The effects of starvation on bioluminescence potential and egg release of the copepod Metridia longa

The effects of starvation on the bioluminescence potential andegg release rate of adult female Metridia longa were examinedduring a cruise in the marginal ice zone of the Greenland Seain April–May, 1989. Periods of starvation of up to 3 weekshad no significant effect on bioluminescence potential, whilerelease of eggs by adult females decreased from a maximum of3 eggs per female per day to no egg release after 10 days ofstarvation. It is suggested that egg release is dependent onrecently ingested food and seasonally timed by the ovanan maturationperiod, while stored food reserves are used for metabolic maintenanceincluding emission of bioluminescence, during periods of lowfood abundance.     

Diet changes in the carbon and nitrogen content of the copepod Metridia lucens

Diel changes in the near-surface (0–50 m) abundance, prosomelength, and carbon and nitrogen content of the copepod Metridialucens were measured in Deep Cove, Doubtful Sound, New Zealand(45°27'S, 167°9'E) between 3 and 6 September 1996. Metridialucens showed maximal abundance a night, suggesting a patternof normal diel vertical migration (DVM). The change in abundancesuggested that the descent of the population occurred –1h prior to dawn and the ascent –1 h after dusk. However,a proportion of the population remained near the surface duringthe day. Although there was no diel pattern in the prosome lengthof M.lucens collected near the surface, there was a marked dielcycle in the measured carbon and nitrogen contents, with maximalvalues being measured towards the end of the night prior tothe downward migration. We suggest that this diet cycle in themeasured elemental content was caused by DVM occurring morestrongly in those animals which had a better body condition,i.e. a higher elemental content per unit length.     

Morphology of genital system and reproductive biology of the arctic calanoid copepod Metridia longa

This paper considers features of the external morphology and internal structure of the genital system of adult males and females of Metridia longa Lubbock from the White Sea, as well as related with them features of reproduction biology of this mass Arctic copepod. Males of M. longa are dimorphically asymmetric. In the population, there are two morphotypes, which are mirror images of each other. Asymmetry manifests in them as the presence of a right (males ??righties??) or left (males ??lefties??) geniculate antenna, structure of the fifth pair of swimming legs, and location of an unpaired gonad and genital opening. The relative position of these structures is linked together. In females, there are two widely spaced copulator pores that lead to the two spermathecae not connected to each other. The latter, in turn, are connected to two gonopores, through which shedding of eggs occurs. The vast majority of the females in the studied population M. longa in all seasons of the year had only one filled spermatheca, either the right or the left. The females with bilateral insemination also occurred, but did not exceed 2.7% of all specimens. The ratio of females with the right and left filled spermathecae and ??right-handed?? and ??left-handed?? males suggests that the male morphotype determines in which spermathecae male gonads are most likely to get into. The presence of females with two filled spermathecae implicates insemination by two males of different morphotypes. Since each spermatheca is connected to only one of the two oviducts, it is assumed that a half of the eggs produced by unilateral inseminated females remains not fertilized. The morphology of genital structures and literature data on the egg production of M. longa indicate that almost a half of eggs produced by females is not viable and thus wasted.     

Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica

We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambdamax, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays.     

Egg and faecal pellet production rates of the marine copepod Metridia gerlachei northwest of the Antarctic Peninsula

Egg and faecal pellet production rates, and their functional response to food and temperature, were measured for the Antarctic copepod Metridia gerlachei during January 1996. The study area comprised the Gerlache and Bransfield Straits and Drake Passage. The highest rates of “in situ” egg and faecal pellet production were observed in Gerlache stations, coinciding with chlorophyll a concentrations approaching food saturation levels. In the Bransfield and Drake stations, with very low chlorophyll concentrations, the rates of egg production were either very low, or no eggs were produced. Egg production rates, although well correlated with “in situ” chlorophyll values, appeared to be independent of food concentration on a short time-scale (24-h incubations), while the production of faecal pellets was closely related to food abundance in the same experiments. In general, the rates of egg production were low, even at food saturation, with a very high individual variability. Although in the majority of Gerlache stations about 50% of total chlorophyll a corresponded to the phytoplankton size-fraction >10 μm, M. gerlachei feeds preferentially on the <10-μm fraction. Temperature had no clear effects on egg production rate but had a significant effect on pellet production rates, with maximum values at 2.5°C. These features appear to agree with the reproductive strategy attributed to the species, based on a relatively low rate of egg production extended over a long reproductive period. Received: 18 November 1996 / Accepted: 24 March 1997     

Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells

Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (λ_max = 480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2–3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis.     

The swimming and feeding behavior of Mesocyclops

The swimming and feeding behaviors of Mesocyclops are described from a review of the literature and personal observations. Mesocyclops exhibits considerable behavioral flexibility in response to environmental stimuli. Mesocyclops edax exhibits an increase in horizontal looping behavior at high prey densities, and performs a tight vertical looping behavior in response to the loss of captured prey. Ingestion rates by Mesocyclops are a complex function of prey density, morphology, and behavior in addition to prey size. Vertebrate predators induce a rapid escape response in Mesocyclops and may be responsible at least in part for their extensive diel vertical migrations. The complex behavioral patterns of Mesocyclops suggest that its distribution and abundance in nature will be distinctly nonrandom and influenced as much by its own behavioral responses as by other external physical factors such as water circulation patterns.     

Effect of salinity on the swimming behaviour of the estuarine calanoid copepod Eurytemora affinis

The calanoid copepod, Eurytemora affinis, is the dominant zooplanktonspecies in most of European and North American estuaries, andtheir population maintenance within an environment of net seawardflow has mainly been explained by endogenous rhythms of circatidalswimming activity. However, no attention has been paid to thepotential link between the swimming behaviour of this speciesand salinity. The swimming behaviour of males, non-ovigerousfemales and ovigerous females from a continuous culture wasinvestigated under different salinity conditions. Increase anddecrease in salinity, respectively, increased and decreasedthe overall swimming activity of both males and non-ovigerousfemales. The complexity of the swimming paths of males and non-ovigerousfemales significantly increased with salinity. In contrast,ovigerous females were less motile and mainly sank. These observationssuggest an endogenous behavioural adaptive strategy to salinityfluctuations and the intrinsic ability of E. affinis to undergoshort-scale vertical migration triggered by changes in salinity.This supports field observations reporting increased abundanceof E. affinis in the water column during flood tides and providesa behavioural basis for the maintenance of viable populationsunder net ouflow conditions.     

The role of photoreception in the swarming behavior of the copepod Dioithona oculata

The copepod Dioithona oculata forms dense swarms near mangrove prop roots that are centered around shafts of light penetrating the mangrove canopy. Swarms can be created in the laboratory within light shafts created with a fiber optic light pipe. Laboratory observations of swarming behavior were recorded using video cameras, and the swimming behavior of the copepods and density of the swarms were quantified using video‐computer motion and image analysis techniques. Swarm formation results from a combination of phototactic and klino‐kinetic behavior. Dark adapted copepods initially exhibit a photophobic response to a light shaft, but become positively phototactic within 3–5 min after exposure to the light. Copepod aggregation rates under the light fit a saturation model, suggesting that copepods are attracted independently to the swarm marker. Copepods reverse their swimming direction when they encounter light intensity gradients near the edge of a light shaft, which aids in maintaining the swarm. Swarm formation can occur in the laboratory at light intensities as slow as 0.1 μM photons m^‐2 s^‐1, which is similar to light intensities at dawn when they are first observed to form in nature. Swarm formation appears to have an endogenous rhythm, as copepods will not form swarms at night under a light shaft.     

Algal toxins alter copepod feeding behavior

Using digital holographic cinematography, we quantify and compare the feeding behavior of free-swimming copepods, Acartia tonsa, on nutritional prey (Storeatula major) to that occurring during exposure to toxic and non-toxic strains of Karenia brevis and Karlodinium veneficum. These two harmful algal species produce polyketide toxins with different modes of action and potency. We distinguish between two different beating modes of the copepod's feeding appendages-a "sampling beating" that has short durations (<100 ms) and involves little fluid entrainment and a longer duration "grazing beating" that persists up to 1200 ms and generates feeding currents. The durations of both beating modes have log-normal distributions. Without prey, A. tonsa only samples the environment at low frequency. Upon introduction of non-toxic food, it increases its sampling time moderately and the grazing period substantially. On mono algal diets for either of the toxic dinoflagellates, sampling time fraction is high but the grazing is very limited. A. tonsa demonstrates aversion to both toxic algal species. In mixtures of S. major and the neurotoxin producing K. brevis, sampling and grazing diminish rapidly, presumably due to neurological effects of consuming brevetoxins while trying to feed on S. major. In contrast, on mixtures of cytotoxin producing K. veneficum, both behavioral modes persist, indicating that intake of karlotoxins does not immediately inhibit the copepod's grazing behavior. These findings add critical insight into how these algal toxins may influence the copepod's feeding behavior, and suggest how some harmful algal species may alter top-down control exerted by grazers like copepods.     

Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme

Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max) = 480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultrahigh throughput screening technologies.     

Cannibalistic feeding behavior of the brackish-water copepod Sinocalanus tenellus

Cannibalistic feeding behavior of the brackish-water copepodSinocalanus tenellus was examined in the laboratory using CI-II,CIII-IV and CVI female as predators and NI-II, NIII-IV, NV-VIand CI-II as prey. In each prey-predator combination, the ingestionrate increased with increasing prey density to an asymptoticvalue. Cannibalism took place even when phytoplankton was availableas an alternative food supply. Based on a daily ration, theoptimal prey stages for CVI females, CIII-IV and CI-II are NI-VI,NI-IV and NI-II respectively. Under average, natural prey density(10 nauplii l^–1), S tenellus can achieve only a smallfraction (max 9%) of the daily minimum food requirement by cannibalisticfeeding. However, the impact of cannibalism on naupliar survivorshipcan be significant. When adult females occur at a density of10 l^–1, the mortality due to cannibalism attains 99.2%during the naupliar stages.     

The role of feeding behavior in sustaining copepod populations in the tropical ocean

A fundamental question regarding marine copepods is how themany species coexist and persist in the oligotrophic environment(i.e. Hutchinson’s paradox). This question is addressedwith a stochastic, object-oriented Lagrangian model that explicitlysimulates the distinct foraging behaviors of three prominenttropical species: Clausocalanus furcatus, Paracalanus aculeatusand Oithona plumifera. The model also individually tracks allprey cells. Each particle’s motion combines sinking, turbulentdiffusion and active swimming when applicable. The model successfullysimulates observed size-partitioned carbon uptake rates. Basedon the model results, the wide-ranging translational ambit employedby C. furcatus is best suited for the acquisition of passiveprey while the relatively stationary behavior of O. plumiferapromotes the capture of larger, quickly sinking cells. The modelresults further suggest that the slow velocities and feedingcurrent employed by P. aculeatus are best suited for acquiringthe smallest cells though it also has a slight advantage overC. furcatus in acquiring the largest prey. A resource threshold,at a prey concentration of 530 cells mL^–1, is consistentlyexhibited by all three modeled species. Overall, these resultsimply that the size-partition preferences due to their differentforaging behavior contribute to the coexistence of these threespecies. This paper is one of six on the subject of the role of zooplanktonpredator–prey interactions in structuring plankton communities.     

Reproductive activity of two dominant Antarctic copepod species, Metridia gerlachei and Ctenocalanus citer, in late autumn in the eastern Bellingshausen Sea

The main reproductive period of herbivorous Antarctic copepods is coupled to the phytoplankton bloom in spring, while omnivorous or carnivorous species apparently reproduce year round. However, our knowledge of the reproduction during autumn and winter is limited. Therefore this study during a cruise with RV "Polarstern" aimed to measure reproductive activity in two dominant copepod species, Metridia gerlachei and Ctenocalanus citer, in late autumn (April/May). For this purpose, gonad development stages were determined from both species, taken with Bongo net in the eastern Bellingshausen Sea (Antarctic Peninsula region), and related to the ambient feeding conditions represented as chlorophyll a concentration. In addition, experiments were conducted with M. gerlachei to study the influence of feeding and starvation on gonad development. Gonad maturity of M. gerlachei differed considerably between stations and decreased over time. The correlation of chlorophyll a and the proportion of mature females, though significant, was weak and highly variable. In experiments, the gonads of feeding females were in better condition than those of their starving counterparts, showing that reproductive activity in M. gerlachei was related to ambient feeding conditions. Mature females of C. citer were found at each station (17-67%). The gonad stage composition was rather similar at all stations, showing no clear temporal and spatial trend. In this species, the proportion of mature females was not related to the chlorophyll a concentration, indicating either that the ambient phytoplankton stock was sufficient to fuel maturation or that other food sources were used. Our study shows that both species are reproducing during the austral autumn, indicating that their reproduction is partially decoupled from the spring phytoplankton bloom.     

The swimming behavior of planktonic crustaceans colonizing algal mats

Laboratory experiments showed that both cladocerans and copepods are able to overcome a filamentous algae barrier, and are able to move within algal mats. This suggests that the crustaceans noted in large numbers in accumulations of algae actively colonize these mats and live inside of them.     

Modulation of swimming behavior in the medicinal leech

The effects of serotonin on the electrical properties of swim-gating neurons (cell 204) were examined in leech (Hirudo medicinalis) nerve cords. Exposure to serotonin decreased the threshold current required to elicit swim episodes by prolonged depolarization of an individual cell 204 in isolated nerve cords. This effect was correlated with a more rapid depolarization and an increased impulse frequency of cell 204 in the first second of stimulation. In normal leech saline, brief depolarizing current pulses (1 s) injected into cell 204 failed to elicit swim episodes. Following exposure to serotonin, however, identical pulses consistently evoked swim episodes. Thus, serotonin appears to transform cell 204 from a gating to a trigger cell.Serotonin had little effect on the steady-state currentvoltage relation of cell 204. However, serotonin altered the membrane potential trajectories in response to injected current pulses and increased the amplitude of rebound responses occurring at the offset of current pulses. These changes suggest that serotonin modulates one or more voltage dependent conductances in cell 204, resulting in a more rapid depolarization and greater firing rate in response to injected currents. Thus, modulation of intrinsic ionic conductances in cell 204 may account in part for the increased probability of swimming behavior induced by serotonin in intact leeches.Abbreviations AHP afterhyperpolarizing potential - DCC discontinuous current clamp - DP dorsal posterior nerve - G2 segmental ganglion 2 - PIR postinhibitory rebound - RMP resting membrane potential     

Modulation of swimming behavior in the medicinal leech

Expression of swimming in the medicinal leech (Hirudo medicinalis) is modulated by serotonin, a naturally occurring neurohormone. Exogenous application of serotonin engenders spontaneous swimming activity in nerve-cord preparations. We examined whether this activity is due to enhanced participation of swim motor neurons (MNs) in generating the swimming rhythm. We found that depolarizing current injections into MNs during fictive swimming are more effective in shifting cycle phase in nerve cords following serotonin exposure. In such preparations, the dynamics of membrane potential excursions following current injection into neuronal somata are substantially altered. We observed: 1) a delayed outward rectification (relaxation) during depolarizing current injection, most marked in inhibitory MNs; and 2) in excitor MNs, an enhancement of postinhibitory rebound (PIR) and afterhyperpolarizing potentials (AHPs) following hyperpolarizing and depolarizing current pulses, respectively. In contrast, we found little alteration in MN properties in leech nerve cords depleted of amines. We propose that enhanced expression of swimming activity in leeches exposed to elevated serotonin is due, partly, to enhancement of relaxation, PIR and AHP in MNs. We believe that as a consequence of alterations in cellular properties and synaptic interactions (subsequent paper) by serotonin, MNs are reconfigured to more effectively participate in generating and expressing the leech swimming rhythm.Abbreviations AHP Afterhyperpolarizing potential - DCC Discontinuous current clamp - DE Dorsal excitor motor neuron - DI Dorsal inhibitor motor neuron - IPSP Inhibitory postsynaptic potential - MN Motor neuron - PIR Postinhibitory rebound - VE Ventral excitor motor neuron - VI Ventral inhibitor motor neuron     

Size-specific swimming behavior of Daphnia pulex

Body length affects several aspects of the behavior of quietlyswimming Daphnia pulex Swimming and sinking rates were measuredat 0.033 s intervals during the ‘hops’ characteristicof Daphnia swimming behavior Larger animals swim faster, covermore distance, and produce more powerful swimming strokes. LargerDaphnia also sink faster, but the sinking rate scales as lengthto the 0 58 power, far lower than the power of 2 00 predictedby Stokes Law considerations. The number of hops s^–1 wasindependent of body size, although a theoretical analysis predictshopping rate (antennal beat frequency) should increase as bodylength squared. Turning behavior, measured as the ratio of displacementto total distance, during 5 s, is also independent of body sizeIndependence of several parameters of body motion and body sizeimplies that factors other than simple mechanics affect Daphniaswimming behavior