Swim Bladder Inflation Research Articles

Effects of photoperiod and night-time aeration rate on swim bladder inflation and survival in Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel), larvae

Success of swim bladder inflation (SBI) is crucial for early survival of Pacific bluefin tuna (PBF) larvae, because it reduces larval sinking death by enhancing buoyancy. In Experiment 1, we examined the effect of photoperiod on SBI and survival in PBF larvae by comparing photoperiods of 9L: 15D (9L), 14L: 10D (14L: natural photoperiod), 19L: 5D (19L) and 24L: 0D (24L) during 2–10 days post hatch (dph). In Experiment 2, the combined effects of photoperiod (24L and 14L) and nighttime aeration rate (enhanced night-time aeration: ENA of 1300 mL min−1 as a countermeasure for sinking death and 130 mL min−1) on the survival and SBI were also examined during 2–10 dph. Moreover, in Experiment 3 the effect of photoperiod on vertical distribution of larvae in night-time was examined on 3–5 dph. Photoperiod of 24L in Experiment 1 significantly inhibited SBI compared with 14L and 19L; nevertheless, it significantly improved survival compared with other photoperiods with a dark period. On the other hand, the shortened light period (9L) showed significantly reduced SBI and also survival. In Experiment 2, the countermeasure for sinking death of ENA under 24L did not further improve the survival; rather it tended to reduce the survival. In Experiment 3, larvae distributed less in the bottom layer in 24L than in 14L, suggesting the reducing effect of 24L on sinking death. The results indicate that 24L without ENA is suitable for survival which is the most serious problem in PBF larviculture.

Zebrafish atoh8 mutants do not recapitulate morpholino phenotypes

Atoh8 is a bHLH transcription factor expressed in pancreas, skeletal muscle, the nervous system, and cardiovascular tissues during embryological development. Although it has been implicated in the regulation of pancreatic and endothelial cell differentiation, the phenotypic consequences of Atoh8 loss are uncertain. Conclusions from knockout studies in the mouse differ widely depending on the targeting strategy used, while atoh8 knockdown by interfering morpholino oligonucleotides (morpholinos) in zebrafish has led to a range of developmental defects. This study characterised zebrafish embryos homozygous for atoh8sa1465, a loss-of-function allele of atoh8, in order to provide genetic evidence for the developmental role of Atoh8 in this species. Embryos homozygous for atoh8sa1465 present normal body morphology, swimbladder inflation, and heart looping, and survive to adulthood. These embryos do not develop pericardial oedema by 72 hpf and are not sensitised to the loss of Fog1 protein, suggesting that this previously described abnormality is not a specific phenotype. Vascular patterning and primitive haematopoiesis are unaffected in atoh8sa1465/sa1465 mutant embryos. Together, the data suggest that Atoh8 is dispensible for zebrafish development under standard laboratory conditions.

Swim bladder inflation as a possible cause of saddleback-like syndrome malformation in hatchery-reared red spotted grouper Epinephelus akaara

Saddleback-like syndrome is one of the most frequently observed malformations in hatchery-reared red spotted grouper Epinephelus akaara. Saddleback-like syndrome is characterized by a V-shaped dorsal-profile depression behind the head region accompanied by deformities or misalignments of the first to fourth neural spines on the vertebral column and the first to fourth pterygiophores of the dorsal fin. Abnormal swim bladder inflation in several finfish species leads to malformations, particularly of the vertebral column. The present study examined the influence of swim bladder inflation on saddleback-like syndrome in hatchery-reared red spotted grouper. The incidence of saddleback-like syndrome increased as swim bladder inflation rate increased. Additionally, the incidence of saddleback-like syndrome among juveniles with inflated swim bladders was significantly higher than that among juveniles with uninflated swim bladders. The larval swim bladder is located just below the first to fifth vertebrae and the first to fifth dorsal spines, where saddleback-like syndrome occurs. Our results suggest that an inflated swim bladder increases the risk of saddleback-like syndrome in hatchery-reared red spotted grouper.

Lethal and sublethal effects of aniline and chlorinated anilines on zebrafish embryos and larvae.

Environmental risk assessments show increased attention to the sublethal effects of chemicals on aquatic organisms. The Organization for Economic Cooperation and Development (OECD) established the "Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages" (OECD test 212) to predict lethal effects. It is still unclear, however, whether this test can predict sublethal effects. Although their sublethal effects are still unknown, chlorinated anilines are widely used in various fields. The purpose of this study, therefore, is to investigate sublethal effects of chlorinated anilines using OECD test 212 with zebrafish, and to examine the correlation of several sublethal effects between embryo and larval stages. Embryos were exposed to aniline and nine chlorinated anilines until 8 days post-fertilization. A delayed lethal effect was observed from three of the 10 anilines tested. In the control group, the swim bladder inflated after hatching, but there was no swim-bladder inflation after exposure to the chlorinated anilines. Fertilized eggs exposed to lower concentrations of test chemicals showed effects during embryogenesis that did not affect mortality rates, such as changes in body curvature and edema. Our results show that chlorinated anilines induce not only lethal effects but also a variety of sublethal effects. Moreover, a detailed estimate of these effects requires study during both embryonic and larval stages. OECD test 212 may therefore prove useful as a method for screening chemicals for lethal and sublethal effects. Copyright © 2017 John Wiley & Sons, Ltd.

Comparative Developmental Toxicity of Desalination Brine and Sulfate-Dominated Saltwater in a Euryhaline Fish.

Desalination is a promising sustainable solution to meet growing water needs of cities across the United States. However, the environmental impacts of the resulting filtrate (brine) discharged to surface water need to be evaluated before large-scale desalination can be successful in the United States. Developing fish are especially sensitive to changes in salinity and varying ionic composition. Limited research is available on the impacts of hypersalinity on chronic vertebrate embryonic development, particularly on sublethal effects. To investigate this, Japanese medaka (Oryzias latipes) embryos were treated with: (1) graphite filtered freshwater; (2) artificial seawater [17, 35, 42, 56, and 70parts per thousand (ppt)]; (3) effluent from a desalination facility at Monterey Bay Aquarium, CA, diluted to 75, 50, and 25% with 35ppt artificial seawater to simulate mixing (39, 42, 46, and 50ppt); (4) artificial San Joaquin River water (CA, USA) (9, 13, and 17ppt); and (5) artificial San Joaquin River water diluted to 75, 50, and 25% with artificial seawater to simulate estuarine mixing in the San Francisco Bay (13, 19, 24, and 30ppt). Percent hatch, survival post hatch, deformities, swim bladder inflation, and median day to hatch were recorded to calculate EC50 (50% effect concentration) and NOEC (no observable effect concentration) values. No significant difference was observed between artificial seawater and Monterey Bay aquarium effluent (EC50=45-55ppt). However, San Joaquin River water decreased survival post hatch and increased deformities in comparison to artificial seawater and San Joaquin River water mixed with seawater, suggesting that unique ion compositions may play a role in embryo and larval toxicity.

P.6.c.006 - Evolution of the composition of cannabis in Spain

The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6–7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.

Physiological and behavioural impacts of Pacific ciguatoxin-1 (P-CTX-1) on marine medaka (Oryzias melastigma)

Ciguatoxins (CTXs) are natural biotoxins produced by benthic dinoflagellates of the genus Gambierdiscus, which are bioaccumulated and biotransformed along food chains in coral ecosystems. They are neurotoxins that activate voltage-gated sodium channels and disrupt ion conductance in the excitable tissues. Pacific ciguatoxin-1 (P-CTX-1) is the most prevalent and potent CTX congener present in fishes from the Pacific Ocean. In this study, P-CTX-1 was administrated to larval marine medaka (2h post-hatch) via microinjection. Exposure to P-CTX-1 at sub-ppb levels led to adverse behavioural changes, altered physiological performances and reduced survivability of the larval marine medaka as early as 24h after exposure. P-CTX-1 decreased the rate of heartbeat and locomotion of the exposed larvae, probably owing to a series of physiological processes and morphological changes such as pericardial oedema, failure of swim bladder inflation and spinal curvature. The exposed larval marine medaka also demonstrated reduced, delayed and paralyzed responses to external stimulations. This may render them more susceptible to predation. P-CTX-1 could be effectively distributed from the yolk sac to all parts of the fish body, including head and trunk, 24h after exposure. Repeated low-dose P-CTX-1 exposure resulted in larval mortality comparable to that of a single high-dose exposure.

Mechanistic Evaluation of Benzo[a]pyrene's Developmental Toxicities Mediated by Reduced Cyp19a1b Activity.

Benzo[a]pyrene (BaP) is a ubiquitous environmental contaminant that is both an endocrine disruptor and a carcinogen. Aromatase (CYP19) is a key enzyme in steroidogenesis that is responsible for conversion of androgens to estrogens and thus plays a key role in steroid homeostasis. We hypothesized that some of the adverse outcomes of early developmental exposure to BaP are the result of reduced Cyp19a1b activity. Our goal was to investigate the role of estrogen homeostasis during early development and determine the role of aromatase inhibition as a relevant mechanism in BaP's developmental toxicities. One-cell zebrafish embryos were injected with a Cyp19a1b-morpholino (MO) or control-MO. Other non-injected embryos were exposed to waterborne BaP, fadrozole (a Cyp19 inhibitor), estradiol (E2), BaP + E2, Cyp19a1b MO + E2, or fadrozole + E2 for 96 hours post-fertilization (hpf). Adverse outcomes were compared between treatments, and the ability of E2 co-exposure to rescue each observed dysmorphology was assessed. BaP significantly decreased cyp19a1b gene expression in 96 hpf zebrafish larvae homogenates. Concentrations of E2 in 48 hpf larvae were significantly decreased by BaP, fadrozole and Cyp19a1b-MO. Cumulative mortality of zebrafish larvae was significantly increased following BaP or fadrozole exposure or Cyp19a1b knockdown compared to controls. E2 co-treatment rescued mortality caused by 10 μg/L BaP, 10 μg/L fadrozole, or Cyp19a1b-MO. In a treatment-blinded morphological assessment of larvae at 96 hpf, several phenotypes were negatively impacted by BaP, fadrozole, or Cyp19a1b knockdown and rescued by exogenous E2 co-treatment; these included body length, optic vesicle size, swim bladder inflation, pericardial and abdominal edema, and incidence of normal larval tail shape. Abnormal pectoral fins were caused by BaP exposure only. Uninflated swim bladders were caused by all treatments including E2 alone. Our results indicate that certain BaP-mediated adverse developmental outcomes were mechanistically in accordance with BaP-mediated Cyp19a1b inhibition.

Developmental toxicity from exposure to various forms of mercury compounds in medaka fish (Oryzias latipes) embryos.

This study examined developmental toxicity of different mercury compounds, including some used in traditional medicines. Medaka (Oryzias latipes) embryos were exposed to 0.001–10 µM concentrations of MeHg, HgCl2, α-HgS (Zhu Sha), and β-HgS (Zuotai) from stage 10 (6–7 hpf) to 10 days post fertilization (dpf). Of the forms of mercury in this study, the organic form (MeHg) proved the most toxic followed by inorganic mercury (HgCl2), both producing embryo developmental toxicity. Altered phenotypes included pericardial edema with elongated or tube heart, reduction of eye pigmentation, and failure of swim bladder inflation. Both α-HgS and β-HgS were less toxic than MeHg and HgCl2. Total RNA was extracted from survivors three days after exposure to MeHg (0.1 µM), HgCl2 (1 µM), α-HgS (10 µM), or β-HgS (10 µM) to examine toxicity-related gene expression. MeHg and HgCl2 markedly induced metallothionein (MT) and heme oxygenase-1 (Ho-1), while α-HgS and β-HgS failed to induce either gene. Chemical forms of mercury compounds proved to be a major determinant in their developmental toxicity.

Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility.

Iodothyronine deiodinases are selenocysteine-containing enzymes that activate or inactivate thyroid hormones (THs). Deiodinase type 2 (Dio2) catalyzes the conversion of the prohormone T4 into the transcriptionally active T3 and is the predominant activating deiodinase in zebrafish. Using zinc finger nucleases, we generated two different dio2(-/-) mutant zebrafish lines to investigate the physiological function of this TH activator. The first line contains a deletion of 9 bp, resulting in an in-frame elimination of three conserved amino acids. The other line is characterized by an insertion of 4 bp, leading to the introduction of a premature stop-codon. Both lines completely lack Dio2 activity, resulting in a strong reduction of T3 abundancy in all tissues tested. Early development is clearly perturbed in these animals, as shown by a diverse set of morphometric parameters, defects in swim bladder inflation, and disturbed locomotor activity tested between 1 and 7 days after fertilization. Permanent Dio2 deficiency also provokes long-term effects because growth and especially fertility are severely hampered. Possible compensatory mechanisms were investigated in adult dio2(-/-) mutants, revealing a down-regulation of the inactivating deiodinase Dio3 and TH receptor transcript levels. As the first nonmammalian model with permanent Dio2 deficiency, these mutant zebrafish lines provide evidence that Dio2 is essential to assure normal development and to obtain a normal adult phenotype.

Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner

6-Formylindolo[3,2-b]carbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching frequency, blocked swim bladder inflation, and strongly potentiated expression of Ahr2-regulated genes. These effects were substantially reduced in embryos with a combined knockdown of Ahr2 and CYP1A, indicating that the toxicity was mediated at least partly by Ahr2. Co-exposure to the CYP1 inhibitor alpha-naphthoflavone (αNF) and FICZ had similar effects as the combination of CYP1A-MO and FICZ. HPLC analysis of FICZ-exposed embryos showed increased levels of FICZ after concomitant CYP1A-MO injection or αNF co-exposure. Together, these results show that a functioning CYP1/AHR feedback loop is crucial for regulation of AHR signaling by a potential physiological ligand in vivo and further highlights the role of CYP1 enzymes in regulating biological effects of FICZ.

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish

Disruption of the thyroid hormone (TH) system, an important mode of action, can lead to ecologically relevant adverse outcomes, especially during embryonic development. The present study characterizes the effects of disruption of TH synthesis on swim bladder inflation during zebrafish early-life stages using 2-mercaptobenzothiazole (MBT), a thyroid peroxidase (TPO) inhibitor. Zebrafish were exposed to different MBT concentrations until 120/168h post fertilization (hpf) and 32days post fertilization (dpf), in two sets of experiments, to investigate the effects of TPO inhibition on posterior and anterior swim bladder inflation respectively, as well as whole body thyroid hormone concentrations (triiodothyronine (T3) and its prohormone, thyroxine (T4)). At 120hpf, MBT did not directly impair posterior chamber inflation or size, while anterior chamber inflation and size was impaired at 32dpf. As previously shown in amphibians and mammals, we confirmed that MBT inhibits TPO in fish. Whole-body T4 decreased after MBT exposure at both time points, while T3 levels were unaltered. There was a significant relationship between T4 levels and the anterior chamber surface at 32dpf. The absence of effects on posterior chamber inflation can possibly be explained by maternal transfer of T4 into the eggs. These maternally derived THs are depleted at 32dpf and cannot offset TPO inhibition, resulting in impaired anterior chamber inflation. Therefore, we hypothesize that TPO inhibition only inhibits swim bladder inflation during late development, after depletion of maternally derived T4. In a previous study, we showed that iodothyronine deiodinase (ID) knockdown impaired posterior chamber inflation during early development. Our findings, in parallel with similar effects observed in fathead minnow (see part I, this issue) suggest that thyroid disruption impacts swim bladder inflation, and imply an important distinction among specific subtypes of TH disrupting chemicals. However, the existence of another – yet unknown – mode of action of MBT impacting swim bladder inflation cannot be excluded. These results can be helpful for delineating adverse outcome pathways (AOPs) linking TPO inhibition, ID inhibition and other TH related molecular initiating events, to impaired swim bladder inflation in fish during early life stages. Such AOPs can support the use of in vitro enzyme inhibition assays for predicting reduced survival due to impaired posterior and anterior chamber inflation.

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow

In the present study, a hypothesized adverse outcome pathway linking inhibition of thyroid peroxidase (TPO) activity to impaired swim bladder inflation was investigated in two experiments in which fathead minnows (Pimephales promelas) were exposed to 2-mercaptobenzothiazole (MBT). Continuous exposure to 1mg MBT/L for up to 22 days had no effect on inflation of the posterior chamber of the swim bladder, which typically inflates around 6 days post fertilization (dpf), a period during which maternally-derived thyroid hormone is presumed to be present. In contrast, inflation of the anterior swim bladder, which occurs around 14dpf, was impacted. Specifically, at 14dpf, approximately 50% of fish exposed to 1mg MBT/L did not have an inflated anterior swim bladder. In fish exposed to MBT through 21 or 22dpf, the anterior swim bladder was able to inflate, but the ratio of the anterior/posterior chamber length was significantly reduced compared to controls. Both abundance of thyroid peroxidase mRNA and thyroid follicle histology suggest that fathead minnows mounted a compensatory response to the presumed inhibition of TPO activity by MBT. Time-course characterization showed that fish exposed to MBT for at least 4 days prior to normal anterior swim bladder inflation had significant reductions in anterior swim bladder size, relative to the posterior chamber, compared to controls. These results, along with similar results observed in zebrafish (see part II, this issue) are consistent with the hypothesis that thyroid hormone signaling plays a significant role in mediating anterior swim bladder inflation and development in cyprinids, and that role can be disrupted by exposure to thyroid hormone synthesis inhibitors. Nonetheless, possible thyroid-independent actions of MBT on anterior swim bladder inflation cannot be ruled out based on the present results. Overall, although anterior swim bladder inflation has not been directly linked to survival as posterior swim bladder inflation has, potential links to adverse ecological outcomes are plausible given involvement of the anterior chamber in sound production and detection.

Early life exposure to PCB126 results in delayed mortality and growth impairment in the zebrafish larvae

The occurrence of chronic or delayed toxicity resulting from the exposure to sublethal chemical concentrations is an increasing concern in environmental risk assessment. The Fish Embryo Toxicity (FET) test with zebrafish provides a reliable prediction of acute toxicity in adult fish, but it cannot yet be applied to predict the occurrence of chronic or delayed toxicity. Identification of sublethal FET endpoints that can assist in predicting the occurrence of chronic or delayed toxicity would be advantageous. The present study characterized the occurrence of delayed toxicity in zebrafish larvae following early exposure to PCB126, previously described to cause delayed effects in the common sole. The first aim was to investigate the occurrence and temporal profiles of delayed toxicity during zebrafish larval development and compare them to those previously described for sole to evaluate the suitability of zebrafish as a model fish species for delayed toxicity assessment. The second aim was to examine the correlation between the sublethal endpoints assessed during embryonal and early larval development and the delayed effects observed during later larval development. After exposure to PCB126 (3–3000ng/L) until 5 days post fertilization (dpf), larvae were reared in clean water until 14 or 28 dpf. Mortality and sublethal morphological and behavioural endpoints were recorded daily, and growth was assessed at 28 dpf. Early life exposure to PCB126 caused delayed mortality (300ng/L and 3000ng/L) as well as growth impairment and delayed development (100ng/L) during the clean water period. Effects on swim bladder inflation and cartilaginous tissues within 5 dpf were the most promising for predicting delayed mortality and sublethal effects, such as decreased standard length, delayed metamorphosis, reduced inflation of swim bladder and column malformations. The EC50 value for swim bladder inflation at 5 dpf (169ng/L) was similar to the LC50 value at 8 dpf (188 and 202ng/L in two experiments). Interestingly, the patterns of delayed mortality and delayed effects on growth and development were similar between sole and zebrafish. This indicates the comparability of critical developmental stages across divergent fish species such as a cold water marine flatfish and a tropical freshwater cyprinid. Additionally, sublethal effects in early embryo-larval stages were found promising for predicting delayed lethal and sublethal effects of PCB126. Therefore, the proposed method with zebrafish is expected to provide valuable information on delayed mortality and delayed sublethal effects of chemicals and environmental samples that may be extrapolated to other species.

A high throughput passive dosing format for the Fish Embryo Acute Toxicity test

High throughput testing according to the Fish Embryo Acute Toxicity (FET) test (OECD Testing Guideline 236) is usually conducted in well plates. In the case of hydrophobic test substances, sorptive and evaporative losses often result in declining and poorly controlled exposure conditions. Therefore, our objective was to improve exposure conditions in FET tests by evaluating a passive dosing format using silicone O-rings in standard 24-well polystyrene plates. We exposed zebrafish embryos to a series of phenanthrene concentrations until 120h post fertilization (hpf), and obtained a linear dilution series. We report effect values for both mortality and sublethal morphological effects based on (1) measured exposure concentrations, (2) (lipid normalized) body residues and (3) chemical activity. The LC50 for 120hpf was 310μg/L, CBR50 (critical body residue) was 2.72mmol/kg fresh wt and La50 (lethal chemical activity) was 0.047. All values were within ranges expected for baseline toxicity. Impaired swim bladder inflation was the most pronounced morphological effect and swimming activity was reduced in all exposure concentrations. Further analysis showed that the effect on swimming activity was not attributed to impaired swim bladder inflation, but rather to baseline toxicity. We conclude that silicone O-rings (1) produce a linear dilution series of phenanthrene in the 120hpf FET test, (2) generate and maintain aqueous concentrations for reliable determination of effect concentrations, and allow for obtaining mechanistic toxicity information, and (3) cause no toxicity, demonstrating its potential as an extension of the FET test when testing hydrophobic chemicals.

Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction.

Thyroid hormone (TH) balance is essential for vertebrate development. Deiodinase type 1 (D1) and type 2 (D2) increase and deiodinase type 3 (D3) decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray), biochemistry, morphology and physiology using morpholino (MO) knockdown. Knockdown of D1+D2 (D1D2MO) and knockdown of D3 (D3MO) both resulted in transcriptional regulation of energy metabolism and (muscle) development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct TH balance during vertebrate embryonic development.

Morphological, Histological, Histochemical and Behavioral Aspects during Early Development of Red Porgy Pagrus pagrus L. Reared in Mesocosm

The present study provides a description of the morphological, histological and histochemical development, and the ecological implications of red porgy larvae reared in mesocosm, from hatching to 30 days after hatching (DAH). Four development stages were defined by body total length (TL), in agreement with major patterns of larval organogenesis. The first stage was characterized by the highest TL relative growth rate (RGR=7.23 % day -1 ) as larvae elongate their body to escape the culture media viscous forces. RGR decreased in the second stage of development (4.34 % day -1 ), as larvae opened the mouth and initiated exogenous feeding. Protein inclusions at the posterior digestive epithelium were indicative of pinocytosis and intracellular digestion. Yolk reserves were consumed at slower rate (until 5 DAH) than larvae in intensive culture, although the peak of swim bladder inflation occur earlier (8-11 DAH). A significant decrease in RGR (1.55 % day -1 ) and the notochord flexion characterized the third stage of larval development. Larvae migrated deeper in the tank (14 DAH) and adopted an aggressive behavior (17-19 DAH). Gastric glands in the forthcoming stomach were common at 20 DAH. In the last stage of larval development the RGR was 3.62 % day -1 . A sharp increase of Artemia consumption from 26 DAH and cannibalism denoted a change in the digestive mode. Results suggest that red porgy larvae growth and development priorities, and behavior patterns at early stages are directed to increase the number of captured prey.

Initial Characterization of Embryonic Development in North American Burbot

AbstractDeclining wild populations and increasing commercial interest are encouraging development of aquaculture methods for the North American Burbot Lota lota maculosa. A current focus of Burbot aquaculture research is reducing high mortality that is typically associated with culture of Burbot embryos and larvae. However, no Burbot embryonic or larval staging systems are currently available to provide a comparative baseline for studies of these life stages. To help address this gap, we examined Burbot embryonic development from egg fertilization until the onset of the larval period at first feeding. Ontogeny was characterized using diagnostic morphological features visible with stereo microscopy. Six developmental periods were characterized (cleavage, blastula, gastrula, segmentation, swim bladder inflation, and first feeding), along with 15 developmental stages. Water temperature ranged from 3°C to 5°C for the duration of this study. Cleavage cycles were approximately 8 h/division. Segmentation began at 12 d postfertilization (DPF) and continued until first hatch at 33 DPF. First feeding was observed at 45 DPF, 12 d after first hatch. Results presented here are expected to help refine Burbot culture methodology and research efforts aimed at conservation, management, and commercial production. Future research should use additional cohorts and expose embryos to a range of culture conditions to better understand the factors governing embryonic survival and variability in the timing of events during development.

Promotion of initial swim bladder inflation and effect of initial inflation failure on skeletal malformation in cultured seven-band grouperHyporthodus septemfasciatus(Thunberg)

The effects of the removal of the oily surface film on initial swim bladder inflation and survival in cultured seven-band grouper Hyporthodus septemfasciatus were investigated during the larval stage. Additionally, the relationship between swim bladder inflation failure and malformations in later stages was examined. To explore promotion conditions for inflation, duplicate experiments were conducted under three different water surface conditions: removal of the oily surface film (ROF group), covering the water surface with an oil film (COF group), and sealing the water surface with liquid paraffin (SLP group). Larval swim bladder inflation was first observed 14 days after hatching (DAH) in the ROF group; thereafter, swim bladder inflation rates increased to 11.1% and 38.7% until the end of the experiments. This indicates that initial swim bladder inflation occurs at the pre-flexion stage in larval development. Conversely, bladder inflation was rarely observed in the COF or SLP groups, indicating the promotional effect of surface film removal on larval swim bladder inflation and suggesting that gulping atmospheric air is necessary for initial inflation. Although larval water surface death occurred frequently in the ROF group, no significant difference existed in any group between the survival rates at 10 DAH and at the end of the experiment. The incidence rate of spinal curvature and deformation of the centra was significantly higher in individuals without initial swim bladder inflation, and these malformations mainly occurred in the caudal vertebra. Therefore, the removal of the oily surface film promotes initial swim bladder inflation and reduces larval malformations.

Effect of water surface condition on survival, growth and swim bladder inflation of yellowfin tuna,Thunnus albacares(Temminck and Schlegel), larvae

Early-stage mortality due to surface water tension-related death and due to sinking to the tank bottom was investigated for yellowfin tuna, Thunnus albacares (YFT), larvae. Different aeration rates and rearing water surface conditions were examined to evaluate the effect on larval survival, swim bladder inflation and growth. The percentage survival of yolk sac larvae was significantly higher when the rearing water surface was covered with fish oil at aeration rates of 0 and 50 mL min−1. The highest mortality occurred at the highest aeration rate of 250 mL min−1 regardless of surface water condition. A second experiment was conducted twice under different water surface conditions: the water surface was covered by fish oil (FO), skimmed of fish oil (SS), and was not treated (NC). The percentage survival was not significantly different between treatments after 7 days of feeding. In contrast to the survival, the proportion of larvae with inflated swim bladders was significantly higher for the NC and SS groups than that of the FO group. Results of these experiments indicate that the addition of oil to the rearing water surface without its removal interferes with the initial swim bladder inflation in YFT larvae. These results also indicate that YFT larvae need to obtain (gulp) air at the water surface for initial swim bladder inflation, and success of initial swim bladder inflation may be crucial for their survival.