Larval rearing of the giant Azorean barnacle,Megabalanus azoricus (Pilsbry, 1916): feeding trials,larval development and settlement on artificial substrata

A series of experiments were conducted to obtain an efficient larval rearing protocol for Megabalanus azoricus. The first part of this study investigates the effect of microalgae‐based diets on survival and larval development. Mono and mixed‐diets were tested at 20 ± 1°C, in a sequence of 11‐day feeding experiments. The second part presents a preliminary study on the influence of a biofilm on recruitment and use of oyster spat collectors in a mass rearing system. A photographic record of larval development and a brief reference to the diagnostic features that enable quick larval staging are also presented, along with morphometric measurements. Of the microalgae tested (Chaetoceros sp., Chloromonas sp., Dunaliella sp., T‐Isochrysis sp. and Skeletonema sp.) the mixed‐diet Skeletonema sp. with T‐Isocrysis sp. showed the highest survival percentages: total survival ranged from 79.7 to 85.7% and 69.7–80.0% of nauplii were in stage VI after 11 days of rearing. Cypris were also present, but only represented 5.3% of the survivors at most. In the mass rearing system juveniles were found settled in the collectors after 25 days, at 20 ± 1°C. However recruitment was less than 1%. Preliminary results showed no settlement preference towards collectors with biofilm. Nevertheless, this study provides the first record of M. azoricus settlement under laboratorial conditions and represent a starting point for future larval rearing studies.     

Photosynthetic and Biochemical Effects of Cold Storage on Marine Benthic Diatoms of the Mexican Pacific Coast

We determined the effects of 16 wk of storage at low temperature (4 C) in darkness on the viability, growth, photosynthetic parameters, and biochemical composition of four diatom cultures. Significant differences in cell density and proximal composition were observed for all diatoms throughout storage. Cell density increased with time of storage for all diatoms. Protein content increased for Navicula incerta, Nitzschia laevis, and Navicula sp., whereas lipid content increased during storage in only N. incerta. When the stored diatoms were used as inocula in fresh medium, they increased their viability, generating a lag phase for Nitzschia thermalis var. minor, N. incerta, and Navicula sp. cultures. There were noted species‐specific modifications in proximal composition, ash‐free dry weight, and photosynthetic parameters in response to storage. We conclude that N. thermalis, N. incerta, N. laevis, and Navicula sp. can be stored at 4 C for 16 wk and are viable in new cultures.     

Effects of diet and temperature on post‐settlement growth and survival of the short‐spined sea urchin Strongylocentrotus intermedius

Effects of diet and temperature on post‐settlement growth and survival of a sea urchin Strongylocentrotus intermedius, which fed on one of six mono‐cultured benthic diatoms, gametophytes of a brown alga Undaria pinnatifida, a green alga Ulvella lens plus mixed benthic diatoms, or no food at 6, 9, 12 and 15°C, were examined. Digestion efficiency (DE), ingestion rate (IR), excretion rate (ER) and digested cell content index (DCCI) of each diatom species were measured. Growth rates largely differed among the dietary treatments from 7 days post‐settlement. Sea urchins fed on a diatom Cocconeis sublittoralis showed considerably higher growth (7.85‐34.67 μm day^?1) than those fed on the other diets and high survival rates (100%). DCCI was also highest in C. sublittoralis. These results suggest that the quantity of diatom cell contents digested is an important factor affecting growth and survival of the sea urchins. Growth rates of sea urchins were higher in higher water temperatures, and significant differences in growth rate were observed between 6‐9 and 12‐15°C. ERs of C. sublittoralis were generally higher at higher temperatures, but DEs did not differ among the temperature treatments. Variations in growth rate among different water temperature appear to be caused by different food intakes of the urchins.     

Optimisation of larval culture of the mussel <Emphasis Type="Italic">Mytilus</Emphasis><Emphasis Type="Italic">edulis</Emphasis> (L.)

The blue mussel Mytilus edulis is a commercially important species whose fishery and culture generally relies on natural spat collection. Hatchery-production could provide an alternative source of seed, enabling reliable expansion of the industry. Mussel spawning and larval rearing trials were carried out to optimise elements of hatchery production. Culturing fertilised eggs at low density (20–200 eggs cm⁻²) rather than high density (400–720 eggs cm⁻²) significantly improved the quality of first veliger larvae and differences in this improvement were evident between the eggs from different females (maternal effects). Veliger larval growth at 17 or 21°C was significantly faster than growth at 14°C. Feeding veliger larvae an identical total ration either daily or at 2–3 day intervals did not significantly affect their growth. Different microalgal diets (1: Isochrysis sp. (clone T-ISO), 2: Chaetoceros calcitrans forma pumilus, 3: C. muelleri, 4: mixed Isochrysis sp. (clone T-ISO) and C. calcitrans f. pumilus, and 5: mixed Isochrysis sp. (clone T-ISO) and C. muelleri) were tested on veliger larval growth and mixed diets outperformed single-species diets.     

Cold Storage of Six Marine Benthic Diatoms Native to the Mexico Pacific Coast

Cultures of six benthic diatoms were maintained in the dark to measure their viability and biochemical composition after 8 wk of storage at low temperature (4 C) in darkness by refrigeration. Cell density, growth rate, and viability for each benthic diatom changed significantly after storage. Significant differences were observed with regard to cell size (length and width) of Nitzschia laevis, Navicula sp., and Amphora tenerrima as a result of storage. In general, the proximal composition of the benthic diatom cultures changed after week 1 of storage and decreased after week 4 of storage for all the diatoms. These results demonstrate that under 1–4 wk of storage these diatoms maintain their viability and had changes in their proximal composition in species‐specific responses. Storage of preserved live microalgae cells is an alternative technique that can be used to reduce the need for continuous maintenance of live cultures and can provide live feed stock for aquaculture applications.     

Effects of addition of Navicula sp. (diatom) in different densities to postlarvae of shrimp Litopenaeus vannamei reared in a BFT system: Growth,survival, productivity and fatty acid profile

The objective of this study was to evaluate the effect of the addition of Navicula sp. on the growth and fatty acids profile of Litopenaeus vannamei postlarvae in a biofloc system (BFT). Four treatments were used: BFT; BFT 2.5N (addition of 2.5 × 10⁴ cells/ml of Navicula sp.); BFT 5N (addition of 5 × 10⁴ cells/ml of Navicula sp.) and BFT 10N (addition of 10 × 10⁴ cells/ml of Navicula sp.), all in triplicate. The shrimp (1 ± 0.01 mg) were stocked at a density of 3,000 postlarvae/m³ and fed with commercial feed. The diatom was added every 10 days, and at the end of 42 days, shrimp performance, water quality and proximal composition were evaluated. The BFT 5N and BFT 10N treatments had higher performance values, highlighting the values of productivity (2.30 and 2.42 kg/m³) and specific growth rate (15.92 and 16.08%/day), which were higher than the other treatments. In addition, the highest levels of fatty acids were observed in treatments with diatom (BFT 5N and BFT 10N), indicating the benefits of Navicula sp. on growth enhancement and fatty acid content of L. vannamei postlarvae grown in biofloc systems.     

The effect of nitrate supplementation on the biochemical composition of benthic diatoms and the growth and survival of post-larval abalone

Elevating the nitrogen concentration in microalgal growth media can elevate the protein content of the algae and consequently increase their dietary value to molluscs. This study examined the protein content, and dietary value to abalone post-larvae, of seven benthic diatoms acclimated to high-(“H” = 49.4 mg NO₃-N l^− 1) and standard-nitrate (“S” = 16.5 mg NO₃-N l^− 1) Walne medium. In Experiment 1, the level of nitrate in the culture medium did not significantly affect the protein content of Cocconeis sp., Cylindrotheca closterium or Nitzschia ovalis, and variation in the growth of post-larval Haliotis iris was not associated with nitrate level or the protein content of the diatoms. When the same six diatom diets were added daily as cell suspensions, growth and survival were much lower than for the equivalent strains fed as established films (P < 0.0001). In Experiment 2a, the protein content of Navicula incerta was elevated by nitrate supplementation, and the high nitrate strain retained a higher protein content (P = 0.005) even after 24 days growing in unenriched, flowing seawater. Haliotis rufescens post-larvae grew significantly faster when fed N. incerta H than when fed N. incerta S (P = 0.006) and survival was also higher with this diet (67 versus 54%). In Experiment 2b, there was no significant effect of nitrate level on protein content of four diatoms or the performance of post-larval H. rufescens. There was, however, a strong positive correlation (r = 0.95, P < 0.0001) between post-larval survival and the lipid content of the diatom diets. Post-larval growth showed positive, but marginally non-significant, relationships with diatom protein and carbohydrate, but no relationship with lipid content. Overall, only one of seven benthic diatom species acclimated to high-nitrate growth medium displayed significantly elevated protein content. Diatom protein content explained relatively little of the variation in food value of diatom strains for post-larval abalone but lipid content correlated strongly with post-larval survival.     

Enhancement of survival and metamorphosis rates of <Emphasis Type="Italic">Penaeus monodon</Emphasis> larvae by feeding with the diatom <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis>

The purpose of this study was to compare the performance of two species of diatoms, Thalassiosira weissflogii and Chaetoceros gracilis, in the larviculture of the black tiger shrimp Penaeus monodon. Shrimp larvae were fed with either C. gracilis, T. weissflogii, or a combination of the two species of diatoms. The larvae fed solely with T. weissflogii or a combination of the two types of diatom had significantly higher survival rates and faster metamorphosis than those fed solely with C. gracilis. The numbers of diatom cells consumed by larvae during 3-h periods were determined, revealing that larvae consumed significantly higher numbers of C. gracilis than T. weissflogii. However, when the protein, total fatty acid, and polyunsaturated fatty acid content of the two species of diatom are compared, significantly higher amounts of each are found in T. weissflogii. Converting the number of diatom cells consumed into equivalent protein, total fatty acid, eicosopentaenoic acid, and decosahexaenoic acid reveals that larvae fed with T. weissflogii received significantly higher amounts of all the nutrients, compared to those consuming C. gracilis. The results showed an advantage of feeding T. weissflogii to C. gracilis in enhancing survival and metamorphosis in P. monodon larvae.     

Identification of facultative anaerobic bacteria isolated from the intestine of the minke whale <Emphasis Type="Italic">Balaenoptera acutorostrata</Emphasis> by 16S rRNA sequencing analysis

There are few reports in the literature about the isolation of bacteria from whale intestine. In this report, we counted colony-forming units in the feces obtained from three female common minke whales (Balaenoptera acutorostrata). The number of colony-forming units ranged from (2.2 ± 0.4) × 10⁵ to (8.9 ± 2.0) × 10⁸ per gram (wet weight) of excrement. 16S rRNA gene sequences of 141 isolates were determined. These strains were identified as Enterococcus faecalis, Enterococcus sp., Enterobacter cloacae, Enterobacter sp., Escherichia coli, Edwardsiella ictaluri or Clostridium sp. The data suggested that the facultative anaerobic population of the intestinal bacterial flora of the minke whale was similar to that of ground mammals.     

Quantitative PCR assay for the detection of the parasitic ciliate <Emphasis Type="Italic">Cryptocaryon irritans</Emphasis>

We developed a quantitative PCR assay for detecting the parasitic ciliate Cryptocaryon irritans, which causes “white spot disease” in marine fishes, from the natural environment. A specific primer set for C. irritans was designed and its high specificity was confirmed in silico: almost all of the sequences deposited in the GenBank nucleotide database were covered, 22/23 for the forward primer and 7/7 for the reverse primer. We estimated that there were 3,415.9 rRNA gene copies per genome of C. irritans. In artificial mixture experiments to validate whether the qPCR assay is applicable to natural samples, the estimated copy numbers showed significantly positive correlations with the number of theronts added (p < 0.001). When we applied this qPCR assay to natural samples collected bimonthly from surface and bottom seawaters at an aquaculture site (water depth, 10 m) from May 2009 to March 2010, we only detected C. irritans (112.0 ± 6.3 cells/l) in the surface seawater sample in November. This qPCR assay is a useful tool for detecting C. irritans rapidly and quantitatively in natural environments; it could also help advance our understanding of the ecology of C. irritans, as well as facilitate the diagnosis of the disease.     

Use of microalgal‐enriched Diaphanosoma celebensis Stingelin, 1900 for rearing Litopenaeus vannamei (Boone, 1931) postlarvae

The present work investigates the effects of Chaetoceros calcitrans, Nannochloropsis oculata, Tetraselmis tetrahele and Isochrysis galbana diets on the lifespan, growth, neonate production and the nutritional profile of Diaphanosoma celebensis. In addition, the effects of enriched D. celebensis on the survival and growth of Litopenaeus vannamei postlarvae (PLs) was compared with Artemia. Results showed that significantly higher (P < 0.05) neonate production of D. celebensis was attained when fed with C. calcitrans compared to the other microalgae. In addition, D. celebensis fed on C. calcitrans had significantly higher levels (P < 0.05) of protein, lipid and carbohydrate compared to the other three microalgae. On the other hand, D. celebensis had a significantly (P < 0.05) longer lifespan when fed on N. oculata and T. tetrahele compared to those fed with C. calcitrans and I. galbana. Shrimp PLs fed D. celebensis enriched with C. calcitrans had higher survival and specific growth rate but it was not significantly different (P > 0.05) from PL fed only Artemia, indicating that D. celebensis has potential to be used as live feed for the hatchery rearing of L. vannamei PLs, in place of Artemia. This study illustrated that the quality of the D. celebensis production and proximate composition was highly correlated with the food type, and it can be used as a valuable live feed for shrimp larviculture.     

Antibacterial activity in microalgae cultures

Five cultures of microalgae (Chlorella minutissima, Tetraselmis chui, Nannochloropsis sp., Arthrospira platensis and Isochrysis sp.) with no culturable bacteria were tested for their ability to inhibit the growth of six Vibrio bacterial strains (V. parahaemolyticus, V. anguillarum, V. splendidus, V. scophthalmi, V. alginolyticus and V. lentus). The influence of light on the antibacterial activity of the microalgae was investigated. All microalgae cultures inhibited the growth of bacteria compared with the control treatments (P < 0.05), and their antibacterial activity was not influenced by the presence or absence of light. In the control groups, the numbers of bacteria increased exponentially during the experimental period in the absence of microalgae cells demonstrating that the bacterial cells were able to utilize the growth medium of microalgae cultures. The present results may explain the low levels or absence of Vibrio strains in microalgae cultures, and the positive effect of addition of microalgae in rearing of fish larvae, and implicate the production of antibacterial compounds by microalgae cells.     

Evaluation of the Microalgae Paste Viability Produced in a Mollusk Hatchery in Southern Brazil

The present study was conducted to define a methodology to produce and store small‐scale microalgae paste to be used in a mollusk hatchery. Microalgae were cultured in 500 L fiberglass tanks, under temperature of 20 ± 2 C, Guillard f/2 culture medium, and continuous light intensity of 203–226 μmol photons/m²/sec. Cultures were centrifuged at 2000 g at the exponential growth phase. Microalgae cell quality after centrifugation and during storage was determined by analyses with Evan’s blue stain and by counting the number of total marine bacteria. Treatments with and without additive were applied to the microalgae paste produced, which was distributed into 100 mL plastic containers, capped, and stored under refrigeration at 4 ± 1 C. Results indicated that in the Chaetoceros muelleri paste, centrifugation did not damage the cells and the number of total marine bacteria reduced significantly from 2.9 × 10⁶ to 8.3 × 10⁵ colony‐forming units per milliliter. Chaetoceros muelleri and Chaetoceros calcitrans pastes stored with addition of 0.1% ascorbic acid had a shelf life shorter than 2 wk. For the treatment without additive, results with Evan’s blue stain showed that cells (99%) remained viable until the sixth week of storage for C. muelleri and seventh week of storage for Skeletonema sp. and C. calcitrans. The number of bacteria did not increase during storage for C. calcitrans and Skeletonema (P > 0.05). For C. muelleri, an increase in bacteria (P < 0.05) was observed after the sixth week of storage. This study demonstrated the feasibility to produce and store microalgae paste for a period of 2–8 wk, which allows it to be used as food source and also optimizes the use of microalgae cultured in laboratory.     

Effects of salinity on the growth and proximate composition of selected tropical marine periphytic diatoms and cyanobacteria

Marine periphytic cyanobacteria and diatoms have been examined as a potential source of feed supplement for rearing aquatic larvae in the aquaculture industry. Culture of the periphytic diatom Amphora sp., Navicula sp., Cymbella sp. and the cyanobacteria Oscillatoria sp. at different salinities showed significant changes in biomass and specific growth rates. Diatoms growth was significantly higher at 35 g L⁻¹, while for cyanobacteria growth was better at 25 g L⁻¹. Significantly higher levels of protein and lipid were found in diatoms at low salinities (15–25 g L⁻¹) and an increase in carbohydrate at high salinities (30–35 g L⁻¹). Conversely, cyanobacteria showed a significantly higher lipid content at 30–35 g L⁻¹ compared with other salinity levels but no significant changes were observed in the protein and carbohydrate contents at different salinity levels. The present findings can be taken into consideration when culturing marine periphytic Amphora sp., Navicula sp., Cymbella sp. and Oscillatoria sp. to provide appropriate levels of protein, lipid and carbohydrate as feed supplement as well as for bioremediation in aquaculture.     

Growth‐promoting bacteria for the green seaweed Ulva clathrata

Seaweed production represents one of the rising activities in the aquaculture industry. This study explores the impact of bacteria associated with the growth of Ulva clathrata, a promising alga in the field of food and bioremediation. Fifty‐six bacteria isolated from four seaweed (Caulerpa sp., Gracilaria sp., Ulva lactuca and U. clathrata) were tested to evaluate their effect on U. clathrata growth. Eight of them showed a significant growth‐promoting effect (p < .05). Isolates Ul‐11 and Ul‐12 from U. lactuca induced an increase of 73 and 81% of growth respectively. They were classified as Reugeria sp. and Alteromonas sp. based on 16S DNA sequencing (>95.0% sequence identity). Multiple combinations of the growth‐promoting bacteria were also tested on U. clathrata, and the best result was achieved by combining Ca‐3/Uc‐18/Ul‐12 isolates with a significant increase in seaweed growth of up to 76%; however, no synergistic effect was observed compared to Ul‐12 alone. Scanning electron microscopy revealed no alteration in the cell wall of U. clathrata when inoculated with selected bacteria, showing an average increase of bacterial density on U. clathrata.     

Asexual propagation of the soft corals Sinularia sp., Cladiella sp. and Sarcophyton sp. (Octocorallia: Alcyonacea) using different methods of attachment in a recirculating seawater system

Three soft coral cuttings (finger soft coral, Sinularia sp.; black finger soft coral, Cladiella sp. and leather mushroom soft coral, Sarcophyton sp.) were selected to distinguish among the effects of five methods of attachment (adhering, containing, impaling, tethering and a natural attachment method used as a control) in a recirculating seawater system on healing time, time of self‐attachment, development of the cuttings and final survival. Three replicate and 30 cuttings per replicate (n = 30) were used for each treatment and the experimental period was 70 days. The results showed that the cuttings of Sinularia sp., Sarcophyton sp. and Cladiella sp. had already healed the wound area with pigmentation between 7 and 12 days for all the methods of attachment. The shortest time of self‐attachment for the Sinularia sp., Sarcophyton sp., and Cladiella sp. cuttings was obtained in the impaling method, with average values of 8.2 ± 0.9 days, 6.1 ± 0.1 days and 9.00 ± 0.8 days respectively. At the end of the experiment, the highest average final survival values of the Sinularia sp., Sarcophyton sp. and Cladiella sp. cuttings were obtained in the impaling method (86.7 ± 5.7%), the containing method (93.3 ± 5.7%) and the containing method (53.3 ± 5.7%), respectively. It was concluded from the study that the suitable methods of attachment for each soft coral were different and these methods are available for use in targeted propagation farms for restoration purposes.     

Cytogenetic analysis of hybrids and hybrid triploids between the river puffer,Takifugu obscurus,and the tiger puffer,Takifugu rubripes

Cytogenetic analyses of the river puffer, Takifugu obscurus, the tiger puffer, Takifugu rubripes and hybrids produced between female T. obscurus and male T. rubripes and their hybrid triploids (produced by cold shock treatment at 4°C) were performed. T. obscurus had 2n = 44 chromosomes and 1.84 ± 0.019 pg DNA/cell, T. rubripes had 2n = 44 and 2.64 ± 0.015, the hybrids had 2n = 44 and 2.15 ± 0.010 and the hybrid triploids had 3n = 66 and 3.22 ± 0.010. The erythrocyte values of the hybrids were more similar to those for T. obscurus, whereas the hepatocyte, midgut and proximal tubule kidney cell values of the hybrids fell down between those for the parental species (p < .05). The erythrocyte, proximal tubule, hepatocyte and midgut epithelial cell sizes for the hybrid triploids were 1.5‐fold larger than those for the hybrids (p < .05). The thickness of retina and each layer in the hybrid triploids were 1.1‐fold larger than those of the hybrids (p < .05) and did not differ significantly among T. obscurus, T. rubripes and the hybrids (p > .05); however, the hybrid triploids had fewer cell nucleus outer layers than the hybrids (p < .05). Gonad development in the hybrids and hybrid triploids was less matured than in T. obscurus and T. rubripes. The metaphase nucleolus organizer regions (NORs) and the gill cells of T. obscurus, T. rubripes and the hybrids contained two satellite telocentrics, whereas the hybrid triploids contained three satellite telocentrics.     

Ingestion,fecundity and population growth of Harpacticus sp. (Harpacticoida,copepod) fed on five species of algae

We investigated the ingestion, fecundity and population growth of Harpacticus sp. fed on monodiet or mixed diets to evaluate the effects of different algae on Harpacticus sp. Harpacticus sp. fed on diatoms (Skeletonema costatum and Chaetoceros curvisetus) had higher ingestion and pellet production. Time to attain 100% in proportions of gravid females differed, with quickest to slowest: S. costatum, C. curvisetus, Gymnodinium sp. and Heterosigma akashiwo, with the exception of Prymnesium parvum (≤16.67%). S. costatum or C. curvisetus produced higher populations than the other three diets, supported complete development to adulthood, and resulted in doubling copepod population within four days, while no population growth occurred for the other three diets. Mixed‐diet experiments showed that egg production and gross growth efficiencies reduced significantly when fed on H. akashiwo, Gymnodinium sp. or P. parvum mixed with Isochrysis galbana, in comparison with 100% I. galbana. Thus, S. costatum and C. curvisetus were beneficial foods while the other three diets were potentially toxic for Harpacticus sp. The data in this article provide further recognition of nutrient deficiency or toxicity of different algae on copepods.     

Fatty acid composition of 12 microalgae for possible use in aquaculture feed

Twelve algal strains representing the classes Cyanophyceae, Prymnesiophyceae, Bacillariophyceae, Rhodophyceae, Cryptophyceae, Chlorophyceae, Xantophyceae and Eustigmatophyceae were selected mainly from the culture collection of the Norwegian Institute for Water Research (NIVA). The algae were grown as continuous cultures in a 1.8 l. reactor, internally illuminated with an 11 W fluorescent tube. The retention time was adjusted in the range 2–4 days to fit the growth rate of the algae. The growth responses and fatty acid composition were analysed. The maximum production rate was obtained with Pseudokirchneriella subcapitata (0.63 g 1⁻¹ day⁻¹) and the lowest with Porphyridium cruentum 0.13 g 1⁻¹ day⁻¹. Arachidonic acid (AA) and eicosapentaenoic acid (EPA) were the dominating polyunsaturated fatty acids (PUFAs) in P. cruentum, while only EPA accumulated in Phaeodactylum tricornutum. Docosahexaenoic acid (DHA) was the major PUFA in Isochrysis galbana, while Pavlova sp. had both EPA and DHA. This is the first report on the fatty acid profiles of Nannochloropsis oceanica, Chroococcus sp., Synechococcus sp. and Tribonema sp.