绢丝丽蚌寄生变态发育的研究

绢丝丽蚌钩介幼虫在雌蚌外鳃中发育成熟后于冬季排放到体外,而后寄生到宿主鱼的鳃丝进行变态发育,寄生期历时近4个月.寄生后3d,内、外足丝消失;35d后,肉足形成;90d后,肠、闭壳肌、肾脏和鳃原基等生成;壳面加厚且外凸.次年春季,钩介幼虫变态发育成大小为253.37×273.26×179.96μm的稚蚌,脱离鱼鳃营独立生活     

氨对褶纹冠蚌钩介幼虫及稚蚌的急性毒性

为探究氨对蚌类早期生活史阶段的毒性效应,研究分别以褶纹冠蚌(Cristaria plicata)的钩介幼虫及稚蚌为实验对象开展了24h和48h的急性毒性实验。结果表明:氨对褶纹冠蚌钩介幼虫的24h半致死浓度(LC₅₀)为0.63 mg NH₃-N/L (NH₃,分子氨)和78 mg TAN_7.0,20℃/L (TAN_7.0,20℃,pH 7.0水温20℃时即标准化的总氨氮);氨对褶纹冠蚌稚蚌的48h LC₅₀为0.60 mg NH₃-N/L和104 mg TAN_7.0,20℃/L;上述阈值高于已有研究中氨对其他淡水蚌类的毒性阈值;褶纹冠蚌钩介幼虫对氨氮的耐受性低于稚蚌,二者均低于幼蚌,因此褶纹冠蚌更早期生活史阶段对氨的耐受性更低。相关研究结果可完善氨对蚌类毒性的理解,为水体氮管理策略的制定和蚌类保护提供一定科学依据。     

蚌科钩介幼虫的比较形态学研究Ⅱ.六个种幼虫的形态

研究了真柱矛蚌、剑状矛蚌,洞穴丽蚌、角月丽蚌、三解帆蚌、褶纹完蚌等六种蚌的育作囊的类型和钩介幼虫的形态,应用光镜及扫描电僮对钩介幼虫的形态结构进行了观察和比较。结果表明 柱矛蚌、剑状矛蚌、三角帆蚌、褶纹冠的育儿囊为外鳃类同生型,洞穴丽蚌、角月丽蚌的育儿囊为外鳃类四生型、钩介幼虫可为二类：真柱矛蚌、剑状矛蚌、褶 纹冠蚌为有钩型,三角帆蚌、油穴丽蚌、角月丽蚌为无钩型。一些细微结构在不同种之间也存在着差     

紫黑翼蚌钩介幼虫寄主鱼的人工筛选及寄生包囊形成观察

选取16种鱼类, 对紫黑翼蚌(Potamilus alatus)钩介幼虫寄主鱼进行人工筛选试验, 结果表明: 仅在淡水石首鱼(Aplodinotus grunniens)上获得变态发育的稚蚌, 寄生变态率为(49.6±9.4)%, 但是过量寄生将导致淡水石首鱼的死亡。除眼斑拟石首鱼(Soiaenops ocellatus)外, 寄生在其他14种淡水鱼类均不可能实现钩介幼虫的变态。进一步扫描电镜观察显示: 钩介幼虫可在与淡水石首鱼同科的眼斑拟石首鱼鳃丝寄生并形成完整的包囊, 且在大规格鱼体形成包囊的速度明显快于幼鱼。但在寄生和淡水低渗双重胁迫下, 眼斑拟石首鱼出现极高的死亡率, 提示应进一步开展淡化驯养以提高眼斑拟石首鱼寄生后的成活率。其中黄颡鱼(Pelteobagrus fulvidraco)、小口黑鲈(Micropterus dolomieu) 、蓝鲶(Ictalurus furcatus)寄生后1—2d内脱落的幼虫几乎全部死亡, 推测3种鱼体中可能存在紫黑翼蚌幼虫的致死因子。综合研究表明: 紫黑翼蚌是目前发现对寄主鱼选择性最为专一的蚌类, 而眼斑拟石首鱼具有作为替代寄主鱼的潜能。     

孕育对褶纹冠蚌滤食率的影响及鳃微结构变化

对比了褶纹冠蚌(Cristaria plicata)孕育蚌、未孕雌蚌和雄蚌的滤食率,并运用组织学、扫描电镜和透射电镜对实验蚌的鳃结构进行比较观察,以此探讨鳃结构变化对滤食功能的影响。滤食实验结果表明:孕育事件显著降低了雌蚌的滤食率,而未孕雌蚌与雄蚌的滤食率无显著差异。孕育雌蚌内外鳃均由两鳃小瓣愈合而成,每一鳃小瓣由成排的鳃丝组成,在中介区鳃丝之间通过丝间隔连接,在内部侧区则通过瓣间隔相连。雌蚌内鳃的鳃间隔为外鳃的2～3倍,而雄蚌的内外鳃无差异。孕育雌蚌外鳃在初级水管、瓣间隔等出现明显的变化,并出现了二级水管结构,而内鳃未发现显著变化。扫描电镜显示:在褶纹冠蚌鳃丝表面存在3种类型的纤毛(前纤毛、前侧纤毛和侧纤毛),其形态结构和分布各具特点,长径58～85μm椭圆形的鳃小孔成排相间分布于鳃丝之间,而3组实验蚌的内外鳃丝之间无明显差异。透射电镜观察发现:孕育雌蚌鳃丝表皮细胞表面形成突起,显著增加了表面微绒毛的数量,可能有利于雌蚌在孕育期间由于初级水管转化成育儿囊后对呼吸、滤食等功能的补偿,与其他蚌科物种报道类似。综合实验表明,孕育雌蚌外鳃结构的变化,尤其是初级水管的结构改变和二级水管、鳃丝表面褶皱的出现可能是影响孕育雌蚌滤食功能的主要原因。     

褶纹冠蚌外套膜组织培养的分泌物的偏光显微镜…

以淡水育珠贝中珍珠形成较快的褶纹冠蚌为材料,用相差显微镜观察组织培养的外套膜的分泌物的形成和变化,用偏光显微镜观察分泌物的双折射现象,并与活体外套膜的分泌物、贝壳的角质层、棱柱层、珍珠层的双折射现象进行比较。结果表明：离体培养的外套膜细胞不仅能产生活体细胞相同的分泌物,而且分泌物还能在培养过程中形成结晶,并逐渐生长。发现外套膜的不同部位分区培养所形成的分泌物的性状与结晶性质和活体有一致性,表明组织     

三角帆蚌钩介幼虫在两种寄主鱼上的寄生效果及其对寄主鱼血浆生化指标的影响

利用黄颡鱼(Pelteobagrus fulvldraco)、尼罗罗非鱼(Tilapia nilotica)对三角帆蚌(Hyriopsis cumingii)钩介幼虫进行了寄生实验,分析了幼虫在寄主鱼上的寄生周期和脱落数量与寄主种类、寄主鱼单尾体重的相关关系.以未寄生幼虫的罗非鱼为空白对照,测定分析了罗非鱼在幼虫寄生前、脱落后主要血浆生化指标的变化.实验结果显示:黄颡鱼、罗非鱼平均每千克体重脱落稚贝的数量分别为(26 435±6 430)只、(26 327±3 091)只,两种寄主鱼间不存在显著差异;脱落的稚贝数量随着寄主鱼体重的增加而增加,并呈显著线性相关.在水温(24±1)℃条件下,黄颡鱼组、罗非鱼组的稚贝脱落高峰分别出现在寄生后的第8 d、第7 d,罗非鱼组表现为明显的"早脱落"现象.罗非鱼在寄生前后血浆总蛋白(TP)、白蛋白(Alb)、总糖(Glu)、总胆固醇(Tc)和高密度脂蛋白(HDL)含量均未出现显著变化,而血浆甘油三酯(TG)、低密度脂蛋白(LDL)含量显著下降(P=0.001,0.020).未寄生组罗非鱼的7项指标均未出现明显变化.实验结果将为三角帆蚌钩介幼虫变态发育相关营养因子的探讨提供理论参考.     

褶纹冠蚌和背角无齿蚌对水体营养盐和浮游植物的影响

2009年5月31日-6月20日在广东省大沙河水库利用微型生态系统比较不同放养密度的褶纹冠蚌(Cristaria plicata)和背角无齿蚌(Anodonta woodiana)对水体氮、磷及浮游植物的影响,探讨两种蚌在控制南亚热带水库富营养化水体藻类水华上的可行性。实验结果表明,在褶纹冠蚌和背角无齿蚌处理组中,总氮、总磷和叶绿素a浓度显著增加,而铵氮的浓度显著下降;褶纹冠蚌和背角无齿蚌导致了浮游植物群落组成结构的改变和数量的增加,实验过程中绿藻所占的比例迅速上升。两种蚌之间没有显著的差异,只是在不同的作用强度下,时间上的响应不同。综合实验结果,褶纹冠蚌和背角无齿蚌难以有效地运用于我国华南地区水库的水质改善与富营养化控制。     

人工采集褶纹冠蚌钩介蚴虫的点滴经验

我们从1972年开始人工繁殖褶纹冠蚌,通过两年的工作认识到褶纹冠蚌的人工繁殖较三角帆蚌要容易得多。但在采集钩介蚴虫的过程中,往往遇到的困难,就是成熟的褶纹冠蚌母体在拿进室内放入盛水的容器中,排蚴的速度极其缓慢,有的母蚌由于长期不排蚴而引起外鳃瓣中钩介蚴虫的萎缩、退化现象,因此采取杀蚌取蚴的方法。我场在1972年初也曾采用此法,后来我们遵照毛主席关于“要认真总结经验”的教导反复实践;不断总结,发现同日成熟的母蚌,当所处的环境条     

褶纹冠蚌外套膜组织培养的分泌物的偏光显微镜观察

以淡水育珠贝中珍珠形成较快的褶纹冠蚌为材料,用相差显微镜观察组织培养的外套膜的分泌物的形成和变化,用偏光显微镜观察分泌物的双折射现象,并与活体外套膜的分泌物、贝壳的角质层、棱柱层、珍珠层的双折射现象进行比较。结果表明;离体培养的外套膜细胞不仅能产生活体细胞相同的分泌物,而且分泌物还能在培养过程中形成结晶,并逐渐生长。发现外套膜的不同部位分区培养所形成的分泌物的性状与结晶性质和活体有一致性,表明组织培养的外套膜小片具有贝体原来的组织结构、分化特征和分泌功能。     

合浦珠母贝幼虫变态中的形态、器官变化及运动与摄食观察

为揭示合浦珠母贝幼虫至稚贝生长发育过程中其外部形态变化及内部器官改变的内在规律, 掌握其形态和器官与运动和摄食行为之间的关联。在光学显微镜下对整个幼虫生长发育及变态过程中的外部形态、内部器官特征进行了系列观察和性状测量; 利用非线性回归参数拟合, 描述各形态性状生长特点及不同属性之间的联系; 观察不同发育阶段其运动与摄食过程。结果显示, 幼虫在正常生长过程中, 其壳长生长方式为加速正增长、壳高为减速正增长、绞合线长为加速负增长, 壳高相对于壳长的生长为快速生长、绞合线长相对于壳长为慢速生长。幼虫生长至壳长为(209.26±9.22) μm时, 内部器官发生改变, 面盘开始逐渐退化从而发育成鳃, 斧足逐渐形成; 壳长生长至(234.30±14.00) μm时, 次生壳开始长出, 外部形态逐渐向稚贝转变。稚贝阶段, 其鳃丝长、鳃丝间距和鳃丝数量相对于壳长的生长均表现为慢速生长。幼虫在水中的运动和摄食过程主要依靠面盘外周纤毛的摆动来完成, 俯视观幼虫绕不规则圆沿顺时针方向运动, 垂直观幼虫螺旋上升或下降。稚贝阶段, 依靠斧足的往复伸缩来完成爬行, 依靠鳃的过滤完成摄食。在幼虫变态过程中, 面盘退化至鳃具备滤食功能期间, 变态幼虫运动功能降低, 摄食能力丧失, 依靠自身能量储备来完成生长和器官发育, 这一过程是苗种培育中的重要关键点。     

Who wins in the weaning process? Juvenile feeding morphology of two freshwater mussel species

The global decline of freshwater mussels can be partially attributed to their complex life cycle. Their survival from glochidium to adulthood is like a long obstacle race, with juvenile mortality as a key critical point. Mass mortality shortly after entering into a juvenile state has been reported in both wild and captive populations, thus weakening the effective bivalve population. A similar phenomenon occurs during metamorphosis in natural and hatchery populations of juvenile marine bivalves. Based on a morphological analysis using scanning electron microscopy of newly formed juveniles of the freshwater species Margaritifera margaritifera (L.) (Margaritiferidae) and Unio mancus Lamarck (Unionidae), we show that a second metamorphosis, consisting of drastic morphological changes, occurs that leads to suspension feeding in place of deposit feeding by the ciliated foot. We hypothesize that suspension feeding in these two species improves due to a gradual development of several morphological features including the contact between cilia of the inner gill posterior filaments, the inner gill reflection, the appearance of the ctenidial ventral groove and the formation of the pedal palps. Regardless of the presence of available food, a suspension feeding mode replaces deposit feeding, and juveniles unable to successfully transition morphologically or adapt to the feeding changes likely perish.     

A review on the “in vitro” culture of freshwater mussels (Unionoida)

Many Unionoida are considered to be extinct, endangered, or of special concern. These bivalves have complex life cycle stages that limit successful culture. In nature, the larvae (glochidia) of these bivalves must successfully parasitize a host (mainly fish) in order to metamorphose into juveniles. The two artificial methods used to obtain juvenile freshwater mussels in laboratory are either by induced attachment to host fish or by in vitro culture of glochidia. This article is focused on the in vitro method that represents a novel and alternative process to fish infestation, offering the ability to obtain larger numbers of juveniles without the need for host fishes and reducing the overall costs of propagation. In vitro culture requires a medium which fulfills the nutritional needs of each glochidia species and avoids microbial contamination. Recently, this methodology has presented excellent results with survival and transformation rates up to 94% using host fish plasma. High efficiencies on growth, and survival rates (84%) of juvenile freshwater bivalve Hyriopsis myersiana (Lea, 1856) up to 120 days were obtained when reared in adequate recirculating aquacultural systems using a very specific diet. More research is still needed to demonstrate successful propagation, mainly concerning the media nutritional composition to increase glochidia transformation and juvenile quality.     

Laboratory evidence suggests glochidia metamorphosis in Sinanodonta japonica (Bivalvia: Unionidae) is supported by gills,but no other tissues of the host Gymnogobius urotaenia (Perciformes: Gobiidae)

We quantitatively assessed the ability of the gills, caudal fin and scales of the floating goby Gymnogobius urotaenia (Hilgendorf, 1879) (Perciformes: Gobiidae) to serve as substrates for the larvae (glochidia) of the freshwater mussel Sinanodonta japonica (Clessin, 1874) (Unionida: Unionidae) by comparing parasitism success and metamorphosis success. We established three experimental treatments with 10 fish per treatment. Twenty glochidia were introduced onto one of the three body parts of each test fish by direct pipette infestation. Glochidia in the gill group had higher parasitism success than those in the fin and scale groups. Juvenile mussels were obtained only in the gill group. We quantitatively assessed the appropriateness of the three body parts as substrates for glochidia on the basis of three indicators: parasitism success; metamorphosis success; and parasitism and metamorphosis success. We conclude from our laboratory experiment that the artificial introduction of S. japonica glochidia onto G. urotaenia gills is a better procedure for obtaining juvenile mussels than the introduction onto fin or scales.     

Encapsulation of attached ectoparasitic glochidia larvae of freshwater mussels by epithelial tissue on fins of naive and resistant host fish

To metamorphose into juveniles and subsequently mature into adults, the glochidia larvae of freshwater mussels in the order Unionoida must temporarily parasitize the gills, fins, or other external structures of fish. Once attached to the fish, the glochidium is encapsulated by host fish epithelial tissue. The migration of epithelial cells of the bluegill sunfish Lepomis macrochirus over glochidia of Utterbackia imbecillis was examined by time-lapse video microscopy, and the morphology was examined by scanning electron microscopy. Initially, the leading edge epithelial cells migrating over the larvae became rounded and the cells moved as a sheet until the attached glochidium was completely covered. Cyst formation on host fish that had been repeatedly exposed to mussel larvae was significantly delayed and morphologically irregular compared to that on na?ve fish. Cyst formation on other species of fish that are less successful as hosts was examined. In general, it took longer for glochidia to become encapsulated on these less suitable potential hosts. The delay and irregularities in cyst formation on resistant fish and nonhost fish species may result in increased mortality and reduced success of metamorphosis of glochidia.     

Influence of cortisol on the attachment and metamorphosis of larval Utterbackia imbecillis on bluegill sunfish (Lepomis macrochirus)

The larvae of unionid freshwater mussels (i.e., glochidia) undergo a parasitic stage requiring their attachment to the external epithelia of fish hosts, where they metamorphose into free-living juveniles. We describe the physiological effects in bluegill sunfish (Lepomis macrochirus) of infection with glochidia from the paper pondshell (Utterbackia imbecillis). Glochidia accumulation on bluegill increased dramatically at concentrations of 2000 glochidia liter(-1) and above, reaching a maximum attachment density of about 30 glochidia g(-1) fish at 4000 glochidia liter(-1). Plasma cortisol was the most sensitive indicator of biological effect to glochidial exposure, increasing significantly in hosts exposed to 2000 glochidia liter(-1) or greater. Glochidia were 31% more likely to undergo successful juvenile metamorphosis when attached to bluegill with elevated plasma cortisol, largely due to the enhanced survivorship of these larvae during the first 48 h after infection. We tested the hypothesis that glochidial attachment and juvenile metamorphosis were stimulated directly by plasma cortisol in fish hosts. Bluegill were given an intraperitoneal injection of cortisol, then infected with 1000 glochidia liter(-1) at 48 h after hormone supplementation. Cortisol-injected fish had a 42% increase in the number of attached glochidia g(-1) fish and a 28% increase in larval metamorphosis compared to sham-injected and control fish. We provide evidence that cortisol enhances glochidial metamorphosis on hosts by improving the retention of attached glochidia. This study gives insights into the influence of host physiology on glochidial attachment and juvenile mussel transformation.     

Glochidium metamorphosis in the endangered freshwater mussel Margaritifera auricularia (Spengler, 1793): a histological and scanning electron microscopy study

The metamorphosis of the glochidium of the critically endangered Margaritifera auricularia in the gills of a host is studied here for the first time. Siberian sturgeon, Acipenser baeri, were infected with glochidia and regularly inspected using scanning and optical microscopy. The mature glochidia immediately attach to the epithelium of the sturgeon gill filaments, piercing the secondary lamellae and the connective tissues, blood cells, and vessels within the lamellae. Once the epithelium is pierced, overlapping host lamellae cover the glochidium and form a cyst. Metamorphosis takes place inside the cyst. Sixteen days after infection the glochidium becomes spherical in shape and the larval muscle is reabsorbed. The two adductor muscles of the juvenile are observed 34 days after infection at 16-20 degrees C. Metamorphosis is complete in approximately 51 days at 18-22 degrees C and in 65 days at 16-17 degrees C. Released juveniles have a spherical shell with a thin rim of new shell material and a finely ciliated foot. Juvenile mean measurements are: length = 190 microm, width = 193 microm, and height = 210 microm.     

In vitro culture of glochidia from the freshwater mussel Anodonta cygnea

Abstract. Larvae of the freshwater swan mussel, Anodonta cygnea , were cultured in artificial media at the controlled temperature of 23°±2°C, with successful metamorphosis for the first time. The artificial medium contained a mixture of M199, common carp plasma, and antibiotics/antimycotics. Glochidia were reared to the juvenile stage in the medium after 10–11 d of culture. After 15 d of controlled feeding with phytoplankton, the juveniles showed an elongated shell with several growth lines. Larval survival was 34.3±9.3%, whereas the proportion undergoing metamorphosis was ≤60.8±4.2%. The ultrastructure of early developmental stages was observed by scanning electron microscopy, from the glochidial to the juvenile stage. Glochidia had a hooked shell, with two equal triangular valves formed by a calcareous layer with numerous pores and covered by a thin cuticle of chitin–keratin. The appearance of the complete foot within 11 d of in vitro culture was considered the final feature of metamorphosis to the juvenile stage. The main alteration during juvenile development was the formation, under the glochidial shell, of a new periostracum with growth lines. The prominent foot, gradually covered by long, dense cilia, showed rhythmical movements involved in the capture of particulate matter. Similarly, cilia and microvilli present in the mantle also performed the same role. Longer cilia, sparsely distributed in the mantle, may function as chemotactile sensors.     

In vitro culture of glochidia of the threatened freshwater mussel Unio crassus Philipsson 1788 – the dilution problem

We applied in vitro techniques in culturing glochidia of the thick-shelled river mussel Unio crassus, seriously threatened European species. Glochidia were freshly isolated from a gravid female. The sterile phase of the cultures was terminated at different time points to assess the optimal length of this phase. We imitated the process of juvenile excision from a fish host by diluting the culture with water at regular time intervals. The metamorphosed juveniles that survived until the end of the experiment and started growing their shells were observed for 24–27 days from the start of the culture in samples diluted for the first time between days 13 and 17. Long-lasting cultures usually became infected and died, whereas in those that were terminated too early, glochidia were unable to develop further in clean water. The transfer of juveniles from an artificial medium to pure water should be done gradually, through a series of dilutions, so that the larvae have the opportunity to feed on the diluted medium after metamorphosis. Only individuals with an active foot capable of operating outside the shell were ready to inhabit water and forage on solid food from the external environment.     

Gill filament differentiation and experimental colonization by symbiotic bacteria in aposymbiotic juveniles of Codakia orbicularis (Bivalvia: Lucinidae)

Summary

Codakia orbicularis is a tropical lucinid harboring gill endosymbionts which are environmentally transmitted from a free living-symbiont form to the new host generation after metamorphosis. Structural changes occurring in the cellular organization from incomplete gill filaments in young aposymbiotic juveniles to full differentiated gill filaments containing bacterial endosymbionts in reared symbiotic juveniles, were analyzed for juveniles from 250 μm to 2 μm shell-length. Aposymbiotic juveniles possess differentiated gill filaments with ciliated, intermediary, and lateral zones similar to those described in wild juveniles, except for the bacteriocytes which are lacking. Granule cells, which progressively differentiate during the morphogenesis of the gill filament, do not appear as a consequence of symbiosis. Experimental colonization of aposymbiotic juveniles by the free-living symbiont form has been obtained through the addition of unsterilized sand collected from the natural habitat of C. orbicularis. Two days after exposure to crude sand, symbiosis-competent bacteria enter by endocytosis at the apical pole of undifferentiated cells which progressively differentiate into classical bacteriocytes similar to those found in the adult gill filaments. Undifferentiated cells of aposymbiotic gill filaments remain receptive to bacteria several months after metamorphosis, and become bacteriocytes when aposymbiotic juveniles get contact with the symbiont free-living form. Therefore, the environmental transmission of symbionts does not appear to be restrained to a defined period of time during post-larval development in C. orbicularis.