Malformed Larvae Research Articles

Environmental microplastics compromise reproduction of the marine invertebrate Mytilus galloprovincialis: A holistic approach

The extensive presence of microplastics (MPs) in marine ecosystems constitutes a major threat to aquatic environments. The gametes of the marine invertebrate Mytilus galloprovincialis, which is essential for coastal ecosystems, are released directly into the water, potentially exposing them to environmental microplastics (EMPs). This study examined the effects of exposing M. galloprovincialis gametes to 50 or 100 µg/L EMP for 1 h on fertilization rates, larval quality, and the molecular mechanisms underlying the induction of apoptosis and shell growth. Our findings show that increased EMP concentrations correlate with reduced fertilization success and higher rates of larval malformations, indicating negative impacts on embryonic development. Additionally, DNA degradation in larvae is related to the EMP concentration. The apoptosis-associated proteins Bax, P53, and Cas-3 are upregulated, whereas Bcl-2 and DNA-ligase are downregulated with increasing EMP concentrations. Prothymosin-ɑ (PTMA), which is crucial for cell proliferation, also decreases with increasing EMP concentrations, contributing to impaired cell proliferation and growth imbalances. Reduced HRG gene expression is correlated with decreased shell growth and larval malformations. This study underscores the detrimental impact of EMPs on bivalve gametes, which impacts fertilization success and larval quality and highlights the potential risks to species survival and marine ecosystem stability.

Applications of Zebrafish Embryo Models to Predict Developmental Toxicity for Agrochemical Product Development.

The development of safe crop protection products is a complex process that traditionally relies on intensive animal use for hazard identification. Methods that capture toxicity in early stages of agrochemical discovery programs enable a more efficient and sustainable product development pipeline. Here, we explored whether the zebrafish model can be leveraged to identify mammalian-relevant toxicity. We used transgenic zebrafish to assess developmental toxicity following exposures to known mammalian teratogens and captured larval morphological malformations, including bone and vascular perturbations. We further applied toxicogenomics to identify common biomarker signatures of teratogen exposure. The results show that the larval malformation assay predicted teratogenicity with 82.35% accuracy, 87.50% specificity, and 77.78% sensitivity. Similar and slightly lower accuracies were obtained with the vascular and bone assays, respectively. A set of 20 biomarkers were identified that efficiently segregated teratogenic chemicals from nonteratogens. In conclusion, zebrafish are valuable, robust, and cost-effective models for toxicity testing in the early stages of product development.

Evaluation of viability, developmental competence, and apoptosis-related transcripts during in vivo post-ovulatory oocyte aging in zebrafish Danio rerio (Hamilton, 1822).

Post-ovulatory aging is a time-dependent deterioration of ovulated oocytes and a major limiting factor reducing the fitness of offspring. This process may lead to the activation of cell death pathways like apoptosis in oocytes. We evaluated oocyte membrane integrity, egg developmental competency, and mRNA abundance of apoptosis-related genes by RT-qPCR. Oocytes from zebrafish Danio rerio were retained in vivo at 28.5°C for 24 h post-ovulation (HPO). Viability was assessed using trypan blue (TB) staining. The consequences of in vivo oocyte aging on the developmental competence of progeny were determined by the embryo survival at 24 h post fertilization, hatching, and larval malformation rates. The fertilization, oocyte viability, and hatching rates were 91, 97, and 65% at 0 HPO and dropped to 62, 90, and 22% at 4 HPO, respectively. The fertilizing ability was reduced to 2% at 8 HPO, while 72% of oocytes had still intact plasma membranes. Among the apoptotic genes bcl-2 (b-cell lymphoma 2), bada (bcl2-associated agonist of cell death a), cathepsin D, cathepsin Z, caspase 6a, caspase 7, caspase 8, caspase 9, apaf1, tp53 (tumor protein p53), cdk1 (cyclin-dependent kinase 1) studied, mRNA abundance of anti-apoptotic bcl-2 decreased and pro-apoptotic cathepsin D increased at 24 HPO. Furthermore, tp53 and cdk1 mRNA transcripts decreased at 24 HPO compared to 0 HPO. Thus, TB staining did not detect the loss of oocyte competency if caused by aging. TB staining, however, could be used as a simple and rapid method to evaluate the quality of zebrafish oocytes before fertilization. Taken together, our results indicate the activation of cell death pathways in the advanced stages of oocyte aging in zebrafish.

Gender-specific effects of polystyrene nanoplastic exposure on triclosan-induced reproductive toxicity in zebrafish (Danio rerio)

Nanoplastics (NPs) and triclosan (TCS) are ubiquitous emerging environmental contaminants detected in human samples. While the reproductive toxicity of TCS alone has been studied, its combined effects with NPs remain unclear. Herein, we employed Fourier transform infrared spectroscopy and dynamic light scattering to characterize the coexposure of polystyrene nanoplastics (PS-NPs, 50 nm) with TCS. Then, adult zebrafish were exposed to TCS at environmentally relevant concentrations (0.361–48.2 μg/L), with or without PS-NPs (1.0 mg/L) for 21 days. TCS biodistribution in zebrafish tissues was investigated using ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry. Reproductive toxicity was assessed through gonadal histopathology, fertility tests, changes in steroid hormone synthesis and gene expression within the hypothalamus-pituitary-gonad-liver (HPGL) axis. Transcriptomics and proteomics were applied to explore the underlying mechanisms. The results showed that PS-NPs could adsorb TCS, thus altering the PS-NPs' physical characteristics. Our observations revealed that coexposure with PS-NPs reduced TCS levels in the ovaries, livers, and brains of female zebrafish. Conversely, in males, coexposure with PS-NPs increased TCS levels in the testes and livers, while decreasing them in the brain. We found that co-exposure mitigated TCS-induced ovary development inhibition while exacerbated TCS-induced spermatogenesis suppression, resulting in increased embryonic mortality and larval malformations. This co-exposure influenced the expression of genes linked to steroid hormone synthesis (cyp11a1, hsd17β, cyp19a1) and attenuated the TCS-decreased estradiol (E2) in females. Conversely, testosterone levels were suppressed, and E2 levels were elevated due to the upregulation of specific genes (cyp11a1, hsd3β, cyp19a1) in males. Finally, the integrated analysis of transcriptomics and proteomics suggested that the aqp12-dctn2 pathway was involved in PS-NPs' attenuation of TCS-induced reproductive toxicity in females, while the pck2-katnal1 pathway played a role in PS-NPs' exacerbation of TCS-induced reproductive toxicity in males. Collectively, PS-NPs altered TCS-induced reproductive toxicity by disrupting the HPGL axis, with gender-specific effects.

Combined Effects of Polystyrene Nanosphere and Homosolate Exposures on Estrogenic End Points in MCF-7 Cells and Zebrafish.

Micro- and nanoplastics (MNPs) and homosalate (HMS) are ubiquitous emerging environmental contaminants detected in human samples. Despite the well-established endocrine-disrupting effects (EDEs) of HMS, the interaction between MNPs and HMS and its impact on HMS-induced EDEs remain unclear. This study aimed to investigate the influence of MNPs on HMS-induced estrogenic effects and elucidate the underlying mechanisms in vitro and invivo. We assessed the impact of polystyrene nanospheres (PNSs; , ) on HMS-induced MCF-7 cell proliferation (HMS: , equivalent to ) using the E-SCREEN assay and explored potential mechanisms through transcriptomics. Adult zebrafish were exposed to HMS () with or without PNSs (, ) for 21 d. EDEs were evaluated through gonadal histopathology, fertility tests, steroid hormone synthesis, and gene expression changes in the hypothalamus-pituitary-gonad-liver (HPGL) axis. Coexposure of HMS and PNSs resulted in higher expression of estrogen receptor (ESR1) and the mRNAs of target genes (pS2, AREG, and PGR), a greater estrogen-responsive element transactivation activity, and synergistic stimulation on MCF-7 cell proliferation. Knockdown of serum and glucocorticoid-regulated kinase 1 (SGK1) rescued the MCF-7 cell proliferation induced by PNSs alone or in combination with HMS. In zebrafish, coexposure showed higher expression of SGK1 and promoted ovary development but inhibited spermatogenesis. In addition, coexposure led to lower egg hatchability, higher embryonic mortality, and greater larval malformation. Coexposure also modulated steroid hormone synthesis genes (cyp17a2, hsd17[Formula: see text]1, esr2b, vtg1, and vtg2), and resulted in higher () release in females. Conversely, males showed lower testosterone, , and gene expressions of cyp11a1, cyp11a2, cyp17a1, cyp17a2, and hsd17[Formula: see text]1. PNS exposure exacerbated HMS-induced estrogenic effects via SGK1 up-regulation in MCF-7 cells and disrupting the HPGL axis in zebrafish, with gender-specific patterns. This offers new mechanistic insights and health implications of MNP and contaminant coexposure. https://doi.org/10.1289/EHP13696.

Photographic characterisation of acidification-induced larval malformations in the European lobster Homarus gammarus (Linnaeus, 1758) (Decapoda: Astacidea: Nephropidae)

Abstract Occurrence of developmental malformations is of interest since they potentially influence organismal performance and fitness. We report an increased incidence (⁓ 46 fold) of physical malformations in the larvae of the American lobster Homarus Gammarus (Linnaeus, 1758) in response to seawater acidification (–0.58 pH units relative to nominal pH 8.0). We observed three malformations under the influence of seawater acidification previously undescribed in lobster larvae: a flared carapace, twisted tail, and cross claw. Larvae reared under seawater acidification exhibit significantly lower survivorship (by ⁓14%) and the occurrence of a malformation decreases survivorship (12.7%). Larvae with four types of malformations did not progress through development to reach post-larval stages. Namely, these malformations were a flared carapace, curled carapace, twisted tail, and cross claw. Results from this study provide photographic documentation of various lobster larval malformations that ultimately affect individual success and can be applied for quality-control in hatcheries.

Effects of incubation temperature and maternal phenotype on Baltic herring (Clupea harengus membras) eggs and larvae: an experimental study

Temperature modifies the reproductive success of fish, yet, in many species, we lack the information on its role in the early development. In this study, the effect of temperature on the relation between maternal traits (length, age, somatic condition, and muscle lipid and ovarian thyroid hormone concentrations), egg quality (fertilization success, development rate, mortality, and hatching success), and offspring traits (size-at-hatch, yolk sac size, and proportion of malformations) were studied in Baltic herring ( Clupea harengus membras) in the northern Baltic Sea. The experiments were conducted at an ambient temperature of 7 °C and at an elevated temperature of 14 °C using 5 to 10 females and 3 replicates per female. The results indicate that elevated temperature may result in a faster developmental rate, a lower early-stage mortality and hatching success, smaller size-at-hatch, a larger yolk sac size, and a higher amount of larval malformations when compared to an ambient temperature. The egg and offspring traits were also associated with the maternal traits, indicating especially that thyroid hormones play a mediating role in the physiological processes.

Effect of non-permeable cryoprotectant sucrose on the development of spotted knifejaw (Oplegnathus punctatus) embryos

In this study, the toxicity of sucrose to Oplegnathus punctatus embryos was evaluated. Embryos at the 4–6 somite, tail-bud, heart formation, and heart-beating stages were exposed to 0, 0.5, 1,1.5, 2, 2.5, or 3 M sucrose for 1 h. Survival rates of embryos at the tail-bud, heart formation, and heart-beating stages after rehydration for 1 h were not affected by treatment with 2 M sucrose (the maximum concentration). Embryos at the tail-bud, heart formation, and heart-beating stages were exposed to 2 M sucrose for 0, 30, 60, 90, 120, 150, or 180 min. Long-term developmental indicators, including rates of survival, hatching, swimming, and malformation, were evaluated for 4 days after rehydration. Based on the survival rates 10 min after rehydration, the longest tolerance time for embryos at the three stages was 120 min. Based on long-term developmental indicators, the longest tolerance times were 60 min at the tail-bud, 60 min at the heart formation stage and 30 min at the heart beating stage. The malformation rates increased as the treatment time increased. The malformation rates were 100% when embryos were exposed to sucrose for ≥120 min. Malformation was divided into larval and embryonic abnormality. As the exposure time increased for tail-bud stage embryos, the rate of larval malformation increased. Treatment at heart formation and heart-beating stages resulted in higher rates of failure to hatch at exposure time. Based on these results, toxicity tests of non-permeable cryoprotectant in embryos requires the observation of development for at least 2 days after rehydration. Based on long-term observation, it was concluded that dehydration before freezing was not the direct cause of larvae deformity that hatched from frozen-thawing embryo. These results provide a reference for the singly use of representative non-permeable cryoprotectant sucrose.

Sperm and egg in vitro storage effects on artificial fertilization and hatching in common carp (Cyprinus carpio L.)

This is the first report showing fertilization of short-term stored eggs (19 °C) with short-term stored diluted sperm (0–2 °C) in common carp (Cyprinus carpio). From 5 males, fresh (control) and 7-day stored sperm were diluted with common carp extender. After collection of eggs from nine females, the eggs were immediately pooled into three groups from females 1–3, 4–6 and 7–9, according to the eggs' quality based on visual criteria. They were placed in beakers, covered and incubated at 19 °C (for each pool, 10 g of eggs from each female were used). The 7-day short-term storage sperm was incubated at 20 °C for 20 min and pooled before being used for fertilization. The eggs were fertilized with fresh and short-term storage sperm after 1, 3 and 6 h post storage of eggs and activated with hatchery water and Perchec solution. Sperm motility and velocity (50–54% and 80–115 µm/s) of 7-day short-term storage sperm at 0–2 °C were ensured by additional incubation of sperm at 20 °C for 20 min before being activated. Principal component analysis showed that the sorting of eggs of different females into three groups according to sperm quality, fertilization ability and production of malformed larvae was successful. It is recommended that overripe common carp eggs are stored for a maximum of 1 h. A period of 3 h can be used for better quality eggs and exceptionally, up to 6 h for very high-quality eggs. Higher larvae malformation was found in the 6 h aged eggs together with fertilized short-term storage and fresh sperm. In all quality groups of eggs, the effect of different sperm, activation medium and egg storage time on the level of malformations was observed. The activation solution for carp did not show any positive fertilization and hatching effects.

Fish with larger pre-seasonal oocytes yields lower egg quality in season – A case study of outdoor-cultured domesticated Pikeperch (Sander lucioperca)

Seasonal reproduction of domesticated pikeperch has been the most critical spawning batch in several European countries. The present study aimed to monitor oocyte growth between mid-November and seasonal spawning to evaluate if oocyte growth trends may predict egg quality. Nineteen sexually mature females were monitored for oocyte sizes every two months. In mid-March, fish were transported to the indoor facility and artificially reproduced. Sixteen females ovulated and egg quality parameters were assessed and further related to oocyte sizes measured in November, January, and March, as well as to size increments between samplings. Based on the oocyte diameters, fish were assigned to two size groups, and the egg quality was compared. Oocyte growth was greater between the first two samplings compared to the consecutive period (150.1 ± 16.5 µm vs 24.7 ± 20.4 µm). Diameters assessed in November and March positively correlated with oil globule fragmentation, while diameters assessed in November and January positively correlated with larval malformation. Although in January larger oocytes showed better embryo survival than smaller ones (60.6 ± 9.5% vs 37.8 ± 23.2%,) they were characterized with greater percent of malformed larvae (25.0 ± 22.0% vs 5.4 ± 3.9%). It appears that fish with bigger oocytes in pre-season have an affinity toward increased embryo survival, however, they seem to have an issue with oil globule fragmentation and larvae malformation in case of seasonal spawning. Therefore, the selection of breeders for either pre-seasonal or seasonal reproduction batch according to the November oocyte size is recommended.

Effects of Exposure to Trade Antifouling Paints and Biocides on Larval Settlement and Metamorphosis of the Compound Ascidian Botryllus schlosseri

To evaluate the effects of antifouling paints and biocides on larval settlement and metamorphosis, newly hatched swimming larvae of the compound ascidian Botryllus schlosseri, a dominant species of soft-fouling in coastal communities, were exposed to (i) substrata coated with seven antifouling paints on the market containing different biocidal mixtures and types of matrices and (ii) sea water containing various concentrations of eight biocidal constituents. All antifouling paints showed high performance, causing 100% mortality and metamorphic inhibition, with ≥75% not-settled dead larvae. All antifouling biocides prevented the settlement of larvae. The most severe larval malformations, i.e., (i) the formation of a bubble encasing the cephalenteron and (ii) the inhibition of tail resorption, were observed after exposure to metal and organometal compounds, including tributyltin (TBT) at 1 μM (325.5 µg L−1), zinc pyrithione (ZnP) at 1 μM (317.7 µg L−1), and CuCl at 0.1 μM (98.99 µg L−1), and to antimicrobials and fungicides, including Sea-Nine 211 at 1 μM (282.2 µg L−1) and Chlorothalonil at 1 μM (265.9 µg L−1). The herbicides seemed to be less active. Irgarol 1051 was not lethal at any of the concentrations tested. Diuron at 250 μM (58.2 mg L−1) and 2,3,5,6-tetrachloro-4-(methylsulphonyl)pyridine (TCMS pyridine) at 50 μM (14.8 mg L−1) completely inhibited larval metamorphosis. These results may have important implications for the practical use of different antifouling components, highlighting the importance of their testing for negative impacts on native benthic species.

Leachate from plastic food packaging induced reproductive and neurobehavioral toxicity in zebrafish

The present study mimicked daily life exposure to plastic food package bags and evaluated its effects on the reproductive and neurobehavioral responses using zebrafish model. Gas chromatography-mass spectrometer (GC/MS) full scan analysis revealed that phthalic acid, isobutyl octyl ester (DEHP) and its metabolites were the main leachate from plastic bags. Our results demonstrated that during the eight weeks exposure, leaching from plastic bags treated with boiling water (P-high group) significantly affected the spawn egg production, embryo hatching and larval malformation rate. Cross-spawning trails between zebrafish collected from the controls and P-high group at the end of eight weeks showed that these adverse effects were more severe in the offspring derived from paternal exposure than those derived from the maternal exposure, suggesting leached chemicals may have a more pronounced effect in sperm than in eggs. In addition, P-high group male testis weight, sperm motility and sperm swimming velocities were decreased significantly. After eight weeks treatment, neurobehavioral tests demonstrated significant changes in the swimming speed during free swimming and light-dark stimulation in the adult zebrafish from P-high group, with the effects being more severe in the males than females. P-high group males also showed altered response in the light/dark explore and mirror attacks assays.

Organophosphate-based flame retardant (tris(1,3-dichloro-2-propyl) phosphate) reduces fecundity and impairs embryonic development in marine invertebrates

Context It is important to understand how marine invertebrates may be affected by tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), as an organophosphorus flame retardant. Aim The present research verified the hypothesis that TDCIPP would adversely affect the growth, development and reproduction of Crepidula onyx (phylum Mollusca) and Tigriopus japonicus (phylum Arthropoda). Methods Effects of chronic exposure for the whole life cycle were examined at two concentrations of TDCIPP chosen on the basis of results from preliminary range-finding test. Key results Exposure to 1 and 10 μg L−1 of TDCIPP significantly reduced larval and juvenile survival rate and shell length in C. onyx. TDCIPP caused adverse reproductive effects, including abnormal embryonic development, which led to non-viable broods and the malformation of larvae. Exposure to TDCIPP resulted in a 100-fold increase in incidence rate of abnormal larva formation. In T. japonicus, exposure to TDCIPP (10 μg L−1) resulted in non-viable broods and a significant reduction in the number of eggs and number of nauplii. Conclusions Larval survival, larval growth, fecundity, and embryonic development were identified as the TDCIPP-affected ecological endpoints in the marine invertebrate C. onyx and T. japonicus. Implications From a management perspective, these results can help us assess the ecological risk posed by TDCIPP.

Potentiality of Novel Chitosan-O-Arginine as Insecticidal and Growth Inhibitory Compound against Diamondback Moth, Plutella xylostella L. on Cauliflower

Chitosan (Cs), a natural biodegradable polysaccharide polymer, prepared from sea waste of crustacean origin, has the potential for plant protection application. In the present study, a novel chitosan derivative, called Chitosan-O-Arginine, synthesized from crude chitosan, was evaluated for its chronic toxicity against second instar larvae of Plutella xylostella by leaf dip method. The results revealed the toxic potential of Chitosan-O-Arginine, which could inflict cent per cent mortality after four days of continuous feeding on the 1000 ppm treated leaf, and the larva could accomplish 0.12 mg weight (99.66% reduction) on the day before death. The next lower concentration (700 ppm) resulted in the maximum larval weight of 0.45 mg (93.71% reduction) after nine days of feeding and resulted in cent per cent mortality after the tenth day. While the untreated larva attained a maximum weight of 5.54 mg. All the larvae were pupated in the next lower concentration (500 ppm) and recorded minimum pupal (1.00 mg; 80% reduction) and adult (0.73 mg; 77.45% reduction) weight, whereas the untreated pupa and adult weighed the maximum of 5.00 mg and 3.20 mg. Considering the developmental period, there was no significant difference in the larval duration and adult life span. While there was an extension of the pupal period by one day at 500 ppm compared to untreated larva. The larval, pupal, and adult malformations were noticed in 300 and 100 ppm concentrations. These findings suggest that chitosan was potent in imposing chronic toxicity and growth inhibitory effect on P. xylostella larvae, and hence it can be recommended as an eco-friendly component in the Integrated Pest Management module.

Oxidative stress response to hydrogen peroxide exposure of Mytilus galloprovincialis and Ruditapes philippinarum: Reduced embryogenesis success and altered biochemical response of sentinel marine bivalve species

The elevated concentration of hydrogen peroxide (H2O2) due to natural and anthropogenic causes is a significant bane to marine and coastal organisms, as it has a prolonged half-life and can inject across lipid bilayers and interfere with aquatic redox processes. The present assessment aimed to determine the acute effect of H2O2 on larval development of the Mediterranean mussel Mytilus galloprovincialis and to observe the oxidative stress (OS) responses, including genotoxicity and neurotoxicity in the clam Ruditapes philippinarum. Two experiments were performed, in which (a) mussel embryos were exposed to the consecutive dilution (0.0, 0.05, 0.5, 5.0, 50.0 and 500.00 μM) of H2O2 for 48 h and (b) adult clams were exposed to similar concentrations - of this reactive oxygen species (ROS) for 21 days. Incremental H2O2 significantly retarded the growth of mussel larvae; the percentage of malformed larvae increased with increasing toxicant concentration (p < 0.01, and r = 0.884). H2O2 also induced oxidative stress (OS) in clams, indicated by the activation/inactivation of antioxidant enzymes (GST, GPx, and GR) with a significant rise in lipid peroxidation (LPO) in the digestive gland tissues of the clams (p < 0.05). No genotoxicity or neurotoxicity on R. philippinarum were evident after chronic exposure. The battery of responses (organismal and cellular parameters) showed embryotoxicity and sublethal effects on bivalves, which confirmed their suitability for assessing the effect of H2O2 mediated OS responses in the marine environment.

Duloxetine ameliorates valproic acid-induced hyperactivity, anxiety-like behavior, and social interaction deficits in zebrafish.

Syndromic autism spectrum disorders (ASDs) are characterized by impaired social communication and repetitive/stereotyped behaviors. Currently available therapeutic agents against ASD have limited efficacy. Thus, searching for novel and effective drugs ameliorating core symptoms, in particular social deficits, is of utmost importance. Duloxetine (DLX), an antidepressant that has been identified as an agonist mimetic for the cell adhesion molecule L1, exhibits beneficial functions in vitro and in vivo. Therefore, in this study, we focused on the rapid and persistent neuroprotective function of DLX following valproic acid (VPA)-triggered hyperactivity, anxiety-like behavior and social deficits in zebrafish. Embryonic exposure to VPA reduced survival in a dose- and time-dependent manner, delayed hatching, and also resulted in a significant number of malformed larvae. After initial dose-response experiments in zebrafish larvae, 10μM VPA exposure between 0.33 and 4.5 days post fertilization (dpf) was identified as an effective concentration that led to an early and persistent ASD-like phenotype in zebrafish. ASD-like elevated acetylcholine esterase (AChE) activity and reduced Akt-mTOR signaling was observed in zebrafish whole brain. Acute administration of DLX (4.5-6 dpf) reduced the VPA-induced ASD-like phenotype in zebrafish larvae. Additionally, such early-life acute DLX treatment had long-term effects in ameliorating social impairments, hyperactivity, and anxiety-like behaviors through adulthood. This was accompanied by reduced AChE activity and by normalized Akt-mTOR signaling. Overall, DLX treatment showed a long-term therapeutic effect on autistic-like behaviors, and alteration of AChE activity and Akt-mTOR signaling were identified as crucial in the VPA-induced ASD zebrafish model.

Effects of short-duration oil exposure on bay anchovy (Anchoa mitchilli) embryos and larvae: mortality, malformation, and foraging.

The Deepwater Horizon oil spill highlighted the need to understand the effects of oil exposure on marine eggs and larvae. To determine how short-duration exposure impacts the survivability of early life stages of the bay anchovy, Anchoa mitchilli, embryos and larvae ≤ 3-days-post-hatch (dph) were exposed to high-energy water accommodated fractions of weathered crude oil for 2 or 6h. Lethal and sublethal effects of short-duration oil exposure were observed, including crippling malformations and altered optimal swimming and foraging behavior of larvae without malformation. The probability of mortality for larvae exposed as embryos (37.37 and 77.31µg L-1 total polycyclic aromatic hydrocarbons or 'TPAH'), assessed 48h after exposure, increased from 0.06 to 0.15 (2h) and 0.10-0.23 (6h) relative to unoiled controls. When exposed as 1-dph larvae (8.80-37.37µg L-1 TPAH) and assessed 24h after exposure, the probabilities increased from 0.20 to 0.76 (2h) and 0.28-0.99 (6h). Among surviving larvae, probabilities of yolk-sac, finfold, notochord, and cranio-facial malformations increased with exposure concentration, duration, and time after exposure by up to 0.07 immediately following exposure of 1-dph larvae and 0.55 24h after exposure. When assessed 48h after exposure as embryos, the probability of larval malformation reached 0.43. First-feeding (3-dph) foraging behavior was altered immediately and 24h after 2h exposures (8.80-77.31µg L-1 TPAH). Time spent in motion and swim speed increased with exposure concentration by up to 331% and 189%, respectively. The number of bursts min-1 increased by 293% immediately and 152% 24h after exposure. Burst distance decreased by 201%. Pause duration and burst speed decreased by 391% and 250% immediately and 124% and 109% 24h after exposure. No effects were found for burst duration or tortuosity. Our results suggest potential cascading effects on fitness and trophic interactions.

Influence of selected precipitating agents used for restoration of water reservoirs on the embryogenesis of pike (Esox lucius L.)

The aim of the study was to investigate the effect of iron coagulant (IC), aluminum coagulant (AC) and lanthanum modified bentonite (LMB) on the embryogenesis of pike. Physicochemical indicators of the water were determined according to the methods recommended by APHA (1999). The applied precipitating agents (IC, AC, LMB) at appropriate concentrations were sprayed on the surface of water in aquariums with the pike eggs placed on nets. The eggs were observed under a microscope, documenting their development using digital photography NIS Elements Br software. The number of fertilized eggs, embryo survival, and the percentage of malformed larvae were determined. The pike eggs and hatched larvae were recorded and measured.The percentage of fertilized eggs was found to be the highest in the control sample (LW) and in the IC sample (94% and 93.3%, respectively), 85.9% in the AC sample and 81.8% in the LMB sample. The pace of pike embryogenesis in the samples was identical – the embryos in both the control and other samples reached individual stages of embryonic development with an equal number of degree days (DD). The IC group hatched the earliest, and the control sample was the last to hatch (LW). In the control sample, the hatched larvae were the longest and had the lowest percentage of malformed individuals. The control sample also had the highest rate of embryo survival (85.9%), while the lowest was in the LMB sample (68.6%). These results indicate that the application of precipitating measures to improve water quality should not coincide with the periods of fish reproduction.

Effects of mine tailing exposure on early life stages of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus)

Mining and processing of minerals produce large quantities of tailings as waste. Some countries, including Norway, allow disposal of mine tailings in the sea. In this study we investigated the impacts of tailings from a calcium carbonate (CaCO3) processing plant on early live stages of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua). Fish eggs (3 days post fertilisation; dpf) were exposed for 48 h to three concentrations of tailings, nominally 1 mg L−1 (low, L); 10 mg L−1 (medium, M) and 100 mg L−1 (high, H); with L and M representing concentrations occurring at tailing release points. Results show that tailings rapidly adhered to eggs of both species, causing negative buoyancy (sinking of eggs) in M and H exposures. While tailings remained on egg surfaces in both species also after exposure termination, adhesion seemed more pronounced in cod, leading to larger impacts on buoyancy even after exposure. Tailing exposure further induced early hatching and significantly reduced survival in M and H exposed embryos in both fish species, and in cod from the L exposure group. Moreover, tailing exposure caused reduced survival and malformations in larvae, potentially related to premature hatching. This study shows that mineral particles adhere to haddock and cod eggs, affecting egg buoyancy, survival and development.

Cooling of Siamese fighting fish Betta splendens (Teleostei, Osphronemidae) embryos at low temperatures

The Siamese fighting fish (Betta splendens) has great importance as an ornamental aquarium fish as well as laboratory model species. Due to its rapid development, a cooling-embryo protocol could provide some advantages in their transportation, embryonic synchronization, and optimization of hatcheries. In this context, this work aimed to develop a protocol to storage B. splendens embryos at two temperatures (5 and 14 °C), testing three cryoprotective solutions (S1: 0.5 M sucrose, 1.5 M methanol; S2: 0.25 M sucrose, 0.75 M methanol; and S3: 0.125 M sucrose, 0.375 M methanol) and evaluating the quality of the larvae obtained. Moreover, a method to isolate the embryos from the bubble nest constructed by the male and to incubate them without parental care was applied in this study. The cooling assays were done using embryos of 24-h-post-fertilization at 26 °C and the results demonstrated that it is possible to store these embryos deprived of cryoprotectants at 5 °C for at least 6-h without negative effects. Meanwhile, S2 and S3 were the most suitable solutions for its storage for 9-h at 5 °C or 24-h at 14 °C, obtaining 77% hatching and 52% normal larvae in the first case or 88% hatching and 81% larvae with mild abnormalities in the second one. Indeed, type and frequency of larval abnormalities were evaluated and, remarkably, a partial recovery was described on malformed larvae from embryo cooled at 14 °C. Finally, this work is the first report about the cooling of B. splendens embryos and establishes the conditions for further studies on this field with this species.