Initial Attachment,Metamorphosis, Settlement,and Survival of Black‐footed Abalone,Haliotis iris,on Microalgal Biofilms Containing Different Amino Acid Compositions

Microalgal biofilms with different amino acid profiles were investigated for their ability to induce abalone, Haliotis iris, attachment, metamorphosis, shell stage, and survival. Twenty microalgal strains, isolated from rocky shores and sandy estuaries, were grown in the laboratory to produce young and matured monospecific biofilms. Abalone larvae were exposed to the different biofilm treatments and controls (no biofilms) for 7 (attachment and metamorphosis) and 14 (shell stage and survival) d. The larvae performed significantly better in biofilms compared with controls across microalgal strains, but attachment, metamorphosis, shell stage, and survival were generally less than about 50, 35, 25, and 25%, respectively. Some microalgal strains belonging to the same species but collected from different sites had different effects on the larvae, likely due to variations in biochemical composition and activity among strains. Although no clear relationship was found between the microalgal amino acid profiles and larval processes, percentage biofilm cover and total amino acids were positively correlated with some of these processes. In addition, abalone performed significantly better when exposed to mature compared with young biofilms. These results may be due to the greater amounts of microalgal cells and their extracellular polymers within older biofilms, which may cue larvae toward more nutritionally favorable environments.     

培养基对微生物被膜形成和厚壳贻贝附着的影响

为探讨微生物被膜生物学特性、培养基以及大型生物附着三者之间的相互关系,本实验通过微生物分子生态学和海洋贝类生态学等方法调查了不同培养基对海洋细菌所形成微生物被膜的影响及其对厚壳贻贝幼虫附着的影响。结果显示,厚壳贻贝稚贝率与培养基类型和细菌初始密度显著相关,进一步分析表明,Zo Bell 2216E和Seawater Luriabertani(SWLB)条件下分别有6株和9株细菌形成的微生物被膜密度与稚贝率显著相关。扫描电镜结果显示,Staphylococcus sp.3在Zo Bell 2216E培养基条件下形成微生物被膜的细菌分布较为紧密,Pseudoalteromonas sp.8则在SWLB培养基条件下微生物被膜细菌分布较为紧密,且形态变为短杆状。SDS-PAGE结果显示,相比Zo Bell 2216E培养基,Staphylococcus sp.3在SWLB培养基条件下9条条带蛋白显著下降,其中2个条带完全消失;Pseudoalteromonas sp.8则在SWLB培养基条件下明显增加5条蛋白条带。研究表明,微生物被膜的形成受到培养基的影响,培养基的不同导致微生物被膜的形态结构、分布和蛋白有所差异,最终导致微生物被膜诱导厚壳贻贝幼虫附着变态的活性差异,本研究为后续开展厚壳贻贝附着的分子机制奠定良好基础。     

Assessment of temperature or salinity effects on larval development by catecholamine‐induced metamorphosis of hatchery‐reared flat oyster,Ostrea angasi (Sowerby 1871) larvae

A need to improve larval rearing techniques led to the development of protocols for catecholamine‐induced settlement of flat oyster, Ostrea angasi, larvae. To further refine these techniques and optimize settlement percentages, the influence of salinity or temperature on development of O. angasi larvae was assessed using epinephrine‐induced metamorphosis. Larvae were reared between salinities of 15–35 and temperatures between 14.5 and 31°C. The greatest percentage survival, growth, development occurred when larvae were reared between 26 and 29°C and between salinities of 30 and 35. Larvae reared outside this salinity and temperature range exhibited reduced growth, survival and/or delayed development. Short‐term (1 h) reduction in larval rearing temperature from 26°C to 23.5°C significantly increased larval metamorphosis without affecting larval survival. Short‐term (1 h) increase in larval rearing temperature from 26°C to 29 and 31°C decreased larval survival and metamorphosis. To ensure repeatability in outcomes, tests showed that larvae sourced from different estuaries did not vary significantly in their metamorphic response to short‐term temperature manipulation and epinephrine‐induced metamorphosis.     

Larval development and fatty acid composition of Ostrea edulis (L.) fed four different single diets from conditioning to pre‐settlement

Survival, growth and fatty acid composition of Ostrea edulis larvae (L.) fed four different single species, microalgal diets, Tisochrysis lutea (T), Chaetoceros neogracile (Cg), Skeletonema marinoi (Sm) or Tetraselmis suecica (Ts) from broodstock to pre‐settlement, were studied. Lower larval growth (5.5 μm to 6.5 μm/d) was recorded in progeny continuously fed single S. marinoi or T. suecica, whereas good growth was achieved with single T. lutea (7.8 μm/d). Larvae, originated from broodstock receiving Sm or Ts, exhibited growth compensation when fed a bispecific balanced diet (TCg). This did not occur when broodstock and larvae were fed Cg or T, for which single or mixed diets led to similar larval growth. Furthermore, survival was high (>90%) regardless of microalgal diet, except for larvae fed from broodstock to pre‐settlement T (53%) or Ts (2%). There were significant differences in 20:5 (n‐3) and 22: 6 (n‐3) contents in polar and neutral fractions of O. edulis expelled larvae dependent on broodstock diet, as well as throughout larval development, but no clear trend was apparent when comparing fatty acid (FA) relative composition of both fractions of O. edulis larvae fed different diets at release or prior to settlement. In contrast, such correlation occurred when FA was expressed in absolute content but exclusively for larvae‐fed single diets and was particularly noticeable between 22: 6 (n‐3) and growth and survival. In the present work, broodstock nutritional deficiencies have been revealed in O. edulis progeny, compensated thereafter by feeding the larvae a mixed diet, and in this balanced condition, no obvious relation with larval development indicators was found with main fatty acid contents.     

脂多糖对细菌生物被膜形成及厚壳贻贝幼虫变态的影响

为探究脂多糖(LPS)对海洋细菌生物被膜形成、海洋贝类幼虫变态所产生的作用,本研究使用不同浓度的LPS直接刺激厚壳贻贝幼虫,观察其对幼虫附着变态的直接作用;同时在海假交替单胞菌形成生物被膜的过程中添加不同浓度的LPS,通过分析生物被膜生物学特性的变化,及变化后的生物被膜对厚壳贻贝幼虫附着变态的影响。研究结果显示,3种浓度的LPS均可直接诱导厚壳贻贝幼虫的变态;10 mg/L浓度LPS处理后的生物被膜其细菌密度、膜厚明显降低(p<0.05),胞外产物中多糖(如可乐酸)、脂类显著增加(p<0.05),同时其对幼虫变态的诱导作用也显著提高(p<0.05),因此推测,在厚壳贻贝幼虫的附着变态过程中,LPS具有直接诱导作用;除此之外,脂多糖还可以通过调控生物被膜胞外物质,特别是可乐酸的生成,从而间接影响厚壳贻贝幼虫的附着变态。     

Growth,settlement and survival of Dicathais orbita (Neogastropoda,Mollusca) larvae in response to temperature,diet and settlement cues

The southern Australian whelk, Dicathais orbita, is a potential candidate for aquaculture, as both seafood and for bioactive compound production. Larval rearing experiments to determine the effects of temperature and diet on the growth and survival of D. orbita larvae under laboratory conditions comprised five different unicellular algal diets of two brown algal species; Isochysis galbana and Chaetoceros muelleri, two green algae; Tetraselmis seucica and Nannochloropsis oculata, and a mixture of all four strains for larvae maintained at 16 and 22°C. Absolute growth, specific growth rate (SGR) and survival were determined regularly. Larvae reared at 22°C on a mixed diet, or brown algae, performed significantly better than those reared on green algal diets alone. Preliminary trials with settlement cues were undertaken on different aged larvae to determine when larvae become competent. An array of natural cues (carrion, Xenostrobus pulex, adult mucus and Ulvella lens), as well as concentrations of KCl was tested. KCl(concentration of 20 mM) induced the greatest settlement, however, no larvae metamorphosed under the conditions provided. This study confirms long‐lived planktotrophic larval development for Dicathais orbita with higher development rates at the higher water temperatures. Further studies will optimize culture conditions and cues for settlement and metamorphosis.     

Experimental Studies to Evaluate Larval Survival of the Fire Shrimp, Lysmata debelius, to the Juvenile Stage

Many coral reefs are threatened because of anthropogenic impacts such as destructive fishing methods for marine ornamentals. The marine ornamental industry is currently almost completely dependent upon wild collections. The development of commercially feasible methods to culture ornamental species could help to reduce the need for wild‐collected specimens and reduce pressure on coral reefs. Advances in larval rearing and nutrition would help overcome the bottlenecks that impede the commercial production of many marine ornamental species. This article focuses on research for improving the production of the fire shrimp, Lysmata debelius. Experiments were performed with larval fire shrimp to evaluate (1) the importance of essential fatty acid enrichment of live prey (rotifers and Artemia) and (2) feeding penaeid shrimp muscle tissue to improve survival to metamorphosis. We also provide the fatty acid composition of Day‐1 posthatch fire shrimp larvae. The enrichment of live prey with docosahexaenoic acid did not improve larval growth, survival, or time to metamorphosis, but larvae fed live prey together with pureed penaeid shrimp muscle had survival rates of 9.8 ± 1.2%. The data and protocols developed in this study provide a foundation and baseline for future fire shrimp larval studies.     

Influence of highly unsaturated fatty acids in live food on larviculture of mud crab Scylla paramamosain (Estampador 1949)

Two experiments were carried out to investigate the effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (ARA) levels in rotifers (Brachionus plicatilis) and Artemia on the survival, development and metamorphosis of mud crab Scylla paramamosain larvae. Five different lipid emulsions, varying in the level of total n‐3 and n‐6 highly unsaturated fatty acids (HUFA), DHA, EPA and ARA were used to manipulate the fatty acid profile of the live food. Fatty acid profiles of the live food and crab larvae at zoea one, three and five stages were analysed to study the HUFA uptake by the larvae. The fatty acid content of the live food affected the fatty acid profiles of the crab larvae. In both experiments, the survival rate in the zoeal stages was not statistically different among treatments. However, larval development rate and metamorphosis success were affected by the dietary treatments. In this respect, the DHA/EPA ratio in the live food seems to be a key factor. Enrichment emulsions with a very high (50%) total HUFA content but a low DHA/EPA ratio (0.6), or zero total HUFA content caused developmental retardation and/or metamorphosis failure. An emulsion with a moderate total HUFA (30%) and a high DHA/EPA ratio (4) was the best in terms of larval development during the zoeal stages and resulted in improved metamorphosis. Dietary ARA seemed to improve first metamorphosis, but its exact role needs further clarification. For the larval rearing of S. paramamosain, an enrichment medium containing about 30% total n‐3 HUFA with a minimum DHA/EPA ratio of 1 is recommended. Further investigation is needed on the total HUFA and optimum DHA/EPA ratio requirements for each crab larval stage.     

Growth and kinetics of lipids and fatty acids of the clam Venerupis pullastra during larval development and postlarvae

This study examines the larval development, metamorphosis and postlarval stage of Venerupis pullastra in relation to growth, lipids content and fatty acid composition, specifically those believed to be essential for most bivalves (i.e. 20:5n‐3 and 22:6n‐3). Clam larvae were fed with two species of microalgae supplied individually or mixed –Isochrysis galbana and Tetraselmis suecica–species normally used in bivalve hatcheries. Larvae fed with T. suecica showed a progressive accumulation of lipids and fatty acids but did not survive to metamorphosis. Contrarily, larvae fed with I. galbana or mixed diet showed a progressive decline in lipids and essential fatty acids (20:5n‐3 and 22:6n‐3) from the pediveliger stage onwards, and a survival rate of 95% until the start of metamorphosis. The lower content in n‐6 and the absence of 22:6n‐3 in T. suecica diet might contribute to the massive mortality observed for larvae fed with this diet. That diet seems to fail in the supply of some particular nutrient that allows energetic transformation of reserves for growth and metamorphosis. Nevertheless, larvae fed on mixture diet showed higher weight growth values at postlarval stage than those larvae fed on I. galbana diet.     

胆碱受体化合物对厚壳贻贝幼虫变态的调控作用

为研究胆碱受体在厚壳贻贝幼虫变态发育过程中的作用,通过药理学手段调查胆碱类神经递质乙酰胆碱、氯甲酰胆碱及其拮抗剂六甲双铵和阿托品对厚壳贻贝幼虫变态发育调控作用。结果显示:在24 h暴露实验中,氯甲酰胆碱在10-5~10-4mol/L浓度表现出诱导活性,乙酰胆碱则无诱导活性。在持续暴露实验中,氯甲酰胆碱在10-5~10-4mol/L浓度表现出诱导活性,乙酰胆碱在10-6~10-4mol/L浓度表现出诱导活性。在拮抗剂实验中,在10-4mol/L氯甲酰胆碱或10-4mol/L乙酰胆碱存在时,N型胆碱受体拮抗剂六甲双铵对厚壳贻贝幼虫的变态均无抑制效果,表明N型胆碱受体可能并不在幼虫的变态发育过程发挥主导作用。M型胆碱受体拮抗剂阿托品在10-4mol/L氯甲酰胆碱存在时表现出抑制作用,且测试液中幼虫的变态率降为0%,表明M型胆碱受体可能参与调控厚壳贻贝幼虫变态发育过程。在整个药理学实验过程中无死亡幼虫出现,因而氯甲酰胆碱和乙酰胆碱可作为有效诱导物来促进厚壳贻贝幼虫的变态发育,尝试应用于该种的水产养殖过程;同时本研究为厚壳贻贝变态发育调控机制的研究提供有效理论依据。     

Spawning and Larval Development of the Four‐Sided Sea Cucumber,Isostichopus badionotus (Selenka 1867), under Controlled Conditions

The objective of this study was to generate information for the development of Isostichopus badionotus culture protocols by determining optimal spawning methods, egg production, length and duration of larval stages, and larval habitat settlement preferences. Studies performed during the spawning seasons of 2010 and 2011 showed that this organism spawns between July and November without mechanical or chemical induction. The larval development has five well‐defined stages: early, mid and late auricularia, doliolaria, and pentactula. Juveniles (654.3 µm length) were obtained on average 25 days after fertilization (DAF), when larvae were incubated at 25 ± 1 C. No preference was observed for any of the materials used for settlement of the larvae. The easy adaptation of I. badionotus to the culture conditions and the competence of the larvae to complete metamorphosis while feeding solely on microalgae concentrates make this species an excellent candidate for farming. However, it is necessary to continue designing techniques for larval culture and to establish appropriate captivity conditions that allow multiannual reproduction.     

Effect of stocking density and feeding regime on larval growth,survival, and larval development of Japanese flounder,Paralichthys olivaceus,using live feeds

This research examined the effect of initial stocking density and feeding regime on larval growth and survival of Japanese flounder, Paralichthys olivaceus. Larval rearing trials were conducted in nine 50‐L tanks with different initial stocking densities combined with different feed rations (20 larvae/L with standard feed ration [LD], 80 larvae/L with standard feed ration [HD], and 80 larvae/L with four times the standard feed ration [HD+]). Larvae were stocked on 0 days posthatch (DPH) following hatching of the fertilized embryos. Larval total length (TL), survival rates, and final densities were observed on larval settlement (32 DPH) to evaluate larval rearing performance. At 32 DPH, there were no significant differences (p > .05) in TL or survival rates between the LD (46.5 ± 17.0%) and HD+ (40.3 ± 9.4%). The TL and survival rate of HD (23.1 ± 3.5%) were significantly lower than that of LD and HD+ (p < .05). However, the larval density of HD was significantly higher than that of LD (p < .05). HD+ achieved the best larvae production (32.27 ± 7.51 larvae/L), supported by sufficient food source, high water exchange, and proper water quality management (routine siphoning, surface skimming). The larval‐rearing protocols and larval development from hatching to metamorphosis is described in detail, with corresponding photographs taken during the experiment.     

Feeding strategies for striped blenny Meiacanthus grammistes larvae

Rotifers and Artemia salina nauplii are the most widely used live prey for newly hatched larvae, but they do not always promote optimal survival and growth. Alternative food sources such as copepods, which bypass these inadequacies and promote adequate growth, are needed and they are viewed with considerable interest by the scientific community. The aim of the present study was to test two different diets [rotifers and A. salina nauplii (group A) and a mixture (group B) of rotifers/Tisbe spp. copepods and A. salina nauplii/copepods] during the larval rearing of the striped blenny Meiacanthus grammistes. The analysis of the survival rate, size (total length and wet weight) and metamorphosis time during the larval phase of this species showed that Tisbe spp. administration can significantly improve larval survival and growth and also reduce the metamorphosis time. The results obtained are related to the fatty acid content of the live prey used and are essential in order to improve the captive production of M. grammistes through a closed system and, in turn, to preserve natural stocks.     

4种饵料对玉足海参幼体生长和发育的影响

为了解单一饵料或混合投喂对玉足海参(Holothuria leucospilota)幼体生长的影响,采用2种单胞藻(牟氏角毛藻、球等鞭金藻)和2种酵母(酿酒酵母、海洋红酵母)单一或组合搭配投喂玉足海参耳状幼体,测定并比较海参幼体的体长、胃宽、水体腔长、变态成活率等指标。试验结果显示：单一饵料组中,牟氏角毛藻对玉足海参幼体体长的生长及变态存活最有利,而球等鞭金藻对其脏器(胃、水体腔)的发育最有利;综合所有处理组的投喂效果看,混合投喂组比单一饵料组更有利于玉足海参幼体的生长和存活,其中酿酒酵母+海洋红酵母的混合投喂组合得到了最高的体长、胃宽和水体腔长特定生长率,牟氏角毛藻+海洋红酵母投喂组的变态成活率最高,变态发育速度最快。结果表明,在玉足海参幼体培养早期适合以混合酵母为主投喂,后期则可较多投喂牟氏角毛藻和海洋红酵母。     

Larval settlement of the silver- or goldlip pearl oyster Pinctada maxima (Jameson) in response to natural biofilms and chemical cues

This paper presents the first laboratory study investigating larval settlement and metamorphosis of Pinctada maxima in response to both natural and artificial inducers. Natural biofilms induced larval settlement and the inductive effect appears to be closely associated with the bacteria in the biofilm. This result indicates that, in nature, bacteria may play an important role in the settlement of P. maxima larvae. Various pharmacological agents were also tested in this study and the results were generally similar to those found with other marine invertebrates, particularly molluscs. The results show that some pharmacological agents are potent artificial inducers of larval settlement for P. maxima. The findings of this study provide valuable information for understanding the mechanism of the larval settlement of this species, which is useful information for the hatchery industry of this pearl oyster.     

肾上腺素对岩牡蛎幼虫变态的诱导

为了得到壳型规则、大小均一的单体牡蛎,本研究采用肾上腺素对岩牡蛎单体苗种生产的诱导条件进行研究,阐明了岩牡蛎单体苗种的最适诱导浓度、诱导时间和诱导密度。结果显示,肾上腺素能显著诱导岩牡蛎幼虫的不固着变态,最适诱导浓度为5×10–5 mol/L,最适诱导时间为1 h,提高诱导浓度和延长诱导时间导致岩牡蛎幼虫的不固着变态率、稚贝壳高和存活率显著降低;肾上腺素对低于8个/mL幼虫密度的诱导效果差异不显著,但稚贝壳高和存活率在8个/mL的培育密度下显著低于0.5～4个/mL,研究表明利用肾上腺素诱导岩牡蛎单体时,可大批量处理眼点幼虫,但稚贝充气培养的最适培养密度应不高于4个/mL。     

Growth and Survival of Larval Sandfish,Holothuria scabra (Echinodermata: Holothuroidea), Fed Different Microalgae

This study assessed the nutritional value of four species of microalgae for larval sandfish, Holothuria scabra (Echinodermata: Holothuroidea). Two‐day‐old larvae were fed either a single diatom (Chaetoceros muelleri, Chaetoceros calcitrans), flagellate (Isochrysis aff. galbana (T‐ISO), Pavlova salina), or a ternary microalgal diet (TMD) comprised of 40% C. muelleri, 40% T‐ISO, and 20% P. salina for 12 d. On Days 4, 6, and 8 length of larvae fed either C. muelleri, C. calcitrans, or the TMD was significantly greater than in T‐ISO and P. salina fed larvae as well as the unfed control larvae. T‐ISO fed larvae had a significantly greater length compared to unfed larvae on Days 4, 6, and 8; however, larvae fed P. salina had a significantly greater length compared to unfed larvae on Days 6 and 8 only. Survival of larvae fed either C. muelleri or the TMD was significantly better than the flagellate fed and unfed larvae on Days 4, 8, and 12. There was no significant difference between survival of unfed larvae and larvae fed either T‐ISO or P. salina during the 12‐d larval culture period. On Day 12, larvae fed either a diatom or the TMD developed in excess of 84% competent doliolariae. Larvae fed C. muelleri had a significantly higher percentage competent doliolariae than larvae from all other treatments except the TMD. Neither larvae fed either T‐ISO or P. salina nor unfed larvae developed any competent doliolariae. In conclusion, this study showed C. muelleri to be the most effective of the four microalgae tested as a single microalgal diet for larval H. scabra and, as such, C. muelleri will be used as a reference diet in future studies.     

Effect of triiodothyronine (T3) in the larval development of the barber goby Elacatinus figaro

The barber goby Elacatinus figaro is an endangered fish, endemic to Brazil and very important to the aquarium trade. One of the bottlenecks for its production in captivity is the larviculture, which is characterized by high mortality rates, especially in the first week after hatching, and prior to metamorphosis. The experiment evaluated the effect of triiodothyronine hormone (T₃) on survival, growth and metamorphosis of barber goby. Larvae of 14 days after hatching (DAH) were immersed in three concentrations of T₃, in triplicate: TC (control) – 0; T01 – 0.01; T025 – 0.025 and T05 – 0.05 mg/L. An additional replicate of each treatment was performed for sampling for the histological analysis of the thyroid follicles in 24 DAH larvae. The survival rates in TC, T01, T025 and T05 were 24%, 54%, 36% and 37% respectively, without significant differences between the treatments (p > 0.05). Regarding larval growth, the highest length values were obtained in TC. In T025 and T05, larval metamorphosis was anticipated in up to 11 days in relation to TC and T01. In T01 larvae, follicles were numerous with the presence of reabsorption vesicles in the colloid periphery, indicating increased production of thyroid hormones (THs), associated with the larval metamorphosis process. In T05, a follicle reduction in number and size was observed, given evidence of the end of metamorphosis. The use of 0.025 and 0.05 mg/L of T₃ anticipated metamorphosis in barber goby larvae, shortening the larviculture period for this species.     

Increasing the stocking density in Paracentrotus lividus larviculture: Effects on survival and metamorphosis rates

The development of sustainable methods for sea urchin juvenile production is currently constrained by high mortality rates during larval growth and the high costs of larval rearing systems management. With the aim of developing a method for the production of juveniles of the purple sea urchin Paracentrotus lividus in a medium‐scale recirculating system, the present study focused on the effects of high stocking densities on larval growth. Plutei larvae were reared at three different densities (up to 7 ind/ml) in a semi‐static culture system. Larval survival and metamorphosis success were evaluated in order to identify the most effective density range. The highest metamorphosis rates (80%–95%) were obtained at 4 and 7 larvae/ml. These results are comparable with (and in some cases higher than) those reported for the same species at much lower larval densities. In conclusion, the rearing conditions tested here show for the first time that a significantly higher (4 ind/ml) stocking density than those of traditional P. lividus rearing methods (based on large volumes and low densities) can be adopted, thus supporting the feasibility of an increase in the final output of competent larvae with no increase in rearing volumes.     

Assessment of Egg and Larval Quality during Hatchery Production of the Temperate Sea Cucumber,Australostichopus mollis (Levin)

A greater understanding of the causes of variation in egg and larval quality during larval rearing of sea cucumbers will assist in the development of methods to further improve production in hatcheries. For Australostichopus mollis the techniques and methods used in other hatcheries during fertilization resulted in polyspermy occurring in 10–60% of spawned eggs. Despite this, hatch rates were usually greater than 90%. By Day 18 when fed the diatom Chaetoceros muelleri at 2000 cells/mL/day, survival in cultures varied between 35 ± 26% and 57 ± 25%. Of remaining larvae, 10–12% remained in the water column for long periods and did not complete metamorphosis. Furthermore, observation of development in relation to growth and feeding rather than gut content was a better indicator of larval competence. Larvae that were between 700 and 1000 ± 50 μm in length had between four and eight hyaline spheres in the folds of the ciliated band and were in various stages of metamorphosis and settlement. Assessment and comparison of current and previous methods enabled a greater understanding of sources of variation in egg and larval quality, impacts on the production cycle, and suggested improvements.