Diurnal variation of tryptic activity in larval stage and development of proteolytic enzyme activities of malabar grouper (Epinephelus malabaricus) after hatching

Diurnal variation in tryptic activity and developmental changes in proteolytic enzyme activities of malabar grouper larvae (Epinephelus malabaricus) were examined. Five different groups were prepared for the experiment of diurnal variation of tryptic activity in larvae: larvae were fed Thai-type rotifers Brachionus rotundiformis from the time of mouth opening, fed rotifers from 6 h after mouth opening, 12 h, 24 h and not fed rotifers (starved control). The experimental tanks were placed in temperature-controlled baths at 28 °C under 24 h light. Developmental changes in proteolytic activity of trypsin and pepsin-like enzyme were measured from hatching to 57 days after hatching (DAH).

The tryptic activity of all fed groups showed the same pattern, and the diurnal variation of tryptic activity was clearly observed from 3 to 6 DAH. The highest tryptic activities were found at 19:00, and the activities were lowest from 01:00 to 07:00. In contrast, that of non-fed larvae was low compared to the fed groups, however the diurnal variation of tryptic activity was shown same tendency to the fed groups. Interestingly, both groups (fed and non-fed) were exhibited a circadian rhythm under the 24 h light conditions and delaying of first-feeding. Tryptic activity of larvae notably increased from 40 to 45 DAH and markedly decreased at 52 DAH. In contrast to the tryptic activity, that of pepsin-like enzyme clearly increased from 47 to 51 DAH. The results suggest that a functional change of protein digestion occurs from 40 to 50 DAH related with metamorphosis in malabar grouper. These results could contribute to determining appropriate feeding schedules, such as feeding time, frequency and optimal time to change food items, in mass-scale production of the present species.     

Isolation and characterization of a regulator of G protein signaling (RGS16) gene homologue in yellow grouper Epinephelus awoara

Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAP) which act as modulators of G protein-coupled receptors. We isolated a RGS16 homologue in yellow grouper (Epinephelus awoara) spleen using suppression subtractive hybridization and RACE-PCR. The nucleotide sequence of yellow grouper RGS16 full-length cDNA was 700 bp and contained an open reading frame of 537 bp, encoding a putative protein of 178 amino acids. The encoded protein shows 47–61% identities to other homologues. RT-PCR analysis demonstrated that RGS16 was expressed in yellow grouper spleen and up-regulated in kidney heart, liver, and anterior kidney by lipopolysaccharide. This study will help towards validating the specific function of RGS in marine fish.     

Isolation and characterization of collagens from the skin of giant grouper (Epinephelus lanceolatus)

ABSTRACT

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of giant groupers (Epinephelus lanceolatus) with yields of 39.51 and 19.12%, respectively. ASC and PSC consisted of two different α chains (α1 and α2) and were characterized to be type I collagen with no disulfide bond. The imino acid contents of the ASC and PSC from giant grouper skin were 189 and 181 per 1,000 residues, respectively. The maximum endothermic temperatures (T_max) of ASC and PSC measured by differential scanning calorimetry (DSC) were 31.71 and 31.33°C, respectively. The denaturation temperatures of ASC and PSC measured by viscometry were 29.84 and 29.05°C, respectively. The maximum solubility in 0.5 M acetic acid was observed at pH 5 and pH 6 for ASC and PSC, respectively. A sharp decrease in solubility was observed for both ASC and PSC in the presence of NaCl above 3% (w/v).     

Requirements of vitamin C (L-ascorbyl-2-monophosphate-Mg and L-ascorbyl-2-monophosphate-Na) and its effects on immune responses of grouper, Epinephelus malabaricus

An 8‐week feeding trial was conducted to evaluate two vitamin C derivatives, L‐ascorbyl‐2‐monophosphate‐Mg (C2MP‐Mg) and L‐ascorbyl‐2‐monophosphate‐Na (C2MP‐Na), to satisfy the vitamin C requirement and to test their effects on the immune responses of juvenile grouper, Epinephelus malabaricus. C2MP‐Mg and C2MP‐Na were each supplemented at 20, 50, 80, 150, 250, and 400 mg kg^?1 diet in the basal diet providing of 7, 18, 31, 51, 93, 145 mg ascorbic acid (AA) equivalent of C2MP‐Mg kg^?1 diet and 4, 10, 17, 31, 47, 77 mg ascorbic acid (AA) equivalent of C2MP‐Na kg^?1 diet, respectively. Basal diet without AA supplementation was included as control. Each diet was fed to triplicate groups of grouper (mean initial weight 3.20 ± 0.05 g). Fish fed diets supplemented with either C2MP‐Mg or C2MP‐Na had significantly (P < 0.05) greater weight gain (WG), feed efficiency and survival than those fed the unsupplemented control diet. Liver ascorbate concentrations in fish generally increased as dietary C2MP‐Mg or C2MP‐Na supplementation level increased. Haemolytic complement activity was higher in fish fed diets supplemented with 92 mg AA equivalent of C2MP‐Mg kg^?1 or 10–17 mg AA equivalent of C2MP‐Na kg^?1 than fish fed the unsupplemented control diet. Lysozyme activity was higher in fish fed ≥51 mg AA equivalent of C2MP‐Mg kg^?1 or ≥47 mg AA equivalent of C2MP‐Na kg^?1 than fish fed the unsupplemented control diet. Analysis by broken‐line regression of WG indicated that the adequate dietary vitamin C concentration from each vitamin C derivative in growing grouper is 17.9 mg AA equivalent of 2MP‐Mg kg^?1 and 8.3 mg AA equivalent of C2MP‐Na kg^?1, and it also indicated that C2MP‐Mg is about 46% as effective as C2MP‐Na in meeting the vitamin C requirement of grouper.     

Effects of feeding frequency on the growth and body constituents of young red-spotted grouper, Epinephelus akaara

In order to obtain optimum feeding frequency in young red-spotted grouper, 25 000 fish averaging 4.7 cm in total length were divided into five groups in duplicate floating net-cages. Each group was reared with the same feeding ration to the fish weight but with different feeding frequencies: one, two, four, six, and eight times daily, respectively. High feeding frequencies resulted in high weight gain, high feed conversion efficiency, and high survival rate. The highest muscle mass and the highest levels of muscle protein were obtained at a feeding frequency of six times daily. While muscle lipid decreased with increasing feeding frequency, lipid levels of the liver and intraperitoneal fat body tended to be elevated by high feeding frequencies. Triglyceride accumulation in the whole body increased with increasing feeding frequency, but was lower in the group fed eight times daily than in the other groups.

Based on the above results, a feeding frequency between four and six times daily is suggested to be optimum for achieving optimum growth rates and lipid reserves in young red-spotted grouper.     

Population age and sex structure of Aphanius fasciatus Nardo, 1827 (Pisces: Cyprinodontidae) in the Mesolongi and Etolikon lagoons (W. Greece)

The population structure of Aphanius fasciatus in the Mesolongi and Etolikon lagoonal system was studied, using 5794 fish. Significant differences were observed in the number of individuals of each sex, the age and size composition and survival. The overall males to females sex ratio was 1:2.44, although there was seasonal variation. During the reproductive period the percentage of males in the population decreased significantly, while after reproduction and during recruitment they increased. The females in each age class were larger than the males (40.33 mm and 36.72 mean total length for females and males, respectively). The survival rate of females was greater than males (0.73 for females and 0.60 for males). The strategy of this species is to invest in female individuals.     

Influence of light intensity on feeding, growth, and early survival of leopard coral grouper (Plectropomus leopardus) larvae under mass-scale rearing conditions

This study investigated the effect of different light intensities on feeding, growth and survival of early stage leopard coral grouper Plectropomus leopardus larvae. Four different light intensities (0, 500, 1000 and 3000 lx) were used and larvae were kept under constant light conditions from 0 day after hatching (DAH) to 5 DAH. The larvae were fed a small S-type of Thai strain rotifers at a density of 20 individuals/mL from 2 DAH. The number of rotifers in larval digestive organ and total length of larvae were examined at 3 h intervals between 04:00 and 22:00 h on 3 DAH, and thereafter at 6 h intervals until the end of the experiment (5 DAH). Four experimental trials of the larval rearing were repeated using by 60 kL mass-scale rearing tanks. The results indicate that coral grouper larvae are visual feeders and their food intake increases with increasing light intensity. Food intake of larvae reared at 3000 lx was significantly higher than those reared at 0–1000 lx on 3 DAH despite being the first-feeding day (P < 0.01). On 4 DAH, total length of larvae reared at 3000 lx was significantly larger than those reared at the lower light intensities (0, 500 and 1000 lx), and thereafter light intensity significantly influenced larval feeding and growth until the end of the experiment. Survival on 5 DAH did not show a significant difference between light intensities, but survival rate at 3000 lx and 1000 lx had a tendency to be higher than those reared at the lower light intensities (0 and 500 lx). In contrast, larvae reared at 0 lx exhibited stagnant and/or negative growth. These results indicate that light intensity is significantly the factor affecting larval feeding, growth, and survival in coral grouper larvae under the rearing conditions.     

Sperm production in the red claw crayfish Cherax quadricarinatus (Decapoda, Parastacidae)

The objective of this study is to evaluate the effect of body size, temperature and annual cycle on sperm production in Cherax quadricarinatus. Sperm count and sperm mortality were estimated, the reproductive system was weighted, and macro and microscopical analysis of the testes and vasa deferentia were conducted. Sperm count and weight of the reproductive system are strongly related to male size, in contrast to sperm mortality. The spermatophore structure presented macro and microscopical differences between sizes. Males higher in size have more adherent spermatophores. This species has a reproductive cycle related to sperm production. Sperm count and weight of the vasa deferentia rise in summer, while the weight of the testes increases in winter. During the spring, the sperm cord presents a higher density than in other seasons. The temperature seems to affect sperm production being 27 and 29 °C the best assayed conditions. The present results are relevant information to obtain the best sperm viability selecting male size, season of sampling and the best temperature for the reproductive stock and future assays of spermatophore cryopreservation for this species aquaculture.     

Parasites of cultured and wild brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) in Lampung Bay,Indonesia

A total of 210 Epinephelus fuscoguttatus (brown‐marbled grouper) was examined for parasites. During three consecutive seasons (two rainy and one dry season from 2002 to 2004), 35 specimens each taken from floating net cages of the National Sea Farming Development Centre (Balai Budidaya Laut) and from wild catches in Lampung Bay, South Sumatra, Indonesia were studied. Twenty‐five (cultured grouper) and 30 (wild grouper) parasite species/taxa were identified, with an infracommunity ranging from one to nine (cultured) and three to 14 parasite species (wild), demonstrating a species‐rich parasite fauna even in the cultured fish. Protozoans (1 species), microsporeans (1), myxozoans (1), digeneans (8), monogeneans (5), cestodes (3), nematodes (8), acanthocephalans (2) and crustaceans (6) were found. The most abundant parasites were the monogeneans Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus lantauensis for both, cultured and wild grouper during all seasons. For the cultured fish, the prevalence of monoxenous ectoparasites (e.g. P. epinepheli, P. lantauensis, Capsalidae gen. et sp. indet., Benedenia epinepheli) was in most cases higher than that of heteroxenous endoparasites. This contrasts the wild grouper, where heteroxenous parasites such as Allopodocotyle epinepheli and Raphidascaris sp. occurred at a similar prevalence compared with the fairly abundant Pseudorhabdosynochus spp. No seasonality of infestation was observed for both cultured and wild fish. The high levels of infestation of potentially pathogenic monogeneans throughout the year could result in significant parasite outbreaks at the locality studied.     

鞍带石斑鱼仔稚（幼）鱼的发育和生长研究

对鞍带石斑鱼的仔稚幼鱼形态发育的各个阶段进行了观察与研究,详细描述从初孵仔鱼到幼鱼各个发育时期的形态特征和发育时间。根据卵黄囊的变化,长鳍棘的长出与收缩,鳞片和体色斑纹的出现,鞍带石斑鱼胚后发育可以划分为仔鱼期、稚鱼期、幼鱼期。仔鱼期又可分为卵黄囊期仔鱼和后期仔鱼。水温27～30℃,盐度27～31,pH值8.0～8.4的海水中培育,初孵仔鱼至孵化后2日龄为卵黄囊期仔鱼。2日龄仔鱼开口,3日龄至20日龄为后期仔鱼,22日龄至30日龄为稚鱼期,31日龄进入幼鱼期。鞍带石斑鱼胚后发育过程中最明显的变化是背鳍棘和腹鳍棘的生长和收缩,也是生产育苗当中比较关键的仔稚幼鱼变态过程。     

Survival and growth of hatchery-reared individuals of the European lobster, Homarus gammarus (L.), in field-based nursery cages on the Irish west coat

At present, one of two strategies is employed by fisheries managers for enhancing wild stocks of homarid lobsters using hatchery-reared individuals. The first is repeated releases of large numbers (>5000 at a time) of postlarvae (stage IV and V; carapace length [CL]=5–7 mm) to selected bottom locations. This option exists primarily because these programs lack space, time, and/or the finances to rear animals to larger sizes that would most likely have initially higher survival rates. The second is to rear animals in the laboratory for 5–8 months to stage XII+ (CL=12–16 mm) and then release small numbers (<1000) of these relatively large juveniles. To date there has been no attempt to release large numbers of relatively large juveniles because the costs are too prohibitive.

We have developed a low-cost, low-maintenance, field-based nursery caging system for rearing cultured lobsters, Homarus gammarus (L). Individuals (780 and ranging in CL from 5.2 to 7.2 mm) were reared in pre-fouled and unfouled containers (360 cm³) fabricated from an extruded plastic netting (3.2 mm aperture) and in pre-fouled plastic petri dishes (200 cm³) that were deployed in five near-bottom cages for 10 months (September 2000 to June 2001) at two subtidal sites located in a shallow, relatively exposed embayment on the west coast of Ireland. Animals apparently were able to survive and grow by suspension feeding on the plankton and/or foraging on the fouling community that settled on and within individual containers. Mean recovery rate (±95% CI) was independent of a priori fouling treatments, but was site-specific (42.1±7.9% and 27.8±13.7%; n=5). These rates are minimal estimates of survival because we found that at least 20% of the animals were capable of escaping from the mesh containers. Mean recovery in petri dishes that prohibited emigration was 53.3±37.02% at one site and 75.0±23.1% (n=5) at the other. These recovery rates compare favorably with survival rates of fed conspecifics held in the laboratory over the same time (54/81=66.7%). At the end of the experiment, animals in field cages had mean CLs that were significantly smaller than the fed controls. Because of costs incurred with maintaining small lobsters under laboratory conditions, results of this short-term, manipulative field experiment indicate that field-based nurseries represent an economically viable, third option for managers of lobster stock enhancement programs.     

Effects of dietary fish oil replacement with vegetable oils on growth and tissue fatty acid composition of humpback grouper, Cromileptes altivelis (Valenciennes)

The replacement of dietary marine fish oil with vegetable oils was examined in fingerling humpback grouper, Cromileptes altivelis, over the course of an 8‐week growth trial. Five isolipidic (10%) and isoproteic (50%) fish meal‐based practical diets were formulated to contain iso‐ingredients but with different sources of lipids [crude palm oil (CPO), refined, bleached and deodorized, palm olein (RBDPO), soybean oil (SBO) or canola oil (CNO)], and their performance was compared with the control diet, which contained cod liver oil (CLO) as the added lipid source. The experimental diets were fed close to apparent satiation twice a day to triplicate groups of fish (10.6 ± 2.2 g). The grouper fingerlings were randomly distributed into groups of 12 fish in cylindrical cages (61 cm depth and 43 cm diameter) that were placed in a 150 tonne polyethylene seawater tank. There were no significant differences (P>0.05) in terms of growth, survival, feed conversion ratio, protein efficiency ratio, net protein utilization, hepatosomatic index and condition factor among fish fed the various dietary treatments. Similarly, the dietary lipid source did not significantly affect the whole body proximate composition of the fish. Muscle and liver fatty acid composition of fish was influenced by the experimental diets. Replacement of dietary CLO with CPO, RBDPO, SBO or CNO produced fish with lower n‐3 highly unsaturated fatty acids and increased levels of 18:2n‐6 in the muscle and liver. The n‐3:n‐6 fatty acid ratio in the muscle of fish fed the CLO‐based diet was 3.0 compared with 0.5–0.8 in the muscle of fish fed the various vegetable oil‐based diets. The present study demonstrated that various vegetable oils can be used in fish meal‐based dietary formulations for humpback grouper without compromising growth or feed utilization efficiency.     

Influence of size, age and maternal condition on the oocyte dry weight of Argentine hake (Merluccius hubbsi)

Effects of maternal age–size and condition on oocyte dry weight were analysed for Merluccius hubbsi. Samples of mature females were collected in the Patagonian spawning area during 10 research surveys carried out between 2000 and 2005. Dry weight of 50 hydrated oocytes ranged from 0.80 to 1.90 mg. Relationships of this variable with total length and age were best described by a logarithmic model during the spawning peak. At the beginning and at the end of the reproductive season no significant relationship between these variables was observed, but a decrease in egg dry weight was detected during the spawning period. In general, young spawners (≤5-year-old) showed more variability in egg dry weight, which was associated with a reproductive season shorter than that of old females. Relative condition factor did not shows a significant relationship with oocyte dry weight, and mean values of this index were similar for different months of the spawning seasons analysed.     

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg)

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.     

Ontogeny of pepsinogen and gastric proton pump expression in red porgy (Pagrus pagrus): Determination of stomach functionality

The appearance of functionally developed gastric glands is commonly considered as the transition from the larval to the juvenile stage in fish, since it means the switch from the less efficient alkaline digestion to a more efficient acid digestion characteristic of adult specimens. From that moment, fish are supposedly able to better assimilate nutrients from inert diets. Acid digestion takes place by the action of pepsin activity and hydrochloric acid, both secreted by the gastric glands of the stomach. Pepsinogen is the precursor of pepsin which is converted into active enzyme by the action of hydrochloric acid secreted by the proton pump. The goal of this work was to asses the ontogeny of pepsinogen and gastric proton pump expression along larval development of red porgy using RT-PCR and in situ hybridization techniques. Firstly, red porgy specific pepsinogen and proton pump partial sequences were isolated. Amplification products presented 615 and 591 bp and were identified as pepsinogen IIb and the -subunit of the proton pump (H⁺/K⁺-ATPase) by sequencing, respectively. Both sequences were aligned to several predicted pepsinogen and proton pump polypeptides from different vertebrate species showing elevated homologies with them, especially in the case of the proton pump, the identity of which was never less than 90%. Pepsinogen and proton pump expressions were detected from 30 days post-hatching (dph), increasing with development. Proton pump expression was localized in the gastric glands of red porgy larvae as revealed by in situ hybridization, showing increasing signal intensity along the digestive system development. Such results indicated that at 30 dph red porgy starts to acquire the adult digestive capacity and therefore inert diets should be better utilized from that time onwards.     

Pigmentation, carotenoids, lipid peroxides and lipid composition of skin of red porgy (Pagrus pagrus) fed diets supplemented with different astaxanthin sources

A feeding experiment was carried out to determine the efficiency of different commercial sources, chemical forms and levels, of dietary astaxanthin, to appropriately pigment the red porgy (Pagrus pagrus) skin. According to this, total carotenoid content, profiles and chemical forms present in the skin were determined. In order to establish the potential for antioxidant protecting role of astaxanthin supplemented diets, peroxide levels and lipid composition of skin were also determined.

Red porgy alevins were fed six dietary treatments in triplicate; a basal diet (B) without carotenoids; two diets (N25 and N50) formulated to supply either 25 or 50 mg kg^− 1 of an esterified source of astaxanthin (Haematococcus pluvialis, NatuRose™); two diets (CP25 and CP50) with either 25 or 50 mg kg^− 1 of unesterified astaxanthin (Carophyll® Pink); and a positive control diet (B + S) proved as a successful pigmenting-diet in previous experiences (B + S, 88% basal diet:12% frozen shrimp) [Cejas, J., Almansa, E., Tejera, N., Jerez, S., Bolaños, A., Lorenzo, A., 2003. Effect of dietary supplementation with shrimp on skin pigmentation and lipid composition of red porgy (P. pagrus) alevins. Aquaculture 218, 457–469].

All fish fed carotenoid supplemented diets displayed a pink-coloured skin after 4 months of feeding in contrast to the greyish appearance displayed by fish fed the basal diet not supplemented with carotenoids (B). Furthermore, astaxanthin diesters were the major carotenoid in the skin of pink fish. A second carotenoid, tentatively identified as tunaxanthin diester, was also detected. The best results in terms of skin natural reddish hue, total carotenoid and astaxanthin contents were found by using the esterified forms of dietary astaxanthin (N25, N50 and B + S). Interestingly, the lowest levels of lipid peroxides were found in the fish fed these three treatments. However, no effect of treatment on lipid composition was found. In conclusion, red porgy alevins are able to efficiently utilise dietary natural or synthetic astaxanthin, and deposit this pigment in its esterified form to acquire an acceptable pink-coloured skin compared to that of the wild fish.     

On the age and growth of bogue,Boops boops (L.), from the Portuguese coast

Abstract Between 1984 and 1990, a total of 4892 bogues, Boops boops (L.) were caught in Peniche and 4352 in Algarve, with the total length varying from 6 to 40 cm. Age determination was carried out using direct reading and back-calculation on the otoliths. Von Bertalanffy growth equations, based on mean lengths per age group, were established for both samples and using both methods. Applying the Hotelling T² test to the growth parameters of those equations, it was concluded that there were no significant differences between them. This conclusion allowed the validation of the two methods employed, and the combination of the data from Peniche and Algarve into one single age-length key from which a growth equation for the Portuguese coast was calculated and described as: L_t = 45.918 {1-exp [-0.124 (t + 2.628)]}.     

Alternative pathways in the life history of Gracilaria gracilis (Gracilariales, Rhodophyta) from north-eastern Sicily (Italy)

The reproductive phenology of Gracilaria gracilis has been followed closely in a brackish water lake in Sicily. The life history of this red alga deviated from the typical Polysiphonia-type cycle and was characterized by the predominance of the tetrasporophytic phase over gametophytes, in situ development of germinated gametophytes and the presence of sterile pleustophytic thalli. Release of spherical agglomerates of cells produced by young tetrasporophytes was observed as well. Their ability to germinate forming new thalli appears to represent an alternative reproductive strategy of this organism. The population structure appears to reflect a predominance of the diploid phase except during the warmer summer season when only vegetative juveniles were found in the lake. Release and early stages of development of carpospores and tetraspores were critically observed in culture. A Dumontia type pattern of germination was observed for both types of spores, showing an erect thallus arising from a multicellular basal disk. Compared to tetraspores, carpospores are less readily released resulting in a subsequent delay in the development of new gametophytes.