Mean Hatching Rate Research Articles

Nematocidal Activity of Ethanol and Aqueous Extracts of Persea americana Seeds against Heligmosomoides polygyrus using the Worm Microtracker Method.

Infections with gastrointestinal helminths constitute a serious obstacle to the good health of the local population in most African Countries. The aim of this study was to evaluate the anthelminthic activity of Persea americana ethanol and aqueous extracts against Heligmosomoides polygyrus using the worm microtracker. Aqueous and ethanolic extracts of P. americana were prepared. Different concentrations of the extracts were tested against the egg and larvae stages of H. polygyrus using an automated high-throughput method. Briefly, embryonated eggs and larvae of this parasite were obtained after the incubation of fresh eggs at 25°C for 24, 48, and 96 hours for embryonated eggs, L1 and L2 larvae, respectively. One hundred microliters of the plant extracts at various concentrations were put in contact in a 96-well microplate with a suspension of 100 embryonated eggs in a total volume of 200 μL and incubated in a worm microtracker where the motility of the worms was recorded every 30 minutes for the ovicidal activity. The final tested extract concentration was 5, 2.5, 1.25, 0.625, and 0.3125 mg/mL, whereas ringer solution (0.95%) and 1.5% Dimethyl sulfoxide (DMSO) were used as negative controls and levamisole as positive control. The same method was used for larvicidal activities. The anthelmintic activity was determined using the average movement of the worms in the tested product compared with the negative control (1.5% DMSO and ringer solution). The egg hatching rates of H. polygyrus had IC50 of 0.49 mg/mL (95% confidence interval: 71.70-92.03) and 0.22 mg/mL (95% confidence interval: 74.28-86.18) for the ethanol and aqueous extract, respectively. These IC50 indicate that the aqueous extract is more active for the inhibition of hatching at a 95% confidence interval. The aqueous and ethanol extracts presented mean inhibitory hatching rates of 78.33 ± 1.67% and 75.67 ± 1.15% at 5 mg/mL, respectively, with no significant differences. The highest percentage of inhibition of L1 larva was observed at 5 mg/mL with 89 ± 2.3%and 85 ± 2.7% for the ethanol and aqueous extracts, respectively. The lowest percentage of inhibition was observed at 0.3125 mg/mL, with 54.67 ± 3.38% and 49 ± 2.64% for the ethanol and aqueous extract, respectively. No significant differences were observed between the two extracts at 5 mg/mL with an inhibitory percentage of 90.67 ± 3.05% (ethanol) and 89.33 ± 2.08% (aqueous). Extracts of P. americana seeds possess nematocidal activity, however, further in silico and in vivo investigations are necessary to confirm their anthelminthic activity.

Reassessment of the olive ridley sea turtle Lepidochelys olivacea nesting population at Nancite Beach, Costa Rica

AbstractNancite is a fully protected beach within the Guanacaste Conservation Area, Costa Rica where olive ridley (Lepidochelys olivacea) sea turtles nest in synchronous mass‐nesting events known as arribadas. Arribadas decreased in magnitude at this beach by approximately 90% during the period 1971–2007 due to unknown causes. The total count estimate of females nesting in a year also decreased over these decades. In the present study, from August 2009 to February 2021, the trend of arribada nesting female abundance and estimated annual production of hatchlings were assessed and compared with previous trend analyses. A total of 62 mass‐nesting events were quantified in the period 2009–2021, with an estimated annual average of 64,694 nesting females. Trend analysis indicated that during our recent study period, the number of females per arribada event increased by an estimated 14% (8%, 20% CI95%). During this period, a mean hatching rate of 33.4% was estimated, corresponding to an overall estimated production average of 2,165,597 per season, which represented an increase of 82.7% compared with a previous estimate for the period 1980–1984, when the arribadas were larger. We suggest that the growth in hatchling production over the past four decades is at least in part responsible for the slight increase in the estimated size of arribadas since 2009/2010. However, because maximal hatching success did not increase above 60%, the beach may be close to reaching carrying capacity, and it seems that high hatchling production may soon level off or begin to decrease. Thus, it is not clear whether current hatchling production will lead to the recovery of the Nancite population to the historical levels documented in the 1970s and early 1980s.

Gene Editing of the Follicle-Stimulating Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Transcription Activator-like Effector Nuclease Technology with Electroporation

Follicle-stimulating hormone (fsh) plays an important role in sexual maturation in catfish. Knocking out the fsh gene in the fish zygote should suppress the reproduction of channel catfish (Ictalurus punctatus). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the fsh gene were electroporated into fertilized eggs with the standard double electroporation technique. Targeted fsh cleavage efficiency was 63.2% in P1fsh-knockout catfish. Ten of fifteen (66.7%) control pairs spawned, and their eggs had 32.3-74.3% average hatch rates in 2016 and 2017. Without hormone therapy, the spawning rates of P1 mutants ranged from 33.3 to 40.0%, with an average egg hatching rate of 0.75%. After confirmation of the low fertility of P1 mutants in 2016, human chorionic gonadotropin (HCG) hormone therapy improved the spawning rates by 80% for female mutants and 88.9% for male mutants, and the mean hatch rate was 35.0% for F1 embryos, similar to that of the controls (p > 0.05). Polymerase chain reaction (PCR) identification showed no potential TALEN plasmid integration into the P1 channel catfish genome. Neither the P1 nor the F1 mutant fish showed any noticeable changes in in body weight, survival rate, and hatching rate when the reproductive gene was knocked out. F1 families had a mean inheritance rate of 50.3%. The results brought us one step closer to allowing implementation of certain genetic techniques to aquaculture and fisheries management, while essentially eliminating the potential environment risk posed by transgenic, hybrid, and exotic fish as well as domestic fish.

Rearing performance of black soldier fly (Hermetia illucens) on municipal biowaste in the outdoor ambient weather conditions of Pakistan and Indonesia.

The availability and continuous supply of black soldier fly larvae (BSFL) is crucial for efficient operation of a BSF biowaste recycling facility. Its rearing performance was for the first time investigated in Pakistan under outdoor ambient weather conditions. Comparison of the findings with the BSF rearing performance of Indonesia's facility highlights the life stages needing special attention. In Pakistan, mean BSF emergence, hatching and survival rate of 58.8% (SD 15.2), 44.5% (SD 21.8) and 91.4% (SD 1.68) were achieved respectively. A positive significant correlation was found between the number of emerged flies and prepupae (R = 0.75) and the number of eggs produced and hatched (R = 0.92). On average, BSF took 49.5 days (SD 3.20) to complete one life cycle (LC) under ambient temperature and relative humidity (RH) between (22-35 °C) and (24.7-89.3%) respectively. The mean duration of eclosion, pre-oviposition, egg hatching, larval feeding and pupation was 15.6 days (SD 1.6), 3.5 days (SD 0.5), 3 days (SD 0.6), 22 days (SD 2.5), 3.8 days (SD 1.2) respectively. In Pakistan, the LC duration was longer with a smaller number of eggs/fly, lower BSF emergence and hatching rates as compared to Indonesia. BSF tolerated the semiarid weather conditions of Pakistan, successfully developed into all instars and completed all LCs under observation. It is suggested to provide controlled environmental conditions at the nursery stage to improve BSFL rearing performance for sustainable biowaste management.

Breeding Biology of the Endangered Burrow-Nesting Lesser Crescent-Chested Puffbird Malacoptila minor

For many birds breeding success may be related to the availability of nesting sites, particularly in ecologically specialized cavity-nesting birds. For burrow-nesting species, the selection of an appropriate substrate may be fundamental to a successful breeding attempt. The Lesser Crescent-chested Puffbird Malacoptila minor is a bucconid with a restricted distribution, which is found in the states of Maranhão and Piauí, in northeastern Brazil. It is the only bucconid listed as threatened with extinction, and little is known about its natural history. A total of 49 nests were found over three breeding seasons (2017–2020). Nests consist of burrows dug in the ground that are used only once for breeding. Incubation lasted 21.2 ± 1.3 days and the nestlings fledged after 19.5 ± 2.9 days. The nests contained two or three eggs. The mean hatching rate was 0.46 ± 0.47 eggs per nest and the fecundity rate was 1.0 fledgling per female. The production was 0.53 fledglings per nest, apparent reproductive success was 22.4% (N = 49), and was 15.7% (N = 49) when based on Mayfield's protocol. The probability of breeding success per period was greater during the incubation phase than during the nestling phase. Predation was identified as the principal cause of nest failure (77.5%), mainly during the incubation phase. Three predators were identified: the Crab-eating Fox Cerdocyon thous, the fire ant Solenopsis saevissima, and a lizard, the Common Tegu Salvator merianae. Breeding season starts with the onset of rainy season and nest flooding did not appear to be a concern, which contrasts with the available data on other bucconids. The results of the present study are the first data on the breeding biology of the Lesser Crescent-chested Puffbird, and represent an important advance in the understanding of breeding patterns in the Bucconidae.

Nesting activity of the Loggerhead sea turtle, Caretta caretta, in Calabria: 2016-2020 reproductive seasons

The nesting activity of the loggerhead turtle along the coast of Calabria during five reproductive seasons (2016-2020) is presented. From May to August, survey methods were conducted using traditional observations on foot and utilizing additional innovative technologies, including electric fat bikes and drones. Monitoring was intensively focused on a key nesting area of approximately 40 km located on the southernmost Ionian coast of Reggio Calabria, and, for only the 2020 season, the area was extended an additional 40 km northeast. In the five nesting seasons, 419 female emergences were recorded, 192 of which were classified as nests. Overall, 65.1% (n = 125) of nests were found along the area regularly monitored in all nesting seasons. The maximum nesting activity was observed during July, and the mean nesting success was 45.8%. The average number of eggs for all seasons was 92.2 (n = 163; S.D.: ± 21.1; range: 39-160). The mean hatching and emergence success rates were 70.8% and 62.5%, respectively. The mean incubation period was 49 days (n = 122; S.D.: ± 5.4; range: 42-82). Considering previous data from the same area, our study shows an actual increase in nesting activity, probably due to the implementation of an effective monitoring method that includes the use of new technologies, which made it possible to expand the regularly monitored area, and a greater awareness campaign in recent years. Regarding the reproductive parameters, our data is similar to that reported for other Mediterranean nesting grounds.

Producing flat-head grey mullet Mugil cephalus (Linnaeus, 1758) fries in captivity from sexually mature adults collected in Sardinian lagoons

Mugil cephalus has been identified as a suitable species for feeding populations in developing countries, and it represents a traditionally harvested and consumed species in various European countries. A decline in wild populations and a growing demand have promoted new interest in mullet production in hatcheries, considered imperative for the production of a steady supply of fry and the expansion of M. cephalus aquaculture. In the present study, wild adults collected in Sardinian lagoons were induced to spawn during their natural reproductive period by treating them with a single dose of a slow release GnRHa preparation (200µgkgbw-1). In 2016, a protocol for reducing stress in capturing, transporting and selecting wild adults was set up, and 64% of the females with an oocyte diameter larger than 550µm were successfully induced to spawn. Five spawning induction trials were carried out in the period 2016–2019. The mean fertilization rate and the mean hatching rate obtained were 83±14% and 76±13%, respectively. The individuals obtained were reared indoor in a recirculating aquaculture system (RAS) at a density of 40 individuals L-1 for 60 days, and their survival ranged from 10±2 to 19±1%. During the whole larval rearing phase, from 0 to 34 days post hatching (dph), the mean standard growth rates recorded were 5.0±0.7% in total length and 14.4±3.9% in body weight. The rearing density used resulted in an average survival rate comparable or even better than those obtained by other authors, thus indicating the possibility of producing M. cephalus fries in captivity from sexually mature adults captured in Sardinian lagoons, reducing the costs and optimizing the production.

EMBRYO-LARVAL TOXICITY OF ANTI-FUNGUS CHEMICALS USED AS PROPHYLACTIC AGENTS ON CLARIAS GARIEPINUS (BURCHELL, 1822)

Embryo-larval toxicity test of selected anti-fungus chemicals used as prophylactic agents on the African catfish (Clarias gariepinus) was conducted to assess the comparative efficacy as well as the toxicity of the different chemicals. Measured endpoints included hatching, embryo and larval survival, and larval growth. Iodine (100 mg L-1), hydrogen peroxide (250 mg L-1), malachite green (5 mg L-1) and formalin (250 mg L-1) were used as prophylactic agents. The eggs were spread on an incubating raft and continuously dipped in the treatment chemical for 15 minutes. After 48 hours, the mean hatch rate was highest in formalin-treated embryos (70%). Iodine treatment resulted in a hatch rate of 36.7%, which was slightly less than half that of the formalin treatment. Both hydrogen peroxide and malachite green resulted in 60.0% hatch rates. The results indicate that formalin-treated embryos performed significantly better than all the other chemicals. The highest percentage survival rate of 63.3% was observed in formalin at 72hph and 56.7% after 168hph. The lowest survival rate (10%) was observed in the iodine treatment group and closely followed by the group treated with malachite green (16.7%). Except for malachite green, which inhibited growth, the prophylactic treatments did not affect the growth performance of fry. This study presents evidence that can be used to support the use of formalin and hydrogen peroxide as prophylactic treatments in the early stages of C. gariepinus, although caution should be exercised due to the possibility of toxicity at higher concentrations and for longer periods of exposure.

Removal of Free‐Living Ciliates from Stock Cultures of Two Calanoid Copepods with Sodium Hypochlorite

AbstractEggs of Acartia tonsa and Parvocalanus crassirostris were contaminated with ciliates (Euplotes sp.) and exposed to five concentrations of sodium hypochlorite (25, 55, 105, 205, and 405 mg/L) for durations of 15 s, 5 min, 15 min, or 30 min to assess the efficacy of household bleach for removing the ciliates. Eggs from each exposure treatment were incubated for 40 h (A. tonsa) or 12 h (P. crassirostris) to assess hatch. The presence of living ciliates was evaluated after incubation, and the egg hatch rate (%) for the copepods was estimated for each treatment and in controls. Exposure to 105 mg/L total chlorine for 15 s killed all of the ciliates and reduced the copepod hatch rate by less than 26% with respect to controls in both species. Exposure to 55 mg/L for 15 min or 25 mg/L for 30 min also killed the ciliates and achieved a mean copepod hatch rate of 38.5% and 58.1% of the control‐group hatch rate, respectively, in A. tonsa. Treatments for 5 min or longer reduced the hatch rate for P. crassirostris eggs by at least 76% at all of the concentrations that we tested.

Comparative study of the breeding performances of five populations of Nile tilapia (Oreochromis niloticus) (F1) in an experimental ongrowing system in Benin (West Africa)

ABSTRACT In order to investigate the breeding performances of five F1 populations of Oreochromis niloticus originated from wild broodstock of Nangbéto, Sohoumè and Togbadji in the Mono basin, Gobé in the Ouémé basin and Gbassa in the Niger basin, breeding tests were conducted for 10 weeks in an ongrowing experimental system. All tested populations were checked daily in order to collect spawn and to compare the reproductive performances. Compared with others, Sohoumè, Togbadji, and Gobé populations significantly (P < .05) displayed the best breeding performances in terms of rate of multi-spawning females (89.3 ± 15.2–92.3 ± 10.8%); mean relative fecundity (4 ± 2–6 ± 3 eggs/g of female), mean gonadosomatic index (3.1 ± 2–4.8% ± 2.6); mean absolute fecundity rates (865 ± 339–989 ± 547 eggs); mean fertilization rates (98 ± 4.3–99.3 ± 1.1%); and mean hatching rates (83.7 ± 10–88.9 ± 9.1%).

Low sperm to egg ratio required for successful in vitro fertilization in a pair-spawning teleost, Senegalese sole (Solea senegalensis).

Cultured Senegalese sole (Solea senegalensis) breeders fail to spawn fertilized eggs. The implantation of large-scale in vitro fertilization protocols, to solve this problem, has been frustrated by low production of poor quality sperm. Cultured females were induced to ovulate with a 5 µg kg−1 single injection of gonadotropin-releasing hormone agonist (GnRHa) and viable eggs (82.6 ± 9.2% fertilization) were stripped 41:57 ± 1:46 h after the injection. Sperm was collected from cultured males, diluted in modified Leibovitz and used fresh to fertilize the eggs. Males were not treated with hormones. A nonlinear regression, an exponential rise to a maximum (R = 0.93, p < 0.0001) described the number of motile spermatozoa required to fertilize a viable egg and 1617 motile spermatozoa were sufficient to fertilize 99 ± 12% (±95% CI) of viable eggs. Similar, spermatozoa egg−1 ratios of 592 ± 611 motile spermatozoa egg−1 were used in large-scale in vitro fertilizations (190 512 ± 38 471 eggs). The sperm from a single male (145 ± 50 µl or 8.0 ± 6.8 × 108 spermatozoa) was used to fertilize the eggs. The mean hatching rate was 70 ± 14% to provide 131 540 ± 34 448 larvae per fertilization. The viability of unfertilized eggs stored at room temperature decreased gradually, and the sooner eggs were fertilized after stripping, the higher the viability of the eggs. The collection of sperm directly into a syringe containing modified Leibovitz significantly increased the percentage of motile spermatozoa (33.4 ± 12.2%) compared with other collection methods. The spz egg−1 ratios for Senegalese sole were at the lower end of ratios required for fish. Senegalese sole have a pair-spawning reproductive behaviour characterized by gamete fertilization in close proximity with no sperm competition. The provision of a large-scale in vitro fertilization protocol (200 µl of sperm per 100 ml of eggs) will enable the industry to operate sustainably and implement breeding programmes to improve production.

Incubation Temperature Influences the Duration of Egg Hatching and Early Development in the Boreal Digging Frog, Kaloula Borealis

This study investigated the boreal digging frog, Kaloula borealis, to determine the egg hatching period and whether the hatching period is affected by incubation temperature. The results of this study showed that all the eggs hatched within 48 h after spawning, with 28.1% (±10.8, n=52) hatching within 24 h and 99.9% (±0.23, n=49) within 48 h after spawning. A significant difference was noted in the mean hatching proportion of tadpoles at different water temperatures. The mean hatching rates between 15 and 24 h after spawning was higher at a water temperature of 21.1 (±0.2) °C than at 24.1 (±0.2) °C. These results suggest that incubation temperature affected the early life stages of the boreal digging frog, since they spawn in ponds or puddles that form during the rainy season.

Induced Spawning of Sahar, Tor putitora (Hamilton-Buchanan) in Terai Region of Nepal

Sahar, Tor putitora (Hamilton-Buchanan) is a high valued indigenous riverine species of Nepal. This is declining in its natural habitat. Tor putitora and Tor tor (Hamilton-Buchanan) have been recommended as vulnerable and endangered species. Limited seed production using natural propagation has restricted its expansion in culture as well as rehabilitation in natural waters. The artificial propagation of sahar using synthetic hormone was conducted in Pokhara, AFU, Chitwan and CARP), Kathar during February to April 2017. Sixty five male (0.5-1.8 kg) and forty five female (1.1-2.1 kg) brood fish were reared in 200 m2 earthen ponds at 1000 kg/ha. Fish were fed with 32% crude protein feed at 3% body weight per day. Maturity was observed regularly for softness of the abdomen. Female brood fish with a soft and extended abdomen were injected with synthetic hormone (Ovaprim) at 0.5 ml/kg body weight. Males were not injected hormone. The fertilized eggs were incubated in Atkin hatching trays. A total of 16 females were induced to spawn, and they produced 1630.80±184.30 (mean±SE) eggs per kg body weight. Mean hatching and larval survival rates were 78.4±1.9 and 74.7±1.1%, respectively.

Influence of Substrate Age on Oviposition Behavior of a Specialist Carrion Colonizer, Cochliomyia macellaria (Diptera: Calliphoridae).

The location and consumption of carrion by arthropods is a process that can be potentially distinguished temporally based on the makeup of the associated community. In fact, succession on carrion is a continuum of different generalist and specialist arthropods entering and leaving the system. Blow flies commonly associated with vertebrate remains are considered specialists due to their reliance on carrion as a source of food for offspring and protein for females. However, this specialization may come at a price; increased competition for resources and greater risk of local extinction. The present study examined the effects of the presence or absence of intraspecific colonization, carcass age, and exposure time on the colonization and oviposition responses of the specialist, primary colonizer, the secondary screw worm, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae). Carcass age, exposure time, and colonization status significantly influenced the ovipositional response of C. macellaria. This species exhibited an oviposition preference for aged carcasses, with a tendency for higher oviposition after 8-h exposure time, but no preference between previously colonized or uncolonized carcasses. Mean egg hatch rate was also shown to be influenced by the aforementioned factors, with mean hatch rates varying between 81.26 and 90.97% across various treatments. These results provide insight into mechanisms driving succession on carrion, as well as highlight the variation observed in successional studies for the targeted species. Investigators relying on arthropod succession to indicate a time of colonization should proceed with caution in relying solely on the assumption primary colonizers only arrive and colonize fresh carrion.

English

This study documented some aspects of breeding biology of Red Wattled Lapwing (Vanellus indicus) such as breeding season, nest structure, clutch size, incubation period, hatching and fledgling success in Bahawalpur District. We selected 6 sites for the observation; barren, open, cultivated, grassy, area along road sides, and constructed areas with the common vegetation of Cynodon dactylon, Ziziphus mauritiana, Albizia procera, Cincrus ciliarus, Cincrus biflorus, Arva jawanica, Eucalyptus cameldulensis, Acacia nilotica, Prosopis juliflora and Conocarpus spp. During the breeding season, the male selects territory. Breeding season starts from April to June. Both male and female participate in nest formation, incubation and other parental duties. Clutch size was mostly 4 and the mean of the clutch size was 4&plusmn;0.0 (range 0-4). The incubation period of the eggs was 25-28 days and the average of the incubation period was 27.1&plusmn;0.4. Total eggs were 24 in 6 nests, out of which 22 were hatched. The percentage of hatched eggs was 91% and the mean hatching rate was 91.6&plusmn;5.2 per clutch. Fledgling success was 79.1&plusmn;7.6 and the percentage of the fledging was 79%. Predation rate in red wattled lapwing was also observed; it was very high due to anthropogenic activities. &nbsp; Key words: Red wattled lapwing, breeding biology, Vanellus indicus, Southern Punjab.

Spawning induction and seed production of Eastern little tuna, Euthynnus affinis (Cantor, 1849), in the post- and pre-spawning seasons by hormonal treatment in a semi-closed recirculation system with elevated temperature

We previously established a method for spawning induction in Eastern little tuna (ELT) Euthynnus affinis (Cantor, 1849) by administering a gonadotropin-releasing hormone analog (GnRHa) during the natural spawning season in Japan (August–October). In order to establish seed production of ELT in the off-spawning season, we first conducted three spawning induction trials by GnRHa administration from October 2011 to January 2012 using ELT broodstock (2 years old; three females and four males) maintained in a 10-m3 tank with a semi-closed recirculation system and static elevated temperature. Average water temperature and daily egg production in three trials lasting 11–15 days were 27.0 ± 0.09°C and 268 173 eggs (Trial 1), 27.0 ± 0.11°C and 277 9098 eggs (Trial 2), and 25.5 ± 0.39°C and 291 113 eggs (Trial 3) respectively. Mean fertilization rate and mean hatching rate were 70.4% and 60.5% (Trial 1), 83.9% and 79.6% (Trial 2), and 62.5% and 57.4% (Trial 3) respectively. We also succeeded in producing ELT larvae in the pre-spawning season (April–July), although the quantity and quality of larvae produced were inferior to those produced in other calendar months. In trials involving periodic GnRHa administration during the off-spawning seasons, hatched larvae were obtained in the 10-m3 tank after six of nine administrations in the 2011–2012 off-spawning season and in 16 of 19 administrations in the 2012–2013 off-spawning season. The findings of this study demonstrated that hormonal treatment and thermal control could be used to extend the spawning period in ELT, potentially allowing larval production in the post- and pre-spawning seasons.

Removal of the adhesive gum layer surrounding naturally fertilised ballan wrasse (Labrus bergylta) eggs

Commercial production of ballan wrasse (Labrus bergylta) as a cleaner fish for the removal of sea lice (Lepeophtheirus salmonis) from farmed salmonids (Salmo salar) has increased due to its proven efficiency. One bottleneck in commercial hatchery production is working with the benthic adhesive eggs, which makes disinfection and incubation of eggs challenging; therefore, this study aimed to find a chemical or enzymatic treatment and process to remove the adhesive gum layer. Naturally spawned eggs were collected from artificial spawning substrates up to 24h post spawning from wild caught broodstock kept in captivity at the Marine Harvest, Machrihanish facility. Four treatments were tested: tannic acid (0.2, 0.1, and 0.05%), sodium sulfite (2, 1, and 0.5%), l-cysteine (2, and 1%), and enzyme alcalase® (4.0, 3.0, 2.0, 1.0, and 0.5%) in vitro. Eggs were exposed for 25min while being continually agitated, and the proportion of “degummed” eggs was counted at the end of each time period. Enzyme alcalase® was the only treatment that proved successful in degumming eggs, with the time to complete degumming (≥96%) inversely related to enzyme concentration. Complete degumming occurred between 15 and 30min for all enzyme alcalase® dose rates. Mean hatch rates for eggs treated with enzyme alcalase ® were not compromised by the treatment and in the highest dose tested were actually found to be higher in treated eggs (78.9±2.4%) than controls (71.3±3.3%). The use of enzyme alcalase® has proven effective in degumming ballan wrasse eggs without affecting hatch rates. However, translation of this method to in situ degumming and thus removal of eggs from spawning substrate on farm remains to be standardised. Statement of relevanceIncreasing efficiency for commercial production of ballan wrasse.

Assessment of nutritional supplementation in phospholipids on the reproductive performance of zebrafish, Danio rerio (Hamilton, 1822)

The objectives of this study were to determine the effects of a tailor made purified diet (PUR) supplemented with phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the reproductive performance, gamete quality, embryo development and growth as well as skeletal malformations of the larvae in zebrafish, Danio rerio. The PUR diet was tested against a commercial diet as a negative control (CD). For each diet, one experimental group of eight adult zebrafish (three males, five females) was maintained in 3-L tanks in a water recirculating system under continuously controlled conditions (photoperiod 14 h light: 10 h dark; water flow rate 7.3 L h−1; 28°C; pH 7.5; 700 μS). Two trials were performed to confirm the results. The CD treatment resulted in the highest final weight of the breeders (0.60 ± 0.18 g); however, the fish failed to spawn. The PC supplemented diet promoted a reasonable final weight of the breeders (0.48 ± 0.10 g) similar to the CD and with a mean hatching rate of 93.80 ± 6.9%. Nevertheless, the PC treatment induced the highest rate of skeletal malformations in progeny (82.40 ± 4.1%), which could be related to the low larval survival at 28 days post-fertilization (dpf) (48.00 ± 11.3%). In comparison to the control groups, broodstock fed the PE diet showed the best results in sperm quality, which was revealed by higher total and progressive motility and higher velocities than sperm from males fed the CD and PUR diets. Furthermore, the PE group produced significantly higher egg diameters (1.20 ± 0.05 mm) when compared to the PUR diet (1.14 ± 0.03 mm). This study highlights the importance of phospholipids in zebrafish gamete quality, and that supplying PE in broodstock diets can improve the reproductive performance of zebrafish.

Biological and genetic characteristics of Glyptotendipes tokunagai (Diptera: Chironomidae) on the basis of successive rearing of forty-two generations over seven years under laboratory conditions.

Members of the nonbiting midge family Chironomidae have been used worldwide as water-quality indicators or toxicity test organisms. The purpose of this study was to establish the chironomid Glyptotendipes tokunagai Sasa as a new test species by conducting successive rearing under laboratory conditions. We monitored biological and genetic aspects of >42 successive generations over 7 yr, and also compared the development of the 39th generation with the fourth generation under five constant temperatures of 15, 20, 25, 30, and 35°C. We observed that the number of eggs in an egg mass and the adult body sizes decreased rapidly in the early generations, and thereafter tended to stabilize from the fifth generation to the 42nd generation. In all generations, the mean hatching rate was >75%. Males were predominant in the early generations, but the sex ratio increased to 0.5 (ranged 0.24-0.61) in later generations. The genetic divergence of the reared generations, analyzed by using the mitochondrial cytochrome c oxidase subunit I gene, decreased from 0.0049 to 0.0004 as the generations progressed. In comparison with the fourth generation, the mortality and developmental time of the 39th generation were generally greater, and the adult body sizes were generally smaller. The estimated low developmental threshold temperatures of eggs, male larvae to male adults, and female larvae to female adults were 9.6, 11.3, and 9.7°C, respectively. The optimal rearing temperature was determined to be 25°C. This is the first record of domesticated rearing of a wild chironomid species under laboratory conditions for >7 yr.

Olive Ridley Mass Nesting Ecology and Egg Harvest at Ostional Beach, Costa Rica

The olive ridley sea turtle (Lepidochelys olivacea), a species listed by the International Union for Conservation of Nature as vulnerable, is characterized by its mass-nesting behavior, also known as arribada. For decades, this phenomenon has facilitated the utilization of this species by coastal communities, much of it illegally. At Ostional beach, Costa Rica, a community-based egg-harvest program operates legally to generate important income for the community while promoting the protection of the ridley nesting assemblage. However, to date, no analysis exists that indicates the long-term viability of the egg harvest program as a sound management tool. To address this void, we generated baseline abundance data of the major arribada events that occurred in the period 2006–2010, as well as egg harvest data, along with preliminary hatching success. Arribadas ranged between 3564 and 476,550 egg-laying females, which indicated a large variability in the magnitude of the mass nesting events. Estimated mean egg harvest was 4746.4 and ranged from 1527 to 8138 total clutches. In relation to the estimated number of clutches laid, the estimated mean of clutches harvested was 21.2%, ranging from 1.5% to 102.4%. Estimated monthly mean hatching rates ranged from 0.0% to 32.6%. It is not clear whether arribadas underwent a significant change in abundance during the study period, although the number of years covered is too short to establish a long-term trend. However, when compared with historical data, the population appears to have declined. Based on our data, we present various management recommendations aimed at increasing hatching rates.