Effects of light and temperature conditions on the expression of GnRH and GtH genes and levels of plasma steroids in <Emphasis Type="Italic">Odontesthes bonariensis</Emphasis> females

In this study we examined the endocrine mediation between environmental factors (temperature and photoperiod) and the brain–pituitary–gonadal axis in females of pejerrey Odontesthes bonariensis. Changes in the expression of brain gonadotropin-releasing hormones (GnRHs) and gonadotropin (GtH) subunit [follicle stimulating-β (FSH-β), luteinizing hormone-β (LH-β), glycoprotein hormone-α (GPH-α)] genes, plasma gonadal steroids [estradiol (E₂) and testosterone (T)], gonadal histology, and gonadosomatic index (GSI) in adult females exposed to combinations of short-day (8 h) or long-day (16 h) photoperiods and low (12°C) or high (20°C) temperatures after winter conditions (8 h light, 12°C) were analyzed. Pejerrey females kept under the short photoperiod had low GSIs, and their ovaries contained only previtellogenic oocytes regardless of the experimental temperature. In contrast, females exposed to the long photoperiod had high GSIs and ovaries with vitellogenic oocytes at both temperatures. These fish also showed a significantly higher expression of sGnRH, pjGnRH, cGnRH-II (the three different GnRH variants found to date in the pejerrey brain), FSH-β, LH-β and GPH-α genes and plasma E₂ levels than those at the shorter photoperiod. No significant changes were observed in plasma T levels. Based on these results, we concluded that the increase in day length but not that of temperature triggers the maturation of pejerrey females after the winter period of gonadal rest and that this occurs by an integrated stimulation of the various components of the brain–pituitary–gonad axis.     

Effect of temperature decrease on oocyte development,sex steroids,and gonadotropin β-subunit mRNA expression levels in female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

To improve understanding of the mechanism of early ovarian development in eels, the effects of water temperature decrease on oocyte development, plasma levels of sex steroids [estradiol 17β (E2), testosterone (T), 11-ketotestosterone (11-KT)], and gonadotropin β-subunit [follicle-stimulating hormone (FSHβ), luteinizing hormone (LHβ)] messenger RNA (mRNA) expression levels were investigated. A total of 27 female Japanese eels Anguilla japonica were divided into initial, control, and test (water temperature decrease) groups. Starting on 22 September 2009, eels in the test group were reared in a tank with gradual temperature decrease from 25°C to 15°C over 39 days, while the control group was maintained at 25°C. The test group accumulated more oil droplets in their oocytes than did the other groups. Levels of sex steroids, especially 11-KT, were higher in the test group. In contrast, FSHβ and LHβ mRNA expression levels were lower in the test group. These results suggest that water temperature decrease only induced an early stage of ovarian development that was partly affected by an 11-KT increase. For further maturation, other environmental factors related to induction of gonadotropin increase appear to be needed.     

The effect of water temperature on gamete maturation and gamete quality in the European grayling (Thymalus thymallus) based on experimental data and on data from wild populations

To investigate the effect of water temperature on gamete maturation and gamete quality, European grayling (Thymalus thymallus) were held under different temperature regimes prior to spawning. Maturation of males and females and their gamete quality depended strongly on temperature regime. The highest percentages of maturing fish and highest gamete quality were obtained under a creek water temperature regime with natural seasonal fluctuations. In warmed creek water (3–4°C), at a constant temperature of 8–9°C, and under an abruptly increasing temperature, regime maturation rates and gamete quality were reduced. The effect was more pronounced in females than in males. The spawning dates of different wild Austrian grayling populations were also correlated with water temperature data collected during the last 10–15 years. The mean spawning date expressed as the number of days from 21 December until spawning ranged from 98 to 111 days for lower elevation populations, while it was considerably delayed for an alpine population. All populations spawned at water temperatures of 5.5–7.2°C. Regression analysis indicated that spawning date correlated with (1) the overall mean water temperature from 21 December until spawning, (2) the mean water temperature during both the last 10 days and 15 days before spawning, and (3) the difference between mean January temperature and that of the last 15 days before spawning. The regression functions indicate that an increase in water temperature from 21 December to spawning of 1°C advances spawning by 5? days, and an increase of 1°C in the last 10–15 days advances spawning by 3? days.     

北太平洋长鳍金枪鱼卵巢的发育特征

根据2013年10月–2014年2月在北太平洋海域(29°08′~41°08′N,163°50′~144°19′W)采集的364尾长鳍金枪鱼的卵巢样本,利用组织学分析,详细描述了长鳍金枪鱼卵巢、卵细胞的发育阶段。结果显示,北太平洋长鳍金枪鱼卵巢内同时存在不同时相的卵细胞,为分批产卵类型;组织学上,长鳍金枪鱼的卵细胞发育过程分为6个时相,卵巢发育过程分为6个时期;卵巢成熟指数在成熟期为Ⅰ~Ⅴ期时逐渐增大,在Ⅵ期时减小;北太平洋长鳍金枪鱼产卵高峰为12月中旬和1月初,其卵巢成熟指数随纬度的升高呈递减趋势,随经度变化规律不明显。研究表明,通过对北太平洋长鳍金枪鱼卵巢的发育特征的分析与探讨,可为北太平洋长鳍金枪鱼的资源状况评估及渔业可持续发展提供生物学信息。     

High water-temperature tolerance in photosynthetic activity of <Emphasis Type="Italic">Zostera marina</Emphasis> seedlings from Ise Bay,Mie Prefecture,central Japan

Photosynthetic activities of seedlings of Zostera marina were successively measured using a gas volumeter for 6 days at seven light (0–400 μmol photons/m² per s) and 11 water temperature conditions (5–35°C). The seedlings were collected from mature plants (Ise Bay, central Japan), and stored and cultured in incubators accurately controlled at each test temperature. The maximum gross photosynthesis (P _maxg) was recorded at an optimal water temperature of 29°C after 0 days. After 6 days, P _maxg appeared at 25°C and most plants cultured at 29–30°C bleached and withered after the drastic increase of light compensation point (I _c). On the contrary, at 5–28°C, the photosynthetic activities either changed little (5–25°C) or recovered after a temporal reduction (26–28°C); seedlings survived and looked healthy after being cultured for 6 days. The recovery was thought to be an acclimation to tolerate higher water temperature. As a result, the critical upper water temperature for Z. marina seedlings was proposed as 28°C. The temperature was consistent with the previously reported maximum water temperature in habitats around the southern boundary of Z. marina in the northern hemisphere.     

黄海南部近岸水域棘头梅童鱼仔稚鱼的分布和漂移趋势

鱼类早期资源特征对鱼类种群的研究和渔业资源的评估管理具有极大的价值.实验于2019年3月—2020年1月在黄海南部近岸水域设置47个站点,每月大潮期间使用仔稚鱼网采集仔稚鱼,揭示了棘头梅童鱼仔稚鱼丰度的时空分布特征和漂移趋势,采用广义相加模型分析环境因子与之关联.调查共采集到棘头梅童鱼仔稚鱼2385尾,出现在6—10月...     

Effects of temperature on the final stages of sexual maturation in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Maturing male and female Atlantic salmon (Salmo salar L.) were held under three temperature regimes for 10 weeks between September and December: warm (constant 14–16 °C), ambient (decreasing from 11 to 5 °C), and cold (decreasing from 7 to 3 °C). Blood samples were analyzed for plasma steroid levels, and the fish were inspected for the presence of expressible milt (total volume and spermatocrit) and ovulation weekly. Samples of eggs were dry-fertilized with milt stripped from three males held at the same temperatures and incubated until the eyed stage. In females, levels of plasma testosterone (T) and 17β-oestradiol (E₂) dropped as ovulation approached, concurrent with a rapid increase in levels of plasma 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). In males, levels of T and 11-ketotestosterone (11-KT) peaked 2–3 weeks after the first appearance of expressible milt, while levels of 17,20β-P increased steadily and did not exhibit a definite peak. Exposure of females to cold water amplified and advanced the profiles of all three steroids compared with the ambient group, and increased the survival rates to the eyed egg stage. Cold water had no immediate effect on the male steroid profiles, but later, higher levels of 17,20β-P were evident compared with both the ambient controls and the warm water group, while the effects on 11-KT and T were more variable. Exposure to warm water completely inhibited both milt production and ovulation. Moreover, warm water modulated the steroid profiles of the males with lower 11-KT levels compared with ambient controls and lower 17,20β-P level compared with cold-water-treated males. In females, warm water resulted in total inhibition of the peri-ovulatory peak in 17,20β-P and prevented the normal decline of T and E₂ levels associated with ovulation. The findings of the present study are highly relevant for broodstock management in aquaculture, as well in understanding the impact of climate change/temperature variability on wild salmon spawning.     

Effect of temperature and photoperiod on the reproductive condition and performance of a tropical damselfish <Emphasis Type="Italic">Chrysiptera cyanea</Emphasis> during different phases of the reproductive season

Effects of temperature and photoperiod on the reproductive activity of a reef associated tropical damselfish Chrysiptera cyanea were evaluated under three phases with different environmental patterns, phase I (April–May; increasing water temperature and photoperiod), phase II (June–July; increasing water temperature and peak/decreasing photoperiod), and phase III (August–September; peak/decreasing water temperature and decreasing photoperiod). When the fish were reared at 20, 25, or 30°C under natural photoperiod, the reproductive conditions differed within and among the phases depending on experimental temperature and environmental patterns. From phases I through III, ovaries with vitellogenic oocytes were notable only at 25°C, whereas regressing and immature oocytes were noticed at 20 and 30°C. The fish underwent active spawning at 25°C, whereas no or few spawnings were observed at other temperatures. In phase III, there was a resultant prevention of decrease in the gonadosomatic index and disappearance of vitellogenic oocytes in the ovaries of fish under a long photoperiod (LD14:10) compared to those under a short photoperiod (LD10:14). These results indicate that a long photoperiod with a suitable range of water temperature is a principal determinant in continuity of reproductive activity and performance, and that a high temperature has a negative impact on their ovarian development.     

Ammonia autointoxication of common carp: case studies

Three case studies of ammonia autointoxication of the common carp (Cyprinus carpio L.) are described. In the first case, carp yearlings with a full digestive tract were transferred during the growing period from pond water (22°C) to tap water (17°C). In the second case, marketable carp were transferred from the fishing ground of a pond (18°C) to a storage pond (10–12°C). Harvest was performed in late September when the fish were still ingesting natural feed. In the third case, marketable carp after storage for 1 month were transferred to storage ponds with markedly lower water temperature. Stress because of harvest and handling also occurred in this case. In all cases, highly increased concentrations of ammonia were found in the blood plasma of the fish (mean ± SD 1760 ± 350 μmol L⁻¹ in the first case, 870 ± 540 μmol L⁻¹ in the second case, and 880 ± 150 μmol L⁻¹ in the third case). Highly congested, dark-red coloured, oedematous gills were observed for all specimens. We can avoid similar cases of ammonia autointoxication by protection of fish from sudden changes of temperature during rearing, harvesting, and handling.     

Effects of temperature and salinity on oxygen consumption and ammonia excretion of juvenile miiuy croaker, <Emphasis Type="Italic">Miichthys miiuy</Emphasis> (Basilewsky)

The metabolic responses of the juvenile Miichthys miiuy in terms of oxygen consumption and ammonia excretion to changes in temperature (6–25°C) and salinity (16–31 ppt) were investigated. At a constant salinity of 26 ppt, the oxygen consumption rate (OCR) of the fish increased with an increase in temperature and ranged between 133.38 and 594.96 μg O₂ h⁻¹ g⁻¹ DW. The effect of temperature on OCR was significant (P < 0.01). Q₁₀ coefficients were 6.80, 1.41, 1.29 and 2.36 at temperatures of 6–10, 10–15, 15–20 and 20–25°C, respectively, suggesting that the juveniles of M. miiuy will be well adapted to the field temperature in the summer, but not in the winter. The ammonium excretion rates (AER) of the fish were also affected significantly by temperature (P < 0.01). The O:N ratio at temperatures of 6, 10, 15 and 20°C ranged from 13.12 to 20.91, which was indicative of a protein-dominated metabolism, whereas the O:N at a temperature of 25°C was 51.37, suggesting that protein-lipids were used as an energy substrate. At a constant temperature of 15°C, the OCRs of the fish ranged between 334.14 (at 31 ppt) and 409.68 (at 16 ppt) μg O₂ h⁻¹ g⁻¹ DW. No significant differences were observed in the OCR and AER of the juveniles between salinities of 26 and 31 ppt (P > 0.05). The OCR and AER at 16 ppt were, however, significantly higher than those at 26 and 31 ppt (P < 0.05), indicating salinity lower than 16 ppt is presumably stressful to M. miiuy juveniles.     

Estradiol-17β suppresses testicular development and stimulates sex reversal in protandrous black porgy,Acanthopagrus schlegeli

Two year old black porgy (Acanthopagrus schlegeli) fed a diet containing 4.0 mg kg^–1 of estradiol-17 (E₂) for 5 months had significantly lower GSI than the control group during the spawning season. E₂ suppressed testicular development, spermiation and plasma testosterone (T) and 11-ketotestosterone (11-KT) and stimulated ovarian development, vitellogenesis and sex reversal. Spermiation in the control group occurred in January and February with the concentrations of 1.08–1.36 × 10¹⁰ sperm ml^–1 of milt. Higher plasma T and 11-KT, but lower E₂ levels were detected in the spermiating fish (control group). Higher plasma E₂ levels were detected in the sex reversing black porgy during the pre-spawning season. A sharp rise in plasma 11-KT and a drop in T levels were detected in spermiating fish (control group) from January to February. Plasma 11-KT levels correlated with the testicular development and spermiation. The data suggest that E₂ plays an important role in controlling the sex reversal of black porgy.to whom correspondence should be addressed.     

Reproductive performance of common dentex, Dentex dentex, broodstock held under different photoperiod and constant temperature conditions

The effect of photoperiod and temperature on the timing and the quality of spawning, and on associated endocrine changes in circulating 17β-oestradiol, 11-ketotestoserone (11-KT) and vitellogenin (Vtg) were investigated in common dentex, Dentex dentex, undergoing their second reproductive cycle. The possibility was also explored of using the measurement of steroids in the culture water of broodstock tanks, rather than in individual blood plasma samples, as a potentially useful tool for assessing the physiological state of a fish without disturbing them. One group of fish was exposed to a simulated natural seasonal cycle and ambient temperature (CONTROL). The other two groups were exposed to simulated seasonal photoperiod cycles (12 month-long) but which were phase-shifted either three months before (ADVANCED) or after (DELAYED) the natural cycle. Temperature was kept at 19.4±0.9 °C all-year-round. In the CONTROL, spawning started in mid-April and lasted until mid-June, while in the ADVANCED group, spawning started 4 months earlier and in the DELAYED group 2 months later than the CONTROL. The total egg production, egg quality, hatching rate, relative fecundity, and spawning index of the experimental groups were similar to the controls. The differences in spawning time induced by photothermal manipulation were associated with a difference in the timing of peak concentrations of plasma E₂, 11-KT and Vtg. In all three groups, the amounts of conjugated 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and free and sulfated 11-KT which could be extracted from the water during the spawning period were significantly higher than those found in the preovulatory period. However, the differences were mostly less than 2-fold suggesting that, at least under the conditions employed in this study, the method was of limited use for non-intrusive detection of gonadogenesis and spawning (as had been hoped). The observed differences in spawning time and in the seasonal changes of sex steroids and Vtg confirm and extend the findings on marine fish. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thermal ecology of subadult and adult muskellunge in a thermally enriched reservoir

The movement of adult muskellunge, Esox masquinongy Mitchill, has been investigated in a variety of systems, but temperature selection by muskellunge has not been examined where well‐oxygenated waters were available over a range of temperatures for much of the year. Thirty subadult and adult muskellunge tagged internally with temperature‐sensing radio tags were tracked from March 2010 to March 2011 in a Tennessee reservoir. Mean tag temperatures were 18.9 °C in spring (March to May), 22.1 °C in summer (June to August), 16.5 °C in autumn and 9.8 °C in winter (December to February). When the greatest range in water temperatures was available (7.1–33.3 °C; May to early August 2010), their realised thermal niche (mean ± 1 SD) was 22.3 °C ± 1.8; the realised thermal niche was affected by fish size (smaller fish selected slightly warmer temperatures) but not sex. An electric generating steam plant discharging warm water resumed operation in January 2011, and most (86%) tagged fish occupied the plume where temperatures were ≈10 °C warmer than ambient water temperatures. No mortalities were observed 15 days later when plant operations ceased. Their affinity for the heated plume prompted concerns that muskellunge will be too easily exploited when the plant operates during winter.     

A preliminary experiment regarding the natural induction of gonadal development in female Japanese eels without hormone treatment

To test whether gonadal development of female eels could be promoted without any exogenous hormone treatments, we observed the effect of water temperature manipulation. After 3–5 months of water temperature treatments, three silver eels showed higher gonadosomatic indices (GSI). In particular, one eel in the 5–15°C fluctuating temperature treatment group (5°C daytime and 15°C night‐time) had the highest GSI of 8.5 with secondary yolk globule stage oocytes of large diameter (OD; 412 μm) after 3 months, which indicated definite gonadal development compared with those in the initial states (mean GSI, 2.4; OD, 226.7 μm). The 5–15°C fluctuating temperature and constant 5°C groups had low oocyte breakdown (atretic) rates. Because daily temperature fluctuations and cold daytime water are experienced by vertically migrating silver eels in the ocean, these temperature conditions may be key maturation process components that could be useful for hormone‐free artificial maturation.     

High water temperature tolerance in photosynthetic activity of <Emphasis Type="Italic">Zostera japonica</Emphasis> Ascherson &; Graebner seedlings from Ago Bay,Mie Prefecture,central Japan

Photosynthetic activity of Zostera japonica seedlings was measured using a gas volumeter at 0 and 6 days in culture under eight light (0–800 μmol photons/m²/s) and ten water temperature conditions (5–35°C). Seedlings from Ago Bay, Mie Prefecture were cultured in incubators accurately controlled at each test temperature for 1 week. After 1 week, maximum gross photosynthesis (P _maxg) appeared at 29°C and most seedlings cultured at 30–35°C bleached and withered. At the same time, the light compensation point (I _c) increased only at 30°C during the culture period. As a result, the upper critical water temperature for survival was 29°C in Z. japonica seedlings, which agrees well with that for the southern boundary of Z. japonica around Japanese coast. It is necessary to monitor this species around this boundary as a bio-indicator for seawater warming.     

Hot-water solubility of mantle collagens in several cephalopod molluscs

The mantle muscles of five cephalopod species, Todarodes pacificus, Photololigo edulis, Sepioteuthis lessoniana, Sepia esculenta, and Sepia longipes, were extracted with 0.1 M NaOH to prepare crude collagen fiber, called ‘residue after alkali extraction’ (RS-AL). Solubility of the collagens in water was examined in the temperature range 20–90°C. The collagens showed a similar tendency in solubility, which gradually increased depending on the treating temperature, and the values at 40–90°C were constantly less than 47.0% for all the species examined. In addition, the collagens were estimated to denature in the approximate temperature range of 37.5–42.5°C. These results suggest that the collagens in RS-AL from these species may have relatively high resistance to hot-water extraction even after their denaturation.     

Live chilling of Atlantic salmon: physiological response to handling and temperature decrease on welfare

Investigation of the physiological effects of live chilling in Atlantic salmon, Salmo salar, has been performed in two experiments. In the first, fish (mean weight 840 g) acclimatized to either 16, 8, or 4°C were directly transferred horizontally or vertically (9 combinations) to water temperatures of 16, 8, 4, or 0°C using a dip net. Blood samples were collected at 1 and 6 h (h) post-transfer. In the second experiment, fish (mean weight 916 g) acclimatized to 16°C were exposed to four temperature-drop regimes (no physical handling): 16–4°C (over 5 h), 16–4°C (over 1 h), 16–0°C (over 5 h), and 16–0°C (over 1 h). Blood samples were collected 1 h post-temperature drop. Physical transfers in the first trial, i.e., temperature drops, resulted in immediate (1 h) increases in blood lactate concentrations at all three temperatures, but levels were significantly reduced and close to pre-transfer levels after 6 h. Horizontal transfers, i.e., 16–16°C, 8–8°C, and 4–4°C, resulted in similar increases and were not significantly different from the groups exposed to temperature drops. The most severe vertical transfer (16-0) resulted in a swift loss of equilibrium and eventually death. In experiment 2, temperature drops from 16 to 4°C and from 16 to 0°C over a period of one or 5 h, without physically handling the fish, resulted in no significant increases in any of the measured parameters 1 h post-transfer, except in the 16–0 (1 h) group. The latter experienced a significant increase in blood sodium, glucose, lactate, and cortisol levels compared to all other groups. The results suggest that salmon are capable of tolerating relatively steep temperature drops without any significant negative effects on blood stress parameters and that physical stress from handling overrides the effect of thermal insults.     

Effects of temperature and adrenaline on the atrial myocardium of the cultured Atlantic salmon (Salmo salar)

The effects of acute temperature changes (2–17°C) on myocardial contractility with or without adrenergic activation were studied in the isolated spontaneously beating atrium of the Atlantic salmon (Salmo salar) reared at 8°C. The atrial frequency was markedly elevated (from 7 to 46 beats/min) by the rise in temperature from 2–17°C. Both the time to peak tension and to relaxation time were shortened. In contrast, the temperature effect on the maximal tension was modest. Exposure to exogenous adrenaline (1.1 nM–11 μM) resulted in a substantial enhancement of the maximal tension, notably at 2°C, while potentiation of the frequency at 2, 8 and 14°C, was less pronounced. The apparent affinity (pD₂) for adrenaline on the chronotropy was higher at 8 and 14°C than at 2°C. For the inotropic responses pD₂ was highest at the acclimation temperature (8°C). By comparison with data for the rainbow trout (Oncorhynchus mykiss) obtained by the same experimental design (Ask et al. 1981), species differences were apparent both in temperature dependence of contractile parameters and in their adrenergic activation. The Q₁₀ for the frequency in absence of adrenaline was higher in the salmon than in the trout for the temperature interval 2–17°C. The apparent affinities for adrenaline for the frequency at 8°C and 14°C and for the maximal tension responses at 2°C and 8°C were also highest for the salmon atrium.     

The interrelation between temperature regimes and fish size in juvenile Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>): effects on growth and feed conversion efficiency

The present paper describes the growth properties of juvenile Atlantic cod (Gadus morhua) reared at 7, 10, 13 and 16 °C, and a group reared under “temperature steps” i.e. with temperature reduced successively from 16 to 13 and 10 °C. Growth rate and feed conversion efficiency of juvenile Atlantic cod were significantly influenced by the interaction of temperature and fish size. Overall growth was highest in the 13 °C and the T-step groups but for different reasons, as the fish at 13 °C had 10% higher overall feeding intake compared to the T-step group, whereas the T-step had 8% higher feeding efficiency. After termination of the laboratory study the fish were reared in sea pens at ambient conditions for 17 months. The groups performed differently when reared at ambient conditions in the sea as the T-step group was 11.6, 11.5, 5.3 and 7.5% larger than 7, 10, 13 and 16 °C, respectively in June 2005. Optimal temperature for growth and feed conversion efficiency decreased with size, indicating an ontogenetic reduction in optimum temperature for growth with increasing size. The results suggest an optimum temperature for growth of juvenile Atlantic cod in the size range 5–50 g dropping from 14.7 °C for 5–10 g juvenile to 12.4 °C for 40–50 g juvenile. Moreover, a broader parabolic regression curve between growth, feed conversion efficiency and temperature as size increases, indicate increased temperature tolerance with size. The study confirms that juvenile cod exhibits ontogenetic variation in temperature optimum, which might partly explain different spatial distribution of juvenile and adult cod in ocean waters. Our study also indicates a physiological mechanism that might be linked to cod migrations as cod may maximize their feeding efficiency by active thermoregulation.     

Effects of aromatizable and nonaromatizable androgens on the sex inversion of red-spotted grouper (Epinephelus akaara)

The effects of aromatizable 17α-methyltestosterone (MT) and non-aromatizable 17α-methyldihydrotestosterone (MDHT) on sex inversion in red-spotted grouper, Epinephelus akaara, were investigated. Fish were implanted with MT, MDHT and MT+AI (aromatase inhibitor, AI) respectively for one month. The results showed that the three treated groups turned into transitional stage with intersex gonads, which contained atretic oocytes and spermatogenic germ cells at all stages of spermatogenesis. The controls did not change sex. The gonads of more than half MT-implanted fish were in early transitional stages of sex inversion, whereas those of more than half MDHT and MT+AI-implanted fish were in late transitional stages of sex inversion. No difference in serum estradiol-17β (E₂) levels between the controls and the treated groups were observed, whereas 11-ketotestosterone (11-KT) and testosterone (T) levels increased in all treated groups. Significantly lower gonadosomatic index (GSI) and gonadal aromatase activity were observed in the treated groups, which were in accordance with the lower mRNA expression of P450aromA. However, P450aromB mRNA expression increased in the MT group, while it did not change in the MDHT group. These results suggest that the sex inversion of red-spotted grouper by MT and MDHT implantation might be due to the suppression of P450aromA gene expression, and resulting in both the decrease of the ovarian estrogen –secretion, as well as the increase in the 11-KT levels. Furthermore, the main reason for MT being less effective than MDHT might be due to partial aromatization of MT to estrogen.