Monsters with a shortened vertebral column: A population phenomenon in radiating fish Labeobarbus (Cyprinidae).

The phenomenon of a massive vertebral deformity was recorded in the radiating Labeobarbus assemblage from the middle reaches of the Genale River (south-eastern Ethiopia, East Africa). Within this sympatric assemblage, five trophic morphs-generalized, lipped, piscivorous and two scraping feeders-were reported between 1993 and 2019. In 2009, a new morph with prevalence of ~10% was discovered. The new morph, termed 'short', had an abnormally shortened vertebral column and a significantly deeper body. This type of deformity is common in farmed Atlantic salmon and other artificially reared fish, but is rare in nature. In the Genale Labeobarbus assemblage, the deformity was present exclusively within the generalized and lipped morphs. The short morph had between seven and 36 deformed (compressed and/or fused) vertebrae. Their body depth was positively correlated with number of deformed vertebrae. In another collection in 2019, the short morph was still present at a frequency of 11%. Various environmental and genetic factors could contribute to the development of this deformity in the Genale Labeobarbus, but based on the available data, it is impossible to confidently identify the key factor(s). Whether the result of genetics, the environment, or both, this deep-bodied phenotype is assumed to be an anti-predator adaptation, as there is evidence of its selective advantage in the generalized morph. The Genale monstrosity is the first reported case of a massive deformity of the vertebral column in a natural population of African fishes.

Identification of reproduction-related genes and pathways in the Culter alburnus H-P-G axis and characterization of their expression differences in malformed and normal gynogenetic ovaries.

This study applied RNA-seq technology to discover reproduction-related genes and pathways in female topmouth culter brain (including pituitary) and ovarian tissues. In functional analysis, 2479 and 2605 unigenes in the brain and ovary tissue were assigned to the "reproductive process" subcategory in addition to the 2660 and 2845 unigenes assigned to the "reproduction" subcategory. Twenty-three complete cDNA sequences were identified through the different gene expression (DGE) approach from five reproduction-related pathways (MAPK signaling pathway, neuroactive ligand-receptor interaction pathway, gonadotropin-releasing hormone signaling pathway, oocyte meiosis pathway, and steroid biosynthesis pathway). The expression levels of 16 candidate genes using qPCR in this study were in accordance with the results of transcriptome analysis. In addition, the expression levels of the FSH, 3ß-HSD, PGR, and NPYR genes in malformed gynogenetic ovaries were considerably low, which was consistent with the progress of oocytogenesis in the ovaries of topmouth culter. The high expression of these four genes in the ovaries of normal topmouth culter suggested they might involve in the preparation for the shift of oogenesis to ovulation. Hence, our work identified a set of annotated gene products that are candidate factors affecting reproduction in the topmouth culter H-P-G axis. These results could be essential for further research in functional genomics and genetic editing for topmouth culter reproduction.

Chronic brain toxicity response of juvenile Chinese rare minnows (Gobiocypris rarus) to the neonicotinoid insecticides imidacloprid and nitenpyram.

Imidacloprid and nitenpyram are widely used neonicotinoid pesticides worldwide and were observed to adversely affect non-target aquatic organisms. In this study, the toxic effect of imidacloprid and nitenpyram on the brain of juvenile Chinese rare minnows (Gobiocypris rarus) was investigated by determining the oxidative stress, 8-hydroxy-2-deoxyguanosine (8-OHdG) content and acetylcholinesterase (AChE) activity. The superoxide dismutase (SOD) activities did not significantly change after long-term exposure to imidacloprid and nitenpyram. A noticeable increase of catalase (CAT) activities was observed on the brain tissues under 0.1 mg/L imidacloprid and under all nitenpyram treatments (p < 0.05). The malondialdehyde (MDA) content increased markedly under 2.0 mg/L imidacloprid and 0.1 mg/L nitenpyram treatments (p < 0.05). The glutathione (GSH) content in the brain significantly increased under 0.5 and 2.0 mg/L imidacloprid (p < 0.05). A significant decrease was observed in the mRNA levels of Cu/Zn-sod under 2.0 mg/L imidacloprid and those of cat under 0.1 and 0.5 mg/L nitenpyram (p < 0.05). The mRNA levels of gpx1 clearly decreased under 2.0 mg/L imidacloprid and under 0.1 mg/L nitenpyram (p < 0.05). The treatments of 0.1 and 0.5 mg/L nitenpyram decreased cat expression levels markedly (p < 0.05). 2.0 mg/L imidacloprid raised the 8-OHdG content. The AChE activities increased markedly under 0.5 and 2.0 mg/L imidacloprid while clearly decreasing under 2.0 mg/L nitenpyram (p < 0.05). Therefore, our results indicate that imidacloprid and nitenpyram might cause adverse effects on juvenile Chinese rare minnows brain. Notably, imidacloprid had greater impacts on juvenile rare minnows compared to nitenpyram.

Maternal Bisphenol A exposure impaired endochondral ossification in craniofacial cartilage of rare minnow (Gobiocypris rarus) offspring.

Bisphenol A (BPA), an endocrine disrupting compound, is present in the aquatic environment. BPA can mimic estrogen and cause adverse effects on development and reproduction in different organisms. As epigenetic modifications due to BPA exposure have been reported, the interest on the effects of this chemical has increased. To assess the potential effects of maternal BPA exposure on offspring bone development, adult Gobiocypris rarus (G. rarus) females were exposed to 15 µg L-1 and 225 µg L-1 BPA for 21 days. Eggs were collected after artificial spawning and fertilized with the fresh milt of non-exposed male fish. The offspring were raised in clean water and randomly selected for examination at different development stages. Our results showed that specific effects including poor quality of the embryos, increased malformation (bent spine and tail), and delayed craniofacial cartilage ossification of the larvae. Additionally, the transcripts of ossification related genes were significantly downregulated in offspring, and the lysyloxidase activity decreased. The present study demonstrated the maternal-mediated skeleton toxicity of BPA and its adverse effects on G. rarus.

Survival rates and the occurrence of larval malformations, including Siamese twins, following fertilization of post-ovulatory aged oocytes in ide Leuciscus idus.

Embryonic development of lower vertebrates can be influenced by many factors, especially when outside of the mother's body and under the influence of variable abiotic and biotic factors. In this study, the effects of fertilization of post-ovulatory aged (overmature) oocytes of ide Leuciscus idus on the risk of developing anomalies in ide larvae were assessed under controlled conditions. There was a negative effect of delayed fertilization of post-ovulatory aged (overmature) oocytes in the ovary on the quality of offspring, which caused reduced survival 3 days post-fertilization and at the hatching stage (68.3 vs. 81.2% and 47.2 vs. 79.7% for the overmature and mature groups, respectively). It also increased the occurrence of numerous developmental defects (19.3 vs. 0.8% for the overmature and mature groups, respectively), including the development of Siamese twins. This is the first reported case of the occurrence of Siamese twins in ide. The ide Siamese twins look like hen eggs with 2 heads and are different from 'typical' Siamese twins in fish.

Toxic effects of triazophos on rare minnow (Gobiocypris rarus) embryos and larvae.

Triazophos (TAP) has been widely used in agriculture for controlling insect pests and is a known organophosphorus pesticide. Due to TAP characteristics, such as high chemical and photochemical stability, its potential toxicity to aquatic organisms has gained great interest. To explore the potential developmental toxicity of TAP, Gobiocypris rarus embryos and larvae were exposed to various concentrations of TAP (0.1-15 mg L(-1)) until 72 h. Results showed that values of 72 h LC50 and EC50 were 7.44 and 5.60 mg L(-1) for embryos, 2.52 and 1.37 mg L(-1) for larvae. Increased malformation, decreased heart rate and body length provide a gradual concentration-dependent pattern. Enzyme activities and mRNA levels were significantly changed even at low concentration (0.05 mg L(-1) for embryos and 0.01 mg L(-(1) for larvae). Overall, the present study points out that TAP is likely a risk to the early development of G. rarus. The information presented in this study will be helpful in better understanding the toxicity induced by TAP in fish embryos and larvae.

Developmental toxicity in rare minnow (Gobiocypris rarus) embryos exposed to Cu, Zn and Cd.

Using rare minnow (Gobiocypris rarus) embryos as experimental model, the developmental toxicity of Cu, Zn and Cd were investigated following exposure to 0.001-1.000mg/L for 72h, and the toxicity effects were evaluated by larval malformation rate, heart rate, pericardial area, spontaneous movements, tail length, enzyme activities and biomarker genes. Our results revealed that increased malformation rate provide a gradual dose-response relationship, and the most pronounced morphological alteration was heart and body malformations. Values of 72h EC50 with their 95 percent confidence intervals on G. rarus embryos were 0.103 (0.072-0.149)mg/L for Cu, 0.531 (0.330-1.060)mg/L for Zn, 0.219 (0.147-0.351)mg/L for Cd. Enzyme activities can be regard as a type of low-dose stimulation and high-dose inhibition. Stress and metabolism-related genes (hsp70, cyp1a and mt) were significantly up-regulated, development-related genes (wnt8a, vezf1 and mstn) were significantly down-regulated after the treatment by Cu, Zn and Cd. Overall, the present study points out Cu, Zn and Cd are highly toxic to G. rarus embryos. The information presented in this study will be helpful in fully understanding the toxicity induced by Cu, Zn and Cd in fish embryos.

Chronic exposure by ingestion of environmentally relevant doses of (226)Ra leads to transient growth perturbations in fathead minnow (Pimephales promelas, Rafinesque, 1820).

PURPOSE: To assess the impact of environmentally relevant levels of ingested (226)Ra on a common freshwater fish species. METHODS: Fathead minnow (Pimephales promelas, Rafinesque) were obtained at the first feeding stage and established on a commercial fish food diet containing (226)Ra in the activity range 10 mBq/g(-1), -10,000 mBq/g(-1). They remained on this diet for 24 months and were sampled invasively at 1,6,18 and 24 months to assess growth, biochemical indices and accumulated dose and non-invasively also at 12 and 15 months to assess growth. RESULTS: Fish fed 10 and 100 mBq/g(-1) diet showed a small transitory deregulation of growth at 6 and 12 months. Fish fed higher activities showed less significant or insignificant effects. There was a trend at 18 months which was stronger at 24 months for the population distribution to change in all of the (226)Ra fed groups so that smaller fish were smaller and bigger fish were bigger than the controls. There were also significant differences in the ratios of protein:DNA at 24 months which were seen as a trend but were not significant at earlier time points. CONCLUSIONS: Fish fed a radium diet for 2 years show a small and transitory growth dysregulation at 6 and 12 months. The effects predominate at the lower activities suggesting hormetic or homeostatic adjustments. There was no effect on growth of exposure to the high activities (226)Ra. This suggests that radium does not have a serious impact on the ecology of the system and the level of radium that would be transferred to humans is very low. The results may be important in the assessment of long-term environmental impacts of (226)Ra exposure.

Normal axial skeleton structure in common roach Rutilus rutilus (Actinopterygii: Cyprinidae) and malformations due to radiation contamination in the area of the Mayak (Chelyabinsk Province, Russia) nuclear plant.

This study was designed to describe normal axial skeletal structure in common roach Rutilus rutilus from putative unaffected environmental conditions, and the occurrence of skeletal malformations in the fish from an area under radiation contamination. Specimens were collected from water bodies of the Techa Cascade Reservoirs located near the Mayak atomic industry plant in the River Ob' drainage, Chelyabinsk Province, Russia. One sample was collected from Lake Irtyash, a reservoir of drinkable water, supplying the town of Ozersk, and the other one from a technical reservoir which is a storage of liquid radioactive waste from Mayak and characterized by high radioactive contamination (mostly (90)Sr and (137)Cs). A comparison was made with historical material collected from the River Ob' before the middle of the 20th century, i.e. before the environment became affected by radioactive contamination. A high number of abnormalities of the axial skeleton were detected in both Mayak samples, in 94 and 97% of examined specimens, in contrast to about 20% in the historical specimens. The abnormalities were in both the unpaired fins and the vertebral column, including the caudal complex and included supernumerary elements, fusions, deformities and displacement of the elements. Most axial skeleton abnormalities, however, were minor, such as splitting, shortening or deformation of spines. Severe defects, such as extensive scolioses, lordoses and kyphoses, were not found. The causes of the abnormalities were not identified in this study, but the high incidence of malformations may be attributed to genetically determined imbalance during development. The almost equal distribution of abnormalities among the fish from non-contaminated and radioactive contaminated reservoirs may be explained by either recent gene flow within the population of R. rutilus in the River Techa system or the effect of unknown unfavourable environmental factors such as chemical pollution.

Predicted exposures to steroid estrogens in U.K. rivers correlate with widespread sexual disruption in wild fish populations.

Steroidal estrogens, originating principally from human excretion, are likely to play a major role in causing widespread endocrine disruption in wild populations of the roach (Rutilus rutilus), a common cyprinid fish, in rivers contaminated by treated sewage effluents. Given the extent of this problem, risk assessment models are needed to predict the location and severity of endocrine disruption in river catchments and to identify areas where regulation of sewage discharges to remove these contaminants is necessary. In this study we attempted to correlate the extent of endocrine disruption in roach in British rivers, with their predicted exposure to steroid estrogens derived from the human population. The predictions of steroid estrogen exposure at each river site were determined by combining the modeled concentrations of the individual steroid estrogens [17beta-estradiol (E2), estrone (E1), and 17alpha-ethinylestradiol (EE2)] in each sewage effluent with their predicted dilution in the immediate receiving water. This model was applied to 45 sites on 39 rivers throughout the United Kingdom. Each site studied was then categorized as either high, medium, or low "risk" on the basis of the assumed additive potency of the three steroid estrogens calculated from data derived from published studies in various cyprinid fish species. We sampled 1,438 wild roach from the predicted high-, medium-, and low-risk river sites and examined them for evidence and severity of endocrine disruption. Both the incidence and the severity of intersex in wild roach were significantly correlated with the predicted concentrations of the natural estrogens (E1 and E2) and the synthetic contraceptive pill estrogen (EE2) present. Predicted steroid estrogen exposure was, however, less well correlated with the plasma vitellogenin concentration measured in the same fish. Moreover, we found no correlation between any of the end points measured in the roach and the proportion of industrial effluents entering the rivers we studied. Overall, our results provide further and substantive evidence to support the hypothesis that steroidal estrogens play a major role in causing intersex in wild freshwater fish in rivers in the United Kingdom and clearly show that the location and severity of these endocrine-disrupting effects can be predicted.

Sublethal toxicity of orchard stormwater runoff in Sacramento splittail (Pogonichthys macrolepidotus) larvae.

The sublethal effects of stormwater runoff from sections of a plum orchard treated with esfenvalerate or diazinon were evaluated in 7-day-old Sacramento splittail (Pogonichthys macrolepidotus) larvae. Fish were exposed to eight runoff samples using the USEPA standard static renewal method for 96 h acute toxicity testing, then transferred to clean water for three-month to assess the survival, growth, histopathological abnormalities, and heat stress proteins (hsp). No significant mortality was observed at 96 h in exposed fish. At one week, histopathological abnormalities included severe glycogen depletion, cytoplasmic protein droplets, vacuolar degeneration, and cell necroses in liver of all exposure groups. Pyknotic nerve cells were seen in brain of one exposure group. Significantly higher cumulative mortality, lower condition factor, and elevated hsp60 and hsp70 levels (p < 0.05) were occurred in several exposure groups. No histopathological abnormalities were observed after three months in any exposure group. This study confirms that standard acute toxicity tests have underestimated the toxicity of stormwater runoff, and although splittail larvae survived the 96 h exposure, they exhibited reduced survival and growth and showed signs of cellular stress even after a three month recovery period.

Selenium-induced teratogenicity in Sacramento splittail (Pogonichthys macrolepidotus).

Selenium is a potent reproductive and teratogenic environmental contaminant and there are concerns over possible reproductive effects of selenium on the Sacramento splittail (Pogonichthys macrolepidotus) population, a threatened species, in California, USA. In this study, the teratogenic effects of selenium were examined in splittail embryos exposed to 0.0, 5.0, and 15.0 mg l(-1) sodium selenite for 48-h at 18.0 degrees C under static conditions, with renewal every 12 h. Embryo development was evaluated daily for abnormalities from initiation of exposure (stage 27) to initiation of exogenous feeding. At the end of evaluation, prelarvae were preserved for histological analysis. There were no significant differences in mortality or hatching success between control and exposed embryos. Exposed fish had pericardial edema and deformities of skeletal tissues (loss of tail, lordosis, scoliosis, and kyphosis). Other histological alterations were limited to dysplasia, hyperplasia and metaplasia of skeletal tissues in the deformed fish. This study showed that a short exposure of embryos during somite development has significant effects on the musculoskeletal development.

Improved effluent quality at a bleached kraft mill as determined by laboratory biotests.

A life-cycle experiment with fathead minnows and Ceriodaphnia survival/reproduction tests were used to evaluate the quality of the effluent from a bleached kraft mill after the implementation of various process modifications and effluent treatment changes. In the life-cycle experiment, the fish were exposed in the laboratory to well water (control) and five concentrations (1.25%, 2.5%, 5%, 10%, or 20%) of effluent from the egg stage to sexual maturity and reproduction (approximately 190 d). None of the effluent concentrations significantly affected the hatching of the eggs, the mortality, weight, length, gender balance, reproduction, and prevalence of visible morphological or histopathological abnormalities of the hatched fish, and the hatchability of the first generation eggs. In Ceriodaphnia tests, the IC25 of the effluent affecting reproduction was approximately 80%. This threshold concentration is well above the 0.7% average yearly concentration of the effluent that exists in the recipient near the point of discharge. The results of these biotests were compared to the results of the same biotests conducted earlier with the effluent from the mill prior to process and treatment modifications. The comparison indicated that since the earlier work, the quality of the mill's effluent improved substantially. Threshold concentrations affecting fathead minnows in the life-cycle experiment and the Ceriodaphnia tests increased by more than eightfold and approximately twofold, respectively. While the most important change in the mill operating conditions responsible for the improvement could not be identified, these results indicate that mills can undertake process and treatment modifications that result in the discharge of effluents seemingly compatible with the aquatic environment.

Tributyltin-induced effects on early life stages of minnows Phoxinus phoxinus.

Toxicity and histopathological effects of tributyltin chloride (TBT) were studied in early life stages of minnows Phoxinus phoxinus. Eggs and yolk sac fry (newly hatched larvae) were exposed in a static-renewal procedure to aqueous TBT concentrations ranging from 0.82 to 19.51 micrograms/L for 3 to 10 days at 16 degrees C and 21 degrees C, respectively. Aqueous TBT concentrations were determined by capillary GC-FPD and revealed a concentration decrease during the static phase. TBT exposure led to mortality, behavioral, gross morphological and histopathological effects. In larvae, increased mortality, deformation of body axis, paralysis and opaque eyes occurred at 4.26 micrograms/L TBT and higher both in the embryonic-larval and larval exposure. Histological changes were evident at initial TBT concentrations of 0.82 up to 19.51 micrograms/L, and were more pronounced after embryonic-larval exposure than after larval exposure. Degenerative alterations occurred in skin, skeletal muscle, kidney, corneal epithelium, lens, pigment layer of the retina and choroid, retina, and CNS including spinal cord. Hydropic vacuolation of the cytoplasm and, in more pronounced cases, irreversible nuclear alterations such as pycnosis, karyorrhexis and karyolysis were also evident. Exposure to 0.82 micrograms/L TBT resulted in alterations in skin, muscle and kidney, with greater effects occurring at 21 degrees C than at 16 degrees C. Toxicity was significantly reduced in the presence of sediment. The observed histopathological effects suggest that early life stages of fish may be negatively affected in environments that are considerably polluted by TBT.