The notochord curvature in medaka (Oryzias latipes)

Journal of Photochemistry & Photobiology, B: Biology 164 (2016) 132–140

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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

The notochord curvature in medaka (Oryzias latipes) embryos as a response to ultraviolet A irradiation Alaa El-Din Hamid Sayed a,b,⁎, Hiroshi Mitani b a b

Zoology Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan

a r t i c l e

i n f o

Article history: Received 22 July 2016 Received in revised form 13 September 2016 Accepted 15 September 2016 Available online 19 September 2016 Keywords: Ultraviolet radiation Malformations Histopathology Vertebral column Medaka

a b s t r a c t In the present work, the destructive effects of ultraviolet A (UVA; 366 nm) irradiation on the developmental stages of Japanese medaka (Oryzias latipes) are revealed in terms of hatching success, mortality rate, and morphological malformations (yolk sac edema, body curvature, fin blistering, and dwarfism). Fertilized eggs in stage 4 were exposed to 15, 30, and 60 min/day UVA for 3 days in replicates. Fish were staged and aged following the stages established by Iwamatsu [1]. We observed and recorded the hatching time and deformed and dead embryos continuously. The hatching time was prolonged and the deformed and dead embryos numbers were increased by UVA dose increase. At stage 40, samples from each group were fixed to investigate their morphology and histopathology. Some morphological malformations were recorded after UVA exposure in both strains. Histopathological changes were represented as different shapes of curvature in notochord with collapse. The degree of collapsation was depended on the dose and time of UVA exposure. Our findings show that exposure to UVA irradiation caused less vertebral column curvature in medaka fry. Moreover, p53-deficient embryos were more tolerant than those of wild-type (Hd-rR) Japanese medaka. This study indicated the dangerous effects of the UVA on medaka. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Development of the notochord of the Japanese medaka (Oryzias latipes) has been studied [2], and provides a means of understanding the developmental basis of functional morphology. The notochord is a fundamental structure that distinguishes chordates from all other animals [2], and it is an essential organ for chordate development [3]. In all vertebrates except Amphioxus, the notochord found in the larva and the adult is postcranial, and severely reduced by the growth of the cartilaginous or bony vertebral column [2]. Many studies have been conducted to summarize the general structure and biochemical characteristics of the notochord and notochordal sheath [4–6,2]. These studies have emphasized that the fate of the layers of the notochordal sheath varies in different groups. They have also stated that the importance of those layers is inversely proportional to the state of the development of the vertebral column; the fibrous sheath is very thin, and only a vestige was left in tetrapods and teleosts.

⁎ Corresponding author at: Zoology Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt. E-mail addresses: [email protected], [email protected] (A. H. Sayed).

http://dx.doi.org/10.1016/j.jphotobiol.2016.09.023 1011-1344/© 2016 Elsevier B.V. All rights reserved.

The normal developmental process of O. latipes has been studied by many investigators [7–10,1]. The normal developmental stages in medaka have been reported in detail [10,1]. Iwamatsu used the development of the notochord as a principle feature in his study. Because O. latipes follows the typical teleostean pattern during development, it has been used as a model animal in a wide variety of research, particularly in embryonic development, because its eggs are transparent and easily observed under a microscope [11,10]. Ultraviolet A (UVA) irradiation is a natural stressor to most forms of life and no one cannot ignore the presence of UVA and its effects, although UVB radiation is of primary interest [12]. The dangerous effects of UVA irradiation have been studied generally in a variety of cell types [13,14], in adult fishes and aquatic animals [15–20], and fish embryos [12,21,22]. Developing fish embryos or larvae are considered the most sensitive life stages because they are particularly sensitive to all kinds of low-level environmental stressors [23,21]. Medaka has been used as a research organism in toxicological studies with adults, embryos, and early larval stages used as sensitive as indicators of aquatic toxicology [24,25,11,26– 32]. The purposes of our study were two folds. First to investigate the effects of UVA irradiation on the developmental stages of medaka. Second, to study the sensitivity of medaka strains wild-type (Wt) and p53 to UVA irradiation.

A.E.-D.H. Sayed, H. Mitani / Journal of Photochemistry & Photobiology, B: Biology 164 (2016) 132–140

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2. Materials and Methods

2.5. Morphology

2.1. Fish

Malformations were documented using a dissecting microscope (M125; Leica) and a digital colored video camera.

Sexually healthy mature wild-type (Hd-rR);(weight = 0.22 ± 0.027 g and length = 2.9 ± 0.057 cm) and homogenic p53-deficient (weight = 0.22 ± 0.020 g and length = 2.86 ± 0.13 cm) Japanese medaka (O. latipes) [31] were bred in 10 L plastic aquaria in water at room temperature at 26–28 °C under a 14 h light: 10 h dark cycle which is required for successful daily breeding [9], fed live brine shrimp (Artemia franciscana) and/or a powdered diet (TetraFin, Spectrum Brands Japan Inc., Tokyo, Japan) three times a day. The eggs, which remain attached to the abdomen of the female fish for several hours after spawning, were removed using brushes and allowed to develop at (pH = 6.75 ± 0.108, dissolved oxygen; DO = 6.37 ± 0.96 mg/L, salinity = 0 ± 0.0%, and temperature = 26 ± 0.35 °C).

2.2. Staging and Experimental Design Fish were staged and aged following the stages established by Iwamatsu [1]. This is the new set of staging criteria available for medaka and is based mainly on external features of live embryos. The stages were used in the present study as follows. Stage 4 Fish are 1 h 45 min old and at a 4 cell stage in which the second cleavage furrows develops on the two blastomeres at a right angle to the first cleavage plane. It deepens until each blastomere is divided into two of the same size. The oil droplets are larger, but fewer gather toward the vegetal pole. Stage 40 Fish are at the first fry stage and the period extends from hatching until the appearance of ray nodes in the caudal and pectoral fins, ribs in the trunk and neural spines on the vertebrate (4.5– 7.0 mm TL). Melanophores distribute bilaterally to the notochord and ventral vein, the dorsal surface of the body and the body cavity. The UVA irradiation doses was selected according to Mekkawy et al. [15]. The fertilized eggs (stage 4) in each group in triplicate were treated as follows at the same time: Group Ӏ: The fertilized eggs were kept for 14 days under the same laboratory conditions as the embryos in the other groups and were considered as control fish. Group ӀӀ: The fertilized eggs were exposed to UVA irradiation for 15 min/day for 3 days. Group ӀӀӀ: The fertilized were exposed to UVA irradiation for 30 min/day for 3 days. Group ӀV: The fertilized eggs were exposed to UVA irradiation for 60 min/day for 3 days.

2.3. UVA Exposure The fish were exposed to UVA irradiation using a UV lamp with 366 nm emission (UVL-56; UVP, San Gabriel, CA, USA). Experimental containers with the fertilized eggs were fitted with the UV lamp at 8 cm above the bottom of plastic petri dishes (10 cm × 2.5 cm). At this level, the intensity of irradiation with this lamp was 2450 μW/cm2 [12] as measured using a UV meter.

2.4. Sampling Exposure was started at stage 4. The first sampling point was at stage 40 (10 and 14 days old). Six embryos were collected at each sampling point and fixed for 24 h in Davidsons's solution (for gross and minor external malformation and histopathology). The embryos were observed daily with a dissecting microscope for mortality and morphological abnormalities.

2.6. Histopathology Fixed specimens were dehydrated and subsequently embedded in paraffin. Sagittal and transverse serial sections were cut at 5 μm and stained with hematoxylin and eosin (H&E). Stained sections were studied using an Olympus microscope (PX50) fitted with a digital Olympus color video camera (DP70). 2.7. Statistical Analysis One-way analysis of variance was conducted to analyze the data using SPSS software [33] at the 0.0001 significance level. Tukey's HSD test was used for multiple comparisons and verification of frequency of hatched, deformed, and dead embryos. Dunnett's t-test was used to compare the three groups of UVA-irradiated fish with a control group. 2.8. Ethics Statement All experiments were performed in accordance with Japanese laws and the guidelines for the care of experimental animals according to The University of Tokyo Animal Experiment Enforcement Rule. 3. Results 3.1. Hatching, Deformity and Mortality Rate The hatching process was started at 9 days (stage 39) after spawning. The total percentage of hatched embryos/fertilized eggs in Wt (Hd-rR) were 98.33 ± 1.67, 98.33 ± 1.67, 95 ± 2.24, and 93.33 ± 3.33, at 12 days (stage 40) after spawning in 0, 15, 30 and 60 min UVA irradiation for 3 days, respectively. The difference between groups was insignificant. Normal embryos and p53 (−/−) embryos exposed for 15, 30, and 60 min UVA irradiation for 3 days showed a different percentage of hatching rate with no significant numbers as 100 ± 0, 98.33 ± 1.67, 98.33 ± 1.67, and 96.67 ± 2.11 spawning in 0, 15, 30, and 60 min UVA irradiation for 3 days, respectively (Table 1; Fig. 1). More malformations were recorded in Wt (Hd-rR) embryos than in p53 (−/−) embryos in all groups exposed to UVA irradiation, but this frequency was not significantly different to that in control embryos in both strains. The percentage of deformed embryos/fertilized eggs in Wt (Hd-rR) and p53 (−/−) embryos were 0.17 ± 0.17, 0.5 ± 0.22, 0.67 ± 0.33, 1.67 ± 0.33, and 0.17 ± 0.17, 0.33 ± 0.21, 0.5 ± 0.22, 0.17 ± 0.17 at 12 days (stage 40) after spawning in 0, 15, 30, and 60 min UVA irradiation for 3 days, respectively. The difference between groups was significant in Wt fish but not significant in fish in the p53 (−/−) group. The same pattern of mortality, where a difference between groups was significant in Wt (Hd-rR), while there was no significant difference in p53 (−/−) embryos. The percentage of mortality rate/fertilized eggs in Wt (Hd-rR) and p53 (−/−) were 0.17 ± 0.17, 0.33 ± 0.2, 0.67 ± 0.33, 1.33 ± 0.33, and 0 ± 0, 0.33 ± 0.21, 0.5 ± 0.22, and 0.67 ± 0.33 at 14 days (stage 40) after spawning in 0, 15, 30, and 60 min UVA irradiation for 3 days respectively (Table 1; Fig. 1). The data indicate that exposure to UVA irradiation caused a time-dependent exposure in decrease hatching rate, increase in both of malformation and mortality rate in embryos that successfully completed the egg stage. 3.2. Morphological Malformations in Posthatched Stages Four gross morphological malformations were observed: yolk sac edema (accumulation of body fluid in the region of the yolk sac), head

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Table 1 Hatching, deformed and mortality rate percentage as mean ± SE (range) after exposure to UVA in embryos of medaka Oryzias latipes.

Wild Type Fish

p53 −/− Mutant Fish

UVA dose

0 (n = 30)

Stage

40⁎⁎

15 min (n = 33)

30 min (n = 33)

60 min (n = 30)

Hatching rate % Deformed rate % Mortality rate %

98.33 ± 1.67a(90–100) 0.17 ± 0.17b (0–1) 0.17 ± 0.17b (0–1)

98.33 ± 1.67a (90–100) 0.5 ± 0.22b (0–1) 0.33 ± 0.21ab (0–1)

95 ± 2.24 a (90–100) 0.67 ± 0.33ab (0–2) 0.67 ± 0.33ab (0–2)

93.33 ± 3.33a (80–100) 1.67 ± 0.33a (1–3) 1.33 ± 0.33a (0–2)

Dose of irradiation Stage Hatching rate % Deformed rate % Mortality rate %

0 (n = 42) 40⁎⁎ 100 ± 0a (100−100) 0.17 ± 0.17a (0–1) 0 ± 0a (0–0)

15 min (n = 30)

30 min (n = 30)

60 min (n = 30)

98.33 ± 1.67 a(90–100) 0.33 ± 0.21a (0–1) 0.33 ± 0.21a (0–1)

98.33 ± 1.67a (90–100) 0.5 ± 0.22a (0–1) 0.5 ± 0.22a (0–1)

96.67 ± 2.11a (90–100) 0.17 ± 0.17a (0–1) 0.67 ± 0.33a (0–1)

⁎Different letters a, b, c and d showed significantly different from non-irradiated controls, P b 0.0001. ⁎⁎ Staging was according to Iwamatsu [1].

edema (accumulation of body fluid in the region of head), notochordal defects (lordosis, dorsoventral curvature; kyphosis, ventrodorsal curvature; scoliosis, lateral curvature; and flat-S-shape curvature), and dwarfism with fin blistering. Some of the affected embryos were recorded with combinations of these malformations.

3.3. Yolk Sac Edema Yolk sac (balloon-shape) edema was observed only in Wt (Hd-rR) embryos exposed to 60 min UVA irradiation for 3 days (Fig. 2e). Yolk sac edema was also associated with notochordal curvature, fin

Fig. 1. Hatching, deformity and mortality rate percentage as mean ± SE (range) after exposure to UVA irradiation in embryos of medaka Oryzias latipes.


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Fig. 2. Notochordal abnormality (body curvature) in the embryos of Oryzias latipes; Wt (Hd-rR) after exposure to UVA irradiation showing: (a) control (stage 40, 14 days); (b) kyphosis in embryos (stage 40, 10 days) exposed to 15 min UVA irradiation; (c) flat-S-shaped curvature in embryos (stage 40, 14 days) exposed to 30 min UVA irradiation; (d) scoliosis in embryos (stage 40, 14 days) exposed to 30 min UVA irradiation; (e) kyphosis with yolk sac (Yso) and head edema (Ho) in embryos (stage 40, 10 days) exposed to 60 min UVA irradiation; and (f) scoliosis, dwarfism, and fin blistering in embryos (stage 40, 10 days) exposed to 60 min UVA irradiation. Scale bar = 1 mm.

blistering, and head edema. Yolk sac malformation caused abnormal growth, so that edematous embryos were usually shorter than normal.

3.4. Body Curvature The most frequently observed gross morphological deformation was notochordal curvature. Different types of notochord curvature were observed in both Wt (Hd-rR) and p53 (−/−) embryos at different doses of UVA irradiation as: (1) lordosis (dorsoventral curvature) (Fig. 3c, d), (2) kyphosis (ventrodorsal curvature) (Fig. 2b, c), (3) scoliosis (lateral curvature) (Figs. 2d, e and 3b, d), and (4) flat S-shape curvature (Figs. 2c and 3e).

3.5. Dwarfism Fin Blistering Fin blistering was observed only in embryos exposed to UVA irradiation for 60 min for 3 days. The membranous fin was blistered and degenerated (Fig. 2e, f). Fin blistering was often associated with yolk sac edema and notochord curvature. Gross anatomy showed dwarfism (the embryos became shorter than unexposed embryos in the same developmental stage). Dwarfism was often associated with yolk sac edema and fin blistering.

3.6. Histopathological Changes in Pot-hatching Stages In this study, the only histopathological alterations we examined were in the notochord collapse (notochordal defects), which was recorded at different ages (10 days and 14 days old) of the medaka O. latipes embryos after exposure to UVA (366 nm) irradiation for 15 min, 30 min, and 60 min for 3 days.

3.7. Malformations of the Notochord The detailed description of the structure of the normal notochord of the medaka was done by [2] and the current results in agreement with that description, where the notochord is consists of highly vacuolated, connected cells enclosed with a sheath (a notochordal sheath), which overlies the most superficial notochord cells that from the notochordal epithelium. The embryos exposed to UVA irradiation showed a variable degree of collapse in the notochord compared with to the control embryos, which have a uniform shape in both Wt (Hd-rR) and p53 (−/−) embryos (Figs. 4a and 5a). The degree of collapse increased with the increasing of UVA irradiation exposure time in both strains, but this degree was high in Wt (Hd-rR) and p53 (−/−) embryos in the postpharyngeal region. The embryos (stage 40, 10 days old) exposed to UVA irradiation for

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Fig. 3. Notochordal abnormality (body curvature) in the embryos of Oryzias latipes: p53(−/−) embryos after exposure to UVA irradiation showing (a) control (stage 40, 10 days); (b) scoliosis in embryos (stage 40, 10 days) exposed to 15 min UVA irradiation; (c) lordosis in embryos (stage 40, 14 days) exposed to 30 min UVA irradiation; (d) scoliosis in embryos (stage 40, 14 days) exposed to 30 min UVA irradiation; (e) flat-S-shaped curvature in embryos (stage 40, 10 days) exposed to 60 min UVA irradiation. (f) lordosis in embryos (stage 40, 14 days) exposed to 60 min UVA irradiation. Scale bar = 1 mm.

15 min had a weak collapsed notochord with partially reduced vacuolated cells (Figs. 4b and 5b). Collapsed notochord with change in vacuolated cells was recorded in the groups exposed for 30 and 60 min for 3 days (Figs. 4c, d and 5c, d). In embryos (stage 40, 14 days old) exposed to UVA irradiation for 15, 30 and 60 min for 3 days, minor degrees of collapse of in the notochord and no damage to vacuolated cells (Figs. 4e–h and 5e–h). Severely collapsed notochord with partially reduced vacuolated cells were observed in trunk and tail regions in embryos (stage 40, 14 days old) exposed to UVA compared with control (Fig. 6), but the degree of this damage was increased by an increase in UVA exposure time. Wt (Hd-rR) embryos showed a higher degree of damage than p53 (−/−) embryos. 4. Discussion Although there are many studies on the impacts of UVB in fish embryos, no detailed information is available on malformation induced by UVA irradiation of fish embryos [12,21,34,35]. The data reported in the present work indicates the dangerous effects of UVA irradiation exposure on the hatching, deformity rate and mortality of medaka embryos. The present study demonstrates that, the UVA irradiation doses used here had acute effects on medaka embryos. Many studies have reported variable levels of damage on the development stages of medaka by other stressors [24,25,11,26–31]. Retardation in the embryo development compared with the normal development was also described

[10]. The delay in embryo development may be a consequence of derangement of cell division and gene expression by the embryos [15]. In the present study, anatomopathological studies were conducted on control embryos and on UVA irradiation-treated embryos (stage 40, 14 days old). Our results showed a quantitative increase of the notochord curvature in a dose-dependent manner. It should also be considered that there are many fish hatching strategies depending on the differences in hatching timing, movements, and method between the same species developing under natural and hatchery conditions [36]. Deformities and hatching failure of medaka embryos were reported after exposure to polycyclic aromatic hydrocarbons [37,38]. Phototoxicity to eggs and larvae of fathead minnow after UVA irradiation resulted in significantly lowered hatching success, and high deformities in larvae [39]. Exposure of medaka eggs and larvae to water-accommodated UVA irradiation was reported to cause observed developmental effects such as yolk sac edema [40]. Ultraviolet radiation inhibited epiboly, which affected notochord development in zebrafish [41]. In the present work, such specific UVA irradiation-induced changes were also recorded under the influence of other types of stress [42,43] and after UVA exposure [21]. Nonspecific UVA irradiation-induced changes recorded in the present work were also recorded by many others after exposure of a variety of fishes to various toxicants [24– 27]. The stress-induced changes after their exposure to UVA irradiation were more pronounced in the early embryonic stages of fishes [21]. The morphological and histological changes were mediated by reactive


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Fig. 4. Notochord malformation in the postpharyngeal region of Oryzias latipes; Wt (Hd-rR) embryos after exposure to UVA (366 nm) irradiation showing (a) control (stage 40, 10 days); (b) collapsed notochord in embryos (stage 40, 10 days) exposed to 15 min UVA irradiation; (c) collapsed notochord in embryos (stage 40, 10 days) exposed to 30 min UVA irradiation; (d) collapsed notochord in embryos (stage 40, 10 days) exposed to 60 min UVA irradiation; (e) normal notochord in embryos (stage 40, 14 days) as a control; (f) normal notochord in embryos (stage 40, 14 days) exposed to 15 min UVA irradiation; (g) collapsed notochord in embryos (stage 40, 14 days) exposed to 30 min UVA irradiation; and (h) collapsed notochord in embryos (stage 40, 14 days) exposed to 60 min UVA irradiation. CC = central canal, SC = spinal cord, N = notochord, VC = vacuolated cells, and NE = notochordal epithelium, Staining: H&E.

oxygen species in addition to DNA damage after exposure to UVA irradiation [15]. The UVA-irradiation induced morphological and pathological changes recorded in the present work confirm the UVA-irradiation induced photoinhibition of the antioxidant defense system of embryo of O. latipes [12]. Similar findings were reported in other publications after embryos of Japanese medaka O. latipes [12], zebrafish Danio rerio [34],

and African catfish Clarias gariepinus were exposed to UVA irradiation [25]. Many alterations have been observed after exposure to UVA irradiation in O. latipes, such as complete mortality at day 9, lack of normal fin development, abnormal swimming movements, spinal column malformation, and hatching success [2]. Although, the study by Bass and Sistrun [12] illustrated the significant influence of UVA irradiation of eggs on development of fish that inhabit shallow and open water, it

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Fig. 5. Notochord malformation in the postpharyngeal region of Oryzias latipes; p53(−/−) embryos after exposure to UVA (366 nm) irradiation showing (a) control (stage 40, 10 days); (b) collapsed notochord in embryos (stage 40, 10 days) exposed to 15 min UVA irradiation; (c) collapsed notochord in embryos (stage 40, 10 days) exposed to 30 min UVA irradiation; (d) collapsed notochord in embryos (stage 40, 10 days) exposed to 60 min UVA irradiation; (e) normal notochords in embryos (stage 40, 14 days) as controls; (f) normal notochords in embryos (stage 40, 14 days) exposed to 15 min UVA irradiation (g) collapsed notochords in embryos (stage 40, 14 days) exposed to 30 min UVA irradiation; and (h) collapsed notochords in embryos (stage 40, 14 days) exposed to 60 min UVA, Staining: H&E.

lacks the morphological and histopathological investigation of consequences resulting from exposure to UVA irradiation. The UVA irradiation doses used in the present study can be considered as sublethal doses for medaka embryos. Our previous publication using African catfish as an animal model showed many embryotoxicological results after exposure to the same UVA irradiation doses as used in the present. The incubation period was prolonged, the hatching process delayed, and

mortality increased after the exposure to UVA irradiation. Moreover, morphological malformations (such as yolk sac edema, body curvature, fin blistering, and dwarfism) and histopathological alterations (such as abnormal gill shape, retina degeneration, villi malformations, degeneration of gray matter and central canal, notochord collapse, and cellular alterations) in liver, skin, and kidney organs after exposure to UVA irradiation were also present. The degree of damage was correlated


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Fig. 6. Notochord malformation at trunk and tail regions in embryos of Oryzias latipes: a, c, e, and g for Wt (Hd-rR) and b, d, f, and h for p53(−/−) embryos after exposure to UVA (366 nm) irradiation showing (a and b) controls (stage 40, 10 days); (c, d) collapsed notochords in embryos (stage 40, 10 days) exposed to 15 min UVA irradiation; (e and f) collapsed notochord in embryos (stage 40, 10 days) exposed to 30 min UVA irradiation; and (g and h) collapsed notochord in embryos (stage 40, 10 days) exposed to 60 min UVA irradiation, Staining: H&E.

with the dose of UVA irradiation, organ location, embryonic stage, and pigmentation [21]. Zebrafish embryos typically exhibited spinal deformities, enlarged pericardial sacs, and severe spinal curling or twisting within 5 days following fertilization as a result of being exposed to UVB (0.93 J/cm2) or large UVA (211.5 J/cm2) irradiation doses at the midgastrula stage [34]. Embryos exposed to UVA irradiation at the midgastrula stage tolerated UVA irradiation doses as high as 635 J/cm2, 761 J/cm2, and 846 J/cm 2 without significant loss of hatching, higher incidence of malformation, or mortality when compared with

unirradiated controls. Dong et al. [34] concluded that zebrafish embryos had high tolerance of UVA irradiation, which confirms their suitability as models photoactivation and photorepair effects. 5. Conclusion Although, medaka embryos were very weak, they were resistant to UVA irradiation at doses higher than African catfish, but less than zebrafish. Accordingly, it appears that the medaka embryos are a

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promising model for studying the effects of UVA irradiation. By contrast, p53 (−/−) embryos showed more resistance to the effects of UVA irradiation than Wt (Hd-rR) embryos confirming our previously published work after gamma and UVA irradiation of medaka [44,45,32]. Beside, this study should be considered by researchers in fish farming and aquaculture sector. Acknowledgments This research was supported in part by the Japan Society for the Promotion of Science (FY2015 JSPS postdoctoral fellowship for overseas researchers) to Alaa El-Din H. Sayed (ID No. P15382). References [1] T. Iwamatsu, Developmental stages in the wild medaka, Oryzias latipes, Bull. Aichi Univ. Edu. 60 (2011) 71–81. [2] S. Ekanayake, B.K. Hall, Development of the notochord in the Japanese medaka, Oryzias latipes (Teleostei; Cyprinodontidae), with special reference to desmosomal connections and functional integration with adjacent tissues, Can. J. Zool. 69 (1991) 1171–1177. [3] D.L. Stemple, Structure and function of the notochord: an essential organ for chordate development, Development 132 (2005) 2503–2512. [4] B.G. Gardiner, Gnathostome vertebrae and the classification of the Amphibia, Zool. J. Linnean Soc. 79 (1983) 1–59. [5] B.K. Hall, Chondrogenesis of the Somitic Mesoderm, Springer-Verlag, 1977. [6] J. Camon, E. Degollada, J. Verdu, Ultrastructural aspects of the production of extracellular matrix components by the chick embryonic notochord in vitro, Acta Anat. 137 (1990) 114–123. [7] H. Gamo, I. Terajima, The normal stage of embryonic development of the medaka, Oryzias latipes, Jpn. J. Ichthyol. 10 (1963) 31–38. [8] T. Iwamatsu, The medaka as a biological material. III. Observations of developmental process, Bull. Aichi Univ. Edu. 25 (1976) 67–89. [9] T. Yamamoto, Stages in development, in: T. Yamamoto (Ed.), Medaka (Killifish): Biology and Strains, Keigaku Publ. Co, Tokyo 1975, pp. 30–58. [10] T. Iwamatsu, Stages of normal development in the medaka Oryzias latipes, Mech. Dev. 121 (2004) 605–618. [11] J.-G. Cho, K.-T. Kim, T.-K. Ryu, J.-w. Lee, J.-E. Kim, J. Kim, B.-C. Lee, E.-H. Jo, J. Yoon, I.-c. Eom, K. Choi, P. Kim, Stepwise embryonic toxicity of silver nanoparticles on Oryzias latipes, Biomed. Res. Int. 2013 (2013) 7. [12] E.L. Bass, S.N. Sistrun, Effect of UVA radiation on development and hatching success in Oryzias latipes, the Japanese medaka, Bull. Environ. Contam. Toxicol. 59 (1997) 537–542. [13] M.J. Peak, J.G. Peak, M.E. Churchill, Cellular and molecular effects of UVA radiation and visible light in mammalian cells, in: F. Urbach (Ed.), Biological Responses to UVA Radiation, Overland Park, Valdenmar 1992, pp. 39–45. [14] B.M. Sutherland, H. Hacham, R.W. Gange, J.C. Sutherland, Pyrimidine dimer formation by UVA radiation: implications for photoreactivation, in: F. Urbach (Ed.), Biological Responses to UVA Radiation, Overland Park, Valdenmar 1992, pp. 47–58. [15] I.A.A. Mekkawy, U.M. Mahmoud, A.G. Osman, A.E.H. Sayed, Effects of ultraviolet A on the activity of two metabolic enzymes, DNA damage and lipid peroxidation during early developmental stages of the African catfish, Clarias gariepinus (Burchell, 1822), Fish Physiol. Biochem. 36 (2010) 605–626. [16] A.G.M. Osman, M. Koutb, A.H. Sayed, Use of hematological parameters to assess the efficiency of quince (Cydonia oblonga Miller) leaf extract in alleviation of the effect of ultraviolet – a radiation on African catfish Clarias gariepinus (Burchell, 1822), J. Photochem. Photobiol. B Biol. 99 (2010) 1–8. [17] A.H. Sayed, H.S. Abdel-Tawab, S.S. Abdel Hakeem, I.A. Mekkawy, The protective role of quince leaf extract against the adverse impacts of ultraviolet-A radiation on some tissues of Clarias gariepinus (Burchell, 1822), J. Photochem. Photobiol. B Biol. 119 (2013) 9–14. [18] A.H. Sayed, A.T. Ibrahim, I.A.A. Mekkawy, U.M. Mahmoud, Acute effects of ultraviolet-A radiation on African catfish Clarias gariepinus (Burchell, 1822), Photochem. Photobiol. B Biol. 89 (2007) 170–174. [19] S. Banerjee, M. Leptin, Systemic response to ultraviolet radiation involves induction of Leukocytic IL-1b and inflammation in zebrafish, J. Immunol. 193 (2014) 1408–1415. [20] J.P. Campanale, L. Tomanek, N.L. Adams, Exposure to ultraviolet radiation causes proteomic changes in embryos of the purple sea urchin, Strongylocentrotus purpuratus, J. Exp. Mar. Biol. Ecol. 397 (2011) 106–120.

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