Development of Expressed Sequence Tags from the Bay Scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value ≤ 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.     

Pecten of the Pigeon's Eye as an Inter-ocular Eye Shade

THE eyes Of all birds have a black pleated structure, known as the pecten, which projects from the retinal surface into the vitreal space, towards the lens. This remarkable structure has provoked many speculations about its function^1,2, but only one has so far been widely accepted : the pecten has a copious blood supply and this is thought to function as a replacement for the capillary bed that, in many other vertebrates, supplies the vitreal surface of the retina^3,4. Its size, shape and heavy black pigmentation have suggested optical functions, some of which centre around its shadow casting potentialities^5,6, but many of these theories do not seem to take account of the way the pecten is placed in the optical system. It is oriented nearly parallel to light entering the centre of the pupil, so that it presents the minimum area to the converging rays forming the retinal image and the folded free border that is its most striking gross anatomical feature approaches close behind the lens. Thus the shadow cast by the incoming light, which we here call the “primary shadow”, will be as small as possible and very blurred. It is, furthermore, centred on the pecten's own base, which is the elongated optic disk or “blind spot”, devoid of receptors, where optic nerve fibres leave the eye. Thus we think the primary shadow has no beneficial functional significance; on the contrary, it is remarkable that such a large nutrient structure can be inserted into a small optical system with so little disturbance to its normal image forming function. There is, however, an optical function that can be performed by an opaque screen in the position of the pecten.     

Light-activated ion channels in solitary photoreceptors of the scallop Pecten irradians.

Retinas from the scallop Pecten irradians were enzymatically dispersed, yielding a large number of isolated photoreceptors suitable for tight-seal recording. Whole-cell voltage clamp measurements demonstrated that the phototransducing machinery remained intact: quantum bumps could be elicited by dim illumination, while brighter flashes produced larger, smooth photocurrents. Single-channel currents specifically activated by light were recorded in cell-attached patches, and were almost exclusively confined to the rhabdomeric region. Their density is sufficiently high to account for the macroscopic photoresponse. Channel activation is graded with stimulus intensity in a range comparable to that of the whole-cell response, and can be recorded with illumination sufficiently dim to evoke only quantum bumps. Light-dependent channel openings are very brief, on average 1 ms or less at 20-22 degrees C, apparently not because of blockage by extracellular divalent cations. The mean open time does not change substantially with stimulus intensity. In particular, since dwell times are in the millisecond range even with the dimmest lights, the channel closing rate does not appear to be the rate-limiting step for the decay kinetics of discrete waves. The latency of the first opening after light onset is inversely related to light intensity, and the envelope of channel activity resembles the time course of the whole-cell photocurrent. Unitary currents are inward at resting potential, and have a reversal voltage similar to that of the macroscopic light response. Voltage modulates the activity of light-sensitive channels by increasing the opening rate and also by lengthening the mean open times as the patch is depolarized. The unitary conductance of the predominant class of events is approximately 48 pS, but at least one additional category of smaller-amplitude openings was observed. The relative incidence of large and small events does not appear to be related in a simple way to the state of adaptation of the cell.     

Spike Potentials Recorded from the Insect Photoreceptor

Slow and spike potentials were recorded from single cells in the receptor layer of the compound eye of the drone of the honeybee. From electron microscopic observation of the drone ommatidium, it was concluded that the response had been recorded from the retinula cell. The following hypothesis is suggested for the initiation of spike potentials in the drone compound eye: Photic stimulation results in a decrease in the resistance of all or part of the retinula cell membrane, giving rise to the retinal action potential. The retinal action potential causes outflow of the current through the proximal process of the cell. This depolarizing current initiates spike potentials in the proximal process or axon of the retinula cell which are recorded across the soma membrane of the retinula cell.     

Identification and Mapping of Amplified Fragment Length Polymorphism Markers Linked to Shell Color in Bay Scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F₁ families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.     

Two Light-Induced Processes in the Photoreceptor Cells of Limulus Ventral Eye

The dark-adapted current-voltage (I-V) curve of a ventral photoreceptor cell of Limulus, measured by a voltage-clamp technique, has a high slope-resistance region more negative than resting voltage, a lower slope-resistance region between resting voltage and zero, and a negative slope-resistance region more positive than 0 v. With illumination, we find no unique voltage at which there is no light-induced current. At the termination of illumination, the I-V curve changes quickly, then recovers very slowly to a dark-adapted configuration. The voltage-clamp currents during and after illumination can be interpreted to arise from two separate processes. One process (fast) changes quickly with change in illumination, has a reversal potential at +20 mv, and has an I-V curve with positive slope resistance at all voltages. These properties are consistent with a light-induced change in membrane conductance to sodium ions. The other process (slow) changes slowly with changes in illumination, generates light-activated current at +20 mv, and has an I-V curve with a large region of negative slope resistance. The mechanism of this process cannot as yet be identified.     

Effects of Sodium, Potassium, and Calcium Ions on Slow and Spike Potentials in Single Photoreceptor Cells

The influence of changes in the ionic composition of the bathing medium on responses of the retinula cell of the honeybee drone to light was examined by means of intracellular microelectrodes. The resting potential of the cell was influenced mainly by the concentration of K. The peak of the receptor potential (the transient), which in a normal solution and with strong light approaches zero membrane potential, overshot this level in a K-rich solution. An increase in the concentration of K also raised the level of the steady-state phase of the receptor potential (the plateau). The amplitude of the receptor potential was decreased and the spike potential rapidly abolished when Na was replaced by either sucrose, choline, or Tris. In a Ca-free solution the amplitude of the response and especially that of the plateau, was increased. An increase in Ca had the opposite effects. All these changes were reversible. An attempt was made to interpret the receptor and spike potentials in terms of passive movements of Na and K across the membrane of the retinula cell. The major difficulty encountered was to find an explanation for the persistence of an appreciable fraction of the transient and the plateau in preparations kept up to 12 hr in a solution in which all the Na had been replaced by choline, Tris, or sucrose.     

Development and Linkage Analysis of 104 New Microsatellite Markers for Bay Scallop (Argopecten irradians)

For genetic analysis and linkage mapping of bay scallop (Argopecten irradians), a set of 120 novel simple sequence repeat markers were developed from microsatellite-enriched libraries and expressed sequence tags. An inter-subspecies hybrid bay scallop family (CC5) of 46 progeny was analyzed as the reference population to confirm polymorphism and test the segregation patterns of these loci. A total of 104 microsatellite markers were polymorphic in the reference family, among which 36 in female, 28 in male, and 40 in both parents, respectively. Linkage analysis allowed mapping these markers to 15 linkage groups, which is close to the haploid chromosome number of bay scallop (n = 16). Analysis of the 40 markers segregating in both parents showed a higher recombination rate in the female parent, with the average of female-to-male recombination ratio of 1.09:1 between linked pairs of markers. When null alleles were considered, there were 17 loci showing segregation distortion at the 5% significance level using the chi-square test. The microsatellite markers developed in this study provide a useful resource for future linkage mapping and quantitative loci analysis in A. irradians.     

The Fine Structure of Photoreceptor Terminals in the Compound Eye of Pandalus borealis (Crustacea)

Seven of the photoreceptor axons of each ommatidium in the compound eye of the prawn Pandalus borealis end in two layers in the optic lamina. They have expanded terminals in the optic cartridges; four distally and three proximally in each cartridge. All seven receptor terminals are presynaptic to one lamina monopolar neuron (M₂) of the cartridge. This monopolar neuron is situated centrally in the cartridge and has a thick axis fibre with radially arranged branches, and its axon has a terminal in medulla externa. At the synapses, an arrowlike presynaptic bar is found facing three postsynaptic profiles. The receptor terminals have several characteristics. Their cytoplasm is filled with empty and coated vesicles, and contains numeorus large mitochondria and clusters of tubular elements. There is a longitudinally arranged fascicle of filaments partly surrounded by electron-dense amorphous material in the terminals. Centrally towards M₂, numerous neural spines invaginate into the terminal. Along the entire terminal periphery, there are invaginations from the glial cells. The terminals also form small knoblike protrusions extending into the surrounding glial cells.     

Thermal and Spectral Sensitivities of Discrete Slow Potentials in Limulus Eye

The discrete, subthreshold, slow potential fluctuations (SPF's) which can be recorded intracellularly in Limulus ommatidia are sensitive to temperature and light wavelength. SPF frequency increases with increasing temperature (Q₁₀ about 3.5) and light intensity. The effects are additive. SPF rise and decay time decrease with increasing temperature (Q₁₀ between 2 and 3). There is a peak, near 520 nm, in the spectral sensitivity of SPF frequency. This peak may correspond to the wavelength of maximum absorption by rhodopsin in the ommatidia. Hydroxylamine produces a rapid, irreversible reduction of SPF frequency and amplitude perhaps owing to its action on the photopigment. The cornea and crystalline cones fluoresce (peak about 445 nm) when excited by near-ultraviolet energy (380 nm peak) and this fluorescence may influence SPF spectral sensitivity measurements. These findings suggest that the SPF's are the results of photolytic and thermolytic reactions occurring in the ommatidial visual pigments and that they have a role in the mechanisms which transduce light to electrical activity in the visual receptors.     

Recording of Retinal Action Potentials from Single Cells in the Insect Compound Eye

Electrical responses were recorded intracellularly from the compound eyes of a fly (Lucilia) and of several dragonflies (Copera, Agriocnemis, and Lestes). An ommatidium of the dragonflies is made up of four retinula cells and a rhabdom composed of three rhabdomeres while the Lucilia has an ommatidium of seven independent retinula cells and rhabdomeres. The intracellular responses presumably recorded from the retinula cell had the same wave form in the two groups of insects: The responses were composed of two components or phases, a transient spike-like potential and a slow one maintained during illumination. The membrane potential, in the range of -25 to -70 mv., was influenced by the level of adaptation, and it was transiently depolarized to zero by high levels of illumination.     

A Consensus Microsatellite-Based Linkage Map for the Hermaphroditic Bay Scallop (Argopecten irradians) and Its Application in Size-Related QTL Analysis

Bay scallop (Argopecten irradians) is one of the most economically important aquaculture species in China. In this study, we constructed a consensus microsatellite-based genetic linkage map with a mapping panel containing two hybrid backcross-like families involving two subspecies of bay scallop, A. i. irradians and A. i. concentricus. One hundred sixty-one microsatellite and one phenotypic (shell color) markers were mapped to 16 linkage groups (LGs), which corresponds to the haploid chromosome number of bay scallop. The sex-specific map was 779.2 cM and 781.6 cM long in female and male, respectively, whereas the sex-averaged map spanned 849.3 cM. The average resolution of integrated map was 5.9 cM/locus and the estimated coverage was 81.3%. The proportion of distorted markers occurred more in the hybrid parents, suggesting that the segregation distortion was possibly resulted from heterospecific interaction between genomes of two subspecies of bay scallop. The overall female-to-male recombination rate was 1.13∶1 across all linked markers in common to both parents, and considerable differences in recombination also existed among different parents in both families. Four size-related traits, including shell length (SL), shell height (SH), shell width (SW) and total weight (TW) were measured for quantitative trait loci (QTL) analysis. Three significant and six suggestive QTL were detected on five LGs. Among the three significant QTL, two (qSW-10 and qTW-10, controlling SW and TW, respectively) were mapped on the same region near marker AiAD121 on LG10 and explained 20.5% and 27.7% of the phenotypic variance, while the third (qSH-7, controlling SH) was located on LG7 and accounted for 15.8% of the phenotypic variance. Six suggestive QTL were detected on four different LGs. The linkage map and size-related QTL obtained in this study may facilitate marker-assisted selection (MAS) in bay scallop.     

Cytogenetic characterization of the bay scallop, Argopecten irradians irradians, by multiple staining techniques and fluorescence in situ hybridization

The chromosomes of Argopecten irradians irradians were studied by various cytogenetic approaches. Conventional chromosome characterization built on C-banding, DAPI-staining, and silver staining was complemented by the physical mapping of ribosomal DNA and telomeric sequence (TTAGGG)n by FISH. Results showed that the constitutive heterochromatin revealed by C-banding was mainly distributed at telomeric and centromeric regions. However, interstitial C-bands were also observed. The pattern of DAPI banding was almost consistent with that of C-banding. Silver staining revealed that NORs were located on the short arms of chromosome 3 and 10, and this was further confirmed by FISH using 18S-28S rDNA. 5S rDNA was mapped as two distinguishable loci on the long arm of chromosome 11. 18S-28S and 5S rDNA were located on different chromosomes by sequential FISH. FISH also showed that the vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome and no interstitial signals were detected. Sequential 18S-28S rDNA and (TTAGGG)n FISH demonstrated that repeated units of the two multicopy families were closely associated on the same chromosome pair.     

Characterisation of the bacterial community associated with early stages of Great Scallop (Pecten maximus), using denaturing gradient gel electrophoresis (DGGE)

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA was used to characterise and compare bacterial communities associated with scallop larvae (Pecten maximus), in different production units in a shellfish hatchery. Water and larvae samples were collected from three different aquaculture systems; stagnant, flow-through and a flow- through system with seawater treated with ozone. Samples were also collected from different algal cultures, inlet tanks and water pipes leading to the different aquaculture systems. Clear differences were seen between the bacterial community associated with the larvae and in the water from the different aquaculture systems. However, there were high similarities in the community composition between different water samples and between larvae samples collected at different time periods, indicating a high stability in the bacterial communities. Fifty three percent of the sequences from these samples were similar to 16S rRNA gene sequences of members of the gamma-subclass of the Proteobacteria. The different algal cultures had different bacterial communities, however 73 percent of the sequences were similar to 16S rRNA gene sequences of members of the alpha-subclass of the Proteobacteria. Differences in the DGGE profiles were also seen between the samples taken from the inlet tanks and water pipes, indicating a change in the bacterial community composition as the water passed through the pipes. To our knowledge this is the first study investigating bacterial communities associated with Great Scallop larvae in different aquaculture systems including noncultured components.     

An investigation of the movement of the scallop,Pecten maximus

Summary 1. In the experimental area specimens of the scallop,Pecten maximus, occur at a density of one every 1 1/2 m². Over 90% are recessed in the bottom.2. Unrecessed specimens remain in the same position on an average for 6 days. Recessed specimens remain on an average 27 days.3. The experimental area has virtually unidirectional tidal flow. The scallops tend to orientate themselves on the bottom so that this flow complements their ciliary currents.

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Eine Untersuchung über die Fortbewegung der KammuschelPecten maximus

Kurzfassung ObgleichPecten maximus (L.) normalerweise im Meeresboden ruht, kann dieses Mollusk recht aktiv schwimmen. Frühere Untersuchungen über die Fortbewegung werden durch Wiederfang markierter Individuen mit Hilfe einer Dredsche durchgeführt; eine derartige Methode kann lediglich etwas über weiträumigere Ortsveränderungen aussagen. In der vorliegenden Studie wurde nun ein kleines Areal mit fixierten Bezugspunkten versehen und — unter Anwendung der Methode des wissenschaftlichen Schwimmtauchens — in bestimmten Zeitabständen 8 Wochen lang überwacht. Auf diese Weise konnte sowohl die Häufigkeit der Ortsveränderungen als auch die Orientierung erfaßt werden. Durchschnittlich verblieben nicht eingegrabene Individuen nur 6 Tage am gleichen Ort, eingegrabene Tiere dagegen auf Grund von Beobachtungswerten 17 Tage (Berechnungen deuten jedoch auf einen korrigierten Endwert von 27 Tagen hin). Es wird angenommen, daß spontanes Schwimmen im wesentlichen nur im Anschluß an Störungen durch andere Tiere erfolgt. Die Orientierung am Boden richtete sich vorwiegend nach dem am Untersuchungsort praktisch nur in einer Richtung laufenden Wasserstrom.P. maximus orientierte sich dabei im allgemeinen derart, daß die Wasserströmung ihre Freßströmungen unterstützt.

     

In Vivo Administration of Scallop GnRH-Like Peptide Influences on Gonad Development in the Yesso Scallop,Patinopecten yessoensis

Existing research on the role of gonadotropin-releasing hormone (GnRH) in bivalve reproduction is inadequate, even though a few bivalve GnRH orthologs have been cloned. The objective of this paper was to elucidate the in vivo effect of GnRH administration in Yesso scallop reproduction. We performed in vivo administration of scallop GnRH (py-GnRH) synthetic peptide into the developing gonad, and analyzed its effect on gonad development for 6 weeks during the reproductive season. The resulting sex ratio in the GnRH administered (GnRH(+)) group might be male biased, whereas the control (GnRH(-)) group had an equal sex ratio throughout the experiment. The gonad index (GI) of males in the GnRH(+) group increased from week 2 to 24.8% at week 6. By contrast the GI of the GnRH(-) group peaked in week 4 at 16.6%. No significant difference was seen in female GI between the GnRH(+) and GnRH(-) groups at any sampling point. Oocyte diameter in the GnRH(+) group remained constant (about 42–45 μm) throughout the experiment, while in the GnRH(-) group it increased from 45 to 68 μm i.e. normal oocyte growth. The number of spermatogonia in the germinal acini of males in the GnRH(+) group increased from week 4 to 6. Hermaphrodites appeared in the GnRH(+) group in weeks 2 and 4. Their gonads contained many apoptotic cells including oocytes. In conclusion, this study suggests that py-GnRH administration could have a potential to accelerate spermatogenesis and cause an inhibitory effect on oocyte growth in scallops.     

Monoamines and reproduction in Pecten maximus,a preliminary approach

Summary

The distribution of serotonin-like immunoreactivity was studied in the central nervous system and the gonad of Pecten maximus. Cerebral and pedal ganglia contain a well developed serotonin-immunoreactive neuronal subpopulation, whereas positive neurons are scarce in the visceral ganglion. The distribution pattern of immunoreactive elements in the gonad indicate that serotonin is involved in peripheral neurotransmission of this organ. Seasonal variations of monoamines (serotonin, dopamine and noradrenaline) have been investigated in the nervous system using HPLC. Lower concentrations of serotonin are observed during winter in the central nervous system; dopamine levels of the visceral ganglion are correlated to gonadal growth.