Cyril Glenn Satuito

Age-related settlement success by cyprids of the barnacleBalanus amphitrite, with special reference to consumption of cyprid storage protein

Newly molted (0-d-old) cyprids of the barnacleBalanus amphitrite Darwin were prevented from settling for 0 to 14 d at four different temperatures (25, 20, 15 and 5°C treatments). The effect on settlement success of prolonging the cyprid lifetime was evaluated using a nitrilocellulose membrane assay. In addition, protein extract prepared from these cyprids was analyzed using gel electrophoresis to characterize the effect of age on protein content and composition. Settlement success was significantly affected for larvae aged at 25 (P < 0.001), 20 (P < 0.001) and 15°C (P < 0.05), while differences in settlement success between age groups was negligible at 5°C (P = 0.09). Settlement success of cyprids increased with time for up to 3 d (P < 0.001, Phase 1), following which settlement success significantly declined (P < 0.001, Phase 11). Temperature had no significant effect on settlement in Phase I (P = 0.17), but did enhance the decline in settlement success with age during Phase II (P < 0.001). Gel electrophoresis revealed a significant decline in the quantity of the cyprid storage protein CMP (Cyprid Major Protein) with increasing age at 25, 20 and 15°C, but CMP levels remained constant at 5°C. These results suggest that, upon molting to the cyprid stage, larvae may still require a settlement-competence attainment period. This may be achieved by CMP utilization during Phase I, depletion of which during Phase II may be responsible for reduction in settlement success with cyprid age such that remaining CMP stores can no longer support the production of adult structures following settlement.

Studies on the factors influencing larval settlement in Balanus amphitrite and Mytilus galloprovincialis

Understanding of factors influencing settlement (attachment and metamorphosis) of marine invertebrate larvae is of great importance in aquaculture and control of biofouling. The influence of two factors on settlement of larvae was assessed from two separate investigations: 1, the influence of age (endogenous factor) on cyprids of the barnacle Balanus amphitrite; and 2, the influence of a microbial film (exogenous factor) on pediveligers of the mussel Mytilus galloprovincialis.

Neurotransmitter blockers as antifoulants against planktonic larvae of the barnacle Balanus amphitrite and the mussel Mytilus galloprovincialis

Since neurotransmitters influence larval settlement and metamorphosis in many species of sessile marine organisms, neurotransmitter blockers were tested as potential antifouling agents against barnacles and mussels. The serotonin uptake blockers amitriptyline and imipramine inhibited attachment of larvae of the barnacle Balanus amphitrite in a dose‐dependent manner in in vitro assays using polyethylene ropes as the settlement substratum. The α‐adrenergic blocker phentolamine inhibited attachment of both B. amphitrite cyprids and pediveligers of the mussel Mytilus galloprovincialis. Although inhibiting attachment, these three compounds significantly induced cyprid metamorphosis into juveniles. The results suggest that these neurotransmitter blockers may find application as a new type of antifoulant against planktonic larvae of both barnacles and mussels.

Induction of metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis Lamarck, 1819 using neuroactive compounds, KCl, NH4Cl and organic solvents

Pediveliger larvae of Mytilus galloprovincialis were subjected to a series of bioassays to investigate the induction of metamorphosis using neuroactive compounds, K+, NH4 + and organic solvents. Growth and survival of post-larvae obtained using ethanol and methanol were also observed. Epinephrine, phenylephrine, clonidine and metanephrine induced larval metamorphosis at 10−6 to 10−4 M in both 24-h and continuous exposure assays. In 24-h exposure assays, α-methyldopa at 5×10−5 M and methoxyphenamine at 5×10−5−10−4 M induced 55−94% metamorphosis. Similarly, excess K+ at 3×10−2 M induced 39% metamorphosis and NH4 + at 1−5×10−2 M induced 63–78% metamorphosis. The EC50s of seven organic solvents ranged from 0.04 to 0.82 M. Post-larvae that metamorphosed using ethanol and methanol survived as juveniles and grew at the same rate as those from microbial biofilm. Thus, the above compounds can be useful inducers of metamorphosis for antifouling studies using larvae and juveniles of M. galloprovincialis.

Larval storage protein of the barnacle, Balanus amphitrite: biochemical and immunological similarities to vitellin.

Biochemical and immunological characterization of cyprid major protein (CMP), the principal protein constituent of cypris larvae of the barnacle Balanus amphitrite (Crustacea: Cirripedia), revealed similarities to egg-yolk protein, vitellin, as follows: CMP and vitellin heavy chain both have a molecular weight of 170 kDa by polyacrylamide gel electrophoresis containing sodium dodecylsulfate; CMP was crossreactive with antiserum against vitellin heavy chain in immunoblot analysis. The sequence of 11 amino acids in the amino-terminus of CMP, however, is not perfectly homologous to that of vitellin heavy chain. Thus, it was deduced that CMP was an isoform of vitellin. Concentration of CMP abruptly increased during the latter naupliar stages, reaching a peak just after metamorphosis to the non-feeding cypris stage, and decreased thereafter with aging of cyprids or during the early juvenile period. Specifically, the concentration of CMP in newly metamorphosed juveniles within one day decreased to 20% that of cyprids. CMP, therefore, appears to function as a storage protein during settlement of cyprids as well as metamorphosis to juveniles. Immunohistochemical analysis using antiserum against vitellin heavy chain on sectioned cyprids suggested that CMP is accumulated in the haemocoel.

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10−4 M epinephrine. The concentration that inhibited metamorphosis by 50% (IC50) for chlorpromazine and amitriptyline was 1.6 × 10−6 M and 6.6 × 10−5 M, respectively. Idazoxan was less effective with an IC50 of 4.4 × 1013 M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K+ was not inhibited by 10−3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.

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

Abstract 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°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.

A Glycoprotein in Shells of Conspecifics Induces Larval Settlement of the Pacific Oyster Crassostrea gigas

Settlement of larvae of Crassostrea gigas on shell chips (SC) prepared from shells of 11 different species of mollusks was investigated. Furthermore, the settlement inducing compound in the shell of C. gigas was extracted and subjected to various treatments to characterize the chemical cue. C. gigas larvae settled on SC of all species tested except on Patinopecten yessoensis and Atrina pinnata. In SC of species that induced C. gigas larvae to settle, settlement was proportionate to the amount of SC supplied to the larvae. When compared to C. gigas SC, all species except Crassostrea nippona showed lower settlement inducing activities, suggesting that the cue may be more abundant or in a more available form to the larvae in shells of conspecific and C. nippona than in other species. The settlement inducing activity of C. gigas SC remained intact after antibiotic treatment. Extraction of C. gigas SC with diethyl ether (Et2O-ex), ethanol (EtOH-ex), and water (Aq-ex) did not induce larval settlement of C. gigas larvae. However, extraction of C. gigas SC with 2N of hydrochloric acid (HCl-ex) induced larval settlement that was at the same level as the SC. The settlement inducing compound in the HCl-ex was stable at 100°C but was destroyed or degraded after pepsin, trypsin, PNGase F and trifluoromethanesulfonic acid treatments. This chemical cue eluted between the molecular mass range of 45 and 150 kDa after gel filtration and revealed a major band at 55 kDa on the SDS-PAGE gel after staining with Stains-all. Thus, a 55 kDa glycoprotein component in the organic matrix of C. gigas shells is hypothesized to be the chemical basis of larval settlement on conspecifics.

A Method for Evaluating the Efficacy of Antifouling Paints Using Mytilus galloprovincialis in the Laboratory in a Flow-Through System

A laboratory test with a flow-through system was designed and its applicability for testing antifouling paints of varying efficacies was investigated. Six different formulations of antifouling paints were prepared to have increasing contents (0 to 40 wt.%) of Cu2O, which is the most commonly used antifouling substance, and each formulation of paint was coated on just one surface of every test plate. The test plates were aged for 45 days by rotating them at a speed of 10 knots inside a cylinder drum. A behavioral test was then conducted using five mussels (Mytilus galloprovincialis) that were pasted onto the coated surface of each aged test plate. The number of the byssus threads produced by each mussel generally decreased with increasing Cu2O content of the paint. The newly designed method was considered valid owing to the high consistency of its results with observations from the field experiment.

Wheat Germ Agglutinin-Binding Glycoprotein Extract from Shells of Conspecifics Induces Settlement of Larvae of the Pacific Oyster Crassostrea gigas (Thunberg)

ABSTRACT Settlement of pediveligers of the Pacific oyster Crassostrea gigas on nitrocellulose membrane, plaster plate, GF/C filter paper, and glass, both as is and with extract from shells of conspecifics (oyster shell extract [OSE]) added to them, were investigated to select a suitable substrate for settlement assays with OSE. Furthermore, the effects of wheat germ agglutinin (WGA), soybean lectin (SBA), lentil lectin (LCA), and concanavalin A (ConA) on the settlement inducing activities of shell chips (SC) of conspecifics and OSE were investigated. Larvae of C. gigas did not settle on any of the substrates tested. When 50 mg SC equivalent (eq) OSE was added to each of these substrates, larvae settled on all substrates, but greatest settlements were on the GF/C filter papers. Settlement of C. gigas on SC and GF/C filter papers containing 100 mg SC eq OSE (OSE paper) decreased in the presence of WGA (0.5 µg/mL) and SBA (5 µg/mL) for SC, and 0.5 µg/mL for OSE paper, but increasing the concentration of SBA in both cases did not result in less settlement. Treating OSE paper with different concentrations of WGA for 2 h reduced C. gigas settlement on OSE paper, with settlement decreasing with the increase in WGA concentration. Treating larvae with 5 µg/mL WGA for 2 h also reduced larval settlement on OSE paper; but, at 50 µg/mL, larval settlement was not significantly different from the control group. Larval settlement was inhibited in the presence of N-acetyl-D-glucosamine (GlcNAc). Furthermore, larval settlement on OSE paper was not affected when OSE papers were treated with mixtures of 50 µg/mL WGA and GlcNAc. Shell chips and GF/C filter papers with OSE dyed with fluorescein isothiocyanate-conjugated WGA exhibited fluorescence under the UV field. Thus, a WGA-binding sugar chain of the glycoprotein in shells of conspecifics may mediate the settlement of C. gigas larvae on conspecifics.