Effects of shell morphological traits on the weight traits of Manila clam (Ruditapes philippinarum)

The shell length, height, and width, live body weight, and edible tissue weight of Manila clam of 1, 2, and 3 years of age were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and live body weight or edible tissue weigh used as a dependent variable for calculating the path coefficients, correlation index and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight or edible tissue weight were all highly significant (P < 0. 01). The shell height at 1-year old clams was highly correlated with the live body weight and edible tissue weight. The shell width of 2- to 3-year-old clams was strongly associated with the live body weight, while the shell length was closely linked to the edible tissue weight. The results of coefficients of determination for the morphological traits against weight traits agreed well with the results of path analysis. The correlation indices for all morphological traits against weight traits were approximately the same as determination coefficients regardless of clam age. The correlation indices (R²) of morphological traits against the live body weight of clams of all ages and edible tissue weight of 1-year-old clams were larger than 0.85, but R² of morphological traits against the edible tissue weight of 2- and 3-year-old clams was smaller than 0.85, indicating that some other factors might be associated with the edible tissue weight of 2- and 3-year-old clams. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), shell width X₃ (cm) against live body weight Y (g), edible tissue weight Z (g): for 1-year-old clams: Y = ?4.317 + 0.18X₁ + 0.147X₂, (X₁ < 0.01, X₂ < 0.01), Z = ?1.011 + 0.095X₂, (X₂ < 0.01); for 2-year-old clams: Y = ?15.119 + 0.249X₁ + 0.176X₂ + 0.688X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?4.248 + 0.198X₁, (X₁ < 0.05, X₃ < 0.01); and for 3-year-old clams: Y = ?25.013 + 0.415X₁ + 1.184X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?7.082 + 0.119X₁ + 0.332X₃, (X₁ < 0.05, X₃ < 0.01).     

Development of microsatellite markers for the Manila clam Ruditapes philippinarum

We isolated nine polymorphic microsatellites from the Manila clam Ruditapes philippinarum. These loci provide one class of highly variable genetic marker as the number of alleles ranged from six to 22, and the observed and expected heterozygosity ranged from 0.136 to 0.909 and from 0.553 to 0.954, respectively. We consider that these loci are potentially useful for detailing the genetic structure and gene flow among R. philippinarum populations.     

大蒜对菲律宾蛤仔早期生长发育的影响

实验了不同大蒜汁浓度(0、2、4、8、16、32 mg/L)对菲律宾蛤仔受精卵孵化率,幼虫生长、存活、变态及稚贝生长与存活的影响,总结了室内大规模人工育苗过程中大蒜防病效果。结果表明:随着大蒜汁浓度的增加,孵化率降低;大蒜汁浓度达到16mg/L,胚胎发育延迟;达到32mg/L,受精卵不能孵化为正常幼虫。浮游期间,幼虫的生长受大蒜汁抑制,幼虫的存活率则随着大蒜汁浓度增加先升高后降低;幼虫的变态率随着大蒜汁浓度的增加先升高后降低,以16mg/L为最适浓度;变态规格随着大蒜汁浓度增加而减小。室内培育期间,稚贝生长与存活随着大蒜汁浓度的增加先升高后降低,以8mg/L为最适浓度;室内大规模人工育苗过程中,使用浓度为8-10mg/L大蒜汁可以起到较好的防病效果。     

Effects of shell morphological traits on the weight trait of the orange strain of the Manila clam

A new strain of Manila clam with orange shell color was produced after selection within a full-sib family for two generations. In the present study, the shell length, height, and width, and the live body weight of the orange strain were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and the live body weight was used as the dependent variable for calculating the path coefficients, correlation index, and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight were all highly significant (P < 0.01). The correlation indices (R²) of morphological traits against the live body weight of clams were larger than 0.85, indicating that the morphology traits were the main factors affecting the body weight. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), and shell width X₃ (cm) against live body weight Y (g): Y = ? 2.62 + 0.34 X₁ + 0.145 X₂, (X₁ < 0.05, X₂ < 0.05). The results suggest that the shell length could be used as the main trait for selective breeding and could indirectly make a large improvement in the weight trait.     

Effects of starvation on growth, survival, and body biochemical composition among different sizes of Manila clam Ruditapes philippinarum

A study was conducted to investigate the impact of starvation on different sizes of Manila clam Ruditapes philippinarum (0.66 ± 0.11, 2.12 ± 0.38, and 11.65 ± 0.84 mm in shell length, respectively) in the summer of 2008. Different size clams were starved for 7, 15, 30, 45, and 60 d, respectively, and followed by refeeding for 30 d. During the study, the water temperature ranged 26.2–28.4 °C, salinity 22–24‰, and pH 7.80–8.12. Compensatory growth occurred in the smallest size-group after 7 and 15 d of starvation, respectively. The point-of-no-return (PNR₅₀) was determined to be 18.7 d. However, no compensatory growth was noted in the medium size-group, and the PNR₅₀ for this group was 25.2 d. The complete compensatory growth was observed for the largest size-group following food depravation for 7 and 30 d, respectively. In the same group, over-compensatory growth occurred 15 d post-starvation. The PNR₅₀ for the largest size-group was 46.3 d. The survival rate of different groups decreased as the starvation time prolonged. To discuss the change in body biochemical composition of individuals in the process of starvation and refeeding, the biochemical composition of the largest group individuals at different stages was determined. There were no significant differences in moisture and ash concentrations of the largest size-group during starvation and refeeding (P > 0.05). The relative body protein content increased as the starvation period prolonged and the level returned to normal after refeeding. The lipid content of the clam at the end of starvation was significantly lower than the initial level (P < 0.05), and remained below the initial level at end of the refeeding period.     

菲律宾蛤仔的精子发生和精子超微结构

用透射电镜研究了菲律宾蛤仔(Ruditapes philippinarum)精子结构和精子发生过程中细胞形态结构的变化及细胞器的演变规律。菲律宾蛤仔雄性生殖细胞的形态由椭圆形渐变为辣椒状,细胞核的形态由椭圆形逐渐拉长,渐变为锥形。染色质的凝集经历：小颗粒团块状一较大颗粒均匀状一粗颗粒均匀状的过程。线粒体在演化过程中数量先增多后逐渐减少,嵴数逐渐增多,电子密度和体积逐渐增大。高尔基体在初级精母细胞期已经发育,随后的各期中发育良好,分泌旺盛。精细胞Ⅱ期,高尔基体分泌的潴泡开始融合,形成前顶体囊。精细胞Ⅲ期,高尔基体的分泌物仍不断融合。精细胞分化的后期,前顶体囊逐渐发育形成顶体。菲律宾蛤仔成熟精子呈长辣椒状,为原生型,由头部、中段和尾部构成。头部的顶体为细长柱形,末端渐细,电子密度较小;细胞核为锥形;中段线粒体4个,尾部鞭毛为典型的“9 2”型结构。此外在成熟精子线粒体环横切面有一特殊“风车状”结构。     

胶州湾菲律宾蛤仔生物量与资源评估

利用1998∽2003年胶州湾10个站中3个站的具有统计学意义的调查数据,对菲律宾蛤仔的生物量、生长期、年龄结构等进行了分析,从而对近期蛤仔的资源进行了初步评估。结果表明,蛤仔种群经过多年的增长延滞期之后,目前开始慢慢恢复。     

庄河海域菲律宾蛤仔底播增殖区自身污染

采用生物沉积物捕集器和封闭式代谢瓶,周年现场研究了庄河海域菲律宾蛤仔的生物沉积速率、排氨率和排磷率.结果表明:菲律宾蛤仔的生物沉积速率、排氨率和排磷率均具有明显的季节变化.生物沉积速率为0.15～1.47 g·ind-1·d-1(年均0.61 g·ind-1·d-1);其排氨率及排磷率分别为0.02～0.40 mg·ind-1·d-1(年均0.17 mg·ind-1·d-1)和0.01～0.39 mg·ind-1·d-1(年均0.13 mg·ind-1·d-1).根据以上结果,估算庄河海域底播增殖菲律宾蛤仔每年产生的生物沉积物达到5.46×107t(干质量),折合有机物9.07×106t、有机碳1.00x106t和有机氮1.18xlO5t;而氨氮和磷酸盐分别为1.49x104t和1.15x104t.表明浅海高密度、规模化菲律宾蛤仔增养殖区自身污染严重,其对环境的影响不可忽视.     

菲律宾蛤仔基因组DNA的提取及其RAPD分析

对菲律宾蛤仔(Ruditapes philippinarum)的三种组织,即性腺、斧足和内脏团的基因组DNA提取方法进行了研究,并对所提取的DNA作RAPD分析。结果表明性腺的DNA的得率明显比斧足和内脏团高。并且探讨了用于对基因组DNA进行RAPD研究的4种引物及最佳PCR条件,得到了较好的RAPD结果。     

Invasion in tidal zones on complex coastlines: modelling larvae of the non‐native Manila clam,Ruditapes philippinarum,in the UK

Aim To evaluate whether natural larval transport and behaviour alone can explain the pattern of invasion and establishment of the non‐indigenous Manila clam, Ruditapes philippinarum ( Adams & Reeve, 1850 ), and its spread beyond the point of introduction in the UK. Location The study is focused on Poole Harbour, south England, the point of introduction of the Manila clam in the UK. Methods We use fine‐resolution hydrodynamic models coupled with a water salinity model and an individual behaviour model of Manila clam larvae. The model was informed by experimental studies on the vertical response of larvae to salinity and field studies of the species in its natural and new environments. Results Variations in the behavioural response of larvae to salinity in the model considerably affected the retention of clam larvae within the harbour. High levels of predicted larval retention occurred in two of five zones in the harbour when the salinity target was set at 17 practical salinity units. Persistently high densities of adult clams and recruits are accurately predicted in these regions. Main conclusions Even within a relatively small region such as Poole Harbour, there is both localized retention of larvae or ‘closed’ areas and areas that are considerably more ‘open’ and potentially connected. The behavioural response of larvae to salinity significantly affected the degree of retention and ‘openness’ of the harbour to this species. Although, through natural transport, larvae could theoretically reach the next available habitat within the duration of their pelagic stage our study indicates that areas of sufficiently reduced salinity may be necessary for sufficient retention, recruitment and establishment of new adult populations in estuaries. High resolution hydrodynamic models, coupled with larval behaviour, can accurately simulate and predict biological invasion along complex coastlines and contribute to risk assessment of the introduction of non‐indigenous species for aquaculture and spatial management of marine protection.     

Reproduction of an alien Ruditapes clam (Bivalvia: Veneridae) on recreational clam-gathering grounds in Tokyo Bay,Japan

The Manila clam Ruditapes philippinarum is an important fisheries resource and popular recreational native species in Japan. To counter the domestic shortage of Manila clams caused by depleted catches, similar clams were introduced into Japan from China and the Korean peninsula. The introduced clam is an unnamed morph closely related to R. philippinarum that originates from southern China. Its shell is morphologically different from that of the typical Manila clam and there are small but distinct genetic differences. There is evidence to suggest that the Chinese morph may hybridize with the Manila clam in the Ariake Sea, but not in Tokyo Bay. We carried out studies to determine the degree of overlap of the breeding seasons of both taxa by collecting samples of the Manila clam and the alien clam released onto recreational clam-gathering grounds in Tokyo Bay and their gonadal maturity was examined histologically. Spawning-stage individuals of the Manila clam appeared from April to August and those of the southern Chinese morph from July to August, indicating overlapping spawning periods and thus the potential for hybridization. We discussed the forming condition of the hybridized clam population in terms of reproduction and recruitment process of the clams.     

Brown muscle disease: Impact on Manila clam Venerupis (=Ruditapes) philippinarum biology

This study assessed the effect of Brown Muscle Disease (BMD) on Manila clam Venerupis philippinarum fitness. BMD was discovered in 2005. It affects the posterior adductor muscle and leads to clam gaping and eventually death. Three statuses of clams were compared: buried individuals with no signs of BMD (BUR); clams at the surface of the sediment with no signs of BMD (SURF) and clams at the surface of the sediment exhibiting signs of brown muscle disease (BMD). Physiological (condition index), immune (hemocyte parameters) and molecular (gene expressions) parameters collected seasonally were analyzed and compared.Results demonstrated a seasonal pattern in condition index (CI) with peaks in spring/summer and decreases in autumn/winter. At each season, the highest CI was observed in BUR and the lowest CI was observed in BMD.In terms of immune response, phagocytosis rate and capacity were higher in clams with BMD whereas the health status of the clams did not influence the total hemocyte count. Genes involved in the immune system (comp, tnf, inter) were upregulated in clams with BMD. The molecular analysis of gill and posterior muscle showed higher mitochondrial metabolism (cox-1, 16S) in cells of infected clams, suggesting a stronger energetic demand by these cells. Finally, genes involved in oxidative stress response (cat, sod), detoxification (mt) and DNA repair (gadd45) were also overexpressed due to reactive oxygen species production.Most of the studied parameters underlined a cause–effect correlation between Manila clam health status (BUR, SUR, BMD) and physiological parameters. An important stress response was observed in BMD-infected clams at different scales, i.e. condition index, immune parameters and stress-related gene expression.     

冬季饥饿再投喂对菲律宾蛤仔生长、存活和生化组成的影响

2007年12月—2008年4月,在冬季低温（水温3.0 ℃～4.2 ℃、盐度25～27、pH 7.90～8.16）条件下,采用饥饿时间不等、恢复投喂时间相同的方式,研究了饥饿再投喂对不同规格菲律宾蛤仔生长、存活及体组分的影响.结果表明：在冬季低温条件下,各试验组菲律宾蛤仔生长性状的差异不明显;其对饥饿的耐受能力随着蛤仔规格的增大而升高;存活率随着饥饿时间的延长而降低,但整个试验过程中未出现不可逆点.从较大规格菲律宾蛤仔（壳长7 mm）的体组分上看：菲律宾蛤仔水分、脂肪含量随着饥饿时间的延长而下降;蛋白质含量在饥饿前期随着脂肪的消耗而上升,后期因脂肪的过度消耗开始被利用而下降;灰分含量在整个试验过程中的变化不显著.     

Effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum

The effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum at the temperature of 19.6–21.6 °C, the salinity of 34‰ and pH of 8.0 were investigated from May 18 to July 18, 2006. In this study, the early, middle and late umbo-veliger larvae with the shell lengths of 100, 140, and 190 μm were subject to temporary food deprivation for up to 4.5, 20, and 25d at 0.5, 4, 5d intervals, followed by refeeding for the remaining of a 24, 20, 25d period, respectively. The results suggested that the larvae should have shown considerable tolerance to starvation due to their endogenous and exterior nutrition material, for larvae and time to the point-of-no-return (PNR: the threshold point during starvation after which larvae could no longer metamorphose even if food is provided) were calculated to be 4.25, 17.54, and 22.17d. As the starvation period prolonged, the mean shell length of larvae starved got close to constants at 1.5, 4, and 15d after starvation, which were different for larvae at different stages when starvation began, survival of larvae decreased, and was lower in treatments starved earlier in development than those starved later, for the early, middle and late umbo-veliger larvae, after 4.5, 20 and 25d of starvation period, few larvaes were alive. After starvation period, the alive larvaes were able to metamorphose and had a capability of compensatory growth when refeeding was given. Starvation not only affected metamorphosis rate, but also caused the delay in the time to metamorphosis and the decrease in the metamorphosed sizes. For example, for the continuously-fed larvae, duration to metamorphosis was 20.7d, for larvae with a size of 100-μm starved for up to 4d, larvae with a size of 140-μm starved for up to 16d, larvae with a size of 190-μm starved for up to 20d, duration to metamorphosis were 29.7, 31.7, and 37.7d, the delay in duration to metamorphosis were 9, 11, and 17d, respectively. Furthermore, importance of nutrition material for maintaining larval survival during starvation and the compensatory growth on larvae at the same feeding time were discussed.     

Spatio-temporal patterns of perkinsosis in the Manila clam Ruditapes philippinarum from Arcachon Bay (SW France)

Pathogens belonging to the genus Perkinsus infect many bivalve molluscan species around the world, including the Manila clam Ruditapes philippinarum. We investigated the spatial distribution of this parasite at 34 stations throughout Arcachon Bay (SW France). Prevalence of perkinsosis was 93% and mean infection abundance was 96 x 10(3) cells g(-1) wet gill. Lowest mean abundances were found close to the Leyre River mouth and a significant negative correlation was observed between mean abundance and salinity. Perkinsosis was rare at the oceanic site where salinities and other environmental parameters were stable. A second aim of this study was to survey perkinsosis during annual cycles at 4 sites within Arcachon Bay. Prevalence and intensities (+/- SE) of the disease were high, on average between 70 and 100%, and 130 x 10(3) +/- 6.7 x 10(3) cells g(-1) wet gill. No seasonal cycle was evident. Clams were infected at 9 mm shell length and infection increased with clam size. The third objective was to determine the disinfection and infection kinetics through a 21 mo reciprocal transplantation between a nearly Perkinsus sp.-free area and a highly affected site. Disinfection appeared to be a very slow process and was similar at the site with favorable conditions for Perkinsus sp. as at the site with unfavorable conditions. Conversely, infection acquisition appeared to be episodic with spatially defined areas. Consequently, the overall lack of a clear seasonal infection pattern is interpreted as the combination of episodic infection events and slow disinfection kinetics.     

菲律宾蛤仔EST_SSR标记与生长性状的相关分析

研究利用20个微卫星标记对菲律宾蛤仔斑马蛤F2代家系107个个体进行遗传多样性分析,并对标记位点与生长相关性状进行分析。在20个微卫星位点共检测到41个等位基因,各位点等位基因数为2—3个,等位基因片段大小为109—430 bp,平均等位基因数为2.05个。平均有效等位基因数为1.71个,观测杂合度平均值为0.504,期望杂合度的平均值为0.431,平均多态信息含量为0.324。经卡方检验,3个位点SSR11,SSR164和SSR213的基因型分布显著偏离了孟德尔定律(P0.01)。运用SPSS 20.0对20个微卫星位点与菲律宾蛤仔斑马蛤家系生长性状的相关性(壳长、壳宽、壳高和体重)进行连锁显著性检验。结果表明,SSR9位点与壳高存在显著的相关关系(P0.05),SSR135和SSR164位点与壳宽呈显著相关(P0.05),SSR142位点与体重呈显著性相关(P0.05)。研究结果可为菲律宾蛤仔的分子标记辅助选育提供参考。     

Molecular cloning and characterization of Y-box gene (Rpybx) from Manila clam and its expression analysis in different strains under low-temperature stress

Manila clam, Ruditapes philippinarum, is an economically important marine bivalve species. Y-box proteins are members of the cold shock proteins family and highly conserved from bacteria to humans. In this study, a novel Y-box gene (Rpybx) was cloned from the Manila clam and gene expression profiling was performed on three shell color strains (white, zebra and white zebra) and two wild populations (Southern and Northern) of R. philippinarum. The complete ORF length of Rpybx is 1367 bp, encoding 253 amino acids residues. Based on the amino acid sequence analysis and phylogenetic analysis, the Rpybx gene was identified as a member of the invertebrate Y-box proteins family. Rpybx has a similar tertiary structure to human Y-box protein YB-1. The Rpybx mRNA levels were analyzed by qPCR under acute and gradually varied cold stress. Under acute low-temperature stress, the expression of Rpybx mRNA in gills and hepatopancreas was significantly increased in all selected strains and populations (P < 0.05). The Northern population showed the lowest relative expression level of Rpybx. The expressions of Rpybx were greatly upregulated in gills and hepatopancreas of different stains and populations at 5 or −2°C under gradually varied temperature stress (P < 0.05). The results shed light on the biological function of the Rpybx gene in defending against low-temperature challenge and further exploring the molecular mechanism of cold tolerance and resistance in R. philippinarum.     

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.     

Doubly Uniparental Inheritance and beyond: The contribution of the Manila clam Ruditapes philippinarum

The Manila clam, Ruditapes philippinarum (Adams & Reeve, 1850), is a widespread and commercially important bivalve species showing a peculiar way of mitochondrial inheritance known as Doubly Uniparental Inheritance (DUI), which is different from the strict maternal inheritance found in the broad majority of metazoans. DUI in R. philippinarum was discovered later than in mytilids and unionids. Nevertheless, this case keeps providing interesting data pertinent to the mechanism of this inheritance system. In this review, we discuss the contribution of this species, both in the context of the available knowledge on DUI and in the broader context of metazoan mitochondrial biology. Indeed, thanks to its unusual features, DUI can shed light on mitochondrial inheritance and biogenesis and, above all, on the relationship between mitochondria and germ line. Moreover, DUI is a unique experimental system for studying mitochondrial heteroplasmy, and two processes that shape genome evolution: genomic conflicts and mito-nuclear coevolution, which are at the very root of eukaryotic life.