Triploid Scallops Research Articles

Triploid-induced complete sterility in the scallop Nodipecten subnodosus might be triggered by an early and sustained DNA damage response

Triploid Nodipecten subnodosus scallops, unlike other mollusks, are completely sterile. In this study we focused on understanding the underlying molecular changes of triploid sterility using a transcriptomic approach. Total RNA from the gonad of diploid scallops in inactive and initial gametogenic stages and triploid scallops of the same cohort and ages were sequenced employing Illumina RNA-Seq. From 68,244 assembled and annotated transcripts, 1120 had terms associated with meiosis checkpoint or arrest, DNA damage response, or recombination. Differential gene expression analyses were conducted by contrasting initial vs. inactive stages of gametogenesis in each ploidy. In diploids, genes participating in homologous recombination during meiosis (msh5 and kdm8), spindle organization (nup62), centrosome formation (cenp-T), and sex differentiation (Ns-dmta2 and pum3), were up-regulated during initial gametogenesis. In triploids, a different set of genes were up-regulated during initial gametogenesis, and included genes involved in the DNA damage response and double strand break repair (rad51-C, xpc, myoVI), in the transition of metaphase/anaphase of mitosis (slp1 and nuf2), as well as genes that trigger both the intrinsic and extrinsic (caspase-3, icad, bmcc1) and extrinsic apoptosis pathways only (tnfr1, dab2ip). The results suggest significant DNA damage in triploids initial gametogenesis, possibly as a consequence of failing to repair double-strand breaks during DNA replication. This coincides with previous observations in which few triploid scallops showed gametic stages more advanced than oogonia or spermatogonia, and when present they were few.

Identification of Male Gametogenesis Expressed Genes from the Scallop Nodipecten subnodosus by Suppressive Subtraction Hybridization and Pyrosequencing

Despite the great advances in sequencing technologies, genomic and transcriptomic information for marine non-model species with ecological, evolutionary, and economical interest is still scarce. In this work we aimed to identify genes expressed during spermatogenesis in the functional hermaphrodite scallop Nodipecten subnodosus (Mollusca: Bivalvia: Pectinidae), with the purpose of obtaining a panel of genes that would allow for the study of differentially transcribed genes between diploid and triploid scallops in the context of meiotic arrest and reproductive sterility. Because our aim was to isolate genes involved in meiosis and other testis maturation-related processes, we generated suppressive subtractive hybridization libraries of testis vs. inactive gonad. We obtained 352 and 177 ESTs by clone sequencing, and using pyrosequencing (454-Roche) we maximized the identified ESTs to 34,276 reads. A total of 1,153 genes from the testis library had a blastx hit and GO annotation, including genes specific for meiosis, spermatogenesis, sex-differentiation, and transposable elements. Some of the identified meiosis genes function in chromosome pairing (scp2, scp3), recombination and DNA repair (dmc1, rad51, ccnb1ip1/hei10), and meiotic checkpoints (rad1, hormad1, dtl/cdt2). Gene expression analyses in different gametogenic stages in both sexual regions of the gonad of meiosis genes confirmed that the expression was specific or increased towards the maturing testis. Spermatogenesis genes included known testis-specific ones (kelch-10, shippo1, adad1), with some of these known to be associated to sterility. Sex differentiation genes included one of the most conserved genes at the bottom of the sex-determination cascade (dmrt1). Transcript from transposable elements, reverse transcriptase, and transposases in this library evidenced that transposition is an active process during spermatogenesis in N. subnodosus. In relation to the inactive library, we identified 833 transcripts with functional annotation related to activation of the transcription and translation machinery, as well as to germline control and maintenance.

Growth and Reproductive Performance of Triploid Yesso Scallops (Patinopecten yessoensis) Induced by Hypotonic Shock

The successful induction of triploid embryos or larvae has been performed in Patinopecten yessoensis during the past 2 decades. However, no research has been reported about the performance of triploid P. yessoensis cultured in the field. This study induced triploidy in P. yessoensis by hypotonic shock and compared the growth and reproductive performance of triploids and diploids reared under commercial conditions for up to 24 mo. The main results of this study are as follows: Triploid scallops were smaller in size and weight compared with diploids and had a retarded absolute growth rate (AGR). After 24 mo of cultivation, the mean shell height, shell length, shell width, and body weight of triploids were 9%, 10%, 9%, and 25% less than diploids, respectively (P < 0.01). Although normal in sex ratio, the reproductive potential of triploids was significantly reduced. Only 87% of the triploids exhibited sex-discernible gonads during the breeding season. None of the male triploids spawned, and the percentage of female spawners among the triploid population was only 27% of that for the diploid population. The relative fecundity of triploid females was only 4% of diploid females. Triploid eggs produced mostly aneuploid larvae and had an extremely small chance of generating viable offspring when fertilized by sperm from diploid males. Most aneuploid larvae died before the D-shaped stage, and no survival exceeded 7 days. The potential to yield viable offspring from the triploid population was estimated to be only 4% × 10-6 of that of the diploid population. Despite the growth disadvantage of triploids, this study may support, in part, the energy reallocation hypothesis, because triploid AGR was similar to diploid AGR (2% variance) during the sexual maturation season. Our results also indicate that there would be no growth advantage, but instead a disadvantage, for triploid P. yessoensis growing at the experiment site. In addition, this research provides the first evidence that viable triploid molluscs can be induced by hypotonic shock, of which the practical and evolutionary implications are also discussed.

MUSCLE AND ROE LIPID COMPOSITION IN DIPLOID AND TRIPLOID SCALLOPS

ABSTRACT The use of triploid bivalves is now common in the market. We tested if there was a difference in lipid and long‐chain highly unsaturated fatty acid (HUFA) accumulation in the muscle of diploid and triploid scallops (Argopecten ventricosus) during maturation, as triploids do not reproduce and lipids are not transferred to the gonad, and thus, could be accumulated in the meat (adductor muscle). Differences in triacylglycerols, phospholipids, esterified and free sterols, as well as HUFA in the reserves of gonads were found between diploid and triploid scallops. In contrast, no differences were found in the muscle for any of the lipid classes or fatty acid content in relation to ploidy, although they varied in relation to the sampling month.PRACTICAL APPLICATIONSThe results obtained indicate that for human consumption purposes, the quality of the meat and roe of scallops follows a temporal pattern of lipid accumulation, with higher levels of total sterols in summer, but a higher proportion of arachidonic acid in winter and lower docosahexaenoic acid in autumn, and that the muscle lipid composition is not affected by the triploid condition.

Comparative biochemical composition of ploidy groups of the lion-paw scallop (Nodipecten subnodosus Sowerby) supports the physiological hypothesis for the lack of advantage in triploid mollusc’s growth in food-rich environments

Induction of triploidy in aquatic organisms has increased worldwide in the last two decades, mostly because triploids have better growth than diploids. According to a physiological hypothesis, partial or total sterility of triploids allows the accumulation of reserves in muscle and other tissues instead of being transferred to the gonad. The present study analyzes lipid, protein, carbohydrate, and fatty acid levels in muscle and gonads of Nodipecten subnodosus triploids and diploids over 18 months from June 2001 to December 2002. An important increase in gonadosomatic index of diploids scallops was observed from May to June 2002 reaching the highest values in August. Such increase was not observed in triploid scallops. Changes in biochemical composition in female gonad were in general related to the accumulation of reserves during gonad development of diploid scallops. This accumulation was lower for triploid scallops, in accordance to their sterility, especially for carbohydrates and acylglycerides. Adductor muscle index as well as protein and carbohydrate levels in muscle increased in both ploidy groups during the reproductive period indicating no mobilization of reserves to sustain gonad development in both ploidy groups. These results partially support the physiological hypothesis on the advantage of triploids: in a rich food locality no mobilization of reserves is needed to sustain gametogenesis. This, together with a possible lower efficiency of energy assimilation at high food concentration for triploids, may be the reason for an apparent lack of superiority of N. subnodosus triploids in terms of adductor muscle growth. Only the levels of particular highly unsaturated fatty acids levels (namely 20:4n-6 and 20:5n-3) in muscle of diploid and triploid decreased during the reproductive period, indicating a possible transfer of selected fatty acids to gonads, even in triploids. The muscle of triploids has a slightly but significantly higher proportion of 22:6n-3 compared to diploids, which can have implications for the nutritional and commercial value of triploid adductor muscle.

Family differences in success of triploid induction and effects of triploidy on fecundity of catarina scallop ( Argopecten ventricosus)

Mass induction of triploidy in the catarina scallop ( Argopecten ventricosus) results in low success in the percentage of triploids produced. To understand whether this is a treatment effect affecting all eggs equally, families were individually induced to triploidy with cytochalasin-B (CB), comparing their survival from egg to D-larvae and spat, and the percent of triploidy within families. Differences in percent triploidy success were evident between families, obtaining some with no triploids, and some with high triploidy. Among the possible causes for these differences are quality of eggs, different developmental rates, and differences in susceptibility to the treatment (CB or DMSO) itself. Regardless of those differences, overall triploidy production was increased by inducing individually eggs of each scallop rather than in mixed egg batches. In the first experiment, it was improved by 17%, and in the second experiment by 42%, as indicated by the weighted mean of triploids among the families, and when compared with previous results with this same species, where triploidy success was 58%. In a second experiment with three different families, the growth and fecundity of triploid and diploid catarina scallops were evaluated. The growth superiority of triploids was confirmed. The results indicated that triploid catarina scallops had a significantly reduced fecundity when compared with diploid scallops. The reduced fecundity appears to be mostly of a random nature, possibly associated with a reduced capability to produce balanced gametes. Whereas the successful production of tetraploid catarina scallop from fertile triploid scallops is in principle possible, the low number of eggs shed by triploid catarina scallops could diminish that success rate, even more if single triploid females are required to optimize tetraploid induction.

Comparative biochemical composition in gonad and adductor muscle of triploid and diploid catarina scallop (Argopecten ventricosus Sowerby II, 1842).

Biochemical components of gonad and adductor muscle for diploid and triploid catarina scallop, Argopecten ventricosus, were evaluated and compared at four periods in 1 year (January, April, June, and October). Two comparisons were done. The first one compared an untreated control (diploid) vs. a triploidy-treated group for which the percentage of triploids was 57%. The second comparison was done on a group derived from within the triploidy-treated group, separating diploids (internal control) from triploids ('putative triploids'). Regardless of which comparison, in the gonad diploid scallops had larger concentrations of proteins, carbohydrates, lipids, and acylglycerols than triploid scallops. This reflects the maturation processes in diploid scallops vs. the sterility seen in most triploid scallops, and it is particularly supported by the consistently larger concentration of acylglycerols in gonads of diploids than in triploids. The gonad index of the internal control (diploid) group was significantly larger than that seen in the putative triploid group at all sampling periods but October, when none of the gonad biochemical components were different between ploidy groups.Triploid scallops had a significantly larger muscle index than diploids from April to October. This can be caused by a larger gain in muscle tissue in triploids than diploids from January to June. However, there were no consistent differences in any of the biochemical components evaluated in adductor muscle of diploids and triploids. The use of freshly ingested food rather than reserve mobilization from muscle in diploids is suggested by these results. Nutrients derived from ingested food are apparently used for muscle growth in triploids, whereas in diploids those nutrients serve primarily for gonad development. The importance of freshly ingested food for maintenance and growth is suggested because the decline in biochemical components seen in October in both muscle and gonad was paired with a decline in weight of those two organs, especially when the control groups are considered, but a decrease was also evident for the triploid groups. This may have been caused by the presence of El Niño, with its characteristic high water temperatures and low productivity.

Triploid and Tetraploid Zhikong Scallop, Chlamys farreri Jones et Preston, Produced by Inhibiting Polar Body I.

Triploid scallops are valuable for aquaculture because of their enlarged adductor muscle, and tetraploids are important for the commercial production of triploids. We tested tetraploid induction in the zhikong scallop by inhibiting polar body I in newly fertilized eggs. The ploidy of resultant embryos was determined by chromosome counting at 2- to 4-cell stage and by flow cytometry thereafter. Embryos from the control groups were mostly diploids (79%), along with some aneuploids. Embryos from the treated groups were 13% diploids, 18% triploids, 26% tetraploids, 13% pentaploids, and 36% aneuploids. Tetraploids, pentaploids, and most aneuploids suffered heavy mortality during the first week and became undetectable among the larvae at day 14. Five tetraploids (2%) were found among a sample of 267 spat from one of the replicates, and none was detected at day 450. The adductor muscle of triploid scallops was 44% heavier (P <.01) than that of diploids, confirming the value of the triploid technology in this species.

Triploid catarina scallop ( Argopecten ventricosus Sowerby II, 1842): growth, gametogenesis, and suppression of functional hermaphroditism

Triploidy was induced in catarina scallop, Argopecten ventricosus, using two concentrations of cytochalasin-B (CB). Growth of triploid scallops exceeded that of diploids in all evaluated traits. The largest percent difference between the diploid control group and the treated groups was seen after diploid scallops reached the peak of sexual maturation and began spawning. The gonad of triploid scallops was easily recognizable by visual inspection because of a brownish discoloration and lack or few egg masses present. That characteristic allowed for the separation of ‘putative triploids’ (PTs), which when compared with diploid scallops from within the same treatment (treated diploids), had a significantly larger muscle weight than the differences seen between treated and control groups. Gametogenesis and the condition of hermaphroditism in this normally functional hermaphrodite were greatly affected by the triploid condition. In the female portion of the gonad, few oocytes developed. The male portion of the gonad was arrested early during development, and the male acini were replaced by female acini, producing all female gonads in increasing percentages during the culture period, suppressing the normal condition of hermaphroditism. Oocytes of diploids were larger than those of triploids early in the culture, probably because of delayed maturation in triploids. Later during the culture, oocytes of triploids were larger than those of diploids only when compared with treated diploids.

Genetic considerations in transfers and introductions of scallops

As the potential of scallop aquaculture becomes increasinglyrealised, transfers (movement within a species' range) andintroductions (movement outside a species' range) of scallops arebecoming more common. To predict the genetic consequences oftransfers, information on genetic differences between source andrecipient populations is vital. Morphological, allozyme and DNAbased data on genetic differentiation of scallop populations andscallop sub-species are presented and discussed. Otherconsiderations are the numbers of individuals transferred andwhether they are wild stock or hatchery product. Loss of geneticdiversity is difficult to avoid in hatchery conditions althoughthere are ecological advantages to using disease-free hatcheryseed. Mitochondrial DNA data indicating significant geneticconsequences of the introduction of Argopecten irradians fromthe USA to China are discussed and compared with data onPatinopecten yessoensis introduced from Japan to Canada.Potential risks and consequences of hybridisation should beexperimentally assessed before introductions of scallops arecarried out. Hybridisation is unpredictable and can lead to lossof genetic diversity or breakdown of co-adapted gene complexes.The use of sterile triploid scallops for introductions to avoid hybridisation and reduce ecological impact has merit butreversion to diploidy may occur.

Triploidy induction in the black scallop ( Chlamys varia L.) and its effect on larval growth and survival

Triploid scallops ( Chlamys varia L.) were produced by treating newly fertilized eggs with cytochalasin B. An exposure of 15 min was applied 10, 20 and 30 min after fertilization. The best results were obtained for a delay of 20 min, both in terms of triploid percentage (78.5%) and 24-h survival (87.5%). Triploidy was detected by karyological examinations of embryos. A karyotype is given for the species. Growth and mortality were compared during larval development for untreated diploid controls and for triploids. No significant difference was found for growth in length, whereas mortality was significantly higher for the triploids, before metamorphosis.

ヒオウギガイ三倍体の配偶子形成と成長

Triploids were induced in the scallop, Chlamys nobilis, by treating the fertilized eggs with cytochalasin B (0.5mg/l) or hydropressure (200kg/cm2). The highest percentage of triploid was 71.4% in 0.5mg/l CB treatment. When diploids were in spawning period, most triploids had spermatocytes or developing oocytes. However, neither matured oocytes nor spermatozoa were observed in triploids. Although at 9 months of age no quantitative characters differed significantly between diploids and triploids, at 14 months the shell width and meat weight of triploids were greater than those of diploids. The relation between gametogenesis and growth in triploid scallops is discussed in this paper.

Detection of induced triploid scallop, Chlamys nobilis, by DNA microfluorometry with DAPI staining

A simple method of detecting the ploidy levels of shellfish by DNA microfluorometry with DAPI staining was demonstrated. Triploid scallops, Chlamys nobilis, were induced by cytochalasin B treatment (0.5 mg/l) of fertilized eggs, commencing 15 min after insemination for 15 min duration. Ploidy levels were determined at 8–13 months of age by microfluorometry of cells from gill tissue and hemolymph stained with DAPI and excited with UV light. Fluorescence intensity per nucleus was measured using a microphotometer. The ploidy levels determined by microfluorometry were verified by both cytogenetic and flow cytometric assays.

Induced triploidy in the bay scallop, Argopecten irradians, and its effects on growth and gametogenesis

Triploidy was induced in the bay scallop, Argopecten irradians, by treating newly fertilized eggs with cytochalasin B. Treatment for 20 min duration 10 min after fertilization resulted in 66% triploidy of scallops in the 0.05 mg/l cytochalasin treatment group and 94% triploidy of scallops in the 0.1 mg/l cytochalasin treatment group, detected by measuring the relative DNA content of scallop granulocytes using flow cytometry. Individually labelled scallops of known ploidy were grown in the Damariscotta River estuary in south-central Maine, U.S.A. from May 24 to September 15, 1982. Mean adductor muscle weight in triploid scallops was 73% greater than that of diploid controls and mean total body tissue wet weight was 36% greater. Shell inflation was significantly greater in triploids, while shell height and length were unaffected. The indexes of weight and glycogen content of the adductor muscle were significantly greater in triploids at the end of the growing season. The majority of triploid scallops failed to ripen during the summer months, while diploid scallops ripened and spawned.