| Marine fouling is found very seriously threatening the development and utilization of marine resources, and results in large losses in environment, economy, and human health. Research and development on non-toxic and effective coatings alternative to traditional toxic one, become a focus topic. Marine fouling organisms include in a wide variety, and barnacle is one of typical fouling invertebrates. Barnacle has stronger adaptability to the environment and is the important species in marine fouling community. The research of searching to the nontoxic and effective antifouing compounds was widely developed in the past decades. And the antifouling mechanisms of those nontoxic antifoulants were very complex, including inhibition against adhesion/settlement/metamorphosis, and interfering in neurotransmission or signal transduction, and so on.In this study, we focused on the settlement and metamorphosis of the barnacle larvae of Amphibalanus(=Balanus) amphitrite as a key example and process of marine fouling. The experimental designing was carried out due to following reasons. Firstly, we tried to find out the optimal growth environment of the larvae through laboratory simulation, and to build up the antifouling bioassay system of the settlement and metamorphosis of the larvae. Secondly, the bioassay system was verified using two compounds of flavone from the metabolites of Streptomyces fungicidicus in previous studies. The anti-settlement activities of these two compounds against barnacle larvae were then determined using this bioassay system. The results show that genistein has strong activity against larval settlement. In addition, a group of functional analogs of mizolastine, which is a kind of H1 receptor antagonist, were found having significant influence on settlement and metamorphosis of the larvae. Therefore, beginning from the function of mizolastine, we collected more functional analogs of mizolastine, and examined their activity against the settlement and metamorphosis of barnacle larvae to screen for optimal antifouling active compounds. In addition, due to the finding in experiment that antihistamine compounds behaved strong antifouling activity, we tried to clarify the roles of histamine in the settlement and metamorphosis of barnacle larvae. The main results of this thesis are as following:1) It was found that barnacle grows in the environment of the water temperature over 22℃.The low temperature inhibits the reproduction and growth of barnacles. Barnacle is hermaphrodite, and mostly cross fertilization. After fertilization, oosperms develops to nauplii and is released to surrounding seawater. Nauplii have small body sizes and poor ability to adapt to the environment. The optimal culture conditions for nauplii in laboratory were found as temperature of 28℃±1℃ and feeding with Chaeloccros gracilis. Under such conditions, the nauplii develop and metamorphosize to cyprids in six days.2) Thirty five signalling interference substances were collected due to the previous study, which shows that mizolastine, an antagonist against the histamine receptor, can prevent settlement and metamorphosis of barnacle larvae. The anti-settlement and anti-metamorphosis activities of these Thirty five signalling interference substances were examined using above bioassay system. The results indicated that eight compounds, including Loratadine, Ondansetron hydrochloride, Antazoline hydrochloride, Clemastine fumarate, Triprolidine hydrochlorise, Azelastine hydrochloride, Propranolol hydrochloride, and Clorprenaline hydrochloride, have extremely strong anti-settlement and anti-metamorphosis activities. They inhibited the larval settlement and metamorphosis completely at concentration of 2.5μg ml-1. Their activities are near to or beyond those of traditional toxic antifoulants, while their toxicities are 10-100. folds lower. There still are four compounds with remarkable activities, which completely inhibited the larval settlement and metamorphosis completely at concentration of 5μg ml-1. The four compounds are Risperidone, Ketetifen fumarate, Tegaserod maleate, and Clenbuterol hydrochloride. And the other 6 compounds, including Amitriptyline hydrochloride, Promethazine hydrochloride, Phenylephrine hydrochloride, Phentolamine Mesylate, Diphenhydramine hydrochloride, and Lafutidine, were found with significant inhibition activities against the larval settlement and metamorphosis at concentration of 5-10μg ml-1 Among the eight extremely active compounds, there are five (Loratadine, Antazoline hydrochloride, Clemastine fumarate, Triprolidine hydrochlorise, and Azelastine hydrochloride) belong to histamine H1 receptor antagonists. Therefore, a hypothesis was deduced that their activities may be due to the interference against the signaling pathway of histamine.3) The eight most active compounds with reduced concentrations were examined in an antifouling bioassay for further study. Their EC50 values were calculated based on the inhibition effects at different concentrations. The results are EC50<0.625μg ml-1, EC50=1.52μg ml-1, EC50 <0.625μg ml-1, EC50<0.2μg ml-1, EC50=0.91μg ml-1, EC50<O.2μg ml-1, EC50=O.73μg ml-1, and EC50=0.72μg ml-1 for Loratadine, Ondansetron hydrochloride, Antazoline hydrochloride, Clemastine fumarate, Triprolidine hydrochlorise, Azelastine hydrochloride, Propranolol hydrochloride, and Clorprenaline hydrochloride, respectively. In addition, no significant enhancement on larval lethality was observed for these eight compounds at concentration of 20 μg ml-1. Above results suggested that these eight active compounds are promising antifouling candidates with high efficiencies and low toxicities.4) It was verified whether histamine involve in the signaling process of the settlement and metamorphosis of barnacle larvae. The verification includes three aspects. The first question is whether histamine exists in the normal barnacle larvae. The positive answer is physically necessary for the hypothesis that histamine involves in the physiological activities of barnacle larvae. The second question is whether additional histamine enhances the settlement and metamorphosis of barnacle larvae. The third question is whether the inhibition effect of antihistamine compounds can be removed or neutralized by histamine. That is to determine whether the barnacle larvae, which were treated by the histamine H1 receptor antagonists, can resume the ability to complete their settlement and metamorphosis after being isolated from the treatment, and being incubated in fresh seawater or the seawater with addition of histamine. The results show that the presence of histamine in both nauplii and cyprids of the barnacle was observed by using the analysis of High Performance Liquid Chromatography with fluorescence detector (HPLC-Fr) after O-phthalaidehyde derivatization. Thus the larvae of two develop stages bioassays both have the physical basis to use histamine as signal carrier. To clarify the functions of histamine in the larvae, two bioassays were further performed, using the nauplii and cyprids, respectively. The addition of 10-8、10-10、10-12 mol/L histamine can all shorten the incubation time for nauplii develop to cyprids stage with higher rates when compared to those of control. The optimal concentration is 10-10 mol/L. The low-energy-containing cyprids were culture, which would result in poor settlement and metamorphosis to juveniles without addition inducers. The enhancement on the metamorphosis was also observed for the treatment of histamine addition. In the presence of the optimal concentration of 5×10-9 M, low-energy-containing cyprids settled and metamorphosed to juveniles with the highest efficiency. The recovery bioassay was conducted to verify whether histamines were involved in barnacle larval settlement and metamorphosis signaling and to examine the anti-settlement effects of the Hi receptor antagonists. An anti-histamine compound of sample No.20 possessing strong antifouling effects against barnacle A. amphitrite. The larvae treated with this sample were selected for recovery bioassays. The results indicated that the inhibition effects against larval settlement were eliminated to some extent if the larvae were removed from environments containing sample No.20. Some of the treated larvae regained their ability to settle and to complete their metamorphosis in fresh ASW. Moreover, this ability was further enhanced with the addition of 5×10-9 mol/L histamine to the fresh ASW. Therefore, the three questions were all have the positive answers, which verified that histamine histamine play an important role in signaling for the larval metamorphosis of barnacle A. Amphitrite.These results in this thesis suggest that some of the compounds influencing neurotransmitter signaling, especially histamine H1 receptor antagonists, are promising new antifouling candidates, and that histamine exists in the larvae of both stages of nauplii and cyprids and play an important role in the signaling for the larvae settlement and metamorphosis. |
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