Competent Larvae Research Articles

Settlement of oyster Magallana bilineata (Röding, 1798) spat in the natural environment: seasonality and substrate texture preference

ABSTRACT This study determined the seasonality of Magallana bilineata spat settlement in a natural oyster bed and established surface texture preference of competent larvae by comparing the numbers of spat settling on the smooth and rough surfaces of ceramic tiles over a period of 31 months. This will guide farmers when to deploy cultches to maximise spat harvest. Regardless of tile orientation, spat numbers were significantly higher on the rough side (129.07 ± 9.3 m−2) than on the smooth side (66.95 ± 7.0 m−2). Spat collection was highest in March, June and July in each of 2015, 2016 and 2017. There was no significant correlation between the number of spat settling in each month with temperature, salinity, pH, dissolved oxygen (DO), sulphide, phosphate, nitrate, nitrite and ammonia concentrations, plankton density measured at deployment and retrieval of tiles, except for DO during deployment. The only significant correlations with mean shell length (SL) were with DO and ammonia during tile retrieval. Mean SL did not significantly differ between tile orientations and surfaces but differed between months, being highest in August 2015 and August 2016. From these results, it is best to deploy roughened cultches in this particular area in March, June and July.

Direct and latent effects of ocean acidification on the transition of a sea urchin from planktonic larva to benthic juvenile

Ongoing ocean acidification is expected to affect marine organisms and ecosystems. While sea urchins can tolerate a wide range of pH, this comes at a high energetic cost, and early life stages are particularly vulnerable. Information on how ocean acidification affects transitions between life-history stages is scarce. We evaluated the direct and indirect effects of pH (pHT 8.0, 7.6 and 7.2) on the development and transition between life-history stages of the sea urchin Strongylocentrotusdroebachiensis, from fertilization to early juvenile. Continuous exposure to low pH negatively affected larval mortality and growth. At pH 7.2, formation of the rudiment (the primordial juvenile) was delayed by two days. Larvae raised at pH 8.0 and transferred to 7.2 after competency had mortality rates five to six times lower than those kept at 8.0, indicating that pH also has a direct effect on older, competent larvae. Latent effects were visible on the larvae raised at pH 7.6: they were more successful in settling (45% at day 40 post-fertilization) and metamorphosing (30%) than larvae raised at 8.0 (17 and 1% respectively). These direct and indirect effects of ocean acidification on settlement and metamorphosis have important implications for population survival.

Effects of currents, waves, and biofilms on motion and surface contacts by tubeworm larvae swimming above or below surfaces

Larvae of many benthic marine invertebrates swim more slowly than the ambient water flow that carries them past surfaces where they might settle. Competent larvae of the tubeworm Hydroides elegans, abundant early colonists in warm-water fouling communities, were used to determine if active behavior by microscopic larvae carried in flowing water can affect their contacts with surfaces representing early stages of fouling community succession (clean flat surface, flat biofilm, biofilm on worm tubes). We studied larvae in waves and in the unidirectional flow used in earlier studies of larval settlement, and near surfaces above them (like those where fouling organisms recruit) or below them. We videotaped larval motion near surfaces in a small flume in which we mimicked fine-scale flow measured near surfaces in harbors. Swimming larvae within millimeters of surfaces moved up and down while being carried by horizontal flow, enhancing their contact rates with surfaces above and below them compared with dead larvae passively carried by the current. After contact, live larvae ‘bounced’ along the surface, sampling it many times per distance they were carried by the current, whereas dead larvae did not. Thus, active behaviors of larvae of H. elegans, which must touch a biofilmed surface to be stimulated to attach and metamorphose, enhance their contact with and exploration of surfaces in flowing water. These behaviors are effective in unidirectional flow and waves, for surfaces above or below larvae, and for smooth or rough surfaces, all of which are conditions that the larvae of fouling-community animals encounter in harbors.

Symbiotic microbiome and metabolism profiles reveal the effects of induction by oysters on the metamorphosis of the carnivorous gastropod Rapana venosa

Most marine mollusks have a pelagic larval phase, and they need to undergo metamorphosis to develop into adults. Metamorphosis is affected by many factors, including abiotic factors such as temperature, salinity and illumination as well as biological factors such as food and microorganisms. In our previous study, we found that the metamorphosis of Rapana venosa requires induction by juvenile oysters, which are the food source of R. venosa. However, the regulatory mechanism of this induction is largely unknown. In the present study, we evaluated the impacts of induction by juvenile oysters on competent larvae of R. venosa. Competent larvae were experimentally divided into two pools, and scallop shells without juvenile oysters and scallop shells with juvenile oysters were added for 2 h and 12 h to monitor alterations in critical gene expression, symbiotic microbiota and metabolomic responses. The carboxypeptidase gene was increased while the cellulase gene was decreased, which may mean that the food habit transition was induced by juvenile oysters. Meanwhile, critical genes in the neuroendocrine system were also significantly altered in juvenile oysters. Furthermore, dramatic changes in the symbiotic microbiota and metabolism profiles were observed, with many of them associated with the digestive system and neuroendocrine system. In conclusion, juveniles as food resources may induce metamorphosis in R. venosa by regulating the neuroendocrine system and promoting the development of the digestive system and changes in digestive enzymes. This study may provide evidence that induction by juvenile oysters can promote food habit transition and metamorphosis in R. venosa by regulating the metabolome and microbiome and further altering the digestive and neuroendocrine systems of R. venosa, which expands our understanding of the regulatory mechanism of metamorphosis in R. venosa. However, further studies are needed to explore the specific substance inducing metamorphosis released by juvenile oysters.

Fatty acid ratio analysis identifies changes in competent meroplanktonic larvae sampled over different supply events

Planktonic communities are a cornerstone of ocean food webs. Early benthic performance of meroplanktonic organisms is shaped by their life stages in planktonic communities. Fatty acid profiles of marine invertebrates are a good indicator of their nutritional state and allow inferring how dietary regimes experienced during larval pelagic life may drive their pre- and post-metamorphosis performance. Fatty acid profiles of Carcinus maenas megalopae were analysed during four larval supply events in two consecutive years to better understand the variability in their nutritional state at settlement. The logratio analysis of fatty acids showed differences between the four larval supply events, with five ratios explaining 83.1% of the variance. The ratios that contributed to separate larval supply events presented a combination of essential, de novo synthetized and diet origin fatty acids (e.g., phytanate/20:4 n-6, 16:0/18:2 n-4). The high fatty acid signature dispersion found within the same supply event suggests that larvae settling at Ria de Aveiro (Portugal) developed through different planktonic feeding zones and experienced contrasting feeding regimes. The fatty acid profile of megalopae demonstrated a high contribution of diatoms, flagellates and bacteria in the larval diet of C. maenas. The present study demonstrated differences between supply events, although a high variability of larval phenotypes was recorded within the same supply event.

Laser ablation of the apical sensory organ of Hydroides elegans (Polychaeta) does not inhibit detection of metamorphic cues.

Larvae of many marine invertebrates bear an anteriorly positioned apical sensory organ (ASO) presumed to be the receptor for settlement- and metamorphosis-inducing environmental cues, based on its structure, position and observed larval behavior. Larvae of the polychaete Hydroides elegans are induced to settle by bacterial biofilms, which they explore with their ASO and surrounding anteroventral surfaces. A micro-laser was utilized to destroy the ASO and other anterior ciliary structures in competent larvae of H. elegans. After ablation, larvae were challenged with bacterial biofilmed or clean surfaces and percentage metamorphosis was determined. Ablated larvae were also assessed for cellular damage by applying fluorescently tagged FMRF-amide antibodies and observing the larvae by laser-scanning confocal microscopy. While the laser pulses caused extensive damage to the ASO and surrounding cells, they did not inhibit metamorphosis. We conclude that the ASO is not a required receptor site for cues that induce metamorphosis.

Dynamic changes in DNA methylation during seahorse (Hippocampus reidi) postnatal development and settlement

IntroductionMost living marine organisms have a biphasic life cycle dependent on metamorphosis and settlement. These critical life-history events mean that a developmentally competent larva undergoes a range of coordinated morphological and physiological changes that are in synchrony with the ecological transition from a pelagic to a benthonic lifestyle. Therefore, transition from a pelagic to a benthonic habitat requires multiple adaptations, however, the underlying mechanisms regulating this process still remains unclear. Epigenetic regulation and specifically DNA methylation, has been suggested to be particularly important for organisms to adapt to new environments. Seahorses (Family Syngnathidae, Genus Hippocampus) are a fascinating group of fish, distinguished by their unique anatomical features, reproductive strategy and behavior. They are unique among vertebrate species due to their “male pregnancy”, where males nourish developing embryos and larvae in a brood pouch until hatching and parturition occurs. After birth, free-swimming offspring are pelagic and subsequently they change into a demersal lifestyle. Therefore, to begin to address the question whether epigenetic processes could be involved in the transition from a planktonic to a benthonic lifestyle observed in seahorses, we studied global DNA methylation profiles in a tropical seahorse species (Hippocampus reidi) during postnatal development and settlement.ResultsWe performed methylation-sensitive amplified polymorphism (MSAP) along with quantitative expression analysis for genes suggested to be involved in the methylation machinery at six age groups: 1, 5, 10, 20, 30 and 40 days after male’s pouch release (DAR). Results revealed that the H. reidi genome has a significantly different DNA methylation profile during postnatal development and settlement on demersal habitats. Moreover, gene expression analysis showed up- and down-regulation of specific DNA methyltransferases (DNMTs) encoding genes.ConclusionOur data show that the differences in the DNA methylation patterns seen among developmental stages and during the transition from a pelagic to a benthonic lifestyle suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. Therefore, epigenetic mechanisms could be necessary for seahorse settlement. Nevertheless, if these epigenetic mechanisms come from internal or if they are initiated via external environmental cues should be further investigated.

Myogenesis of Siboglinum fiordicum sheds light on body regionalisation in beard worms (Siboglinidae, Annelida)

BackgroundMany annelids, including well-studied species such as Platynereis, show similar structured segments along their body axis (homonomous segmentation). However, numerous annelid species diverge from this pattern and exhibit specialised segments or body regions (heteronomous segmentation). Recent phylogenomic studies and paleontological findings suggest that a heteronomous body architecture may represent an ancestral condition in Annelida. To better understand the segmentation within heteronomous species we describe the myogenesis and mesodermal delineation of segments in Siboglinum fiordicum during development.ResultsEmploying confocal and transmission electron microscopy we show that the somatic longitudinal musculature consists of four separate strands, among which ventrolateral one is the most prominent and is proposed to drive the search movements of the head of the late metatrochophore. The somatic circular musculature lies inside the longitudinal musculature and is predominantly developed at the anterior end of the competent larva to support the burrowing behaviour. Our application of transmission electron microscopy allows us to describe the developmental order of the non-muscular septa. The first septum to form is supported by thick bundles of longitudinal muscles and separates the body into an anterior and a posterior region. The second group of septa to develop further divides the posterior body region (opisthosoma) and is supported by developing circular muscles. At the late larval stage, a septum reinforced by circular muscles divides the anterior body region into a forepart and a trunk segment. The remaining septa and their circular muscles form one by one at the very posterior end of the opisthosoma.ConclusionsThe heteronomous Siboglinum lacks the strict anterior to posterior sequence of segment formation as it is found in the most studied annelid species. Instead, the first septum divides the body into two body regions before segments are laid down in first the posterior opisthosoma and then in the anterior body, respectively. Similar patterns of segment formation are described for the heteronomous chaetopterid Chaetopterus variopedatus and serpulid Hydroides elegans and may represent an adaptation of these annelids to the settlement and transition to the sedentarian-tubiculous mode of life.

The Characteristic of Critical Genes in Neuroendocrine System and Their Regulation on Food Habit Transition and Metamorphosis of Veined Rapa Whelk Rapana venosa (Valenciennes, 1846)

Metamorphosis is a critical developmental event in mollusks, and neuroendocrine system plays an essential role in this process. Rapana venosa is an economically important shellfish in China, but the artificial technology of R. venosa aquaculture is limited by metamorphosis. As a carnivorous gastropod, food habit transition makes the mechanism of R. venosa metamorphosis more complex. To investigate the changes in the neuroendocrine system and to reveal its role in regulating the food habit transition and metamorphosis of R. venosa, we cloned the cDNA sequences encoding 5-hydroxytryptamine receptor (Rv-5HTR), nitric oxide synthetase (Rv-NOS) and cholecystokinin receptor (Rv-CCKR), and investigated their expression by quantitative real-time PCR analysis, and explore the spatio-temporal changes of 5-HT protein expression using Immunohistochemical (IHC) analysis. The expression of the three geens was significantly increased in the early intramembrane veliger stage, which indicates that the three genes are related to the development of digestive system. Additionally, expression of the three genes was decreased after metamorphosis, while Rv-NOS and Rv-CCKR were increasingly expressed in competent larvae, which may help the larvae find suitable environments and promote digestive system development for metamorphosis, and the result of 5-HT IHC analysis also reflects the development of neuroendocrine system. Furthermore, results show that CCK can effect the expression of digestive enzyme, NOS and 5-HT receptor. Finally, based on the present results, we hypothesized that CCK and CCK receptor may be critical regulatory factors of food habit transition and metamorphosis. These results might provide information on the development of neuroendocrine system of R. venosa, and new insight into the regulation of the food habit transition and metamorphosis of gastropods.

A novel system for intensive Diadema antillarum propagation as a step towards population enhancement

The long-spined sea urchin Diadema antillarum was once an abundant reef grazing herbivore throughout the Caribbean. During the early 1980s, D. antillarum populations were reduced by > 93% due to an undescribed disease. This event resulted in a lack of functional reef herbivory and contributed to ongoing ecological shifts from hard coral towards macroalgae dominated reefs. Limited natural recovery has increased interest in a range of strategies for augmenting herbivory. An area of focus has been developing scalable ex situ methods for rearing D. antillarum from gametes. The ultimate use of such a tool would be exploring hatchery origin restocking strategies. Intensive ex situ aquaculture is a potentially viable, yet difficult, method for producing D. antillarum at scales necessary to facilitate restocking. Here we describe a purpose-built, novel recirculating aquaculture system and the broodstock management and larval culture process that has produced multiple D. antillarum cohorts, and which has the potential for practical application in a dedicated hatchery setting. Adult animals held in captivity can be induced to spawn year-round, with some evidence for annual and lunar periodicity. Fecundity and fertilization rates are both consistently very high, yet challenges persist in both late stage larval development and early post-settlement survival. Initial success was realized with production of 100 juvenile D. antillarum from ~ 1200 competent larvae. While the system we describe requires a significant level of investment and technical expertise, this work advances D. antillarum culture efforts in potential future hatchery settings and improves the viability of scalable ex situ production for population enhancement.

Effect of larval density on growth and survival of the Pacific oyster Crassostrea gigas in a recirculation aquaculture system

The present study determined the effect of larval stocking density on larval yield (number of live larvae at the end of the experiment), competent larvae (number of live larvae at the end of the experiment retained on a sieve with mesh size of 236 μm (1st experiment) and 239 μm (2nd experiment)), and larval shell length of Crassostrea gigas in a recirculation aquaculture system (RAS). Two experiments were performed at different larval stocking densities: 50, 100 and 150 larvae mL−1 (1st experiment) and 50, 75 and 100 larvae mL−1 (2nd experiment). We used 250 L tanks and a randomized block design with three replicates per treatment (larval density). The physical and chemical parameters of the water were stable in all treatments during the experimental period. Our results demonstrated the feasibility of producing Crassostrea gigas larvae in a RAS at larval densities of 50 and 75 larvae mL−1; however, rearing larvae at densities of 50 larvae mL−1 resulted in the best growth and highest proportion of competent larvae. This RAS approach could be used to optimize larval rearing and improve both the resource efficiency and profitability of commercial hatcheries.

Modelling regional and local-scale larval seeding strategies for abalone (H. midae) ranching in South Africa

Understanding connectivity between abalone populations and residence times for pelagic larvae within suitable settlement habitats is critical for sustainable management of wild abalone stocks and ranching schemes. In the Eastern Cape region of South Africa, poaching has severely depleted wild abalone Haliotis midae populations. Rebuilding stock through larval seeding is being considered as a potentially viable method, though is hampered by a limited understanding of abalone habitat connectivity through larval dispersal, both at a regional and local scale. To address this shortcoming, we apply a multi-scale approach to investigate potential larval dispersal along the coastline of Eastern Cape, South Africa, examining both intra- and inter-site connectivity, with the primary focus at the local nearshore scale (< 40 m water depth) where surf zone processes have a strong influence. Delft3D is applied at a regional scale (400-km domain) and XBeach is used at a local scale (5-km domain) to model a known abalone habitat at Cape Recife at high-resolution (5-m cell size). Models were validated using field observations. Complex topo-bathymetry data for the local-scale model domain around Cape Recife were obtained using a state-of-the-art low-cost multi-method surveying approach. At a regional scale, sites with abalone habitat have the potential to be connected across distances of 50–100 km. However, a large sandy embayment (Algoa Bay; 100-km arc length) is likely to present an obstacle to larval transport in all but the most extreme and sustained wind and wave forcing conditions. The central Cape Recife abalone ranching area has the potential to act either as a self-seeding or meta-population connected site, depending on forcing conditions. At the local scale around Cape Recife, a zone of shallow gullies, under lower-energy waves, was determined to be the optimal release point to maximise residency times within the preferred rocky, shallow subtidal habitat of H. midae. Competent larvae released in this zone are predicted to have an order of magnitude greater residency than randomly seeded larvae. This study provides site-specific connectivity and residence time information for the Eastern Cape abalone fishery, but more generally it provides a novel multi-scale method to aid in planning and managing of abalone larval seeding for ranching and stock enhancement programs that could be applied in other locations.

Reduced seawater pH alters marine biofilms with impacts for marine polychaete larval settlement

Ocean acidification (OA) can negatively affect early-life stages of marine organisms, with the key processes of larval settlement and metamorphosis potentially vulnerable to reduced seawater pH. Settlement success depends strongly on suitable substrates and environmental cues, with marine biofilms as key settlement inducers for a range of marine invertebrate larvae. This study experimentally investigated (1) how seawater pH determines growth and community composition of marine biofilms, and (2) whether marine biofilms developed under different pH conditions can alter settlement success in the New Zealand serpulid polychaete Galeolaria hystrix. Biofilms were developed under six pH(T) treatments (spanning from 7.0 to 8.1 [ambient]) in a flow-through system for up to 14 months. Biofilms of different ages (7, 10 and 14 months) were used to assay successful settlement of competent G. hystrix larvae reared under ambient conditions. Biofilm microbiomes were characterized through amplicon sequencing of the small subunit ribosomal rRNA gene (16S and 18S). Biofilm community composition was stable over time within each pH treatment and biofilm age did not affect larval settlement selectivity. Seawater pH treatment strongly influenced biofilm community composition, as well as subsequent settlement success when biofilms were presented to competent Galeolaria larvae. Exposure to biofilms incubated under OA-treatments caused a decrease in larval settlement of up to 40% compared to the ambient treatments. We observed a decrease in settlement on biofilms relative to ambient pH for slides incubated at pH 7.9 and 7.7. This trend was reversed at pH 7.4, resulting in high settlement, comparable to ambient biofilms. Settlement decreased on biofilms from pH 7.2, and no settlement was observed on biofilms from pH 7.0. For the first time, we show that long-term incubation of marine biofilms under a wide range of reduced seawater pH treatments can alter marine biofilms in such a way that settlement success in marine invertebrates can be compromised.

Enhancing coral recruitment through assisted mass settlement of cultured coral larvae.

The escalating rate at which coral communities are declining globally requires urgent intervention and new approaches to reef management to reduce and halt further coral loss. For reef systems with limited natural larval supply, the introduction of large numbers of competent coral larvae directly to natural reef substrata provides a potentially useful approach to replenish adult coral populations. While few experiments have tested this approach, only one experiment has demonstrated its long-term success to date. Given the differences in life-history traits among corals, and different sensitivities of larvae to abiotic and biotic factors, coupled with the dynamic nature of post-settlement survivorship and recruitment processes, trials of the larval enhancement technique with larvae of different coral species are needed to test the broader applicability and viability of this approach. Accordingly, in this paper we examine the applicability of the larval enhancement technique to restore a population of Acropora loripes in the Bolinao-Anda Reef Complex, Pangasinan, northwestern Philippines. Larvae were cultured ex situ following spawning of collected A. loripes colonies in June 2014. Competent larvae were transported to degraded reef areas and approximately 300,000 larvae were introduced in each of three 6 × 4 m plots directly on the reef. Fine mesh enclosures retained the larvae inside each treatment plot for five days. Three adjacent 6 × 4 m plots that served as controls were also covered with mesh enclosures, but no larvae were introduced. Each plot contained ten 10 × 10 cm conditioned settlement tiles cut from dead tabulate Acropora that were used to quantify initial larval settlement. After allowing larval settlement for five days, mean settlement on tiles from the larval enhancement plots that were monitored under stereomicroscopes was significantly higher (27.8 ± 6.7 spat per tile) than in control plots, in which not a single recruit was recorded. Post-settlement survivorship and growth of spat and coral recruits on tiles and reef substrata inside the experimental plots were monitored periodically for 35 months. After 35 months, the mean size of each of the remaining 47 A. loripes coral colonies surviving on the reef substrata was 438.1 ± 5.4 cm3, with a mean diameter of 7.9 ± 0.6 cm. The average production cost for each of the surviving A. loripes colonies at 35 months was USD 35.20. These colonies are expected to spawn and contribute to the natural larval pool when they become reproductively mature, thereby enhancing natural coral recovery in the area. This study demonstrates that mass coral larval enhancement can be successfully used for restoring populations of coral species with different life-history traits, and the techniques can rapidly increase larval recruitment rates on degraded reef areas, hence catalysing the regeneration of declining coral populations.

Bivalves are NO different: nitric oxide as negative regulator of metamorphosis in the Pacific oyster, Crassostrea gigas

BackgroundNitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish.ResultsIn this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot’s smooth muscle relaxation.ConclusionTogether, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

Spawning and larval rearing of red sea urchin Salmacis bicolor (L. Agassiz and Desor, 1846;Echinodermata: Echinoidea)

Gonads of sea urchin attract consumers due to its high nutritional value than any other seafood delicacies. Aquaculturists are also very keen on developing larval culture methods for large-scale cultivation. The present investigation systematically examined the larval rearing, development, survival and growth rate of Salmacis bicolor fed with various microalgal diets under laboratory condition. Fertilization rate was estimated as 95%. The blastula and gastrula stages attained at 8.25 h and 23.10 h post-fertilization. The 4 - armed pluteus larvae were formed with two well - developed post-oral arms at 44.20 h following post-fertilization. The 8 - armed pluteus attained at 9 days post fertilization. The competent larva with complete rudiment growth was developed on 25th days post - fertilization. Monodiet algal feed - Chaetoceros calcitrans and Dunaliella salina resulted medium (50.6 ± 2.7%) and low survival rate (36.8 ± 1.7%) of S. bicolor larvae. However, combination algal feed – Isochrysis galbana and Chaetoceros calcitrans has promoted high survival rate (68.3 ± 2.5%) which was significantly different between the mono and combination diet. From the observations of the study, combination diet could be adopted as an effective feed measure to promote the production of nutritionally valuable roes of S. bicolor.

Successful rearing of Ostrea edulis from parents originating from the Wadden Sea, the Netherlands

Since 2017, the European flat oyster (Ostrea edulis) reappeared in the Wadden Sea after decades of absence. In several countries, restoration and reinforcement projects have been initiated. Better insight in Ostrea behaviour is needed to ensure sustainable conservation in the future. In order to acquire substantial numbers of oysters for eco-physiological and transplant experiments without threatening the small local population, indoor rearing facilities were set-up at Royal NIOZ. Here, a broodstock of 38 adults originating from the western Wadden Sea, produced over 21 million larvae in 10 batches within 3 months (Sept-Nov 2018). Release of larvae and rearing took place at a water temperature of 19.7 ± 0.6 °C and a salinity of 30.5 ± 0.5 PSU. Outbreaks of Vibrio bacteria did not occur. The growth rate before settlement was 1.5–1.7 μm per day, which was at the low end of growth rates reported in previous studies. The first competent larvae were observed 17–22 days after release. Size at settlement varied between 255−325 μm. Survival until settlement was 0.96−0.98 per day. After settlement, growth rates increased to 30−47 μm per day, with batches that were released in September having a significantly higher growth rate and initial size compared to larvae that were released later. Mortality from release to the end of the experiment (Feb 2019) was 0.04−0.05 per day. A cryopreservation trial did not result in living larvae after thawing, most likely due to their advanced development when preserved. The larvae and juveniles were negatively tested for the parasite Bonamia ostrea and can thus be used in oyster conservation and restoration projects. Recommendations for future breeding attempts include the use of a higher water temperature and slightly higher food concentrations.

Species level identification of Antillogorgia spp. recruits identifies multiple pathways of octocoral success on Caribbean reefs

Successful recruitment is critical to the maintenance and resilience of populations and may be at the core of the transition from scleractinian- to octocoral-dominated faunas on some Caribbean reefs. For sessile invertebrates, recruitment incorporates the composite effects of larval supply, settlement and survival. The relative success of these processes differs between species, and successful recruitment may be achieved through different life history strategies. Recruitment of six abundant and widespread Antillogorgia spp. was assessed at six sites on Little Bahama Bank from 2009–2012. Identification of recruits to species level, based on microsatellite analyses, revealed differences in recruitment and survival between species, sites and years. The broadcast spawning species, A. americana and A. acerosa, had low rates of early recruitment and post-settlement survival. Higher levels of recruitment success were achieved among brooding and surface brooding species following somewhat different patterns of early recruitment and survival. The internal brooder A. hystrix had the highest recruitment at five of the sites, but low survival dramatically reduced its abundance and after a year it had similar densities as the surface brooding species, A. elisabethae and A. bipinnata. The brooders have smaller colonies and produce fewer larvae than the broadcast spawning species, but they release competent larvae, which may account for their higher recruitment rates. The Antillogorgia illustrate the diversity of successful reproductive strategies exhibited by octocorals, and differences in the life history strategies among these congeners are best characterized by their mode of larval development.

Cloning and characterisation of NMDA receptors in the Pacific oyster, Crassostrea gigas (Thunberg, 1793) in relation to metamorphosis and catecholamine synthesis

Bivalve metamorphosis is a developmental transition from a free-living larva to a benthic juvenile (spat), regulated by a complex interaction of neurotransmitters and neurohormones such as L-DOPA and epinephrine (catecholamine). We recently suggested an N-Methyl-D-aspartate (NMDA) receptor pathway as an additional and previously unknown regulator of bivalve metamorphosis. To explore this theory further, we successfully induced metamorphosis in the Pacific oyster, Crassostrea gigas, by exposing competent larvae to L-DOPA, epinephrine, MK-801 and ifenprodil. Subsequently, we cloned three NMDA receptor subunits CgNR1, CgNR2A and CgNR2B, with sequence analysis suggesting successful assembly of functional NMDA receptor complexes and binding to natural occurring agonists and the channel blocker MK-801. NMDA receptor subunits are expressed in competent larvae, during metamorphosis and in spat, but this expression is neither self-regulated nor regulated by catecholamines. In-situ hybridisation of CgNR1 in competent larvae identified NMDA receptor presence in the apical organ/cerebral ganglia area with a potential sensory function, and in the nervous network of the foot indicating an additional putative muscle regulatory function. Furthermore, phylogenetic analyses identified molluscan-specific gene expansions of key enzymes involved in catecholamine biosynthesis. However, exposure to MK-801 did not alter the expression of selected key enzymes, suggesting that NMDA receptors do not regulate the biosynthesis of catecholamines via gene expression.

Comparative Analysis of Mono and Combined Microalgal Diets on Growth Parameters of Salmacis bicolor Larvae

The present study investigates the effect of four microalgal diets in two dietary modes viz. mono diets (Dunaliella salina, Isochrysis galbana, Chaetoceros calcitrans) and combined diet (Isochrysis galbana and Chaetoceros calcitrans) on growth parameters such as morphological development, larvae survival and metamorphosis of Salmacis bicolor species grown under laboratory conditions for a period of 25th days post fertilization. Micoralgal fatty acids compositions was determined by Gas chromatography - mass spectrometry (GC-MS). Significant difference (p < 0.05) in the growth response was observed between the mono and combined diets. Among the mono diets, better growth in terms of survival rate (45.6 ± 1.2%), larvae length (468.1 ± 3.1 μm), post oral arm length (191.5 ± 3.3 μm) and metamorphosis rate (42.41 ± 2.4%) was achived in larvae fed with mono diet: C. calcitrans. Beside, combined diet has promoted high survival rate (62.3 ± 2.1%), larvae length (642.12 ± 3.6 μm), post oral arm length (232.1 ± 2.5 μm), larvae width (410.48 ± 1.8 μm), stomach length (336.8 ± 2.6 μm) and metamorphosis rate (53.8 ± 3.2%) with high significant level (p < 0.05) compared to the mono microalgal diets treatments. The fatty acid analysis revealed high concentration of vaccenic acid (C18:1–12.34 ± 0.11 mg/ml), linoleic acid (C18:2–15.78 ± 0.25 mg/ml) n-6 PUFA and alpha-linolenic acid (C18:3–19.4 ± 0.41 mg/ml) n-3 PUFA levels in combined diet. From the observations of the present study, it was concluded that combined diet using I. galbana and C. calcitrans could be adopted as best candidate microalgal species for promoting large scale production of competent larvae of S. bicolor.