Short Communications

Convenient Assay for Settlement Inducing Substancesof Barnacles

Hiroyuki Kawahara,1,* Ryo Tamura,2 Seiko Ajioka, and Yoshikazu Shizuri

Marine Biotechnology Institute, Shimizu Laboratories, 1900 Sodeshi, Shimizu, Shizuoka 424, Japan

Abstract. A convenient assay method for estimation of barnacle, Balanus amphitrite, settlement inductive

activity was developed. To avoid the inductive effect by comrades and laborious observation requirements,

cyprid larvae were put individually into wells of a 96-well plate. The settlement ratio from the experiment

without any inducers was quite low; therefore this assay allowed easy estimation of settlement inductive

activities. Some known inductive agents, such as serotonin and barnacle extracts, clearly showed inductive

activities. This assay method is proven to be suitable for estimation of barnacle settlement-inducing activities

of both water-soluble and -insoluble compounds.

The barnacle is one of several fouling animals that cause

serious problems on ship hulls and other marine infrastruc-

tures. Organotin compounds such as TBTO [bis-(n-

tributyltin)-oxide] have been used as effective chemicals to

prevent biofouling. However, use of these compounds has

been restricted in recent years because heavy metals may

have detrimental effects on the marine environment. Now

safer compounds that are effective against fouling organ-

isms but have less influence on the marine environment are

required. To find new inhibitory compounds that are not

toxic, it is important to investigate the mechanism of bar-

nacle settlement.

It is known that barnacles produce a pheromonelike

substance called arthoropodin in order to arrange them-

selves (Larman et al. 1982), and some neurotransmitters,

such as serotonin (5-HT) and dopamine, were also found to

act as barnacle settlement inducers. To understand barnacle

settlement, it is necessary to investigate the inductive com-

pounds and the mechanism of the settlement induction.

Some assay methods were reported to estimate settlement

inductive activities, but there were some problems, such as

uneven results or restricted test samples. Assays that include

several barnacles placed in the same vessel (Kon-ya et al.

1995; Kon-ya and Endo1995) are not appropriate to esti-

mate the inducing abilities of test compounds because the

first settled barnacle may induce settlement of other bar-

nacles. A published method using a nitrocellulose mem-

brane with adsorbed test sample allows easy estimation of

inductive activities, but this method can only be applied to

samples that are carried by the nitrocellulose membrane

(Matsumura et al. 1994). Here we report a convenient assay

system that is suitable for estimation of the settlement in-

ductive activities for barnacles, Balanus amphitrite, without

interference of factors such as the inductive effect by com-

rades and laborious observation requirements.

MATERIALS AND METHODS

Cyprid Larvae of Barnacles

Cyprid larvae were prepared according to the method de-

scribed previously (Kado 1991; Kon-ya and Miki 1994).

Received October 31, 1997; accepted July 10, 1998.1Present address: Nippon Suisan Kaisha, Ltd., Central Research Laboratory, 559-6

Kitano, Hachiouji, Tokyo 192-0906, Japan.2Present address: Ihara Chemical Industry Co., Ltd., Research & Development Dept.,

2256 Nakanoko, Fujikawa-cho, Ihara-gun, Shizuoka 421-33, Japan.

*Corresponding author. Fax: +81-426-56-5188; email: hkawa@mars.dti.ne.jp

Mar. Biotechnol. 1, 98–101, 1999

© 1999 Springer-Verlag New York Inc.

Nauplii were obtained from reared barnacles, Balanus am-

phitrite and fed with Artemia salina at 23°C. Nauplii were

reared to cyprids by feeding with Cheatoceros calcitrans at

23°C. Aged cyprids were prepared by storage at 4°C for

seven days.

Assay for Estimation of Self-Inducing Activity onBarnacle Settlement

To estimate the self-inducing activity, 10 cyprids were put

into a 35-mm-diameter polystyrene vessel in triplicate and

50 cyprids were put alone into 50 of the same vessels con-

taining 5 ml of 90% seawater. All vessels were incubated at

23°C in the dark. The barnacles in the vessels were observed

for 24 days and settled barnacles were counted.

Assay to Determine Effect of Vessel Size onSettlement Ratio

To compare the effect of vessel size on settlement ratio,

three different sized vessels, a 96-well plate, a 24-well plate,

and a 35-mm-diameter Petri dish were tested. Twenty-four

wells were used for each size, and cyprids were put alone

into each well with 200 µl, 1.4 ml, and 5 ml of 90% seawater

for the 96-well plate, 24-well plate, and 35-mm-diameter

Petri dish, respectively. These were incubated at 23°C in the

dark and observed for five days.

Assay for Estimation of SettlementInducing Activity

Test samples were dissolved in 90% seawater at specific

concentrations. The sample solutions were put into wells of

a 96-well plate in aliquots of 200 µl. Immediately after

metamorphosis, cyprids were put alone into each well.

Cyprids in wells were incubated at 23°C in the dark and

settled barnacles were counted at 24-h intervals until the

settlement ratio observed exceeded 60% at the highest con-

centration of inducers.

Preparation of Boiled Extracts (Arthoropodin)

Boiled extract from Balanus amphitrite was prepared ac-

cording to previous work (Larman et al. 1982). Barnacle

tissue from 30 adult barnacles was homogenized with 10 ml

of 0.02 M phosphate buffer, pH 7.0, containing 0.15 M

NaCl, in a glass homogenizer. Homogenate was coarsely

filtered and autoclaved at 121°C for 15 min. The precipitate

was removed by filtration and the supernatant was treated

with an equal volume of saturated ammonium sulfate (0–

2.0 M) at 25°C. The solution was allowed to stand overnight

at 4°C and then centrifuged at 800g for 30 min. The pre-

cipitate was resuspended in 0.02 M phosphate buffer, pH

7.0, and centrifuged at 38,000g for 30 min. The soluble

fraction was dialyzed against 0.02 M phosphate buffer, pH

7.0, for 24 h at 4°C. The final protein concentration was

determined by the Bradford method as 0.3 mg/ml (Brad-

ford 1976).

RESULTS AND DISCUSSION

Differences in the settlement ratio between the experiment

using 10 cyprid larvae in one dish and the experiment in

which 10 larvae were separated into 10 dishes are shown in

Fig. 1. In the former experiment, the result suggests that

barnacles, after settlement and metamorphosis, induce the

settlement of other barnacles in the same dish, and the

inducing abilities of the initially settled barnacle differ be-

tween individuals. Cyprid larvae to be used in the assay with

Figure 1. Difference in the settlement ratio between isolated lar-

vae and collective larvae: s: isolated larvae, d: collective larvae.

Settlement ratio was calculated as the number of settled barnacles

divided by the number of larvae used.

Assay for Barnacle Settlement Inducers 99

10 cyprid larvae in one dish should be prepared with great

care to avoid uneven results. A low settlement ratio value

was observed in the experiment in which 10 larvae were

separated into 10 dishes. Cyprid larvae rarely settle without

external inducers.

The influence of vessel diameter or volume of seawater

on the settlement ratio was examined. The results suggest

the effect of vessel size on barnacle settlement is not sig-

nificant (data not shown). The 96-well plate, which has

small diameter wells, is suitable for our assay and very small

amounts of sample are needed.

Cyprid larvae stored for 7–10 days settled at a higher

ratio than postmetamorphosis cyprids (Branscomb and

Rittschof 1984). This increment of the settlement ratio was

also observed in our experiment using isolated larvae (data

not shown). We interpret this increment in settlement ratio

as a function of endogenous larval maturity for settlement

and metamorphosis rather than other external factors.

Overall the settlement ratio observed for isolated larvae was

not high. Therefore, we can use cyprid larvae prepared just

after metamorphosis or several days after metamorphosis in

order to estimate inducing activity of candidate substances.

Some known inducing substances were tested in this

assay (Fig. 2). 5-HT is a known neurotransmitter reported

as a settlement inducer for barnacles (Kon-ya et al. 1995).

The results of the experiment using 5-HT clearly showed

inducing activity in the same concentration range as re-

ported. The adult extract from barnacle tissues also showed

inducing activity as described previously (Branscomb and

Rittschof 1984; Matsumura et al. 1994). Larvae placed in

conditioned seawater (collected from culture of adult bar-

nacles after 20 h) also showed an increased settlement ratio.

Results from this series of experiments confirm that

this assay method using multiwell plates is suitable for es-

timation of inducing activities. Moreover, the assay requires

a very small amount of sample and is hardly influenced by

larval conditions, such as ages and uniformity.

ACKNOWLEDGMENTS

We wish to thank Mrs. S. Kikuta and Miss M. Umehara,

Marine Biotechnology Institute, for their technical assis-

tance. We also thank Dr. K. Kon-ya, Ihara Chemical Indus-

try Co., Ltd., for his critical discussions. This work was

performed as a part of the Industrial Science and Technol-

ogy Frontier Program supported by the New Energy and

Industrial Technology Development Organization.

REFERENCES

Bradford MM (1976) A rapid and sensitive method for the quan-

titation of microgram quantities of protein utilizing the principle

of protein-dye binding. Anal Biochem 72:248–254

Branscomb ES, Rittschof D (1984) An investigation of low fre-

quency sound waves as a means of inhibiting barnacle settlement.

J Exp Mar Biol Ecol 79:149–154

Kado R (1991) Effect of light on the larval development of Balanus

Figure 2. Estimation of settlement induction for known inducers

using the multi-well method. A: The effect of 5-HT at different

concentrations (j: 10−4 M; s: 10−6 M; d: negative control). B:

The effect of the boiled extract (arthoropodin) at different con-

centrations (h: 12 µg/ml; j: 3 µg/ml; s: 0.6 µg/ml; d: negative

control). C: The effect of conditioned seawater (s) versus a nega-

tive control (d). Settlement ratio was calculated as indicated in the

legend of Fig. 1.

100 Hiroyuki Kawahara et al.

amphitrite Darwin (Cirripedia). Nippon Suisan Gakkaishi 57:

1821–1825

Kon-ya K, Endo M (1995) Catecholamines as settlement inducers

of barnacle larvae. J Mar Biotechnol 2:79–81

Kon-ya K, Miki W (1994) All-seasonal assay for antifouling sub-

stances using reared barnacle larvae. J Mar Biotechnol 1:193–195

Kon-ya K, Miki W, Endo M (1995) L-Tryptophan and related

compounds induce larval settlement of the barnacle Balanus am-

phitrite Darwin. Fish Sci 61:800–803

Larman VN, Gabbott PA, East J (1982) Physico-chemical proper-

ties of the settlement facter proteins from the barnacle Balanus

balanoides. Comp Biochem Physiol 72B:329–338

Matsumura K, Mori S, Nagano M, Fusetani N (1994) A protein

complex, inducing larval settlement, from the barnacle, Balanus

amphitrite. Zool Sci 11:42

Assay for Barnacle Settlement Inducers 101