Survival, growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays

Straight-hinge veliger and pediveliger larvae of the mussel Mytilus galloprovincialis were refrigerated for varying periods for use in bioassays. Straight-hinge veliger larvae grew to the umbo-veliger stage after 2 months in the refrigerator, but no pediveligers were observed during the 3-month refrigeration period. The average survival rate of larvae in the refrigerator was 79% after 1 month, but gradually decreased with the refrigeration period, and was as low as 22% after 3 months. All refrigerated larvae grew to the pediveliger stage in the incubator at 17 degrees C at the same rate as that of the control larvae that were not refrigerated. Settlement and metamorphosis of pediveligers from both refrigerated and control groups were facilitated by microbial film and epinephrine and inhibited by phentolamine. Thus, refrigeration can be used as an effective method of storing larvae of M. galloprovincialis for use in assays to assess candidate settlement inducers and antifouling substances.     

Where to Settle—Settlement Preferences of Mytilus galloprovincialis and Choice of Habitat at a Micro Spatial Scale

The global mussel aquaculture industry uses specialised spat catching and nursery culture ropes made of multi-filament synthetic and natural fibres to optimise settlement and retention of mussels for on-growing. However, the settlement ecology and preferences of mussels are poorly understood and only sparse information exists in a commercial context. This study quantified the settlement preferences of pediveligers and plantigrades of Mytilus galloprovincialis on increasingly complex surfaces and settlement locations at a micro spatial scale on and within ropes under commercial hatchery operating conditions using optical microscopy and X-ray micro-computed tomography (µCT). M. galloprovincialis has clear settlement preferences for more complex materials and high selectivity for settlement sites from the pediveliger through to the plantigrade stage. Pediveligers of M. galloprovincialis initially settle inside specialised culture ropes. Larger pediveligers were located close to the exterior of ropes as they increased in size over time. In contrast, smaller individuals were located deeper inside of the ropes over time. This study demonstrates that X-ray µCT is an excellent non-destructive technique for mapping settlement and attachment sites of individuals as early as one day post settlement, and quantifies the number and location of settled individuals on and within ropes as a tool to understand and optimise settlement in complex multi-dimensional materials and environments.     

Hybridisations between Mytilus edulis and Mytilus galloprovincialis and performance of pure species and hybrid veliger larvae at different temperatures

The mussels Mytilus edulis L. and Mytilus galloprovincialis Lamark hybridise naturally in the wild along the Atlantic coast of Europe producing a patchwork of mixed pure species and hybrid populations. Individuals of both species were spawned in the laboratory and were hybridised in a series of reciprocal crosses. After 72 h, the proportion of eggs which developed into larvae (%yield) and the proportion of those larvae which had a normal veliger morphology (%normality) were estimated and compared between pure species and hybrid families. There were no significant differences in %yield or %normality between pure species and hybrids, but significant differences were evident between the offspring from different parents irrespective of whether they were hybrids or pure species. Therefore confirmation of hybrid heterosis in laboratory studies should not be based on a single, or a few reciprocal crosses. Hybrid and pure species veliger larvae were grown for approximately 4 weeks at 10, 14 or 20 °C. In all trials, pure M. galloprovincialis larvae grew significantly faster at 20 °C than either reciprocal hybrid or pure M. edulis larvae. Irrespective of temperature, in general, hybrid larvae grew slower than larvae of either pure species. Increased exposure to planktonic predation due to slow growth can be interpreted as selection against hybrids and this may play a role in the structure and distribution of mixed pure species and hybrid populations.     

Effects of low light and high temperature on pediveligers of the fluted giant clam Tridacna squamosa

ABSTRACT

This paper examines the responses of the fluted giant clam Tridacna squamosa pediveligers to elevated temperature and reduced light levels. In a light reduction experiment, a total of 104,000 T. squamosa pediveligers were exposed to four different levels of shading for approximately one month. The most heavily shaded treatment, at 0.4% of ambient light, had significantly lower survival than the other groups, which all received 1% or more of ambient light. In a second experiment, for approximately two weeks 13,000 T. squamosa pediveligers were divided among three treatments: one at ambient temperature averaging 29.5 °C, and two with elevated temperatures averaging 32.2 °C and 34.8 °C. The elevated temperature treatments resulted in near total mortality. The highest temperature survived by any pediveliger was 32.8 °C. Our results indicate a potential synergetic effect, with turbidity causing giant clam pediveligers to settle in shallower water―where they will likely be exposed to higher temperatures.     

Study on the Transfer of Foreign Genes into the Mussel Mytilus galloprovincialisLam. Eggs by Spermatozoa

The possibility of transferring exogenous DNA into eggs by mussel Mytilus galloprovincialisLam. sperms both with the use of certain methods of transfection and without them was studied. The efficacy of egg fertilization by sperms treated with foreign DNA and the development of larvae at early stages of embryogenesis were evaluated. Negative effects of the contact between mussel sperms and exogenous DNA on fertilization and subsequent development were noted. The proportion of developing larvae decreased with increasing DNA concentration and sperm exposure. Transfer of plasmids pCMVlacZ and pMTbGHinto eggs was observed in group crosses. With the use of PCR, foreign DNA sequences were found in the larvae at the stage of veliger 48 h after fertilization. An intense signal was recorded after sperm electroporation in 10% DMSO.     

Optimising settlement assays of pediveligers and plantigrades of Mytilus galloprovincialis

The mussel Mytilus galloprovincialis is a common aquaculture species, and also a major fouling organism that has negative economic impacts. There are no standard assay conditions for this important species and therefore, this study quantified the effect of key factors on the settlement of pediveligers and plantigrades. Density dependent settlement did not occur for either pediveligers or plantigrades. Settlement increased in drop assays in a 12 h light:12 h dark cycle, while bottom shade had no effect of any magnitude. In addition, settlement was significantly enhanced by storing pediveligers for between 4 and 24 days at 4°C. Overall, these data provide the template to optimise and standardise static laboratory settlement assays for mussels in order to develop materials that either enhance settlement for the aquaculture industry, or deter settlement for antifouling applications. Furthermore, simple mechanisms such as storage at 4°C can enhance settlement beyond current methods used in aquaculture hatcheries.     

Influx and net flux of amino acids into larval and juvenile European flat oysters, Ostrea edulis (L.)

Influx and net flux of amino acids into veliger, pediveliger, and juvenile stages of the oyster, Ostrea edulus (L.), were examined. Influx of alanine and glycine was determined using ¹⁴C-labelled substrates and monitoring the disappearance of radioactivity in the medium. Net influx was determined fluorometrically by following the disappearance of primary amines from solutions containing known substrates. Rates of influx and net influx are comparable for juvenile Ostrea down to ambient substrate levels of 2 μM. Net influx of amino acids into veliger and pediveliger larvae occurs at all concentrations examined (9 μM minimum). Rates of maximum influx (J_maxⁱ) and ambient substrate concentration at which influx is half-maximal (K_t) are reported for each stage. The K_t for glycine and alamine influx is 35–40 μM, decreasing to ≈ 15 μM in 1.5 mm sized juvenile oysters.     

Enhancing the settlement and attachment strength of pediveligers of Mytilus galloprovincialis bychanging surface wettability and microtopography

Surface wettability and microtopography can either enhance or deter larval settlement of many sessile marine organisms. This study quantifies the effect of these surface properties on the settlement of pediveligers of Mytilus galloprovincialis, using polymers spanning a range of wettability and microtextured polydimethylsiloxane (PDMS). Furthermore, the adhesion strength of settled pediveligers on microtextured PDMS surfaces was quantified using a flow chamber. Settlement was enhanced at the hydrophilic end of the wettability spectrum, where mean settlement on nylon reached 33.5 ± 13.1%. In contrast, mean settlement on the most hydrophobic polymer (PDMS) was 4.2 ± 3.2%. Microtopography had a much stronger effect compared to wettability, where 400 μm textured PDMS enhanced settlement above 90%. Settlement preferences were also positively correlated to adhesion strength at flow rates of 4 knots, with all initially settled pediveligers on smooth PDMS detaching, while 79.9 ± 5.7% of pediveligers remained on the 400 μm texture.     

Veligers of an introduced bivalve,Limnoperna fortunei,are a new food resource that enhances growth of larval fish in the Paraná River (South America)

1. Larvae of ‘sábalo’, Prochilodus lineatus, whose adults represent over 60% of overall fish biomass in the Río de la Plata Catchment, have been observed to feed intensively on veligers of the exotic bivalve Limnoperna fortunei. 2. To assess the effects of this dietary shift on the growth of P. lineatus, 28‐day laboratory experiments were carried out feeding newly hatched P. lineatus larvae with three diets: zooplankton artificially enriched with L. fortunei veligers; natural zooplankton; and zooplankton artificially enriched with cladocerans and copepods. The average length, weight and gut contents of the fish larvae were assessed weekly and metabolic rates of fish larvae were measured. 3. Proportions of veligers in gut contents were always higher than those in the experimental diet: 100, 76 and 21% for veliger‐enriched, natural and low‐veliger diets, respectively. Larvae fed a veliger‐enriched diet grew to a significantly larger size than larvae fed the other two diets. In energetic balance comparisons using metabolic rates and prey energy content, all three diets were sufficient to support metabolism and growth. The greatest values of excess energy at the end of each week were in the veliger‐enriched experiments. 4. Feeding on veligers of L. fortunei significantly enhances the growth of P. lineatus larvae and supports the idea that this new and abundant resource is selectively preyed upon by P. lineatus during its larval stage. Higher growth rates may stem from the higher energy contents of veligers compared to crustaceans and/or from the lower energy costs of capturing slower prey.     

Modulation of Mytilus trossulus (Bivalvia: Mollusca) larval survival and growth in culture. Short communication

Commercial importance and ability to live in a wide range of salinities have made the common mussel, Mytilus trossulus, a relevant model to study modulation of larval growth and development. We investigated the effects of various salinities combined with neomycin and ampicillin application on Mytilus larvae survival and growth. Both neomycin and ampicillin enhanced trochophore and veliger survival under condition of low salinity. The average veliger size was increasing in accordance with the increase of salinity. In case of neomycin treatment 3.6% of the larvae reached the pediveliger stage. No abnormalities of larval morphology of the FMRFamide and 5-HT systems occurred after 7 days of culturing with both antibiotics.     

Possible functions of Dpp in gastropod shell formation and shell coiling

We examined dpp expression patterns in the pulmonate snail Lymnaea stagnalis and analyzed the functions of dpp using the Dpp signal inhibitor dorsomorphin in order to understand developmental mechanisms and evolution of shell formation in gastropods. The dpp gene is expressed in the right half of the circular area around the shell gland at the trochophore stage and at the right-hand side of the mantle at the veliger stage in the dextral snails. Two types of shell malformations were observed when the Dpp signals were inhibited by dorsomorphin. When the embryos were treated with dorsomorphin at the 2-cell and blastula stages before the shell gland is formed, the juvenile shells grew imperfectly and were not mineralized. On the other hand, when treated at the trochophore and veliger stage after the shell gland formation, juvenile shells grew to show a cone-like form rather than a normal coiled form. These results indicated that dpp plays important roles in the formation and coiling of the shell in this gastropod species.     

Neural correlates of settlement in veliger larvae of the gastropod,Crepidula fornicata

Settlement behavior of molluscan veliger larvae prior to metamorphosis requires cessation of swimming, accomplished by arrest of prototrochal cilia on the margin of the velum (the larval swimming organ). Ciliary arrest in larvae of gastropods is mediated by an action potential that occurs synchronously across the velum as a consequence of electrical coupling between the prototrochal ciliated cells. We developed a preparation for extracellular recording of such ciliary arrest spikes from intact swimming and crawling veliger larvae of the caenogastropod Crepidula fornicata, using a fine wire electrode. Ciliary arrest spike rates during bouts of substrate crawling were significantly higher than those recorded during preceding swimming periods in larvae that were competent for metamorphosis, but not in precompetent larvae. Spike rates were similar on clean polystyrene substrates, and on substrates that had been coated with a natural cue for metamorphosis (mucus from conspecific adults). We used immunohistochemical methods to localize neuromodulators that might regulate the function of velar cilia. Labeled terminals for serotonin, FMRFamide, and tyrosine hydroxylase (an enzyme for catecholamine synthesis) were located in positions consistent with modulatory effects on the prototrochal ciliated cells. Prototrochal ciliary arrest spike rates and beat frequencies were measured in isolated velar lobes from competent larvae, which were exposed to serotonin, FMRFamide, and dopamine (10^?5 mol L^?1). Serotonin abolished arrest spiking and increased beat frequency; dopamine also increased beat frequency, and FMRFamide depressed it. Competent larvae tested in a small static water column swam to the top of the column when exposed to serotonin, but occupied lower positions than controls when in the presence of dopamine and FMRFamide. The larval nervous system appears to regulate velar functions that are critical for settlement behavior, and is likely to do so by integrating different sensory modalities in an age‐dependent manner.     

The biology of the Northeastern Pacific Turridae. V. Demersal development,synchronous settlement and other aspects of the larval biology of Oenopota levidensis

Summary

The larval development and metamorphosis of a turrid gastropod is described for the first time. This snail, Oenopota levidensis, is typical of the boreal genus Oenopota, which has over 150 described species. Development to a veliger occurs within a lenticular capsule in about 50 days. The capsules hatch to release veligers which swim for less than a week. The remainder of their planktotrophic larval period is spent demersally. Demersal veligers assumed one of two characteristic postures; they remain on the bottom with the velum either extended laterally or folded over the shell. These demersal veligers continue development and metamorphose after another 25 days. The majority of the veligers in the 55 cultures examined metamorphosed and settled within a 96-h period, even though their oviposition occurred over a 47-day period. Potential selective forces leading to this synchronous settlement are proposed.     

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.     

Effect of varying dietary levels of ω6 polyunsaturated fatty acids during the early ontogeny of the sea scallop, Placopecten magellanicus

This study investigated the effects of selected microalgal diets on growth, survival and fatty acid composition of sea scallops, Placopecten magellanicus, from egg hatching to postlarvae. Scallops were offered Pavlova lutheri (MONO) and Pavlova sp. (strain CCMP 459, P459) in combination with Chaetoceros muelleri (CHGRA) and P459 alone. These diets exhibited varying proportions of ω6 docosapentaenoic acid (22:5ω6, DPA) which represented 8.9% of total fatty acids in P459 compared to 5.4% in P459-CHGRA and only 0.5% in MONO-CHGRA. For experiment I, larvae were fed three diets (MONO-CHGRA, P459-CHGRA or P459 alone) for 35 days after fertilization. For experiment II, larvae were fed P459-CHGRA for 21 days and split among the three diets until 39 days post-fertilization. For experiment III, 4-day-old larvae were exposed to P459-CHGRA until they become postlarvae. MONO-CHGRA failed to produce pediveligers when fed to early veligers but yielded competent larvae when fed to late veligers. This may be related to the low ω6 DPA content of this diet. High accumulation of DPA by early veligers suggests a nutritional requirement for this fatty acid. Unialgal P459 supported lower pediveliger production and lower growth rate than other diets containing CHGRA. This may be the effect of higher levels of energetic substrates in CHGRA-containing diets. Finally, P459-CHGRA yielded the best survival and growth rate during the whole larval period. All three experiments showed marked arachidonic acid (20:4ω6, AA) accumulation in early veligers (4 to 21 days) and in pediveligers (32 to 43 days). Accumulation of AA during metamorphosis might reflect stimulation of immune functions due to pathogen infection and degradation of larval organs or the role of AA in cell signalling processes associated with tissue reorganization. Early veligers markedly accumulate DPA likely in response to low docosahexaenoic acid (22:6ω3, DHA) concentrations in the three tested diets. The potential role and effect of DPA as a substitute for DHA is discussed.     

Effect of Copper Ions on Early Developmental Stages of the Mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Bivalvia)

The effect of exposure to copper in seawater (0.005, 0.01, and 0.02 mg/liter) on the development of Mytilus trossulus was examined in the stages of fertilization, blastula, early veliger, veliger, and veliconch. Copper in a concentration of 0.01 and 0.02 mg/liter inhibited the development and growth of the embryos and larvae. At 0.005 mg Cu/liter, the embryos and larvae were capable of adaptation. If M. trossulus embryos and larvae were maintained for 1–2 days in seawater containing 0.01 mg Cu/liter and then transferred to clean water, the consequences were largely dependent on the developmental stage at which the exposure to copper took place. The early developmental stages were more sensitive to the effect of copper than veliger larvae.Original Russian Text Copyright ¢ 2005 by Biologiya Morya, Yaroslavtseva, Sergeeva.     

The presence and location of small cardioactive‐like peptides in larvae of Crassostrea virginica

We examined the presence and location of small cardioactive peptide (SCP)‐like neuropeptides in both the central and peripheral nervous systems of D‐hinge, newly eyed, and pediveliger larvae of Crassostrea virginica. Results indicate that SCP‐like substances are present early in development (D‐hinge larvae), and that presence in the central and peripheral nervous systems increases as the larva develops toward metamorphic competence (pediveliger larvae). In addition, in newly eyed and pediveliger larvae, SCP‐like labeling is found in a varying number of neurons within all central ganglia, with the possible exception of the accessory ganglia. Varicose labeling of axons is also documented within the ganglia, commissures, and connectives, as well as in peripheral nerves. Peripheral tissues innervated by axons exhibiting SCP‐like immunoreactivity include the velum, foot, esophagus, mantle, and various musculatures. As indicated by the location of SCP‐like labeling in various organs and tissues, it is likely that these neuropeptides modulate muscle contraction or ciliary beating in molluscan larvae such as those of C. virginica.     

Embryo-larval tolerance of Mytilus galloprovincialis,exposed to the elevated sea water metal concentrations—I. Toxic effects of Cd,Zn and Hg in relation to the metallothionein level

Developing eggs of Mytilus galloprovincialis were exposed to elevated concentrations of sea water cadmium, zinc and mercury, 48 hr post-fertilization, in order to study the toxic effects at the morphological and biochemical level comparatively. Decreasing order of metal toxicity (Hg > Zn > Cd) was based on the proportion of normally formed straight-hinge veliger larvae and upon the growth inhibition of the primary larval shell. Metal-induced proteins isolated from homogenate extract of the straight-hinge veliger larvae by Sephadex G-75 chromatography were considered to belong to the metallothionein class due to their high metal content as well as according to their chromatographic and spectral properties. The inverse relationship between relative toxicity level and amounts of total and induced metal bound to MT has been found to be described by the function of a potency type. Combined exposure to zinc and cadmium simultaneously, resulted in an antagonistic effect, showing a larger proportion of normally formed veliger larvae and a smaller growth inhibition in comparison with the sum of single metal effects. The induction of MT in larvae subjected to cadmium and zinc mixture was markedly larger than the addition of individual metal effects.     

Freshwater invasibility level depends on the population age structure of the invading mussel species

Environmental conditions may affect invasibility and potentially prevent successful invasions of the freshwater bivalve Limnoperna fortunei Dunker, 1857. However, even though the larval stage may be considered an important species’ characteristic, the invasion processes of L. fortunei are mainly evaluated using only the adult stage. Therefore, the aim of this study was to identify what, and how, environmental filters might predict the likelihood of occurrence of each of the L. fortunei larval stages. Logistic regressions were applied using the larval stages of L. fortunei as a surrogate of population age structure and the environmental variables as the main filters that potentially predicted the likelihood of the occurrence of each larval stage of L. fortunei. The turbidity predicted the occurrence of the D-shaped larvae and straight-hinged veliger stages, while the umbonated-veliger and pediveliger stages were predicted by the pH and conductivity. Finally, the phytoplankton density (diatoms) predicted the occurrence of the umbonated-veliger, pediveliger and mainly the plantigrade stages. Our findings suggested that, during larvae development, from younger to older stages, the main environmental predictor of larvae occurrence shifted from abiotic to biotic variables, indicating that larval stages are indeed an important factor that helps in the evaluation of freshwater invasibility. These findings are particularly important for floodplain systems, where flood pulse dynamics may increase propagule pressure, leading to a successful spread among habitats. Then, even though environmental filters were important to predict successful invasions, we highlight that a successful invasion might depend on the population age structure of the invading species arriving in the new habitat. Thus, successful L. fortunei invasions in floodplain systems may depend on the interaction between invasiveness, invasibility and propagule pressure.