Hitoshi Kitamura

The induction of larval settlement and metamorphosis of two sea urchins, Pseudocentrotus depressus and Anthocidaris crassispina, by free fatty acids extracted from the coralline red alga Corallina pilulifera

Lipophilic inducers of larval settlement and metamorphosis of Pseudocentrotus depressus and Anthocidaris crassispina, two commercially important sea urchin species in Japan, were isolated from the foliose coralline red alga Corallina pilulifera (collected in 1990 near Saga, Japan) and identified. Larval assays of the fractions obtained by silica gel column chromatography of the total lipids showed that non-polar groups of lipids were effective at inducing larval settlement and metamorphosis. The effective fractions were further subjected to gel filtration (Sephadex LH-20) and also to silica gel column chromatography, and the effective components isolated as single spots by thin-layer chromatography. The components at a concentration of ca. 0.4 mg paper-1 (sample was adsorbed on a paper with 20 cm2) induced high rates of larval settlement of both P. depressus and A. crassispina. Chemical analyses of the components revealed a mixture of free fatty acids (FFAs), dominated by eicosapentaenoic acid (20:5, 41 to 50%), palmitic acid (16:0, 11 to 17%), arachidonic acid (20:4, 9 to 15%), and palmitoleic acid (16:1, 4 to 5%). In assays with the four standard FFAs, only 20:4 and 20:5 induced larval settlement and metamorphosis of the two species, while 16:0 and 16:1 were ineffective. The larvae underwent significant rates of settlement and metamorphosis in response to the two former FFAs at levels as low as 0.18 mg paper-1. Amongst the free fatty acid components of the alga, 20:5 was isolated as the chemical inducer of larval settlement and metamorphosis of the sea urchins in the laboratory.

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 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.

The effect of mono‐amino acids on larval settlement of the barnacle, Balanus amphitrite Darwin

Settlement of barnacle larvae is believed to be induced by the chemical cues present in their surrounding environment. Here, an investigation was carried out on the effects of sixteen different mono‐amino acids with acidic, basic, uncharged polar and nonpolar side chains, and GABA on larval settlement of the barnacle, Balanus amphitrite. Settlement inducing activity by nine mono‐amino acids, viz. asparagine, glutamine, tyrosine, serine, glycine, tryptophan, leucine, isoleucine and valine (but not phenylalanine) with uncharged polar and nonpolar side chains was observed. Of these, the most active mono‐amino acids were serine, leucine and isoleucine, which were effective at a threshhold of 1.0 × 10‐7 M. On the other hand, aspartic acid, glutamic acid, GABA, and the basic mono‐amino acids lysine, arginine and histidine did not have any inducing effect. These results suggest that uncharged polar and non‐polar end group of the amino acid chain play an important role in inducing the settlement process in cyprids.

Volatile substances from adult extracts induce larval settlement of the barnacle balanus amphitrite

The volatile substance extracted from conspecific adults induces larval settlement of the barnacle Balanus amphitrite. The settlement inducing activity of the volatile fractions was checked monthly from June, 1997 to December, 1998. Both water soluble extracts and volatile fractions from the barnacle were prepared by the steam distillation. The active cue in the volatile fraction was always extracted with n‐pentane under acidic conditions, although settlement inducing activity varied with the sample. GC‐MS analysis of the active and inactive pentane fractions revealed 1, 2, 3‐trimethyl‐benzene as the settlement inducing substance. Commercially available 1, 2, 3‐trimethylbenzene also showed high settlement inducing activity at a concentration of 0.8 × 10‐12 M (100 pg 1‐1). This substance was detected at concentrations of more than 7 ng g‐1 of wet barnacle (equivalent to 0.6 × 10‐12M, equal to 70pg1‐1) by GC analysis. These results indicate that 1,2,3‐trimethylbenzene in the volatile fractions acts as a chemical cue for larval settlement. Monthly variation in the settlement inducing activity was observed, which synchronized with the breeding season of the barnacle. This observation suggests that the barnacle produced the chemical cue in the gonad during maturation or accumulated it from the environment.