Embryonic and Larval Development of the Asian ... - Springer Link

ISSN 10623590, Biology Bulletin, 2015, Vol. 42, No. 4, pp. 334–346. © Pleiades Publishing, Inc., 2015. Original Russian Text © A.M. Shadrin, D.S. Pavlov, 2015, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2015, No. 4, pp. 401–414.

ZOOLOGY

Embryonic and Larval Development of the Asian Seabass Lates calcarifer (Pisces: Perciformes: Latidae) under Thermostatically Controlled Conditions A. M. Shadrina and D. S. Pavlovb a

b

Moscow State University, Moscow, 119992 Russia Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 119071 Russia email: shadrin[email protected] Received October 9, 2014

Abstract—Material for this study was obtained from the hatchery with brood stock of Lates calcarifer that originated from a natural population living in inshore waters off Central Vietnam. Commercial interest in L. calcarifer as an object of mariculture and wildstock fishery has resulted in several publications on its early life history; nevertheless, comprehensive description of early development of L. calcarifer based on controlled incubation of embryos and larvae has remained absent. In the present paper embryonic and larval develop ment to the stage of anlage of pelvic fins is described in detail and illustrated with original drawings of live material on the basis of thermostatically controlled incubation of embryos at 27°C and larvae at 26.8°C (26.5–28.0°C). The first cleavage furrow appeared at the age of 33.5 min. The duration of synchronous cleav age cycle was 16 min. About 80% of all embryos hatched at the age of 18 h. The length of newly hatched larva during the first hour after emergence from the egg shell was 1.63 ± 0.016 mm (1.50–1.75 mm). Chronology of development of the organs, early circulatory system, and pigmentation pattern is given. The dynamics of change in the trunk and caudal body segment number in larva from hatching to the moment of anlage of pel vic fins is shown. The total number of body segments reached the maximum value of 26–27 soon after hatch ing and then decreased to 20–21 segments. Newly received data are discussed in a comparative context of development of some other teleosts. DOI: 10.1134/S1062359015040123

INTRODUCTION Barramundi Lates calcarifer (Blotch, 1790) (Perci formes: Latidae) is one of four currently distinguished species in the genus Lates Cuvier et Valenciennes, 1828 (Katayama and Taki, 1984; Otero, 2004; Pethiy agoda and Gill, 2012). This fish occurs in the Indo West Pacific, to the east of the Persian Gulf, in South eastern Asia, off Papua New Guinea, and off Northern Australia. L. calcarifer has considerable commercial value as an object of mariculture and wildstock fishery. For this reason different aspects of its biology have been studied intensively including the early live history (Jerry, 2014). Nevertheless, comprehensive descrip tion of early development based on controlled incuba tion of embryos and larvae is still absent and its mor phology at different stages and the chronology of early ontogeny of L. calcarifer remain poorly known. Papers on embryonic development of L. calcarifer (Manee wongsa and Tattanon, 1982; Konsutarak and Watanabe, 1984), contain information that can be considered only as a general background for further detailed investigation. In this work we present a detailed description of embryonic and larval development of L. calcarifer to the stage of anlage of pelvic fins with precise temporal

characteristics of the developmental sequence at a constant temperature of 27°С. The description is illus trated with original drawings made on the basis of live material. MATERIAL AND METHODS This study was conducted at the Coastal Division of the Joint Russian–Vietnamese Science and Tech nological Tropical Center in Nha Trang, Vietnam. The material was obtained from the Research Institute for Aquaculture No. 3 in Nha Trang. Spawners of L. cal carifer used at this farm are descendants of fish from a wild local population. The material was received from four spawnings after stimulating injections: on Octo ber 13, 2009, October 28, 2009, October 27, 2010, and November 3, 2010. Spawning started between 18:40 and 19:30, at a temperature about 27°С. After collection from the tank, fertilized eggs were immediately transported to the research site in thermostatically controlled con tainers and further incubated under laboratory condi tions at 27 ± 0.5°С and salinity 32‰. When larvae switched to external feeding, they were kept in the Research Institute for Aquaculture No. 3 in Nha

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Table 1. Size of the fertilized egg and of oil globule in different batches of eggs of L. calcarifer Spawning data (of material getting)

Diameter of the fertilized egg, mm

Mean diameter of the oil drop, mm

n

0.81 ± 0.002 (0.79–0.82) 0.83 ± 0.002 (0.81–0.85) 0.7 ± 0.006 (0.75–0.8)

0.25 ± 0.001 (0.25–0.26) 0.27 ± 0.001 (0.27–0.28) 0.25 ± 0.002 (0.24–0.26)

16 39 16

October 13, 2009 October 28, 2009 November 3, 2010 n—number of measured specimens.

Trang at a temperature of 26.5–28°С (average 26.8°С) following the standard procedures for this species. Larvae were transferred to the laboratory for study as needed. Drawings of the live embryos and larvae were made with the aid of a camera lucida. The age of every stage of embryonic development is indicated at the beginning of its description in minutes (min), hours (h), and τ0, where τ0 is the duration of the synchronous cleavage cycle. This is a dimensionless time unit proposed by Detlaff (1964, 1977) for the measurement of different developmental processes in amphibia and adapted for teleost fishes by Ignatieva (1976a, 1976b). The value of τ0 was based on cytokine sis during the first four cleavage cycles. The age of every stage of larval development is indicated in hours (h) and days (d) after hatching (AH). The developmental stage in this work was defined as any morphological state during ontogeny that had specific morphological characteristics differentiating it from any other states. To determine the chronologi cal characteristics of different developmental stages during cleavage and at the beginning of organogenesis, we selected a sample containing at least 60–70 eggs. This sample was then examined at a low magnification ensuring that most of these eggs appeared in the visual field. At later stages, the eggs were observed at a larger magnification. The age of a particular developmental stage was defined by the time when its characteristic traits were developed in 70–80% of individuals. In determination of the beginning and end of blastulation, we follow Lentz and Trinkaus (1967). The beginning of gastrulation was defined as the moment of directed individual migrations of deep cells, forming the germ ring and embryonic shield (Ballard, 1973a, 1973b). In our study we used only those larvae that demon strated high locomotor activity and displayed quick positive phototaxis. Measurements were made for lar vae obtained from the spawning of October 14, 2009. The measurements are presented as the mean ± SD. The sizes of larvae refer to their total length (TL). RESULTS Embryonic Development Fertilized eggs of L. calcarifer are characterized by a smooth, unstructured, and transparent envelope. BIOLOGY BULLETIN

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The yolk is segmented into large granules, transparent and colorless. There is a single oil globule inside the yolk. The cytoplasm is transparent, almost colorless, with a slight tint of yellow. The size of fertilized eggs of L. calcarifer from different parents and in different spawnings ranged from 0.75 to 0.85 mm, and the diameter of oil globules ranged from 0.24 to 0.28 mm (Table 1). Cytoplasm aggregation at the animal pole starts after fertilization. Cytoplasm movement occurs both at the surface and from inside the yolk. This process is accompanied by development of cytoplasm streaming, which can be observed until the onset of the second cleavage. Cleavage The duration of synchronous cell cleavage cycle is about 16 min. The first cleavage furrow yielding the stage of two blastomeres (Fig. 1a) appears at the age of 33.5 min. The first four cleavage furrows leading to the stages of 2, 4, 8 and 16 blastomeres are meridional and appear simultaneously in every blastomere at an inter val of about 16 min (Figs. 1a–1d). Starting with the stage of 32 blastomeres (Fig. 1e), the furrows are not only meridional, but also lattitudinal; as a result a morula is forming. From this moment, some asyn chrony is observed in the cytokinesis in the blas tomeres located in various areas. During the stage of 64 blastomeres (Fig. 1f), the cytoplasm of some marginal blastomeres is confluent with the yolk cell cytoplasm. Trinkaus (1993) refers to them as open blastomeres. At the following two stages (128 and 256 blastomeres) (Figs. 1g, 1h), cellular nuclei not divided by membranes could be clearly seen at the periphery of the blastodisk. Some of the mar ginal blastomeres collapse and contribute their nuclei and cytoplasm to the yolk syncytial layer. The base of the blastodisk was almost flat without pronounced relief at the 8 and 9 fission cleavages (Figs. 1h, 1i). A yolk syncytial layer developed between the blastomeres of the lower layer and the yolk. During the following cleavages, the germ assumed the shape of biconvex lens, which is evidence of the final differen tiation of the yolk syncytial layer and the enveloping layer, reduction of the adhesive properties of the deep cells' membranes, and start of blastulation (Trinkaus,1963; Lentz and Trinkaus,1967).

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(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(k)

(l)

ysn

ysn

(j)

dsb

ysn

1 mm

Fig. 1. Embryonic development of L. calcarifer. (a) 2 blastomeres; (b) 4 blastomeres; (c) 8 blastomeres; (d) 16 blastomeres; (e) 32 blas tomeres; (f) 64 blastomeres; (g) 128 blastomeres; (h) about 256 blastomeres; (i) about 512 blastomeres; (j) blastulation; (k) begin ning of gastrulation; (l) gastrulation. ysn, Yolk syncytial nuclei; dsb, dorsal sector of the blastodisc. Scale bar 1 mm. BIOLOGY BULLETIN

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Blastulation 3 h 20 min (12.5 τ0) (Fig. 1j). Onset of blastulation. The germ has the shape of a biconvex lens. Gastrulation 4 h 15 min (15.9 τ0) (Fig. 1k). The base of the germ is flattened. Nuclei are clearly seen in the yolk syncy tial layer. 4 h 35 min (17.2 τ0). The base of the blastodisc starts bending inside. About 15–20 min after this pro cess has started, the center of this rise gets noticeably shifted towards its ventral sector from the center. Thus, the bilateral axis of the embryo is defined. 5 h 05 min (19.1 τ0). (Fig. 1l). Continuing migra tion of deep cells towards the dorsal sector of the blas todisc and its periphery (Ballard, 1973a, 1982) results in formation of the germ ring and the embryonic shield. 5 h 25 min (20.3 τ0) (Fig. 2a). The cell ball begins to spread over and around the yolk towards the vegetal pole. Epiboly has begun. The blastoderm covers about 25–30% of the yolk surface. Organogenesis 6 h 05 min (22.8 τ0) (Fig. 2b). The blastoderm over laps with about 40% of the yolk surface area. As a result of convergent migrations of the hypoblast, a consider able part of the cellular material is aggregated in the dorsal sector of the blastodisc. The anterior part of the embryo body is forming. It is submerged into the yolk to 1/3 of the body height. 7 h 05 min (26.6 τ0). The blastoderm overlaps with nearly 50% of the yolk surface area. 7 h 45 min (29.1 τ0). Epiboly about 70–80%. Ante rior and medial parts of the embryo deeply suppress the yolk underneath. Structures of the axial complex start differentiation. Dorsal and ventral relief of the embryo anterior part is defined by differentiation of the primary brain vesicles, the prosencephalon, mes encephalon, and rhombencephalon. The notochord rudiment consisting of loosely aggregated cells becomes noticeable. Its optical density reaches the maximum value in the medial part of the body. The bands of the paraxial mesoderm are formed on both sides of the notochord. 8 h 05 min (30.3 τ0) (Figs. 2c, 2d). Epiboly about 80–90%. The prosencephalon differentiated into the telencephalon and diencephalon. A longitudinal medial depression is formed at the dorsal surface of the embryo; it is located over the mesencephalon and anterior part of the rhombencephalon. The notochord rudiment becomes denser and can be clearly traced from the posterior margin of the mesencephalon almost to the blastoderm margin. The rhombenceph alon and medula spinalis are situated directly above BIOLOGY BULLETIN

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the notochord. The notochord rudiment and the spi nal cord are laterally embraced by the paraxial meso derm. There are two bands of cells on both sides along the forming embryo’s body. In the caudal part these bands widen and turn into the germ ring embracing the open part of the yolk cell surface. 8 h 45 min (32.8 τ0). Epiboly about 95%. 9 h 05 min (34.1 τ0). Epiboly 98–100%: the com pletion of epiboly. The first somite furrows appear in the paraxial mesoderm. One to three pairs of somites are visible. There are many actively migrating fusiform and tearshaped cells, and precursors of melanophores at the dorsolateral surfaces of the yolk sac appeared. 9 h 55 min (37.2 τ0) (Fig. 2e). 4–5 pairs of somites have developed in the paraxial mesoderm. The hind brain (rhombencephalon) differentiated into the metencephalon and myelencephalon. The fourth and the fifth encephalic vesicles have differentiated. The cerebellum formed as the upper part of the meten cephalon flexure. Optic placodes appear as two ellipti cal bulges. Kupffer’s vesicle can be seen in the lower part of the tail bud. A large number of migrating color less melanophores is retained on the lateral and dorsal surfaces of the yolk sac. 10 h 45 min (40.3 τ0). 7–8 pairs of somites have developed. Most melanophores remain colorless; however, some of them contain granules of brown melanin, which results in a yellowbrown color. Some of these cells have moved from the yolk sac to the embryo’s body surface. 11 h 55 min (44.7 τ0). 10–12 pairs of somites have developed. The otic placodes have become slightly vis ible on either side of the myelencephalon. The ante rior 5–7 segments are becoming Vshaped. Kupffer’s vesicle reaches the maximum size. Most melano phores have now moved from the yolk sac to the body of the embryo and to the surface of the yolk globule. Most of them become weakly brownyellow although some are pale gray or remain colorless. The embryo is pigmented unevenly. The middle part of the body and dorsolateral parts of the head above the cerebellum are pigmented the most intensely. 12 h 25 min (46.6 τ0) (Fig. 2f). 13–14 pairs of somites have formed. Epiphysis developed in the dien cephalon and can be seen at the boundary of the telen cephalon and mesencephalon. In otic placodesformed cavities, and they were transformed into more clearly visible otic vesicles. The amount of pigment increased in brown and black melanophores. Black melano phores have pronounced homogeneous gray colora tion. They are located predominantly on that part o thef surface of the oil globule that is submerged in the yolk as well as on the dorsolateral surface of the embryo body. Melanophores localized on the oil glob ule are significantly branching, treelike in shape. Most melanophores located on the embryo body are slightly

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(b)

es

(c) mpb fb ppb

ape

m gr

pc gr

(d)

(e)

c

op

(f) e

ph

pc

Kv pm gr

Kv (g)

(h)

ph

ph

(i) hs

1 mm

Fig. 2. Embryonic and larval development of L. calcarifer. (a) Epiboly 25–30%; (b) epiboly about 40%; (c) epiboly 80–90%, orga nogenesis; (d) epiboly 80–90%, organogenesis, view from a vegetative pole; (e) embryo 50 min after termination of epiboly; (f) 13–14 somite stage, start of melanine pigmentation; (g) 18–19somite stage, the beginning of tail bud separating from the yolk sack; (h) 26– 28somite stage, beginning of skeletal muscle contraction; (i) newly hatched larva 1.63 mm. gr, Germ ring; es, embryonic shield; ape, anterior part of the embryo body; t, telencephalon; d, diencephalon; m, mesencephalon; r, rhombencephalon; e, epiphysis; n, noto chord; op, optic placode; c, cerebellum; Kv, Kupffer’s vesicle; pm, promelanophores; ht, heart; hs, hydrosinus. Scale bar 1 mm. BIOLOGY BULLETIN

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elongated, roundish or fusiform cells with one or two philopodia. Brown melanophores are colored hetero geneously and have a shape similar to black melano phores from the body surface. Uncolored and trans parent melanophore cells in most cases have one light brown melanin granule. 13 h 50 min (51.9 τ0) (Fig. 2g). 18–19 pairs of somites have formed. The tail bud begins to separate from the yolk sack. A heart rudiment appears under the head region at the level of the cerebellum. The eye vesicle became cupshaped and the lens placodes dif ferentiated inside. Kupffer’s vesicle wholly reduced. Beginning of the Motile State 14 h 50 min (55.6 τ0). There are 22–24 muscular segments in the body. Optic lenses are spherical. Two otoliths can be seen at each otic vesicle. Weak and hardly detectable heartbeats occur in most embryos with an estimated average frequency of about 50 per minute. 16 h 00 min (60 τ0) (Fig. 2h). There are 26–28 muscular segments in the body. Heartbeats become more frequent and regular. The frequency of beating is 60–80 beats per minute (bpm). The trunk myomeres produce weak muscular contractions. The muscular contractions produce lashings from side to side with a frequency of about 2–3 times per minute. A brown color dominates in the embryo’s colora tion. Melanophores with some amount of black form a wide belt in the middle part of the body and make welldeveloped aggregations in the dorsolateral area and above the margin of the mesencephalon and metencephalon, as well as in front of the olfactory capsules. The pattern of oil globule pigmentation has not changed. 16 h 30 min (61.9 τ0). First cases of hatching are recorded. 16 h 50 min (63.1 τ0). About 5% of embryos hatched. 17 h 30 min (65.6 τ0). About 50% of embryos hatched. 18 h 00 min (67.5 τ0) About 80% of embryos hatched. Larval Development Endogeneous nutrition. 1 h AH (Fig. 2i). The length of newly hatched larvae during the first hour after hatch ing is 1.63 ± 0.016 mm (1.50–1.75 mm, n = 19). The larvae remain almost motionless for 1–1.5 hours after their emergence. They are located almost vertically underneath the surface film, touching it with the ante rior or anterior–ventral part of the yolk sac. The body includes 28–29 segments, 12 (rarely 13) of them being trunk, and 16–17 being caudal. The periderm, or enveloping layer, forms the hydrosinus, or dorsal sinus, which is more pronounced in the trunk BIOLOGY BULLETIN

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region. The frequency of heart beating is about 110 bpm. The pigmentation pattern has not changed signifi cantly. Weakly branching brown melanophores form a dense aggregation in the form of a belt at the body sur face, from the 2nd to the 16th segment. Pigment cells of the same type cluster in groups over the optic lobes and cerebellum border. They can also be found above the myelencephalon, intensely pigmenting the outer part of the oil globule that is not submerged into the yolk, and additionally can be seen upon its submerged side in a small quantity. Significantly branched treelike black melanophores pigment the part of the oil globule that is submerged in the yolk. Some weakly branched black melanophores can also be found among the brown ones. 1 day 6 h AH (Fig. 3a). The length of the yolk sack larva is 2.53 ± 0.25 mm (2.35–2.65 mm, n = 13). There are 26–27 muscular segments: 10–11 trunk and 16–17 caudal. The oil globule diameter ranges from 0.25 to 0.26 mm; it has not changed from the start of development. The yolk is resorbed to 80–90% of the volume at hatching. Rudiments of pectoral fins can be seen. Mesenchymal fin buds protrude distally from the ventrolateral region adjacent to the level of the seg ments 1–4. The blood flow with a few colorless blood elements in a simple circulatory system has developed. The mandibular, hyoid, and four pairs of gill arches have differentiated. However, only mandibular arteries are actually functioning. Weak peristalsis observed in the middle and caudal parts of the intestine. Pigmentation becomes more pronounced. At this stage, significant pigmentation is observed from the first to the 21st segment with particular strengthening from the 3rd–4th to the 18th–19th segments. Branched brown melanophores dominate in the body pigmentation. Most pigment cells shifted from the oil globule to the yolk sac, to the pericardium region, in the area of eyes, olfactory capsules and above the optic lobes. Larvae have neutral or weak negative buoyancy. When immobile, they either slowly fall to the bottom of the tank or hang in midwater. Many larvae demon strate weak positive phototaxis. After approximately 5–10 minutes, most of them tend to aggregate in a more illuminated part of their tank. Mixed (Endogenous–Exogenous) Nutrition. 3 days 3 h AH (Fig. 3b). The length of larvae is 2.72 ± 0.02 mm (2.55–2.85 mm, n = 17). There are 26–27 muscular segments: 9–10 trunk and 16–17 caudal. The oil glob ule is usually slightly deformed by nearby organs and pericardial cavity. Its volume is reduced by 50–60% to the volume at hatching. Only a very thin layer of the remaining yolk is retained around it. The primordia of the swim bladder is beginning to develop above the posterior part of the esophagus. The liver rudiment with a differentiated gall bladder joins

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SHADRIN, PAVLOV (а) ppf

(b) eph

sb gb

og

liv

stm sp gr (c) sb

pgf

og

liv gb

sp (d)

mh sp

1 mm

Fig. 3. Larval development of L. calcarifer. (a) Yolk sac larva 2.53 mm 30 h AH; (b) larva 2.72 mm 3 days AH; (c) larva 2.93 mm 5 days AH; (d) larva 3.25 mm 5 days AH. pf, Pectoral fins; og, oil globule; eph, esophagus; stm, stomach; int, intestine; rec, rec tum; sp, spleen; liv, liver; gb, gall bladder; gr, germ ring; pgf, primordial gill filaments; ppf, primordial pectoral fin; sb, swim blad ders; mh, material of the hypurals, parhypural, and haemal spines. Scale bar 1 mm. BIOLOGY BULLETIN

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the yolk globule at the rear underneath. The gall blad der represents a large colorless thinwalled bag having the shape of an elongated and slightly curved tear drop. The jaw apparatus and pectoral fins are motile. The frontal part of the lower jaw projects forward more than the upper. Differentiation of the digestive tract becomes more pronounced. The relatively thickwalled esophagus, which is getting narrower in the caudal direction, goes to the stomach, which is notably widened and com paratively thinwalled. The stomach is directed down wards and to the right, forward up and backwards, thereby forming a loop, and then narrows and transits to the intestine. The rectum is clearly differentiated in the intestine. Peristaltic undulations occurred through the entire intestine. An anlage of the spleen attaches to the back part of the outer wall of the stomach flexure. The circulatory system also becomes more com plex. The mandibular, hyoid, and all gill arteria are functioning. Arterial branches to the brain can be found. Roots of the aorta, dorsal aorta, caudal artery, and caudal vein are functioning. There are also branches of the blood vessels that embrace the poste rior part of the intestinum, stomach, and the oil glob ule along with the nearby liver. The number of blood elements has increased significantly. Details of the pigmentation altered. The pigment layer of the eyes is fully pigmented with black melanin and contains guanine granules. The ratio of brown and black melanofores involved in larva pigmentation has changed. Dominance of brown melanophores becomes less pronounced. Most brown and black branching melanophores aggregated into a few pig ment spots that form the dorsal and lower lateral rows transiting to ventrocaudal pigment rows. All these rows are incomplete. A large number of black (gray) melanophores of unusual shape appeared at the lateral surfaces of the body from the 3–4th to 22nd–23rd seg ments. They form a pattern of longitudinal rows of dorsoventrally oriented streaks (Fig. 4). The pigment cells above the optic lobes and cerebellum disap peared. A pronounced ventral row of exceptionally black melanophores appeared. There are also groups of black melanophores on elements of the lower jaw and olfactory capsules and on the operculum. Larvae actively swim, hunt, and demonstrate pronounced positive phototaxis. They aggregate in a more illumi nated part of the tank within a few minutes. 4 days AH. About 50% of larvae have swim bladder full of air. 5 days 5 h AH (Fig. 3c). The length of the larvae is 2.93 ± 0.03 mm (2.7–3.15 mm, n = 20). In the body, 25–26 muscular segments was differentiated: 9–10 trunk and 16–17 caudal. Swim bladders of all larvae filled with air. Simple fingerlike gill filaments primordia are formed on the first pair of gill arches. BIOLOGY BULLETIN

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The residuals of the oil globule with a diameter of 0.05–0.07 mm are still seen. It is embraced by the liver which has increased in size. The epithelium of the stomach and intestine is thrown into deep folds. The anlage of the spleen became larger. A group of branching black melanophores is arranged into an incomplete lateral pigment row situ ated straight along the lateral line. Melanofores from the dorsal row frequently branch to the dorsal part of the fin fold. The dorsal wall of the body cavity over the esophagus, the swim bladder, and the intestine, as well as the upper part of the swim bladder, are intensely pigmented. Exogenous Feeding. 6 days 7 h AH. The oil globule is completely utilized. 7 days 16 h AH (Fig. 3d). The length of the larvae is 3.25 ± 0.02 mm (3.1–3.4 mm, n = 17). There are 25– 26 muscular segments in the body: 9–10 trunk and 16–17 caudal. The folds of the digestive tract have become more pronounced. The primordial gill filaments can be found on all gill arches and many of them begin to develop secondary lamellas. A teeth are formed on the upper jaw. The preanal fin fold is significantly reduced. Material of the hypurals, parhypural, and haemal spines is visible as an aggregation of mesenchymal cells in the fin fold under the terminal part of the tail. Pigmentation with brown branched melanophores is accompanied almost everywhere by black dendritic melanophores. Branching of the dorsal melanophores into the cavity of the dorsal fin fold increased. a few separately located yellow or orangeyellow cells, pre sumably xantophores, appeared on the dorsal side of the trunk and head. 9 days 10 h AH. The caudal part of the notochord in most larvae begins to flex. 11 days 17 h AH (Fig. 5a). The length of the larvae is 3.59 ± 0.04 mm (3.25–3.85 mm, n = 15). 22–23 muscular segments can be distinguished in the body: 9–10 trunk and 13–14 caudal. The notochord flexion is completed. Areas of the development of definitive unpaired fins are clearly differentiated in the medial fin fold. The pterygophores primordias, the inner sup ports of elements of the dorsal and anal fins, are devel oping on the bases of their rudiments. Mesenchimal cells in areas of formation of the dorsal and anal fins became oriented as future fin rays in their distal parts. Sixteen rays differentiated in the caudal fin. Most of them are already segmented. Rudiments of spines appeared on the preoperculum; one being anterior preopercular and three posterior preopercular. All gill filaments have secondary lamellas, but their shape, number, and location are far from a definitive state. The branchyostegal rays supporting the gill membrane developed. The number of bright yellow xanthophores increased significantly. Separate cells occur in various regions of the larvae body surface. The most intense

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Dorsal part of protopterygium Rows of ventrally oriented melanophores

Anus

Postanal part of protopterygium

Fig. 4. L. calcarifer larva 3 days AH. Fragment of pigmentation.

yellow pigmentation is observed at the upper and upperlateral surface of the head and anterior trunk. 17 days 2 h AH (Fig. 5b). The size of larvae is 4.70 ± 0.05 mm (4.35–5.20 mm, n = 20). 20–21 muscular segments are distinguished in the body: 9–10 trunk and 11–12 caudal. Vertebral bodies with neural and haemal processes developed in all regions of the spinal column. Ribs are developing. Four rigid rays, two undefined, and 12 already segmented soft rays are differentiated in the dorsal fin. In the anal fin, three rigid and 7–8 soft and already segmented rays developed. The anlagen of pel vic fin buds is clearly differentiated. There are no structural elements. The number of spines on skeletal elements of the gill cover increased. There are six posterior preopercu lar spines and one anterior preopercular spine, and one minute spine is located in the posterior angle of the interoperculum. Gill filaments structure is similar to the definitive state. The pattern of the xanthophore distribution has not changed, although their number increased. The maxi mum density is observed at the base of the caudal and dorsal fins, at the caudal peduncle and along the dorsolateral surface of the trunk and head. These parts have become yellowish. The proximal parts of most soft rays of the dorsal and anal fins are pigmented with black melanophores. Longitudinal rows of verti cally oriented gray melanophores have disappeared.

DISCUSSION The average values of the sizes of eggs and oil glob ules varied across the egg batches obtained from differ ent spawnings and parents (Table 1). Our data also dif fer from that previously published (Table 2). Many investigations (Solemdal et al., 1995; Kjesbu et al., 1996; Brooks et al., 1997; Marteinsdottir and Steinars son, 1998; etc.) have indicated that the size and other characteristics of eggs and sperm might vary depend ing on the age of the spawners, their physiological condition, and the sequential number of the egg batch. These factors would account for the observed differ ences in egg diameters and the sizes of the newly hatched larva observed in our study. Most previous studies of the early ontogeny of L. calcarifer were conducted without strict tempera ture control (thermostating) with significant fluctua tions of temperature. That considerably complicates comparative analysis of developmental chronology. However, Maneewongsa and Tattanon (1982) con ducted their observations at a prevailing temperature of 27°С, which allows some approximate comparison with our data. Time interval from fertilization to the first cleavage fur row appearance in L. calcarifer is about 34 min (2.1 τ0), which agrees with the results of Maneewongsa and Tattanon (1982) and corresponds to the data on the relative duration of this period in some other teleosts. According to Maneewongsa and Tattanon (1982), the BIOLOGY BULLETIN

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(а)

(b)

sp

pf

1 mm

Fig. 5. Larval development of L. calcarifer. (a) Larvae 11.5 days AH, TL = 3.59 mm; (b) larvae 18 days AH, TL = 4.70 mm. pf, Pelvic fin; sp, spleen. Scale bar 1 mm.

duration of subsequent cleavage cycles is very irregular and significantly differs from our data. This may reflect the inconstancy of the temperature regime in their experiments. In our study, cleavage in the L. calcarifer continued about 12.5 τ0, which agrees with the results for Cypri nus carpio (Cyprinidae, Cypriniformes), Misgurnus fossilis (Cobitidae, Cypriniformes), Esox lucius (Eso cidae, Esociformes), Coregonus peled, C. lavaretus, C. nasus, Salmo gairdneri, and S. trutta (Salmoni formes). However, the beginning of gastrulation and the stage of 10 pairs of somites in L. calcarifer occurred ear lier than in all these species. Maneewongsa and Tattanon (1982) documented hatching at the age of 17 h 30 min after fertilization, which is in good coincidence with our data. In our experiments, the first hatchings occurred at the age of 16 h 30 min and 80% of individuals emerged by 18 hours. The age of the transition to the motile state is also similar to that documented in this work. According to Leis and CarsonEwart (2000), larvae with a body length of 3 mm already have two posterior preopercular spines and also there are aggregations of mesenchymal cells in the areas of formation of the BIOLOGY BULLETIN

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dorsal and anal fin anlage. In our study larvae with an even larger body length (3.25 mm) have no spines on the elements of the operculum and lack any indica tions about the formation of dorsal and anal fin anlage, so in our material these organs started to develop in larger larvae. We recorded some more differences associated with correlation of the rate of linear growth and differentiation of some organs. For instance, Leis and CarsonEwart (2000) reported that pelvic fin buds become visible by the completion of notochord flex ion, while according to our results the rudiments of the future pelvic fin appeared approximately six days after completion of the notochord flexion. Furthermore, all larvae in our study with a body length of 3.59 mm or more have a single minute anterior preopercular spine, while in Leis and CarsonEwart (2000) and Konishi et al. (2012) there is no indication of this structure in larvae of the same size and age. We did not specifically analyze the linear growth rate of the larvae. However, it is easy to see that the maximum daily increase was recorded on the first day of their life after hatching. Then, a certain decrease was observed, as was noted by Kohno et al. (1986),

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Table 2. Size of fertilized eggs and of larvae of L. calcarifer immediately after hatching by data of various authors Diameter of the fertilized eggs, mm

Diameter Size of prolarva immediately of the oil globule, mm after hatching, mm

0.7

0.2

0.6–0.8 ~0.8 0.74–0.8 0.8 – 0.776–0.782 (mean 0.78)

0.25–0.35 ~0.2 0.20–0.28 – – –

– 1.5 1.5 (TL) – 1.72 ± 0.02 (SL) 1.40 (from 1.3) (TL) 1.62 ± 0.04 (TL)

Source Dunstan, 1959 (cited by: Copland, Grey, 1987) Moore, 1982* Maneewongsa and Tattanon, 1982 Maneewang and Watanabe, 1984 Bagarinao, 1986 Kohno et al., 1986 Kailasam et al., 2007

TL is total length of a larva, SL is length up to the posterior end of chorda, “–“ means no data. * Measurements are made on items preserved with formalin (a considerable distortion of size is possible).

who explained it by the onset of active differentiation of many organs involving significant energy cost. Larvae from experiments of Kohno et al. (1986) and Konsutarak and Watanabe (1984) at an age below 5 days were smaller than larvae in our experiments but grew faster and at the age 8–9 days exceeded our fish in body length. This difference can be explained by the different temperature regime and feeding patterns. Up to now there was no published information on pigmentation of larvae with a body length of less than 3 mm, which would be explicit enough to conduct a comparative analysis with our data. As for the pigmen tation pattern in larger larvae, most detailed data are presented in Leis and CarsonEwart (2000) and gener ally agree with newly received data in our study. How ever, we could not find in the literature any indications of the presence in larvae (body length 2.55 mm and more) on the lateral body surfaces of longitudinal rows of black (gray) melanophores of unusual shape. Changes in the number of body segments during embryonic and larval development had specific fea tures. The total number of segments increased from the beginning of somitogenesis, reaching the maxi mum value of 28–29 ((12–13) + (16–17)) in newly hatched larva. After this, the number of segments begun to decrease, and in larvae one day after hatching became 26–27 ((10–11) + (15–16)). Reduction of the number of myomeres from head to anus reached the final value of 9–10 at the transition to mixed nutrition simultaneously with the differentiation of the digestive system to the functional state. The number of caudal myomeres at this time did not change. It begun to decrease with differentiation of the caudal fin skele ton, reaching 11–12 by the time of its complete devel opment, with the total number equal to 20–21. The described dynamics is related to differentiation of the organs of the body cavity and fixation of position of its caudal border in the definitive positon in relation to body segments and to reduction of some terminal caudal segments in the course of differentiation of the caudal fin. Thus, reduction of the number of segments

in the trunk region occurs due to change in position of the anus and reduction of their total number—only due to reduction in the caudal region. However, the mechanism itself of displacement of the caudal border of body cavity in relation to body segments requires separate investigation. According to our estimations, reduction of the total number of muscle segments and of duration of the body cavity from hatching to transition to mixed feeding takes place in many teleosteans. For example, it is well expressed in several representatives of Scor paeniformes, in particular in Dendrochirus zebra (Scorpaenidae, Pteroinae), Inimicus sp. (Scor paenidae, Choridactylinae), and is also noted in fishes from the fam. Carapidae (Ophiddiiformes), Mugilidae (Mugiliformes), Fistulariidae, Centrisccidae, Car angidae (Perciformes) and in other taxonomic groups (Okyama, 1988; Shadrin et al., 2003). Mechanism of these processes may be connected with species speci ficity of ontogenesis too, as further investigations may demonstrate. In conclusion, early ontogeny of L. calcarifer is typical of many known pelagic spawners Percomorpha Neoteleostei (Rosen, 1973), spawning large numbers of small eggs during the extended spawning period. The onset of gastrulation linked with individual directed movements of deep cells (Ballard, 1973a, 1973b) occurs somewhat earlier than the beginning of the epiboly. Organogenesis begins approximately in the middle of epiboly, whereas segmentation of the lat eral mesoderm starts slightly earlier or almost simulta neously with its termination, usually corresponding to the middle of the embryonic period. Embryos become motile a short time before hatching. The level of mor phological development in newlyhatched larvae is very low and similar to embryos. Such larvae have large yolk reserves and are quite slowmoving immediately after hatching. After yolk reabsorption, the oil globule, if present, is retained for a longer time and is reab sorbed already with involvement of the circulatory sys BIOLOGY BULLETIN

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tem and probably partially shares the function of a swim bladder before its development. ACKNOWLEDGMENTS This study is supported in part by the Program “Scientific Schools” (grant NSh2666.2014.4). We are very grateful to the staff of the Joint Rus sian–Vietnamese Science and Technological Tropical Center Hai Thanh Nguyen and Luong Thi Bich Thuan and the administration of the Joint Russian– Vietnamese Science and Technological Tropical Cen ter for their help in this work. All experiments comply with the current laws of Vietnam. REFERENCES Bagarinao, T., Yolk desorption, onset of feeding and survival potential of larvae of three tropical marine fish species reared in the hatchery, Mar. Biol., 1986, vol. 91, pp. 449– 459. Ballard, W.W., Morphogenetic movements in Salmo gaird neri Richandson, J. Exp. Zool., 1973a, vol. 184, pp. 381– 426. Ballard, W.W., A new fate map for Salmo gairdneri, J. Exp. Zool., 1973b, vol. 184, pp. 49–73. Ballard, W.W., Morphologenetic movements and fate map of the cypriniform teleost, Catostomus commersoni Lace pede, J. Exp. Zool., 1982, vol. 219, pp. 301–321. Brooks, S., Tyler, C.R., and Sumpter, J.P., Egg quality in fish: what makes a good egg?, Rev. Fish. Biol. Fish, 1997, vol. 7, pp. 387–416. Copland, J.W. and Grey, D.L., Management of wild and culture sea bass/barramundi (Lates calcarifer), in Proceed ings of an International Workshop Held at Darwin, N.T. Aus tralia, September 24–30, 1986, no. 20, Canberra: Australia: ACIAR, 1987, vol. 7, pp. 387–416. Detlaff, T.A., Cell divisions, duration of interkinetic states and differentiation in early stages of embryonic develop ment, Adv. Morphog, 1964, vol. 3, pp. 323–362. Detlaf, T.A., Some temperature–temporal patterns of embryonic development of poikilotherms, in Problemy eks perimental’noi embriologii (Problems of Experimental Embryology), Moscow: Nauka, 1977, pp. 269–289. Dunstan, D.J., The barramundi in Queensland waters, Technical paper no. 5, Australia: CSIRO Australia Division of Fisheries and Oceanography, 1959. Ignat’eva, G.M., Rannii embriogenez ryb i amfibii (sravni tel’nyi analiz vremennykh zakonomernostei razvitiya) (Early Embryogenesis of Fish and Amphibians (Comparative Analysis of Temporal Patterns of Development)), Moscow: Nauka, 1979. Jerry, D.R., Biology and Culture of Asian Seabass Lates cal carifer, Boca Raton, FL: USA: CRC Press, 2014. De JesusAyson, G.E., Ayson, F.G., and Thepot, V., Early development and seed production of Asian seabass, Lates calcarifer, in Biology and Culture of Asian Seabass Lates Cal carifer, Jerry, D.R., Ed., Boca Raton, USA: CRC Press, 2014, pp. 16–30. BIOLOGY BULLETIN

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Translated by N. Smirnov

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