2 Tianjin Research Institute for Water Transport Engineering, Ministry of Transport, Tianjin 300456, China;
3 Ocean University of China, Qingdao 266100, China
During the early life history, especially the embryonic stage, the morphology and physiology of fish may change dramatically in a short period, and be very sensitive to the environmental changes. Thus, fish embryos are often selected to study the toxicological effects of organic pollutants and heavy metals (Ishibashi et al., 2004; Faria et al., 2017). Compared with adult fish, embryos can respond to environmental pollutants more sensitively and quickly, so they can be used as an effective means of evaluating water pollution (Hong et al., 2014). So far, embryos of Danio rerio (Wilkinson and van Eeden, 2014) and Oryzias latipes (Bo et al., 2011) have been widely used in toxicological studies for freshwater environments. In recent years, with the rapid development of molecular genetics combined with genomics, fish embryos have also been applied in drug development and human disease research. For example, it was discovered that a variety of mutations in D. rerio are similar to those that cause human diseases. Hence, the embryos of D. rerio can be used as a classic model for studying human vascular development and hematopoietic disorders (Gore et al., 2018).
For the use of fish embryos as models for either environmental pollution assessment or medical research, it is fundamental to understand normal and abnormal embryogenesis, including the distinction between pathological and non-pathological abnormalities. So far, many studies have focused on the impact of external environmental factors on abnormal development of fish embryos or gene expression (Scholz and Gutzeit, 2000; Yu et al., 2006). However, a growing number of studies have suggested that birth defects are an often-overlooked feature of heredity (Negrín-Báez et al., 2015; Kyriakis et al., 2019). In this case, it is very important to study the abnormal development of non-pathological embryos in model fish due to genetic factors (aging, low fertility or low matching rate) without external impact.
Recent works have showed that embryos abnormalities of fish are related to the age of the broodstock, egg, and sperm quality, and reproductive strategies. Duangkaew et al. (2020) found that type-A spermatogonia (ASG) numbers in medaka (O. latipes) increased continuously from pre-puberty stage until adolescence stage, but decreased in senescence stage. Riesco et al. (2019) used zebrafish as a model, explored the impact of low sperm quality on offspring. They found that the fertilization and malformation rates of offspring with low-quality sperm were significantly worse than samples with high-quality sperm.
Oryzias melastigma, a new model animal for marine ecotoxicology (Wittbrodt et al., 2002; Bo et al., 2011), has many advantages. First, the small-sized adult body length of about 2–3 cm makes it easy to cultivate indoor on large scale. Secondly, the rapid sexual maturity allows it to lay eggs at 3–4-month-old with the capability of daily spawning. Thirdly, the transparent eggs with a slow embryo development process favor observations in detail. Fourthly, it is sensitive and responds quickly to the external environment. Furthermore, it has irreplaceable advantages as a key species for studies of pollution indicators and endocrine disruptors in the context of marine ecological degradation. So far, preliminary studies have been performed on early development, toxicology, and the gene expression of O. melastigma. However, no results have been reported on the abnormal development of non-pathological embryos.
Oryzias melastigma was selected for 185-day microscopic observations of embryogenesis. Remarkable anomalies during the main stages of embryogenesis were observed and analyzed, and the results were compared to several other economic fish with similar egg types. The findings may provide the further understanding of the early life history of this model animal, provide evidence for comparative analysis in toxicology and molecular biology research, and provide an important theoretical basis for the breeding of these economic fish.2 MATERIAL AND METHOD 2.1 Medaka fish maintenance
Oryzias melastigma was used as the model fish and was cultivated in a climate chamber with constant temperature for generations. Four month-old O. melastigma with a body length of 28±2 mm were used as the broodstock and incubated in a glass tank (30 cm×20 cm×25 cm) with 12-L water. Two females and two males were cultivated as a group in one tank and five groups were cultivated. Each group was fed with nauplii of Artemia salinat twice every morning and evening. The light and dark period was set as 14 h and 10 h, respectively. Water for the experiment was filtered from Shazikou of Qingdao and controlled at a salinity of 31 and a temperature of 27±1 ℃. Water was half exchanged daily to remove feces and eggs from the tank bottom.2.2 Eggs collection and hatching
The experiment was carried out in 2019 from Apr. 9 to Oct. 10 (185 d in total). The spawning of broodstock was observed from 7:00 AM to 10:00 AM every day. Once clusters of fertilized eggs were observed near the cloaca of female, broodstock were caught and placed in a plastic bowl lined with cotton. Subsequently, fertilized eggs were transferred by a plastic pipette and placed on a Petri dish for the direct observation of embryos. The embryos were transferred to 500-mL beakers by pipette and cultivated in filtered seawater with spawning recorded daily.2.3 Data analysis
Data of egg production per day, fertilization rate, hatching rate, and the abnormality rate were analyzed using Excel 2013 and Origin 8. The abnormality rate of pseudo-fertilization was calculated by dividing the number of pseudo-fertilized eggs by the total number of eggs of the day. The ratio of blastomere separation and dislocation was equal to the number of eggs of the abnormal shape that divided by the number of fertilized eggs. In addition, the calculation of the ratio of more Kupffer's vesicles, double blastoderms was the same as above.
The abnormality types is described as follows. (1) Pseudo fertilization (PSF): unlike unfertilized eggs, oil droplets in the pseudo-fertilized eggs could gathered in plant poles and coalesced into one single oil droplet in 10 h, which was faster than normal embryos. Subsequently, the blastoderm formed, but could not start cell division as normal fertilized eggs did. (2) Blastomeres separation (BLS): blastomeres were excessively separated from each other. (3) Blastomeres displacement (BLD): the blastoderm lacked a symmetry axis during cleavage, and did not form bilaterally symmetrical rows of blastomeres. (4) More Kupffer's vesicles (MKV): two or more Kupffer's vesicles appeared on the underside of the caudal end of the body. (5) Double blastoderms (DOB): one egg has two separated blastoderms. (6) Double embryonic bodies (DEB): some double blastoderms eventually developed into double embryonic bodies, that is, two embryos in one egg. Some double embryonic bodies had only the tail connected and the head separated from the trunk, and some double embryonic bodies shared the heart with only the head separated.2.4 Image capture and process
The development of fish eggs was observed with an Olympus SZX16 stereo microscope according to the published reports on the embryo development of O. melastigma and the abnormality in the development of the embryos was screened and imaged by the SONY H-50 camera. Accuracy of the experiment was assured by reverse tracing of observed abnormal embryos.3 RESULT AND DISCUSSION 3.1 Spawning of O. melastigma
Oryzias melastigma bred every morning and laid eggs naturally. Fertilized eggs were suspended near the cloaca of the broodstock by filaments for several hours followed by fall-off. Broodstock in the prosperous period of spawning completed fertilization within an hour in the morning under light. However, broodstock in the senescence period needed 3–4 h or more to complete breeding under the same conditions.
Oryzias melastigma quickly reached sexual maturity within 90–100 d at 27±1 ℃. Initially, females typically laid 2–3 eggs with low fertility. Within 7–10 d of development, female O. melastigma laid more than 10 eggs and this ability lasted for about 100 d. Females that were 7- and 8-month-old entered a prosperous period and laid 50–70 eggs per day (up to 102 eggs recorded). After the initial, rising, and prosperous periods of spawning, most O. melastigma entered a senescence period under laboratory conditions with a quick decline in spawning ability (Fig. 1a).
During the pre-puberty and adolescence stage, the fertilization rate maintained at a high level (71%±6% to 96%±3%) (Fig. 1b). Until Day 165 of the experiment, the 9-month-old female of O. melastigma entered the senescent stage, and the fertilization rate showed a fluctuating decline down to as low as 34%. Unlike the egg production rate and the fertilization rate, the hatching rate of normally fertilized eggs was high ranging from 81%±4% to 92%±7%. The most representative abnormalities in the development of O. melastigma were pseudo-fertilization, blastomeres separation and displacement, more Kupffer's vesicles, double blastoderms, and two embryonic bodies. The highest daily pseudo-fertilization rate was 47.73% recorded on May 18 (Day 40), while the highest blastomeres separation to displacement ratio was 11.46% on April 13 (Day 5) (Fig. 1c). The occurrence of abnormality with more Kupffer's vesicles and double blastoderms was relatively lower than that of other abnormalities, being 3.47% and 0.29%, respectively. Nine conjoined twins were found during the 185-day experiment, accounting for 0.02% of normal fertilized eggs (39 286). Nearly half of the conjoined twins occurred after Day 168 in the senescent stage.
The proportion of conjoined twins (0.02%) was much lower than double blastoderms (0.29%). By tracking the development of the eggs with double blastoderms, we found that most separated blastomeres (>90%) can be fused to normal embryos. Only a few double blastoderms failed to fuse and eventually formed a conjoined twin. The double blastoderms were observed in the eggs produced by females at various ages, but the conjoined twins occurred in the senescence stage mainly. We speculate that the ability to regulate the gastrulation is weakened with age (Zhu, 1982; Spencer, 2001), so the isolated blastoderm cells cannot fuse again and eventually form a conjoined twin.
Morphological abnormalities are common in fish embryos development. Pseudo-fertilization could occur during embryos development of cold-water fish such as Verasper moseri (Xiao et al., 2008; Du et al., 2010) and Gadus macrocephalus (Bian et al., 2014; Yusupov, 2016). Moreover, Kraeussling et al. (2011) found that the medaka embryos lacked axial symmetry in the four-cell stage, resulting in the blastomeres dislocation. Like O. melastigma, fertilized eggs of these fish require 9–14 d to hatch. The relatively long incubation time provides an opportunity for embryo self-regulation. Even if the embryo develops abnormally in early stage, it can be self-fixed later.3.2 Morphology abnormalities of O. melastigma embryos
The embryo development of O. melastigma can be divided into eight stages in chronological order and morphological features, namely, activated egg stage, blastodisc stage, cleavage stage, blastula stage, gastrula stage, neurula stage, organogenesis stage, and hatching stage (Iwamatsu, 2004; Chen et al., 2016; Wang et al., 2017). The observed morphological abnormalities at all stages of embryogenesis are described below.3.2.1 Activated egg stage and blastodisc stage
Under normal conditions, females laid mature fertilized eggs, which appeared as translucent spheres (Fig. 2a). The eggs were followed by oil droplets (Ishigaki et al., 2016), which gathered to plant poles within 10 min (Fig. 2b). However, unfertilization or pseudo-fertilization frequently occurred in cases where females had initially reached the pre-puberty or senescence stage or males had a low sperm quality. Oil droplets were sparsely distributed around yolk spheres in unfertilized eggs without aggregation (Fig. 2c & d). Unlike unfertilized eggs, in pseudofertilized eggs, oil droplets gathered around plant poles and coalesced into one oil droplet within 10 h, which was faster than in normal embryos (Fig. 2e & f). Blastoderm was formed subsequently. However, this could not be divided as in normal cells.
At the beginning of the study, O. melastigma females were sexually mature in about 10 d and a low fertilization rate was observed. Pseudo-fertilized embryos became pseudo-blastocysts (Fig. 2e & f). On Day 140, broodstock were 8-month-old, and the spawning ability reached 60–100 eggs per day, while a large number of pseudo-blastocysts remained. On Day 180, the broodstock entered a senescent stage and a large number of unfertilized eggs were observed in addition to the pseudo-blastocyst fertilization rate below 30%. Preliminary analysis showed that the fertilization rate was closely correlated to the reproductive ability of the broodstock. Either sexual immaturity or senescence affected the fertilization of eggs. The formation of pseudo-blastocysts was likely to be related to the age of fish and the egg quality (Brooks et al., 1997; Bobe and Labbé, 2010).
In aquacultural practice, the successful culture of any fish species depends always on stable supply of quality fertilized eggs, i.e. good sperm and oocyte and high gamete matching. In some flatfish, e.g., summer flounder (Paralichthys dentatus), barfin flounder (V. moseri), and half-smooth tongue-sole (Cynoglossus semilaevis Gunther), the acquirement of quality fertilized eggs during the embryogenesis of their ontogeny remains unresolved, which often come with the of unfertilized or pseudo-fertilized eggs. Therefore, we should pay attention to the factors that affect the reproductive ability of broodstock to improve the quality of eggs.3.2.2 Cleavage stage
Similar to other teleosts, O. melastigma embryos started to form blastoderm 1 h after fertilization, followed by cell division. In this study, various abnormal embryogenesis processes were observed in the two-cell stage or beyond. The most common observation was blastomeres separation, which was first observed in the two-cell stage. In the normal twocell stage, there were two blastomeres of equal size in hemispheric shape, and closely stick to each other (Fig. 3a). The abnormally developed blastomeres were spherical and separated from each other (Fig. 3b & c). The excessive blastomeres separation was closely related to the subsequent formation of the conjoined twins.
The second cleavage of a fertilized egg forms normally four blastomeres of similar size and shape (Fig. 4a). However, we observed abnormities during cultivation, including separation (Fig. 4b) and displacement (Fig. 4c–e). The abnormal blastomeres were distinct in size and shape indicating asynchronous division. Recent works have found that the first desynchronization of blastomere divisions in some medaka (Kraeussling et al., 2011) and zebrafish (Olivier et al., 2010; Mendieta-Serrano et al., 2013) embryos may appear in the four-cell stage, even in the two-cell stage. This desynchronization was also observed in the embryonic development of marine fish such as V. moseri and G. macrocephalus, and some eventually formed normal embryos (Xiao et al., 2008; Yu et al., 2014). In the four-cell stage, recleavage was also observed in a single blastomere, and small cells were found within each blastomere (Fig. 4f).
Unlike the large fish that lay eggs once a year, such as Pagrus major and Paralichthys olivaceus, O. melastigma lay eggs every day. O. melastigma is a typical small fish (body length is only 2.8–3.0 cm), and its maximum spawning can reach 100 per day. To ensure the growth, O. melastigma reproduce as much offspring as possible at a time. Unlike red sea bream P. major and P. olivaceus, the energy allocated to each fertilized egg is limited, resulting in the imperfect synchronization of fertilized egg development. The fertilized eggs of O. melastigma can only be "regulated" or "fixed" (if problems occur) by themselves during the development. Results show that indeed, many abnormal fertilized eggs in the early stage could eventually develop normally. However, if the age of a broodstock was not suitable (e.g., in pre-puberty stage or senescence stage), and the quality of its sperm or eggs were not in the optimal state, this self-regulation ability of fertilized eggs could likely to decrease, and the abnormalities in the development of O. melastigma embryos could appear.
About 2 h after fertilization, normal fertilized eggs cleaved for the third time, forming eight blastomeres of slightly different sizes in two rows with four in each row (Fig. 5a). Specific cases of an abnormal sixcell process were observed in the four- and eight-cell stage, in which the blastomeres were partly or completely separated (Fig. 5b & c). In the eight-cell stage, abnormal blastomeres separation (Fig. 5d) or dislocation (Fig. 5e & f) was also observed. In the 16–32-cell stage, blastomeric separation was the most common abnormality in the development of normal embryos (Fig. 6a–c).3.2.3 Blastula stage
Normal fertilized eggs were divide into small semicircle blastoderm and slowly expanded to the edge of the eggs in about 5 h of development (Fig. 7a). In contrast, those abnormally developed fertilized eggs presented a variety of phenomena, including diapause, abnormal increase in the volume of cell differentiation, narrow and long blastoderm, and abnormal blastoderm epiboly. It was observed that some fertilized eggs underwent normal cell division, but stagnated in the morula stage or blastula stage, whereby oil droplets left the vitellus and were half extruded (Fig. 7b). Another phenomenon was that abnormally large cells appeared at the edge of normally divided cells, leading to the loss of development of some organs in the later stage (Fig. 7c & d). In addition, we observed that blastoderm of fertilized eggs was thin in the middle and thick at the edges (Fig. 7e), or had inclusion with wave-like epiboly (Fig. 7f).
Diapause is a metabolic arrest usually expressed in insect embryos as an extreme adaptation to persist in environments that show alternate periods between favorable and adverse conditions in annual cycle (Levels and Denucé, 1988). Hand et al. (2016) proposed that diapause in the annual killifishes was arguably the best known and most studied among the examples of vertebrate diapause. Hu and Brunet (2018) found that the African turquoise killifish embryos could enter diapause to suspend their development over the dry season. The embryos are able to stay in diapause for months, even several times longer than the fish lifespan, then turn back from diapause, and resume their compressed lifecycle in the following rainy season.
In the experiments, electricity in the laboratory was accidently power off for several hours for device maintenance. The temperature decreased a little, while the photoperiod changed considerably, especially in the morning when the brookfish began to spawn. On the next day, when we checked the eggs that already collected for more than 24 h, we found that some eggs remained in the blastula stage, and then continue their ontogeny. Thus, we assumed that the special developmental strategy was diapause. To verify that, embryos of the two–four-cell stage were placed in refrigerator (4 ℃) for 2.5 h, and then restored into ambient temperature (20 ℃). The eggs continued its ontogeny as the normal eggs did.
Therefore, we were sure that the embryos could reverse from arrests (diapauses) in different stages under various circumstance in, at least temperature and photoperiod, as several other annual fish do (Arezo et al., 2005; Domínguez-Castanedo et al., 2018). Understanding the relationship between diapause and environmental factors shall be an issue for us in the future studies.3.2.4 Gastrula stage
About 17 h after fertilization, layer cells in the fertilized egg continued to expand to the plant pole and then entered the gastrula stage. The abnormal embedding of layer cells was observed, which is quite common in the senescent stage. As shown in the Fig. 8, the layer cells did not expand at the edges, but were embedded in the vitellus with obvious notches formed at the edges. This phenomenon was due probably to the quick absorption of layer cells, resulting in an abnormal development rate. In addition, the oil droplets were found dislocated. Connection between the oil droplets and the blastoderm did not pass through the center, which was probably one of the reasons for the abnormal development.3.2.5 Organogenesis stage 126.96.36.199 The Kupffer's vesicle
At 26–28 ℃, O. melastigma entered the organogenesis stage at 26–28 h after fertilization and the Kupffer's vesicle began to appear. This course lasted for about 24 h and disappeared soon after the heartbeat started. Two (Fig. 9a & b) or more (Fig. 9c & d) Kupffer's vesicles commonly appeared during the embryogenesis of O. melastigma as observed in other fish species such as P. major and P. olivaceus (Xiao et al., 2008; Yu et al., 2014). Further tracking showed that the development of these embryos continued normally. Furthermore, it was observed that normal Kupffer's vesicles generally disappeared soon after the heartbeat began. However, the disappearance of the Kupffer's vesicles in some fertilized eggs might lag as the tailbud separated from the embryo later than regular ones in lower wobbling frequency.
The Kupffer's vesicle, a transitory organ peculiar to teleost embryos, is commonly observed in embryos of Scophthalmus maxima, G. macrocephalus, Larimichthys crocea, and Brachydanio rerio (Brummett and Dumont, 1978; Laale, 1985; Navis et al., 2013). Kreiling et al. (2007) found that Kupffer's vesicle is an ellipsoidal fluid-filled cavity that appears approximately 12 h post fertilization in zebrafish embryo, and enclosed by curved ciliated epithelial surfaces on all sides. Previous studies have pointed out that the function of the Kupffer's vesicle is to start the asymmetric development of fish, but this conclusion remains controversial (Okabe et al., 2008; Smith et al., 2014). Taking G. macrocephalus as an example, the Kupffer's vesicle is only present in about 10% of the embryos, and fertilized eggs without Kupffer's vesicle can still develop into normal juveniles. In this study, fertilized eggs with two or more Kupffer's vesicles developed normally. Therefore, the function of the Kupffer's vesicle requires further exploration.188.8.131.52 Conjoined twins
We observed a unique phenomenon in our study: conjoined twins. In the initial cleavage stage, blastomeres were separated from each other in the fertilized eggs (Fig. 10a). The follow-up observations showed that some separated blastomeres developed independently, forming double blastoderms, and eventually developed into double embryonic bodies (Fig. 10b). Some double embryonic bodies had double heads and were separated from the main bodies with only the tail unseparated; some double embryonic bodies even shared the same heart. Clear blood circulation was observed in the double embryonic bodies, but almost none of them could break through the egg membrane and thus eventually died.
The double embryonic bodies were observed together with multiple oil droplets, suggesting that in the organogenesis stage, oil droplets did not completely fuse into a single oil droplet. Before blood circulation was established, embryos of O. melastigma were specifically absorbed on to the surface of vitellus to form primitive blood vessels and blood cell clusters. The primitive circulation systems helped to establish the initial blood circulation and the later heartbeat. A single oil droplet generally made it easier to establish the blood circulation of the initial embryo during this process. In contrast, multiple oil droplets might hinder normal blood circulation and force blood channels to "detour", which was likely to be one of the main reasons of abnormal embryonic body development.
In this study, we observed some cases where separated blastomeres fused into normal embryos, which is consistent with the discovery in the study by Zhu (1982) on Clarias fuscus, indicating that the eggs of O. melastigma and C. fuscus had a strong regulatory capability. Separated cell clusters may approach and fuse again through blastocyst and gastrulation movement to form one embryo and develop into normal larvae (Spencer, 1992). This implies the undifferentiation of blastomere in an early stage. On the other hand, if the two divided blastomeres were founded in early stage, the embryo have no chance to develop into normal larvae; in other words, the blastomeres lost cell totipotency to some extents in a certain duration. Meantime, gastrula blastomeres were directionally differentiated ready for the next ontogenetic process (Laale, 1984; Arbuatti et al., 2011).
Some double embryonic bodies with double heads and one tail indicated that the already-differentiated blastomeres also have the potency to fuse up each other in later stages (Spencer, 2001; Lanteri et al., 2013), when the blastomeres cell totipotency alter during embryogenesis or proceed in ontogeny and differentiation, the cell totipotency would directionally differentiate into special "totipotency". The two mechanisms both clarify why the trunk could fuse together and the head remained separate, and partially explain the phenomenon in the animal kingdom including human being, e.g., Siamese twins, from the ontogenetic perspective.4 CONCLUSION
We explored the spawning features of O. melastigma during the pre-puberty stage, adolescence stage, and senescence stage in 185 consecutive days of experiment. We observed that the brood fish in the prosperous stage could lay 50– 70 eggs per day on average. However, the spawning capability declined quickly once O. melastigma entered the senescent stage. Abnormal developmental process was frequently observed in fertilized eggs, including pseudo-blastocysts, blastomere separation or dislocation, abnormal blastoderm expansion, two Kupffer's vesicles, and conjoined twins. The abnormal position and number of oil droplets were observed in many processes of fertilized egg embryogenesis, which was likely to hinder blood circulation in a later stage. The above-mentioned embryonic abnormalities were more common in the development of fertilized eggs produced by broodstock in the early or senescent stages and were probably related to poor egg development and low gamete matching.5 DATA AVAILABILITY STATEMENT
The data of this study are available from the corresponding author on reasonable request.
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