This study aims to reveal the first report of the natural spawning of F1 hybrid grouper (TGGG), a crossbreed between the tiger grouper, Epinephelus fuscoguttatus 9 giant grouper, E. lanceolatus, since its first production in 2006. This marks the completion of its full cycle after a 10-year period. In order to establish a seed rearing protocol for a novel F2 hybrid TGGG, natural spawning, embryonic and larval developments were thoroughly observed. Five batches of natural spawning were recorded with an average of 1.50-15.3 kg eggs collected, while fertilization and hatching rates were recorded at 85.3-97.6%, and 63.0-98.3%, respectively. F2 larvae hatched out at 17:50 hours with an average body size of 1.74 ± 0.01 mm, and a yolk sac volume of 0.85 ± 0.197 mm 3. The first feeding was initiated 3 days after hatching, which coincided with the onset of functional feeding apparatus and active swimming behavior. Larval dorsal and pelvic spines were formed at 6 days AH coupled with dynamic feeding activity, as more food was found in the digestive tract. Meanwhile, the F2 hybrid grouper shifted habitat from pelagic to benthic as early as 25 days AH, and entered a juvenile stage at 35 days AH, attaining a skin coloration similar to that of the F1 juvenile. This study concluded that naturally spawned eggs of F2 hybrid TGGG were exceptionally high in quality, although larvae were small and fragile, and performed vigorous feeding activities and cannibalistic behavior. Thus, these findings can serve as primary data to further develop the optimal rearing protocol to enhance the overall rearing performance. Keywords Hybrid grouper Á Natural spawning Á Embryonic and larval development Á Morphology Á Sensory organ Á Behavior changes

The e¡ects of delayed ¢rst feeding on the survival and growth of tiger grouper, Epinephelus fuscoguttatus (ForsskÔl 1775), larvae were examined under controlled conditions. The total length, yolk sac volume, oil globule volume, yolk sac absorption time and nutritional transition period (NTP) of the larvae fed at di¡erent ¢rst times (0, 6, 12, 18 and 24 h after the mouth opening stage; h AMO) were compared. Larval ¢rst feed intake was observed at 54 h after hatching (h AH) at 27.5 AE 0.5 1C. The yolk sac was consumed more rapidly with an increase in delayed ¢rst feeding and was signi¢cantly di¡erent among treatments (Po0.05). Larvae ¢rst fed at 0 h AMO had the longest yolk sac absorption (72 h AH) and NTP (20 h) times and had the highest survival and growth rates at the end of the experimental period (360 h AH), being signi¢cantly higher (Po0.05) than the other treatments. First mortality was observed at 69 h AH, approximately 2 h after point of no return (PNR) occurred. This study suggests that ¢rst feeding of tiger grouper larvae should commence at 0 h AMO for enhancement of larval survival and growth.

Zeylanicobdella arugamensis (Annelida: Hirudinea), a marine parasitic leech, is currently affecting different species of cultured groupers, hybrid groupers, snappers and sea bass in Malaysia. Dillenia suffruticosa (Dilleniaceae), a medicinal plant found in Sabah, has been selected in our experiment to kill the leeches as a natural control method. The leech‐infested hybrid groupers were collected from aquaculture facilities, and the isolated leeches were challenged against methanol extract of D. suffruticosa leaves. The experiment was carried out using various concentrations of the extracts such as 25, 50 and 100 mg/ml. The methanol extract showed significant antiparasitic activity against Z. arugamensis with 100% mortality at a concentration of 100 mg/ml in 14.39 ± 3.75 min., followed by 50 and 25 mg/ml in 32.97 ± 9.29 and 41.77 ± 5.40 min., respectively. The phytochemical composition of the extract was determined using GC‐MS analysis to understand the nature of the principal compounds responsible for its antiparasitic properties. The leaves of D. suffruticosa demonstrated the presence of different bioactive compounds of various natures with varying percentages. Thus, it could be revealed that the methanol extract of D. suffruticosa mainly contains vital phytochemical compounds and showed an effective antiparasitic activity against the harmful leeches of hybrid groupers.

No abstract

Marine leech Zeylanicobdella arugamensis (Piscicolidae), an economically important parasite is infesting predominantly cultured groupers, hybrid groupers and other fish in Southeast Asian countries. In this study, we tested the anti-parasitic potential of a medicinal plant Nephrolepis biserrata found in Sabah, East Malaysia against Z. arugamensis. Various concentrations of methanol extracts of the plant were tested experimentally against Z. arugamensis and disinfestation of the leech from its primary host hybrid groupers. The composition of methanol extract of N. biserrata was determined through LC-QTOF analysis. The significant anti-parasitic activity of 100% mortality of leeches was observed with the exposure of N. biserrata extracts. The average time to kill the leeches at concentrations of 25, 50 and 100 mg/ml was 25.11 ± 3.26, 11.91 ± 0.99, and 4.88 ± 0.50 min., respectively. Further, at various low concentrations of N. biserrata 2.5, 5 and 10 mg/ml, hybrid groupers were disinfested in an average time of 108.33 ± 12.65, 65.83 ± 9.70 and 29.16 ± 5.85 min., respectively. The tandem mass spectrometry data from LC-QTOF indicated some hits on useful bioactive compounds such as terpenoids (ivalin, isovelleral, brassinolide, and eschscholtzxanthin), flavonoids (alnustin, kaempferol 7,4′-dimethyl ether, and pachypodol), phenolics (piscidic acid, chlorogenic acid, and ankorine), and aromatic (3-hydroxycoumarin). Thus N. biserrata can act as a potential biocontrol agent.