Tsuneo Honjo

Effect of 17β-estradiol on the reproduction of Japanese medaka (Oryzias latipes)

Abstract Estrogenic compounds such as 17β-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis–ova. Males treated with E2 concentrations=55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis–ova in male medaka exposed to environmentally relevant concentrations of E2.

Acute effects of triclosan, diclofenac and carbamazepine on feeding performance of Japanese medaka fish (Oryzias latipes)

The toxicity of three pharmaceutical and personal care products (PPCPs)--carbamazepine (CBMZ), diclofenac (DCF), and triclosan (TCS)--was examined by measuring their effects on feeding behavior and swimming speed of adult Japanese medaka fish (Oryzias latipes). Medaka were exposed to 6.15 mg L(-1) CBMZ, 1.0 mg L(-1) DCF, 0.17 mg L(-1) TCS, or no PPCP (control) for 9 d. Fish behaviors were monitored during days 5-9 of the exposure period. Feeding behavior (time to eat midge larvae, TE) and swimming speed (SS) of individual exposed and control fish were tracked in two dimensions, using an automated system with a digital charge-coupled device camera. As a result, feeding behavior was affected by exposure to CBMZ and DCF, while SS was altered by exposure to CBMZ and TCS. Thus, TCS, DCF and CBMZ appear to affect fish behaviors through different mechanisms. Overall, the results suggest that behavioral changes may provide a sensitive indicator for assessing the toxicity of PPCPs to aquatic organisms.

Interactions between the red tide dinoflagellates Heterocapsa circularisquama and Gymnodinium mikimotoi in laboratory culture

Abstract Interactions between Heterocapsa circularisquama and Gymnodinium mikimotoi, causative red tide dinoflagellates, were investigated using bialgal cultures. G. mikimotoi was killed by H. circularisquama when the initial cell density of each species was set at 200 cells ml−1. However, cells of H. circularisquama transformed to temporary cysts when the initial cell density of G. mikimotoi was increased to 2000 cells ml−1. Thus the interaction between H. circularisquama and G. mikimotoi was found to be dependent upon the initial cell densities of the two species. Culture filtrate of H. circularisquama induced no inhibitory effect on the growth of G. mikimotoi. Similarly when separated by a membrane filter, G. mikimotoi grew well when cultured with H. circularisquama. G. mikimotoi appear to be killed by cell contact with H. circularisquama. In growth experiments using a culture filtrate of G. mikimotoi and cultures separated by a membrane filter, G. mikimotoi was shown to secrete a substance that inhibited the growth of H. circularisquama. However, the inhibitory effect of the medium was found at higher cell densities of G. mikimotoi than in the bialgal cultures at which the growth of H. circularisquama was suppressed and formed temporary cysts. It is likely that the inhibitory effect of G. mikimotoi on H. circularisquama in the bialgal cultures occurred mainly by direct cell contact. The growth of H. circularisquama and G. mikimotoi in the bialgal cultures was simulated using a mathematical model to quantify the interaction. The degree that G. mikimotoi was inhibited by H. circularisquama was found to be three times larger than the inhibitory effect of G. mikimotoi on H. circularisquama.

Suppression of sexual behavior in male Japanese medaka (Oryzias latipes) exposed to 17β-estradiol

Mating pairs of Japanese medaka (Oryzias latipes) received 17beta-estradiol (E2; dose, 3 and 30 microg/g body weight daily) in their diet and E2-free diet (control) for two weeks. The effect on reproductive success was assessed in the mating pairs, and sexual and nonsexual behaviors in the male were quantified. The fecundities of the E2-treated groups were reduced significantly (p < 0.05) compared with those of control, and the sexual behaviors (following, dancing, floating, and crossing) of male fish in response to prostaglandin F2alpha (PG)-injected E2-unexposed females were suppressed dramatically. While the fertility of spawned eggs and the frequencies of the nonsexual behaviors of resting and swimming in a straight line performed by male were unchanged in E2-exposed groups. Therefore, sexual behavior in male may be linked with reproductive success in mating pair of madaka exposed to E2. This assay for sexual behavior using PG may provide a useful tool for assessing the effect of endocrine-disrupting chemicals.

The biology and prediction of representative red tides associated with fish kills in Japan

Abstract The damage to marine resources caused by red tides represents a serious problem for fisheries and allied industries. In Japan, most fish farms have suffered heavy losses from red tides of the dinophyte Gymnodinium nagasakiense and of the raphidophytes Chattonella antiqua, C. marina, and Heterosigma akasbiwo. Therefore, an extensive series of studies has been conducted in Japan. Elucidation of the mechanisms of outbreak of harmful red tides associated with fish kills and the development of countermeasures for these red tides are urgently needed. Recently, Heterosigma red tides have caused severe losses for salmon culture operations in several places around the world. The development of fish farming and the progress of eutrophication on a global scale will accelerate the incidence of harmful red tides. This article is a review of the physiological ecology of these organisms and of the techniques proposed in Japan to predict the outbreaks of harmful red tides in areas that have experienced damage fr...

Possible factors responsible for the toxicity of Cochlodinium polykrikoides, a red tide phytoplankton

Cochlodinium polykrikoides, a harmful red tide dinoflagellate, is highly toxic to fish, but the toxic mechanism is still unknown. Recent study has suggested that C. polykrikoides generates reactive oxygen species (ROS) such as superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and the ROS-mediated ichthyotoxicity has been proposed. In this study, we found that the levels of O(2)(-) and H(2)O(2) detected in C. polykrikoides were trace levels as compared with those of Chattonella marina which is well-known to produce ROS. Furthermore, no significant increase in O(2)(-) generation by C. polykrikoides was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus prepared from skin and gill of yellowtail, whereas C. marina generated increased level of O(2)(-) responding to these stimuli. Interestingly, the cell-free aqueous extract prepared from C. polykrikoides showed toxic effect on the HeLa cells, but the extract of C. marina had no significant effect. Furthermore, gradual accumulation of polysaccharides in the medium was observed during the growth of C. polykrikoides, and the medium gradually became viscous, but no such changes were observed in the medium of C. marina. These results suggest that multiple factors may be responsible for the toxic mechanism of C. polykrikoides.

Effects of 4‐nonylphenol on reproduction of japanese medaka, Oryzias latipes

We exposed mature Japanese medaka (Oryzias latipes) to 4-nonylphenol (4-NP) to elucidate its reproductive effects and estrogenic responses. For each treatment, eight breeding pairs were exposed for 3 weeks at measured 4-NP concentrations of 24.8, 50.9, 101, and 184 micrograms/L. Egg production of the pairs during the third week of exposure (15-21 d) was significantly decreased in the > or = 101-microgram/L treatment group, and fertility was significantly decreased in the 184-microgram/L group. Induction of testis-ova was observed in male fish at all 4-NP concentrations, whereas abnormality of spermatogenesis was found only in those receiving the 184-microgram/L treatment. The gonadosomatic index of the males in the 184-microgram/L group was also remarkably reduced. Hepatic vitellogenin (VTG) levels were significantly increased in both sexes receiving 4-NP treatments of > or = 50.9 micrograms/L. Overall, the lowest-observed effective concentrations (LOECs) of 4-NP for decreasing reproductive capacity (fecundity and fertility) and eliciting estrogenic responses (induction of testis-ova and VTG) were 101 and < or = 24.8 micrograms/L, respectively. The LOEC for reduced reproduction in medaka was at least four-fold greater than that required to elicit estogenic responses in male fish. Thus, we suggest that the estrogenic responses expressed in male fish are not responsible for the reduction in reproductive capacity.

Toxicity and LD50 levels of the red tide dinoflagellate Heterocapsa circularisquama on juvenile pearl oysters

Abstract Laboratory experiments were conducted to clarify the mechanism of the mass mortality of pearl oysters ( Pinctada fucata ) associated with the red tide dinoflagellate Heterocapsa circularisquama such as occurred in Ago Bay, Japan, in 1992. Two-month-old pearl oysters were treated with various densities of H. circularisquama, Heterosigma akashiwo and a sterile control. Immediately after the exposure to H. circularisquama cells, juvenile pearl oysters rapidly contracted their mantles and closed their shell valves. Then, they contracted their gills and experienced irregular heartbeat patterns until the heart stopped permanently. The mortality of the juveniles was closely correlated with the cell density of H. circularisquama . The LD 50 was approximately 20 000 cells ml −1 after 24 h and 10000 cells ml −1 after 48 h from the start of the experiments. However, in the presence of either H. akashiwo at 100 000 cells ml −1 or supernatant of H. circularisquama culture, juvenile pearl oysters did not die within 72 h. During the red tide in Ago Bay, 1992, the maximum cell density of H. circularisquama reached over 80 000 cells ml −1 . Therefore, we consider that the mass mortality of cultured pearl oysters during this red tide was caused by the direct action of H. circularisquama cells.

Early–life‐stage toxicity in offspring from exposed parent medaka, Oryzias latipes, to mixtures of tributyltin and polychlorinated biphenyls

The present study examined the effects of tributyltin (TBT), polychlorinated biphenyls (PCBs), and mixtures of both chemicals on reproduction in Japanese medaka, Oryzias latipes. For 21 d we gave groups of medaka freeze-dried brine shrimp flakes contaminated with a mixture of either 0, 1, 5, or 25 microg TBT g(-1) plus 0 or 25 .micro PCBs g(-1). We measured the fecundity and fertility of the parent fish and assessed the deformity, hatchability, time-to-hatching, and swim-up failure rate of the next generation. Fertilization success in the third week of the administration period was significantly decreased by administration of 25 microg TBT g(-1) (77%) compared with the control group (87%). Both TBT and PCBs were transferred maternally into the eggs of the next generation, causing early life-stage toxicity. Administration of 1 microg TBT g (-1) was not toxic to embryological development, but abnormal eye development (i.e., small eyes or no eyes) occurred when TBT at the same concentration was mixed with PCBs (6.4%). Administration of TBT alone significantly decreased hatchability and increased swim-up failure, and administration of PCBs alone significantly increased time-to-hatching. Statistical analysis by two-way analysis of variance detected an interaction between TBT and PCBs in these three parameters. TBT induces abnormal development of the eyes, reduced hatchability, and increased swim-up failure, whereas PCBs delay time-to-hatching. Administration of mixtures of TBT and PCBs has more adverse effects on the developmental stage of medaka than does that of each chemical alone.

Extracellular polysaccharide-protein complexes of a harmful alga mediate the allelopathic control it exerts within the phytoplankton community.

The goal of this study was to examine the significance of allelopathy by the raphidophyte Heterosigma akashiwo in a multispecies phytoplankton community in the field. Towards this aim, we sought allelochemicals of H. akashiwo, which had allelopathic effect both in laboratory experiments and in the field. As an initial step, we showed that the allelopathic effects of H. akashiwo filtrate were both species-specific and dependent upon the cell density of the target species. Secondly, we found for the first time that extracellular, high-molecular weight allelochemicals [that is, polysaccharide-protein complexes (APPCs)] were produced by a marine phytoplankton species, H. akashiwo. Thirdly, we indicated that the purified APPCs selectively inhibited the growth of the diatom Skeletonema costatum that is a major competitor of H. akashiwo, and thereby tended to promote the formation of monospecific H. akashiwo blooms. Furthermore, we demonstrated that the inhibitory effect of APPCs on the growth of the diatoms was determined by binding to the cell surface of the target species. Finally, we succeeded in the detection of APPCs in the field samples at concentrations exceeding their experimentally determined action threshold during the H. akashiwo bloom. Strategies for ecosystem control, including mitigation of harmful algal blooms (HABs), should take into account that red-tide organisms like H. akashiwo are already part of complex webs involving inter-specific allelopathic inhibition and ecosystem control during their dense blooms.