Histological Development of the Long‐Snouted Seahorse Hippocampus

Histological development of the long-snouted seahorse Hippocampus guttulatus during

ontogeny

C. OFELIO*A, A. O. DÍAZB, G. RADAELLIC, M. PLANASA.

Running head: H. GUTTULATUS HISTOLOGICAL DEVELOPMENT

a Departamento de Ecología y Recursos Marinos, Instituto de Investigaciones Marinas, Consejo

Superior de Investigaciones Científicas (IIM-CSIC), Eduardo Cabello 6, 36208 Vigo, Spain,

bDepartamento de Biología. Instituto de Investigaciones Marinas y Costeras (IIMyC), FCEyN,

CONICET- Universidad Nacional de Mar del Plata. Funes 3250, 7600 Mar del Plata, Buenos Aires,

Argentina and c Dipartimento di Biomedicina Comparata e Alimentazione, Universitá di Padova,

Viale dell’Universitá 16, 35020 Legnaro , Italy.

Article

*Author to whom correspondence should be addressed. Tel +34 986 23 19 30; email [email protected]

The objective of the present study was to describe histological development of the European long- snouted seahorse Hippocampus guttulatus, to increase understanding of the biology and physiology of the species. Most vital organs were present in juveniles by the time of their release from the male’s pouch. Digestive tract specialization occurred at 89 effective day-degrees (Dºeff), corresponding to 15 Accepted

This article has been accepted for publication in the Journal of Fish Biology and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jfb.13668

Dºeff (20 dpp), lipids were being mobilized from the liver and oocytes attained the perinuclear stage.

The fovea emerged at 177 Dºeff (30 dpp), contemporaneous with the shift from pelagic to benthic behaviour in juveniles. At this stage, the most interesting feature was the formation of the second intestinal loop. Male gonads were never observed during the study (from 0 to 354 Dºeff; 0–60 dpp), but the first oogonia were present at 30 Dºeff (5 dpp). In 354 Dºeff (60 dpp) juveniles, oocytes were observed in a cortical alveoli stage, indicating maturity. Low digestive efficiency was observed at early stages, which was due to a poorly developed gastrointestinal tract and an immature digestive tract prior to 89 Dºeff . The present study demonstrates that approximately 89 and 177 Dºeff represent two important transitional stages in the early development of H. guttulatus. At a temperature of approximately 19 ± 1º C and an age of one month (177 Dºeff), main organs were fully functional, suggesting that the adult phenotype was largely established by that age, with females becoming

matureArticle at the age of two months (354 Dºeff).

Key words: development; fishes; Hippocampus; histology; ontogeny; Syngnathidae.

Accepted

Research into the biology and rearing techniques of seahorses (Syngnathidae: Hippocampus

Rafinesque 1810) has increased substantially in recent years (Cohen et al., 2016) with an aim to contribute to the conservation of wild populations and to satisfy an increasing demand from the international aquarium trade (Foster & Vincent, 2004). The long-snouted seahorse Hippocampus guttulatus Cuvier 1829 is a recent candidate in the ornamental trade and its rearing will both help in the experimental assessment of ecological hypotheses and aid conservation of the species. In spite of recent studies carried out on H. guttulatus, many aspects of their biology and physiology remain unknown, including many developmental features.

Teleost early ontogeny can be direct or indirect, depending on the complexity of morphological, physiological, metabolic and behavioural changes, which will also determine the

beginningArticle of the juvenile period (Balon, 1999; Koumoundouros et al., 1999; Falk-Petersen, 2005;

Gisbert & Doroshov, 2006). Hippocampus spp. anatomical and morphological ontogenesis differ in many aspects from other teleosts with one of most distinguishing characteristics being development of the male brood pouch, where fertilized eggs are incubated and embryogenesis takes place

(Kornienko, 2001; Porter et al., 2015). Seahorse eggs hatch before embryogenesis in complete, with the embryos remaining inside the male’s brood pouch until full yolk resorption (Wetzel & Wourms

2004). Hatched embryos continue developing inside the male pouch for several days before they are released (Sommer et al., 2012). Embryonic and larval (post-hatch–pre-release) periods overlap in seahorse species and free-swimming offspring emerge later from the pouch (Pham & Drozdov, 1998;

Kornienko, 2001; Wetzel & Wourms, 2004). Studies on the biology, morphology, anatomy and histological development in Hippocampus spp. are scarce (Kornienko, 2001; Wetzel & Wourms, Accepted 2004; Planas et al., 2012; Sommer et al., 2012; Willadino et al., 2012; Palma et al., 2014; Novelli et al., 2015).

Hippocampus guttulatus is one of two seahorse species inhabiting the north-eastern Atlantic

Ocean and Mediterranean Sea (Foster & Vincent, 2004; Planas et al,. 2008) and it has recently been

This article is protected by copyright. All rights reserved. introduced into the ornamental aquarium trade (Olivotto et al., 2011). Many aspects of its ontogeny, such as the effect of temperature, diet and digestive capabilities in early ontogeny, have been investigated (Planas et al., 2012; Palma et al., 2014; Blanco et al., 2015), but its morphological development is almost unknown, despite the fact that development is a very important aspect to consider for rearing and conservation programmes. The analysis carried out in the present study provides a global description of the histological development in H. guttulatus during early ontogeny, contributing to the understanding of the biology and physiology of the species.

MATERIAL AND METHODS

ETHICS STATEMENT

In the present study, sampling methods, animal maintenance and manipulation practices were

conductedArticle in compliance with all bioethics standards of the Spanish Government and approved by the Consejo Superior de Investigaciones Científicas bioethics committee.

BROOD STOCK

Brood stock of H. guttulatus consisted of an F1 generation of adults maintained in four 630 l aquaria assembled as autonomous semi-open systems. Water temperature was maintained at 19 ± 1°

C and a 16L:8D photoperiod regime was applied. Breeding individuals were fed three times daily on adult enriched Artemia sp. (EG Grade INVE Aquaculture; www.inveaquaculture.com) and wild mysidaceans Leptomisys sp. and Sirella sp. Artemia was ongrown for 15–25 days in 100 l units at 26º

C, by using a mixture of 107 cells ml–1 of both Phaeodactylum tricornutum and Isochrysis galbana Accepted microalgae and a daily dose of spirulina (0.04 g l–1) and red pepper (0.1 g l–1; Bernaqua, Belgium; www.invivo-group.com). Uneaten food and faeces were siphoned daily before feeding and 10% of aquarium volume replaced daily.

Five batches of newborn H. guttulatus were distributed in thirteen 30 l pseudo-Kreisel aquaria

(Planas et al., 2012; Blanco et al., 2014) and raised at initial densities of 5 individuals l–1 (Blanco et al., 2014). The aquaria were connected to a semi-open recirculation system, including a degasifying column and two 50 l biofilters containing mechanical (up to 20 µm) and biological filters, aerators and skimmers. Seawater was pumped from the biofilters to 36 W UV sterilization units and then to

50 l reservoir tanks. Water temperature was maintained at 19 ± 1° C by using an inlet heating system

(HC300/500 A; Hailea; www.hailea.com). A 16L:8D photoperiod regime was applied and the light source consisted of 20 W fluorescent lamps (Power Glo, Valencia, Spain). Gentle aeration was supplied as described by Blanco (2014). The daily feeding regime of seahorse juveniles from 0 to 59 effective day-degrees (Dºeff, corresponding to 10 days post partum; dpp) consisted of two doses of

–1 –1 ArtemiaArticle nauplii (1 Artemia ml per dose) and one dose of cultivated copepods (0.7 copepods ml ).

–1 From 65 to 177 Dºeff, two daily doses of 24 h enriched Artemia metanuplii (1 Artemia ml ) were delivered. A mixture of metanauplii enriched for 24, 48 and 72 h was supplied from 183 Dºeff until the end of the study (354 Dºeff). Uneaten food and faeces were daily siphoned before each feeding dose and 30% of aquarium volume was replaced daily.

LIVE-PREY PRODUCTION

Acartia copepods were cultivated at low densities (1–3 copepods ml–1) in 500 l tanks and feed on mixtures of the microalgae Isochrysis galbana and Rhodomonas lens. Artemia cysts were incubated daily at 28° C for 24 h and hatched nauplii transferred to 5 l buckets (100 Artemia ml–1). Accepted Artemia enrichment was carried out providing daily mixtures of 107 cells ml–1 of both P. tricornutum and I. galbana microalgae, spray-dried spirulina (0.02 g l–1) and red pepper (0.1 g l–1, Bernaqua) for at least 3 days (Planas et al., 2017).

Hippocampus guttulatus were sampled at 0, 30, 59, 89, 118, 177 and 354 Dºeff, corresponding to 0, 5, 10, 15, 20, 30 and 60 dpp. For that, 121 individuals (9–34 specimens, depending on the survival and availability of individuals at each sampling time) were sampled and submitted to histological characterization. All samples were taken before the first daily feeding dose. Sampled individuals were anesthetized with 0.1 g l–1 MS-222 and photographed for further standard length

(LS) measurements (Lourie et al., 1999) using an image analysis software (NIS Elements Nikon; www.nikoninstruments.com/en_GB/Products/Software/NIS-Elements-Documentation).

Subsequently, the H. guttulatus were washed with tap water, gently dried to remove excess water and weighted on a MC210P Sartorius (www.sartorius.com) microbalance (± 0.01 mg).

o Effective day-degrees (D eff) was used as an index of developmental progress based on a

Article (2)(1) species-specific threshold temperature (T0) at which development is arrested (Planas et al., 2012).

o o The correspondence between dpp and D eff was calculated as: D eff = ΔtTeff = Δt(T – T0), where Teff is the biological effective temperature, Teff = (T – T0) and T is the rearing temperature in ºC and T0 the temperature at which development is arrested (13.1 ºC, Planas et al., 2012).

Daily mass specific-growth rates (G% day–1) were calculated as: G = 100(eg – 1), where g is

–1 the instantaneous growth coefficient, obtained by g = (lnM2 – lnM1)(t2 – t1) , and M1 and M2 are the wet mass (MW, mg) of H. guttulatus at day t1 and t2 respectively. Fulton's condition factor (K) was

3 calculated as: K = 100MWLS .

After weighing, anaesthetized H. guttulatus were submitted to an overdose of 0.1 g l–1 MS-

222 and quickly fixed by immersion in 4% buffered formaldehyde (pH 7.2). Fixed samples were Accepted individually transferred to histology cassettes, washed overnight with tap water to remove formalin residues, then transferred to a bath of distilled water. Decalcification was performed by immersion of cassettes in 10% formic acid (Sigma-Aldrich Co.; www.sigma-aldrich.com) for at least 5 days.

Subsequently, tissues were dehydrated in a graded series of ethanol and embedded in paraffin wax.

This article is protected by copyright. All rights reserved. Thin sections (5 µm thick) were cut with a rotary microtome and finally stained with haematoxylin- eosin (H-E). Image analysis was performed using CorelDraw graphic Suite X8

(www.coreldraw.com), ImageJ-Fiji (www. imagej.net) and Acrobat Photoshop CS 6.0

(www.adobe.com).

RESULTS

ENDOGENOUS RESERVES AND GROWTH

At male pouch release, remnants of yolk were still present around the intestinal tube as a fragmented and acidophilic mass enveloped by a thick layer of squamous syncytial epithelium [Fig.

1(a)]. Heterogeneous oil droplets were observed on the periphery of the yolk remnants. No signs of

yolkArticle were observed at 30 Dºeff. The onset of exogenous feeding, occurred immediately after pouch release and growth showed an exponential trend from 59 Dºeff (Table I and Figs 2 and 3]. In the present study, histological development is described according to ontogenetic changes observed at each sampling stage (Table II).

NERVOUS SYSTEM AND PITUITARY GLAND

In newborns, different regions of the brain became recognizable. At 30 Dºeff, small packed cells covered the periphery of the optic tectum. By then, it was possible to recognize the forebrain

(secondary prosencephalon, formed by the telencephalon and hypothalamus), the midbrain

(mesencephalon, which includes the optic tectum) and the rhombencephalon (located in the Accepted hindbrain), in which a metencephalon and a myelencephalon were discerned. The spinal cord extended along the body, dorsally to the notochord. It was lined centrally by grey matter and on the periphery by white matter. Different nerve cell types were distinguished in the grey matter whereas the white matter was characterized by the presence of myelinated nerve fibres [Fig. 1(b)]. At 89 Dºeff,

This article is protected by copyright. All rights reserved. a laminar architecture layer characterized the optic tectum. The pituitary gland, located below the hypothalamus, was composed of both neural and glandular tissue. Dorsal to the pituitary gland, a vascularized folded epithelium, the saccus vasculosus, was observed [Fig. 1(c)]. At 354 Dºeff, the cerebellum comprised most of the metencephalon. The cerebellar cortex of grey matter included an outer molecular layer and an internal granular layer. At the boundary between those two layers, the

Purkinje neurons were observed, with many dendrites extending into the molecular layer [Fig. 1(d)].

The open nostrils, located rostral on each side above the mouth, were already functional at birth [Fig.

1(e),(f)]. The spinal nerves and ganglions of peripheral nervous system (PNS) emerged from the spinal cord between the vertebrae.

GILLS AND PSEUDOBRANCH

Article Gills consisted of very thin and vascularized lamellar structures located in bilateral pharyngeal cavities. At birth, a pair of gill filaments was projected from four cartilaginous gill arches, with secondary lamellae still under development [Fig. 1(b)]. At 30 Dºeff, a layer of stratified epithelium covered the distal end of primary lamellae. Several erythrocytes prevailed in the central venous sinus, with a few chloride cells at the edge of the filament [Fig. 1(g)]. The secondary lamellae originated perpendicularly to gill filaments, with slender pillar cells delimiting the capillary blood channels and thus forming lacunae, which regulate the blood flow. At 59 Dºeff, mucous cells appeared in the epithelium of primary lamellae and the secondary lamellae increased in length and number from 89 Dºeff. At Dºeff 177, mucous cells expanded over the epithelium of secondary lamellae and chloride cells proliferated in the interlamellar space of filaments [Fig. 1(g)].

At birth, the pseudobranch appeared as a small, red, round-shape structure located in the inner Accepted side of the operculum, with undifferentiated cells. At 89 Dºeff, the pseudobranch expanded towards the gill cavity and increased in vascularization, acquiring a lamellar shape at 177 Dºeff. During ontogeny, the pseudobranch developed similarly to gills [Fig. 1(h)].

Eyes in newly released juveniles were pigmented and functional, consisting of two spherical lenses, a cornea and sclera (fibrous tunica), a choroid body and iris (vascular tunica) and by retina

(nervous tunica), which consisted of a pigmented epithelium and an inner retinal nerve. At birth, a thin layer made by cones and rods of photoreceptor cells extended from the outer layer of the nervous tunica to the pigmented layer of retina. A thick and homogeneous inner plexiform layer was identified in the central region of the retina, followed by rounded cells forming the ganglia, which connected with the nerve fibres [Fig. 4(a)]. From 89 Dºeff, the vascular choroid that surrounded the optic nerve formed the rete mirabile, a complex network of arteries and capillaries [Fig. 4(b)]. At 118

Dºeff, the thickness of photoreceptors and retinal layers increased [Fig. 4(c)]. A 177 Dºeff the fovea appeared as a well-defined convexiclivate pit with sloping edges [Fig. 4(d),(e)]. In coincidence with

theArticle pit, retinal layers were thinner, while the thickness of photoreceptor and ganglion layers on the fovea slope increased. A thinning of those layers characterized the centre of the pit, providing the highest visual resolution of all retinal areas. At this stage, apical displacement of the outer photoreceptors segment also took place, indicative of scotopic vision improvement. At 354 Dºeff, to maintain the temporal location for the fovea, the addition of new cells in the margin of the growing retina induced a lateral shift of the cells born in the periphery, resulting in irregular growth [Fig.

4(f)].

DIGESTIVE SYSTEM

The mouth and anus were open at birth and four regions were differentiated in the gut of Accepted newborn H. guttulatus: headgut, foregut, midgut and hindgut. A stomach was not present. From the proximal end of the oesophagus to the distal end of the anus, the digestive tract showed the same basic structural composition: mucosa, with lamina propria, submucosa and visceral peritoneum.

Conversely, the muscularis externae was a circular muscle adjacent to the submucosa. The headgut

This article is protected by copyright. All rights reserved. included mouth and pharynx, comprising the region where acquisition and mechanical breakdown of food takes place. The pharynx was located at the level of gill arches and continued posteriorly, developing into an oesophagus (foregut), which expanded considerably with juvenile growth. A single layer of columnar epithelial cells with conspicuous nucleus and thick brush border lined the mucosa layer of midgut, which consisted of a simple straight tube separated from the hindgut by the ileocaecal valve [Fig. 5(a)]. Finally, the hindgut comprised the posterior region of the intestine and the anus. Several longitudinal folds lined by a simple cubic epithelium provided with microvilli were located at the junction between oesophagus and midgut [Fig. 5(b)]. Small sensory cells forming taste buds were randomly scattered in the mouth and oropharyngeal cavity, which were devoid of tongue and teeth [Fig. 5(c)]. A layer of striated muscle fibres and thin lamina propria enveloped the oesophageal lumen, which was lined by a stratified squamous epithelium rich in goblet cells. The goblet cells present in the oesophagus, together with longitudinal folds, increased noticeably in

numberArticle and length with development. In newborns, the nuclei of enterocytes were still under mitosis

[Fig. 5(d)], with few mucous secreting cells interspersed in the midgut. However, numerous mucous cells were abundant in the hindgut. The rectum showed a cuboidal epithelium without any folds or mucous cells. At that stage, the hindgut showed pinocytotic activity marked by apical acidophilic inclusions and large supranuclear vacuoles. From 30 Dºeff, large vesicles were present in the midgut

[Fig. 5(e)], accompanied by undigested Artemia nauplii and copepods being digested.

Initially, the gut appeared as a syphon-like snout with short tubular shape [Figs 5(h),(i),(g) and 6(a),(b),(c)], which later became longer and more slender [Fig. 5(j),(k),(l),(m), 6(d),(e),(f),(g)].

At 89 Dºeff, with the formation of the first intestinal loop [Figs 5(j) and 6(d)], supranuclear vesicles were gradually reabsorbed in the hindgut and lipid vacuoles were also found in the mucosa of the midgut as well as acidophilic granules in the longitudinal folds of oesophagus [Fig. 5(f)]. From 118 Accepted

Dºeff, gut contents become broken-down, digested and almost unrecognizable. At 177 Dºeff, the digestive tract already showed the definitive adult shape, with the formation of a second loop and the accumulation of adipose perivisceral tissue around the midgut [Fig. 5(l), 6(f)]. In addition, intestinal folds elongated considerably. In 354 Dºeff H. guttulatus, following a sharp increase in size, a

This article is protected by copyright. All rights reserved. noticeable expansion in length of the digestive tract was observed [Fig. 5(m), 6(g)]. In addition, the lamina propria became thicker, with a further increase of the midgut absorptive surface, by specialization of cells and tissues of the submucosa and mucosa. Intestinal wrinkles appeared as transverse folds that extended around the lumen of the intestine. Those folds became larger as ontogeny progressed, in accordance with gut elongation and expansion.

ACCESSORY DIGESTIVE GLANDS

Accessory digestive glands included the liver, gallbladder and pancreas. At birth, the liver and pancreas were already formed and functional. The liver was located cranially to the midgut, connected with the posterior portion of the heart through the venous sinus [Fig. 5(b)] and extended longitudinally along the peritoneal cavity [Fig. 5(g)]. Packed hepatocytes with central nuclei were

organizedArticle in a chord-like pattern, separated by a network of sinusoid capillary and biliary canaliculi, with some rounded spaces interspersed in the cytoplasm, which were considered former lipid inclusions [Fig. 7(a)]. The exocrine pancreatic tissue was located adjacent to the liver and lined by acinar cells with dark basophilic cytoplasm, basal nucleus and several eosinophilic zymogen granules

[Fig. 7(b)]. The large gallbladder, characterized by a simple columnar epithelium, was located in the apical region of the swim bladder, between the liver and the pancreas [Fig. 7(c)]. At 30 Dºeff, the liver parenchyma showed a significant increase in lipid deposits, which persisted until Dºeff 89 [Fig.

7(d),(h)]. From Dºeff 118 onwards, lipid deposits were no longer observed in the hepatic parenchyma.

At 177 Dºeff, the liver showed hepatic lobules on both sides of the midgut [Fig. 7(i)]. Moreover, extrahepatic pancreas expanded longitudinally, being able to accommodate at 354 Dºeff the large arterial tree formed by the portal afferent vein [Fig. 7(j)]. At this age, the pancreas was Accepted interconnected with the liver, forming an hepatopancreas. The gallbladder did not undergo significant changes with H. guttulatus ontogeny, whereas bile ducts increased progressively in size.

The heart was located caudally to the gill cavity and separated at birth into four chambers: venous sinus, atrium, ventricle and arteriosus bulb. All chambers were enclosed into the pericardium, a fibrous tissue filled with fluid. In newborns, ventricle and atrium occupied most of the cardiac cavity, whereas the venous sinus was the smallest chamber, with the sinoatrial valve already present.

At birth, first trabeculae appeared into the ventricle [Fig. 7(e)]. The atrioventricular valve appeared at

30 Dºeff, leading to complete compartmentalization of the heart and active blood circulation [Fig.

7(f)]. From 89 Dºeff, the venous sinus expanded equalising the volume of the atrium. At that age,

earliest atrial trabeculae also proliferated and a fully developed heart was observed at 177 Dºeff, with the atrium filled by mature erythrocytes [Fig. 7(g)]. As H. guttulatus developed, the heart grew proportionally without detectable changes.

Article

SWIM BLADDER AND LOCOMOTOR SYSTEM

The swim bladder comprised several layers; from outside inwards, we differentiated the fibromuscular tissue, the tunica externae, the submucosa, the muscularis mucosae (a smooth muscle) and the tunica interna, which includes a columnar secretory epithelium. Such epithelium is slightly modified into the gas gland; a folded columnar epithelium highly vascularized by a dense network of capillaries that forms the rete mirabile. The swim bladder, located dorsally to the midgut, was already differentiated in newborns, but not inflated in all individuals [Fig. 7(c), 8(a)]. The swim bladder was not connected with the foregut through the pneumatic duct. At birth, a functional gas gland and the rete mirabile were identified. At 354 Dºeff, the rete mirabile increased noticeably, forming a counter- Accepted current arrangement of arterial and venous capillaries.

Cartilaginous pectoral, dorsal and anal fins were also present in newborns. At 30 Dºeff, eighteen cartilaginous rays were present in the dorsal fin. From this age, the formation of the

This article is protected by copyright. All rights reserved. vertebral column began in the caudal area by the compression of the hyaline substance of the notochord and the formation of primordial vertebrae.

ENDOCRINE GLANDS

At birth, a thick epithelium bounded by an extracellular colloid lumen marked two thyroid follicles in the foregut, at the level of gill arches. An endocrine pancreas appeared as an islet organ scattered in the exocrine pancreas at 30 Dºeff [Fig. 8(c)]. At 89 Dºeff, a corpuscle of Stannius was observed on the lateroventral surface of the kidney [Fig. 8(d)]. A columnar epithelium lined several lobes, separated each one by loose connective tissue. In 177 Dºeff, a pair of corpuscles of Stannius were observed in the excretory kidney [Fig. 8(e)]. At that age, the number of thyroid follicles increased to nine: thus, generating the thyroid gland [Fig. 8(f),(g)]. The size of the follicles increased

atArticle 354 Dºeff, without changes in number. The endocrine pancreas persisted as a single Langerhans islet with small cells and a rich capillary network.

LYMPHOID ORGANS

The thymus appeared as a large lymphoid organ located dorsolaterally to the gill cavity and delimited by a connective tissue capsule. At birth, few basophilic round cells were interspersed into the thymus, which was surrounded by epithelial cells [Fig. 8(b)]. As development proceeded, the thymus increased in size, becoming an elongated organ at 89 Dºeff, filled with packed thymocytes and melanomacrophage centres. No difference between cortical and medullary regions was noticed. On the other hand, the spleen at birth developed as a compacted eosinophilic organ surrounded by a Accepted fibrous capsule and smooth muscle enclosed in the pancreatic tissue. At 30 Dºeff, ellipsoid capillaries were observed, with formation of primitive splenic sinusoids. During ontogeny, the spleen increased proportionally in size, acquiring an elliptic shape on Dºeff 177, with increased ellipsoid capillaries in the parenchyma [Fig. 8(h)].

KIDNEY AND URINARY BLADDER

The kidney extended along the body, just below the notochord axis. At birth, hemopoietic tissue was observed in the cranial region, lacking renal tubules [Fig. 5(b)]. Excretory activity was observed in the posterior half of the kidney, where primary renal tubules lined by a simple cuboidal epithelium with a brush border were present. Such renal tubules became more convoluted after 30

Dºeff. From 177 Dºeff, blood cells and the haemopoietic portion expanded throughout the kidney [Fig.

8(i)]. As ontogeny progressed, kidney size and number of renal tubules increased.

The urinary bladder, located adjacent to the hindgut, consisted of a thin-walled sac, differentiated from the ureter. The inner layer consisted of one or two layers of epithelial cells lining the inner lumen [Fig. 9(a)]. At birth, the bladder appeared distended, surrounded by smooth muscle

layersArticle and replaced by connective tissue around the urethra. In an empty bladder, several mucosal folds were detected surrounding the lumen. Morphological changes in the urinary bladder were not observed with development.

REPRODUCTIVE SYSTEM

The reproductive system generated from the mesothelium of the excretory system, developing as a paired structure in the dorsal region of the peritoneal cavity. At 0 Dºeff, oogonia were already present [Fig. 9(b)] and a few developed into primary growing oocytes (chromatin–nucleolus stage) at

89 Dºeff [Fig. 9(c)]. The second stage of primary oocyte growth (perinucleolar stage) was observed at

118 and 177 Dºeff. The nucleus enlarged considerably, being bounded by strong basophilic ooplasm Accepted and containing several nucleoli in the periphery of the nucleoplasm [Fig. 9(d),(e)]. At 354 Dºeff, oocytes were significantly larger. Alveoli were observed in the periphery of the cytoplasm (cortical alveoli stage) [Fig. 9(f)], representing the first stage of secondary growth. Oocytes in the cortical

DISCUSSION

Considering the limited information available on the morphophysiology of H. guttulatus seahorses, this study provides important observations of the histological development of this species during early ontogeny. Major developmental changes occurred during the first month of development, coincident with the duration of the planktonic phase of the species after male pouch release (Olivotto et al. 2011; Blanco & Planas 2015).

An indirect development through the production of lecithotrophic larvae (yolk sac present) is typical in the majority of teleosts. A yolk sac is commonly present in newly hatched larvae, which

developArticle and grow in a relatively short time (up to 30 days after hatching) before metamorphosing into juveniles, whose definitive phenotype will be similar to adults but still lacking mature gonads

(Balon, 1999). Conversely to indirect ontogeny, some teleost species have direct ontogeny, generally characterized by the production of larger and high density yolk eggs and longer embryogenesis

(Balon, 1999; Falk-Petersen, 2005). Direct ontogenesis is observed throughout the family

Syngnathidae (seahorses, pipefishes, seadragons, pipehorses) (Kornienko, 2001; Sommer et al.,

2012; Wootton & Smith, 2015). Compared with indirect ontogenesis, direct development is safer, without the cost of metamorphosis and the need of forming temporary organs.

Hippocampus spp. embryos develop into free embryos before being released to the external environment from the male brood pouch. Such embryogenesis is a long process where duration differs among species, particularly when comparing tropical v. temperate species ( Wetzel & Accepted Wourms, 2004; Silveira & Fontoura, 2010; Sommer et al., 2012; Novelli et al., 2015).

In H. guttulatus, newborns are free-swimming and morphologically similar to adults but some yolk remnants are still present surrounding the intestine. Similarly, yolk residuals have been reported in newborn long-snout seahorse Hippocampus reidi Ginsburg 1933 by Novelli (2015), even though

This article is protected by copyright. All rights reserved. that author defined those individuals as larvae with indirect development. From a functional level, the capability to start feeding soon after hatching or after male pouch release is species-specific and determined in teleost fish by the presence of taste buds, open nares, functional visual system, mucous cells in the oesophagus, open mouth and anus, differentiated digestive tract and functional liver and pancreas (Rønnestad et al., 2013). A functional locomotor system is also a prerequisite for effective feeding (Rønnestad et al., 2013). In newborn Hippocampus spp. the presence of cartilaginous fins allows immediate active swimming. In addition, gas transferred from the blood into the swim bladder is responsible for buoyancy control, improving energy-efficiency in prey capture (Ortiz-Delgado et al., 2012). Unlike what has been suggested in a previous study by Palma et al. (2014), a diaphragm regulating contractions between the gas resorbing and the gas producing regions in the swim bladder was not observed in the present study. Therefore, due to the lack of a pneumatic duct connecting the swim bladder with the digestive tract and in agreement with previous findings in H. reidi (Novelli et

alArticle ., 2015), H. guttulatus should be considered a physoclistous species (Genten et al., 2009). The swim bladder is a hydrostatic organ whose functionality has been studied in other teleosts such as the zebrafish Danio rerio (Hamilton 1822) (Finney et al., 2006). As a response to water pressure changes, the release and absorption of gas into this organ is driven by inflation and deflation reflexes controlled by sympathetic and parasympathetic system stimulation via the autonomous nervous system (Finney et al., 2006). Nutritional or physical factors might determine correct development of the swim bladder, or the occurrence of undesirable hyperinflation, as reported in H. guttulatus

(Blanco et al., 2014; Palma et al., 2014).

Some teleost species do not develop a stomach during ontogeny (Wilson & Castro, 2010;

Rønnestad et al., 2013). In such agastric species, lacking a functional stomach and pharyngeal teeth, digestion takes place mainly in the intestine (Rønnestad et al., 2013). A first chemical digestion Accepted begins in the oesophagus and continues in the midgut, where the absorption of nutrients occurs.

Oesophageal goblet cells are directly linked to the ability of fish larvae to start exogenous feeding, improving food ingestion. They play an important role lubricating and protecting the digestive mucosa from chemical and physical damages, thus facilitating the transit of food (Sarasquete et al.,

This article is protected by copyright. All rights reserved. 2001; Sarasquete et al., 2014). Moreover, goblet cells take part in physiological processes such as nutrient absorption, antimicrobial function, binding of hormones and protection of cells from phagocytosis and dehydration (Domeneghini et al., 2005; Bansil & Turner, 2006; Díaz et al., 2008).

Intestinal wrinkles in the midgut are involved in the absorption of nutrients, whereas the absorption of water and electrolytes and the elimination of undigested foods occur in the hindgut (Wilson &

Castro, 2010).

Profiles of digestive enzyme activities have suggested limited digestive capabilities accompanied by low growth rates in early developing H. guttulatus (< 89 Dºeff) (Olivotto et al., 2011;

Blanco et al., 2015). A reduced initial growth in early H. guttulatus life stages is related to the simplicity of their gut anatomy, which develops with age from a short and straight tube into a long and segmented duct. The presence of acidophilic granules and supranuclear vacuoles in the intestine has been described in larvae of many species (Boulhic & Gabaudan, 1992; Luizi et al., 1999; Gisbert

etArticle al., 2004; Hachero-Cruzado et al., 2009; Sarasquete et al., 2014; Yúfera et al., 2014; Park et al.,

2015), resulting from protein pinocytotic uptake and lipid accumulation before stomach development and lipid metabolism becomes functional (Gisbert et al., 1998; Izquierdo et al., 2000). Protein absorption in larval stages is faster in the posterior intestine. Nonetheless, in advanced developmental stages, the absorption occurs mostly in the anterior intestine rather than in the distal (Wilson &

Castro, 2010). Due to the stomach development, acidophilic granules in the teleost intestines become indistinguishable during the transition from larvae to juveniles, while in agastric fishes these are maintained (Govoni et al., 1986; Gisbert et al., 2004). The transport of lipids in the intestine at the onset of exogenous feeding is reduced and only a small proportion of absorbed lipids incorporate into lipoprotein particles, mainly in the distal region (Izquierdo et al., 2000). The presence of lipid droplets in the midgut of first-feeding fishes may be due to enterocyte immaturity. Thus, lipids would Accepted be mobilized when the capacity of enterocytes to synthesize lipoproteins is improved (Deplano et al.,

1991). Intestinal loops commonly develop after the increase in intestinal surface (Wilson & Castro,

2010). In the present study, the first intestinal loop emerged close to 89 Dºeff and the second close to

177 Dºeff, resulting in an increase of the absorptive surface.

This article is protected by copyright. All rights reserved. Excretion of live brine shrimps Artemia nauplii has been reported in early developmental stages of Atlantic herring Clupea harengus L. 1758, pipefish Syngnathus fuscus Storer 1839 and

Atlantic halibut Hippoglossus hippoglossus (L. 1758), due to the short gut passage times of the prey, impeding proper digestion (Govoni et al., 1986; Luizi et al., 1999). However, digestive capabilities might differ from one prey type to another. The retention time of copepods in the chambers of the intestine in young H. reidi is higher than for Artemia nauplii (Govoni et al.1986); therefore, digestion and nutrient absorption improved. A limited initial feeding efficiency (differing with prey offered) was also observed in young H. guttulatus (Palma et al., 2011; Planas et al., 2012), probably due to low enzymatic secretory organ activity and limited digestive capabilities (Blanco et al., 2015). The potential mechanical processing of a given prey would be another important factor regarding digestive capabilities. Young H. guttulatus are able to break copepods into small pieces (pers. obs.), facilitating the action of digestive enzymes in the gut. This has not been observed with Artemia

nauplii.Article Hippocampus spp. ingest prey by suction, thus only the pharynx is involved in the mechanical processing of softer prey before entering the intestine.

Accessory digestive glands (liver, pancreas and gall bladder) were already present in H. guttulatus newborns, being connected to the intestine by bile and pancreatic ducts. Lipids began to accumulate in the liver at 30 Dºeff, indicating the functional development of the digestive tract and the synthesis and storage of macromolecules. As described in other teleosts (Luizi et al., 1999; Diaz et al., 2002; Liu et al., 2013), lipoprotein accumulation increased with growth, reaching maximum levels about Dºeff 89. Another important function of the liver is the production of bile, which will be stored in the gall bladder for the digestion of lipids via biliary and pancreatic enzymes (Diaz et al.,

2002; Rønnestad et al., 2013). The presence of enzymatic precursors, revealed by the existence of zymogen granules, suggests that the pancreas was also functional in newborn seahorses, highlighting Accepted the importance of the pancreatic secretion in agastric fishes (Gisbert et al., 2004). In contrast to other teleosts (Chen et al., 2006; Galaviz et al., 2011; Sarasquete et al., 2014), a recent study on H. guttulatus (Blanco et al., 2015) reported that trypsin levels do not increase during early development and that chitinase and α-amylase activities increase from 89 and 177 Dºeff, respectively. Such findings

This article is protected by copyright. All rights reserved. would support the hypothesis that digestive enzyme dynamics correspond to the anatomical development of the digestive system (Chen et al., 2006; Álvarez-González et al., 2010; Galaviz et al.; 2011).

The central nervous system (CNS) and PNS are the two basic components of the nervous system. The CNS includes the brain and the spinal cord, while the PNS is composed of ganglia and spinal nerves. The nervous system was completely formed in H. guttulatus newborns, with a progressive increase in size of the optic tectum and differentiation of pituitary gland and saccus vasculosus. A recent study on the evolution of the brain in Sygnathidae (Tsuboi et al., 2017) supports the hypothesis that brain development is based on evolutionary capabilities adapted to feeding on highly mobile prey, so that the processing of visual information led to larger brain sizes compared with species that feed on less mobile prey (Tsuboi et al., 2017). Environmental adaptation also contributes to modifications in the brain structure, resulting in increased eye size in planktivorous

fishArticle and an enlargement of the optic tectum in species feeding on fast-moving prey (Kotrschal et al.,

1998). The pituitary gland in H. guttulatus was highly recognizable at the second stage of development (from 35 to 89 Dºeff). This endocrine gland is involved in teleost growth and reproduction, through liberation of hormonal stimuli and gonadotropin activation (Patiño & Sullivan,

2002; Falk-Petersen, 2005; Piferrer & Guiguen, 2008; Piccinetti et al., 2012). In addition, development of the saccus vasculosus allowed juveniles to perceive seasonal changes in day length

(control of photoperiod) (Nakane et al., 2013; Nishiwaki-Ohkawa & Yoshimura, 2016) and is related to reproduction and growth, influencing the metabolic rate, body pigmentation, behavioural activity and feeding performance (Crovatto-Veras et al., 2013).

At birth, eyes were already pigmented and the visual system was fully functional, allowing H. guttulatus to be excellent visual predators (Van Wassenbergh et al., 2009; Roos et al., 2011; Celino Accepted et al., 2012; Blanco & Planas, 2015). The fovea, a depression in retinal tissue, is a specialized area for high acuity vision in many vertebrates. It facilitates accurate fixation on objects and image focussing (Harkness & Bennet-Clarck, 1978). The structure of the fovea observed in adults of the big-belly seahorse Hippocampus abdominalis Lesson 1827, spotted seahorse Hippocampus kuda

This article is protected by copyright. All rights reserved. Bleeker 1852, west Australian seahorse Hippocampus subelongatus Castelnau 1873 and Barbour’s seahorse Hippocampus barbouri Jordan & Richardson 1908 (Lee, 2013; Mosk et al., 2007; Olivotto et al., 2011), also appeared in H. guttulatus at 177 Dºeff, coinciding with a shift from pelagic to benthic habitats. Asymmetrical retinal growth observed in the following stage (354 Dºeff), permits maintenance of the fovea’s temporal position, impeding that portion being covered by retina cells, which are constantly regenerated (Easter, 1992). Moreover, the direct vascular connection of the pseudobranch with the choroid of the eye suggest that it is responsible for control of ocular blood pressure (Genten et al., 2009).

Gill arches and filaments are responsible for ion exchange and develop earlier than lamellae, which regulate gas exchange (Rombough, 2007). The presence and distribution of mucous cells vary among species, being more abundant in primary than secondary lamellae (Díaz et al., 2008;

Srivastava et al., 2012). Primary gill filaments were observed in newborn H. guttulatus. Chloride

(ion-rich)Article and mucous cells developed in a later stage, prior than secondary lamellae. Hence, the primary role of gills in early developmental stages are ionic regulation and osmoregulation, whereas respiratory function develops in later stages (Hachero-Cruzado et al., 2009; Brauner & Rombough,

2012). Considering that secondary lamellae are responsible for oxygen uptake, active respiration in

H. guttulatus starts in the third stage of development (from 94 to 177 Dºeff) (Rombough, 2007).

Organs such as the heart, kidney and haematopoietic kidney are also involved in oxygen transportation, developing early in the ontogeny of H. guttulatus and being functional close to 89

Dºeff. This finding is in agreement with observations in Atlantic Bluefin tuna Thunnus thynnus (L.

1758), a teleost with an advanced developmental larval stage (Yúfera et al., 2014). At 89 Dºeff, corpuscles of Stannius, the primary function of which is the control of calcium regulation, were also present in the kidney. Glomeruli have not been reported in the excretory kidney of H. guttulatus Accepted (Genten et al., 2009), but some adaptations to the lack of glomeruli, such as osmoregulatory adjustments in body fluid compositions, have been observed in H. kuda, a species tolerating salinities between 15 and 50 (Hilomen-Garcia et al., 2003).

This article is protected by copyright. All rights reserved. Endocrine glands and follicles in teleosts are involved in peptide and thyroid hormone secretion (Santamaría et al., 2004; Hachero-Cruzado et al., 2009; Sarasquete et al., 2014; Yúfera et al., 2014); thus, regulating protein synthesis, metabolism and growth (Genten et al., 2009). The endocrine pancreas (Langerhans islet) and thyroid follicles were already developed in newborns of H. guttulatus.

The spleen, pronephron (head kidney) and thymus are organs involved in the immune system of fish (Falk-Petersen, 2005; Mahabady et al., 2012). In marine larvae, the head kidney is the major haemopoietic-lymphoid organ and the first to develop (Falk-Petersen, 2005; Geven & Klaren, 2017).

Lymphomieloid cells were observed in the pronephron tissue of H. guttulatus throughout development, whereas lymphoid organs (spleen and thymus) emerged later. In H. guttulatus, the thymus was the first organ to become lymphoid, in agreement with observations in the platyfish

Xiphophorus maculatus (Günther 1866) (Leknes, 2003). An increase in size was the only trait

observedArticle in the spleen of seahorses during the study period.

Sex in marine fish differentiates in different ways (Devlin & Nagahama, 2002; Piferrer &

Guiguen, 2008). In agreement with Novelli (2015), male gonads were not observed in the present study, but the first oogonia were present from 30 Dºeff. The second stage of primary oocyte growth

(perinucleolar stage) appeared at the third stage of development (from 94 to 177 Dºeff), whereas oocytes in the cortical alveoli stage became gonadotropin-dependent at 354 Dºeff, indicating that individuals were mature. Such oocytes would be released during the current breeding season (Brown-

Peterson et al., 2011).

Considering the ecology and the mating system of seahorses (Foster & Vincent, 2004), the idea that seahorses could be protogynic hermaphrodites, as proposed by Novelli (2015), is not supported by the present study. However, this subject deserves further consideration in order to Accepted ascertain the factors involved in seahorse sex determination (e.g. environmental factors such as temperature) and how wild population would be affected in a global scenario of increased temperature.

This article is protected by copyright. All rights reserved. In summary, H. guttulatus juveniles are characterized by direct development and the lack of a stomach (Wilson & Castro, 2010). Most vital organs were already present upon release from the male brood pouch and they continue developing throughout ontogeny. The low digestive efficiency observed at early life stages is probably due to a poorly developed and immature gastrointestinal tract prior to Dºeff 89. The present study demonstrates that developmental stages around 89 and 177 Dºeff are important transitional stages in early development of the species. At a temperature of 19 ± 1º C, main organs were fully functional at 177 Dºeff (30 dpp), suggesting that the beginning of the definitive phenotype could be established around that stage, with females becoming mature at an age of two months (354 Dºeff).

This study was partially financed by the European Union (Erasmus traineeship) and by a grant awarded by the Spanish

Ministry of Education, Culture and Sport within the Campus do Mar International program, University of Vigo. We Article express our gratitude to J. Pintado, M. Pombal and F. Saborido-Rey for scientific support and to C. Gestal for technical assistance in microscope image acquisition. We are also grateful to A. Chamorro and G. Caporale for technical support in the experimental work and to A. Paltrinieri for his contribution to image processing.

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effective day degrees (Do) at 19 ± 1o C. (a) Endogenous reserves remaining in newborn surrounding the digestive tract; (b) central nervous system and cranial organ distribution; (c) central nervous system and pituitary gland; (d) cerebellum filling the metencephalon; (e) olfactory bulb in the forebrain; (f) nasal aperture located laterally above the mouth; (g) gill morphology; (h) pseudobranch morphology. Af, afferent artery; BV, blood vessel; CR, choroid; CS, cartilaginous skeleton; E, eye;

Ef, efferent artery; FB, forebrain; FG, foregut; GA, gill arches; GC, gill cavity; H, heart; HB, hindbrain; HG, hindgut; hp, hypophthalmus; L, liver; La, lacunae; MC, mucous cells; MF, muscular fibres; my, myelencephalon; MG, midgut; mt, metencephalon; N, notocord; Nr, nare; O, otholith;

OB, olfactory bulb; OE, oesophagus; ON, optic nerve; OC, optic capsule; OD, oil droplet; ot, optic tectum; Pa, pancreas; PG, pituitary gland; Pkj, Purkinje cells; PL, primary lamellae; tl, Article telencephalon; PSB, pseudobranch; sv, saccus vasculosus; SL, secondary lamellae; VS, venous sinus; YSL, yolk syncytial layer; YS, yolk sac; 1 molecular layer; 2, granular layer. →, Pillar cells;

►, chloride cells.

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FIG. 2. Mean (± S.D.) wet mass (MW) in Hippocampus guttulatus during ontogeny over a period of (a)

0.064x 2 60 days (y = 6.996e , r = 0.973, P < 0.05) and (b) 354 effective day degrees (Dºeff; y =

6.996e0.011x, r2 = 0.973, P < 0.05).

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FIG. 3. Mean (± S.D.) standard lengths (LS) in Hippocampus guttulatus during ontogeny over a period

0.022x 2 of (a) 60 days (y = 16.647e , r = 0.972, P < 0.05) and (b) mean (± S.D.) wet mass (MW) against LS

(y = 0.002x2.910, r2 = 0.977, P < 0.05) over a period of 60 days.

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FIG. 4. Sagittal histological section of Hippocampus guttulatus visual system (H&E) over a period of

0–354 day degrees (Dº). (a) Functional eyes; (b) vascularization of retina by rete mirabile; (c) retinal layers (d) temporal location of fovea; (e) temporal location of fovea; (f) lateral displacement of retina. A, artery; BR, brain; C, cornea; CR, choroid rete; F, fovea; gcl, ganglionic cell layer; in, inner nuclear layer; I, iris; ip, inner plexiform layer; Ls, lens; MF, muscular fibres; nf, nerve fibres; on, outer nuclear layer; pr, photoreceptors; R (→), retina; S, sclera; 1, photoreceptor layer; 2, outer nuclear layer; 3, outer plexiform layer; 4, inner nuclear layer; 5, inner plexiform layer; 6, ganglionic cell layer; 7, layers of optic nerve fibres.

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FIG. 5. Sagittal histological section of Hippocampus guttulatus digestive system (H&E) over a period of 0–354 day degrees (Dº). (a) Midgut and hindgut separated by ileocecal valve. Lipid

This article is protected by copyright. All rights reserved. vacuoles (*); (b) anterior region of digestive system and associated organs. Magnification shows the transition between stratified and columnar oesophageal epithelium; (c) taste buds in buccopharyngeal cavity, magnification of a sensory cell; (d) midgut with nucleus of enterocytes under mitosis (*); (e) acidophilic droplets in the midgut included in a circle; (f) longitudinal folds with acidophilic granules included in the circles; (g)–(m) digestive tract during ontogeny, where (l) and (m) show adipose tissue (**). Glycoconjugates scattered in the midgut and hindgut (►). BB, brush border; BR, brain;

G, gonads F, faeces; FG, foregut; FR, fins ray; G, gills; GB, gall bladder; H, heart; Hg, headgut; HG, hindgut; IC, intestinal content; ICV, ileocecal valve; K, kidney; L, liver; LF, longitudinal folds; MF, muscular fibres; MG, midgut; OE, oesophagus; Pa, pancreas; Ph, pharynx; TB (→), taste bud; SB, swim bladder; Sc, spinal cord; UP, urinary pore; VS, venous sinus; YS, yolk sac; 1, first intestinal loop; 2, second intestinal loop. Article Typesetter

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FIG. 6. Illustration of the development of the intestinal tract during early ontogeny of Hippocampus guttulatus over a period of 0–354 day degrees (Dº).

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FIG. 7. Sagittal histological section of Hippocampus guttulatus, extramural organs (H&E) over a period of 0–354 day degrees (Dº). (a) Hepatic parenchyma of a newborn seahorse. Packed hepatocyte, sinusoid capillary (long →) and biliary canaliculi (short →); (b) exocrine pancreas with

This article is protected by copyright. All rights reserved. dark basophilic cytoplasm and zymogen granules; (c) gallbladder located between pancreas and liver, cranially to the swim bladder; (d) lipid deposits (*) in hepatic parenchyma; (e) heart compartmented with open senoventricular valve; (f) heart with atrioventricular valve open; (g) heart with mature erythrocytes and trabeculae in the atrium; (h) liver, pancreas, spleen, gall bladder and islet of Langerhans included in the exocrine pancreas; (i) differentiation of two hepatic lobules surrounding the midgut; (j) hepatic arterial tree and hepatopancreas. At, atrium; av, atrioventricular valve; BA, arteriosus bulb; ep (→), hepatocyte; IL, islet of Langerhans; GB, gall bladder; gg, gas gland; K, kidney; L, liver; MG, midgut; Pa, pancreas; Pv, portal vein; SB, swim bladder; SP, spleen; sv, senoventricular valve; Tr, trabeculae; Ve, ventricle; VS, venous sinous; Zy, zymogen granules; 1, tunica externae; 2, submucosa; 3, tunica interna.

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FIG. 8. Sagittal histological section of Hippocampus guttulatus, endocrine gland, lymphoid organs and kidney (H&E) over a period of 0–354 day degrees (Dº). (a) Swim bladder before inflation; (b) thymus adjacent to the gill cavity; (c) islet of Langerhans (endocrine pancreas) included in exocrine pancreas; (d) corpuscle of Stannius scattered in the excretory kidney; (e) paired corpuscles of

Stannius (*); (f) thyroid follicles filled with extracellular colloid lumen; (g) thyroid gland; (h) spleen with ellipsoid capillaries in the parenchyma; (i) kidney filled with haemopoietic tissue and blood cells. A, artery; bc (→), blood cells; el (→), ellipsoid capillary; GB, gallbladder; GC, gill cavity; gg, gas gland; He, hemopoietic kidney; IL, islet of Langerhans; K, kidney; mm, muscularis mucosae; Pa, Accepted pancreas; RM, rete mirabile; RT, renal tubules; sb, submucosa; SP, spleen; te, tunica externae; TF, thyroid follicle; TG (↔), thyroid gland; Ty, thymus; UB, urinary bladder.

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FIG. 9. Sagittal histological section of Hippocampus guttulatus, excretory system and gonads,

(H&E) over a period of 0–354 day degrees (Dº). (a) Excretory system; (b) oogonia in the ovary; (c) first stage of primary growth, chromatin-nucleus oocytes; (d)–(e) second stage of primary growth, perinucleolar oocytes; (f) first stage of secondary growth, the cortical alveoli oocytes; (g) oocytes in the ovary organized in progressive order of development. A, alveoli; CA, cortical alveoli; CN, chromatin nucleus; HG, hindgut; k, kidney; n, nucleus; nl, nucleoli; np, nucleoplasm; OG, oogonia;

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TABLE I. Growth and Fulton’s condition index (K) of Hippocampus guttulatus during the

ontogeny as days post partum (dpp) and effective degree days (Dºeff)

dpp Dºeff MW LS g G K (19 ± 1ºC) (mg) (mm) (% day–1) 0 0 5.5 ± 1.0 15.1 ± 1.1 1.59 5 30 7.5 ± 2.2 17.4 ± 1.6 0.06 6.40 1.42 10 59 15.2 ± 3.4 21.2 ± 2.0 0.14 15.22 1.60 15 89 23.0 ± 7.1 25.3 ± 2.4 0.08 8.55 1.41 20 118 29.7 ± 9.2 27.7 ± 2.5 0.05 5.26 1.40

Article 30 177 59.6 ± 22.7 34.4 ± 4.1 0.07 7.04 1.42 60 354 272.8 ± 57.1 58.5 ± 4.0 0.05 5.27 1.58 MW, wet mass; LS, standard length; g, instantaneous growth coefficient; G, daily mass-specific growth rates.

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TABLE II. Anatomical changes in Hippocampus guttulatus during the ontogeny (from 0

to354 Dºeff). Number in brackets indicate the effective day-degrees (Dºeff) at which histological changes appeared

Morphological observations Organ Stage 1 Stage 2 Stage 3 Stage 4 (0–30 Dºeff) (35–89 Dºeff) (94–177 Dºeff) (183–354 Dºeff) Eyes Pigmented (0) Rete mirabile (89) Fovea (177) Retina growth (354) Nervous Few cells in optic Laminar architecture of Size increase Size increase system tectum (30) optic tectum; pituitary gland, saccus vasculosus (89) Gills Gills arch (0) Mucous cells in primary Mucous cells in secondary Well developed lamellae (59) lamellae (177) Heart 4 chambers (0) Atrial trabeculae (89) Completely developed Size increase (177) Atrio-ventricular valve – – – (30) Headgut Taste buds, 2 thyroid – 9 Thyroid follicles (177) – follicle, mouth opened (0) Foregut Oesophagus with CM, Acidophilic granules in Longitudinal folds Longitudinal folds

Article longitudinal folds (0) oesophagus (89) elongation (177) elongation (354) Yolk Homogeneous – – – acidophilic (0) Reabsorbed (30) – – – Midgut Mitotic enterocytes (0) First intestinal loop (89) Second intestinal loop, Digestive tract adipose tissue expansion (354) accumulation (177) Supranuclear vacuoles – – – (30) Hindgut Ileocecal valve, mucous Supranuclear vesicles – – cells, acidophilic reabsorbed (89) supranuclear granules, lipid vacuoles, anus open (0) Extramural Extended liver, Liver with lipids droplets Lipid mobilization (118) Hepatopancreas organs pancreas and gall (89) Two hepatic lobules (354) bladder functional (177) (0), Lipid vacuoles in the liver (30) Swim bladder Bladder with rete – – – mirabile (0), functional bladder (30) Endocrine 2 thyroid follicles (0), Corpuscle of Stannius (89) 9 Thyroid follicles, 2 –

Accepted glands Langerhans islet (30) corpuscles of Stannius (177) Linfoid Hemopoietic thymus Limphoid thymus (89) Increased size in splenic – organs and spleen (0) capillaries (177) Kidney Lympho-myeloid tissue – Blood and haemopoietic Increase in size and red blood cells cells (177) (354) (0), renal tubules (30) Gonads Oogonia (0) Oocytes at chromatin- Oocytes at perinucleolar Oocytes at cortical nucleolus stage (89) stage (118) alveoli stage (354) Locomotor Cartillaginous fins (0) – – – system Habitat Pelagic (0) – Benthonic (177) – Thisbehaviour article is protected by copyright. All rights reserved.