarly orientated, pointing anteriorly in the anterior half and posteriorly in the posterior half of the body. They probably have a sensory function as distance receptors, monitoring water currents and disturbances in the environment surrounding the shrimp.     

Identification of sex, age and species-specific proteins on the surface of the harpacticoid copepod Tigriopus japonicus

 Behavioral experiments have shown that male copepods of the species Tigriopus japonicus (Nori) can distinguish species, sex, and developmental stage of potential mates using contact chemoreception. Lectin-binding patterns on the body surface of females have indicated that surface-bound glycoproteins may be important signals in mate choice. In the present study, the proteolytic enzyme trypsin was used to cleave surface proteins from females, reducing their attractiveness to males. The protein fragments released were used to make monoclonal antibodies. Three levels of screening were used to identify monoclonal antibodies that recognized proteins involved in mate recognition. One monoclonal antibody bound to the terminal urosome and lateral prosome of CV females, and its binding significantly decreased female attractiveness to males. Western blotting showed that this antibody bound the trypsin-cleaved fragment and proteins of homogenized CV females and virgin adult females, but did not bind proteins of homogenized males, CIII females, or females of T. californicus or T. fulvus. This antibody recognized proteins on the surface of females that may enable males to discriminate conspecifics, sex, and age. It is likely that this molecule has a central role in the evolution of reproductive isolation in this group. Received: 22 July 1999 / Accepted: 21 March 2000     

Phylogeography of the rock-pool copepod Tigriopus brevicornis (Harpacticoida) in the northern North Atlantic, and its relationship to other species of the genus

We investigated relationships among North Atlantic Tigriopus brevicornis populations and their relationships to Mediterranean T. fulvus and North American T. californicus, using crossing experiments and mitochondrial DNA sequencing. All T. brevicornis populations tested were interfertile, while interspecific crosses produced either no offspring or offspring that did not survive past the larval stage, with the exception of a few T. brevicornis × T. californicus crosses that produced mature adults. DNA sequencing of a fragment of mitochondrial cytochrome oxidase I (COI) showed that samples of T. brevicornis from Iceland, the Faroes, Ireland, Scotland and Nova Scotia formed a single shallow clade. In contrast, T. brevicornis from more southern populations in France and Portugal formed a clade with substantially greater branch lengths. Tigriopus brevicornis was monophyletic, and T. brevicornis plus Mediterranean T. fulvus were together also monophyletic. The phylogeography of T. brevicornis closely mirrored that found in T. californicus, with substantially reduced interpopulation divergence at northern latitudes. The known distribution of T. brevicornis in Iceland and the Faroes is shown and dispersal mechanisms and habitat selection briefly discussed.     

Feeding ecology of Spinocalanus antarcticus, a mesopelagic copepod with a looped gut

Spinocalanus antarcticus, an abundant mesopelagic copepod in polar seas, has a greatly elongated and looped midgut, contrary to most other copepod species. The total gut length is 1.77, 1.86 and 1.90 times the total body length in adult females, CV and CIV, respectively. Gross morphology of the midgut is similar in all copepodite stages and adults. It is described here from specimens collected in the Arctic Ocean. In stratified samples from the deep Amundsen and Makarov Basins S. antarcticus showed a clear preference for the depth layer between 100 and 500 m. Generally, the guts were packed with material, but most of it was impossible to identify. In most specimens the digestive tract was filled with undefined detritus particles ("detritus balls"). They were almost spherical, heterogeneous organic aggregates of 40-100 µm diameter, with small clay-sized mineral flakes imbedded. Mineral particles in the size range of 1-10 µm were found in large quantities in the guts of many specimens. Cysts of Chrysophycea and dinoflagellates and fragments of dinoflagellates, diatoms, tintinnids and radiolarians, as well as skeletons of silicoflagellates, were rather rare; some animal remnants were also found. A high carbon/nitrogen ratio (8.9) and very high lipid content (54% of dry weight) indicated a very good nutritional state. The adaptive significance and possible feeding strategy of this deep-water copepod is discussed.     

Feeding of marine planktonic copepods on mixed phytoplankton

The feeding of juveniles and adults of the copepods Eucalanus pileatus, Temora stylifera and T. turbinata fed a mixture of the phytoplankton Skeletonema costatum, Leptocylindrus danicus and Rhizosolenia alata f. indica was studied at 20°C. E. pileatus nauplii, copepodids and adult females ingest similar percentages of the 3 algae in terms of carbon. Temora juveniles younger than CII ingest mainly S. costatum; at more advanced developmental stages, the ingestion rate on S. costatum remains constant whereas feeding on L. danicus and R. alata f. indica increases with increasing body weight. Feeding on high concentrations of large particles reduces the grazing pressure on small particles, thus favoring zooplankton which require small-sized food.     

Experimental study on acute effects of the combined exposure to temperature increase and chlorination upon the marine copepod Acartia omorii

The acute effects of combined exposure to temperature increase and chlorination on a neritic marine copepod, Acartia omorii Bradford (collected offshore of Onjuku, Japan in 1982), were investigated in the laboratory. Continuous flow exposure and batch exposure modes were compared. Based on the results of continuous flow experiments, the 24-h median lethal concentration (24-h LC₅₀, in mg l^-1) of total residual chlorine was estimated using the multiple regression equation below, with a multiple correlation coefficient of 0.955: 24-h LC₅₀=2.988-0.034 dT-1.611 log₁₀ t where dT is temperature rise (°C) and t is exposure duration (min). In batch experiments, the predictive power of the multiple regression equation was reduced, probably due to variations in chlorine concentration during exposure duration.     

Polychlorinated biphenyl (PCB) effects on marine phytoplankton photosynthesis and cell division

Inhibition of photosynthesis and cell division by polychlorinated biphenyls (PCBs) was studied using 7 marine phytoplankton species representing 4 algal classes. PCB concentrations as low as 1.0 g l^-1 reduced cell division of Thalassiosira pseudonana 3H and Isochrysis galbana. Both photosynthesis and cell division of T. pseudonana 3H, Chaetoceros socialis, Skeletonema costatum, T. pseudonana 13-1, Monochrysis, lutheri and I. galbana were inhibited at a PCB concentration of 10.0 g l^-1. The effects on photosynthesis were immediate and probably resulted in reduced rates of cell division. Interspecific differences in susceptibility were observed. These differences have significance with respect to primary production and the species composition of phytoplankton communities. The initial slopes of photosynthesis-irradiance (P-I) curves for the diatoms S. costatum and T. pseudonana 3H were reduced in the presence of PCBs. These results suggest that PCBs affect the photosynthetic light reactions.     

Diet and respiration of the small planktonic marine copepod Oithona nana

The functional responses of Oithona nana (Giesbr.) to various phytoplankton and zooplankton food species are described. The food species were divided into three size categories, the seasonal abundances of which were measured in Loch Turnaig, a Scottish sea loch in 1977. The seasonal variations in feeding rates in the sea for each size class were derived. The seasonal variation in respiration rate of O. nana was measured, and metabolic requirements were claculated as between 6 and 40% of the food material estimated as being eaten. O. nana differs from other common copepods in having a wide food-particle size spectrum and a low metabolic rate. It is suggested that these adaptations constitute the strategy whereby O. nana maintains its population levels throughout the year.     

Acute toxicity and sublethal behavioral effects of copper on barnacle nauplii (Balanus improvisus)

This study documents the effects of short-term (24h) sublethal copper exposures on undirected swimming activity and photobehavior of Balanus improvisus stage II nauplii. All Cu treatments were static, with temperature and salinity conditions at 20°C and 15 or 30. The 24h LC 50 estimate for Cu is 88 ppb at 15 and >200 ppb at 30. Sub-lethal Cu concentrations cause reductions in swimming speed, which decrease progressively with increasing Cu dose. At 50 ppb Cu, this was significant primarily at light intensities below the phototactic threshold. At higher Cu concentrations, significant reductions in mean linear velocity occurred at most light intensities tested. At 30, 50 and 100 ppb Cu also reduce the positive phototactic response and 150 ppb Cu causes reversal of phototaxis at optimal intensities. Photokinesis is reduced at 100 ppb Cu and disappears at 150 ppb Cu. At 15, the behavioral effects of 50 ppb Cu resemble those occurring with 150 ppb Cu at 30. Swimming speed and photobehavior show promise as sensitive behavioral indicators of copper toxicity. Additional research is required to determine if these responses apply to a broad range of pollutants and to other planktonic organisms. There is also a need to further evaluate the significance of these behavioral effects ecologically.Contribution No. 181 from the EPA Environmental Research Laboratory, Narragansett, RI 02882, USA     

Non-consumptive effects of predator presence on copepod reproduction: insights from a mesocosm experiment

Reproduction in planktonic animals depends on numerous biotic and abiotic factors. One of them is predation pressure, which can have both direct consumptive effects on population density and sex ratio, and non-consumptive effects, for example on mating and migration behaviour. In copepods, predator vulnerability depends on their sex, motility pattern and mating behaviour. Therefore, copepods can be affected at multiple stages during the mating process. We investigated the reproductive dynamics of the estuarine copepod Eurytemora affinis in the presence and absence of its predator the mysid Neomysis integer in a mesocosm experiment. We found that the proportion of ovigerous females decreased in the presence of predators. This shift was not caused by differential predation as the absolute number of females was unaffected by mysid presence. Presence of predators reduced the ratio of males to non-ovigerous females, but not by predation of males. Our combined results suggest that the shift from ovigerous to non-ovigerous females under the presence of predators was caused by either actively delayed egg production or by shedding of egg sacs. Nauplii production was initially suppressed in the predation treatment, but increased towards the end of the experiment. The proportion of fertilized females was similar in both treatments, but constantly fell behind model predictions using a random mating model. Our results highlight the importance of non-consumptive effects of predators on copepod reproduction and hence on population dynamics.     

On the occurrence of a double parasitism (copepod and isopod) on the anchovy fish in India

A double parasitism (isopod and copepod) on anchovy fish, Stolephorus commersonii Lacepede, 1803 was recorded from Parangipettai (India) coastal waters during June, 2004. This is the first report from this region and the infestation is discussed in relation to environmental parameters in this short communication.     

Growth rate and reproductive differences in a widespread estuarine harpacticoid copepod (Scottolana canadensis)

We demonstrate the presence of significant genetically based differentiation in growth rate (g dry weight d^-1) and reproductive traits (percent reproductive females and mean clutch size g dry weight^-1) among females of an harpacticoid copepod (Crustacea),Scottolana canadensis (Willey), taken from a broad range of latitudes and reared in the laboratory under the same conditions. As temperature increases (15°–25° C), the growth rate of southern-derived copepods continues to increase, while that of northern-derived copepods levels off or decreases. Southern-derivedS. canadensis also have a higher percentage of reproducing females at high temperature (25°C) when rations (cells ml^-1) are reduced, while northern-derived females are at an advantage at low temperature (15°C). Both life-history traits indicate local adaptation to maximize scope for growth and reproduction at prevailing temperatures. The data support our hypothesis that evolution has occurred to maximize feeding minus metabolic energy expended, and that this maximization requires changes in feeding efficiency with differing temperatures.