sm80n4-4404

INTRODUCTIONTop Among the brachyurans, portunids stand out as highly valued fisheries and aquaculture species on account of their export potential and high nutritional value (). Since worldwide captures of this resource reached their asymptote at 90 million tonnes 20 years ago (), unmet demands have led to overexploitation of several local resources. This has promoted research on breeding techniques to restock overfished areas () and to enhance production through aquaculture (, , , ). While China, Indonesia and USA report the highest captures worldwide (), India, Micronesia, Philippines and Vietnam are pillars of the breeding industry, where hatcheries have achieved efficient development and growth (, , ). Crustaceans show great variability in stages and total duration of their embryonic development, resulting from evolutionary adaptations (, , ) and from physiological modulation by environmental conditions (, ). The knowledge of this part of the ontogeny is not only of intrinsic scientific interest but also a prerequisite for developing hatchery and nursery practices (, , ). Also, it can be used in forecasting temporal patterns of larval production for fisheries management purposes (). In most species, embryonic development is one of the phases of the life cycle that are most sensitive to environmental stressors (, , so its characterization under normal conditions is required to understand their effects. Although embryogenesis in brachyurans is a continuous process, partition in steps or stages of development, defined by the most important morphological changes, is a useful tool for their characterization (). The transit through successive stages of embryonic development includes changes in both shape and size (). Eggs vary in shape, from spherical to elliptical, and can change from one to the other during development (). In studied portunids, eggs sphericity is maintained throughout development, except in some species where slight ellipticity is observed at the onset of eye formation (, ). In some members of this family, the size of eggs right after spawning is positively correlated with the size of the progenitor female, but this is not a general rule (, , ). On the other hand, egg size increase is an intrinsic ontogenetic process occurring gradually through embryogenesis and culminating at a faster rate at the end of development (, ). The southern surf crab, Ovalipes trimaculatus (de Haan, 1833), is a portunid with continuous distribution on the mid-latitude (25-45°S) marine coasts of the Southern Hemisphere, reported in Argentina, Australia, Brazil, Chile, Japan, South Africa, Australia, Peru, Uruguay and some islands of the Indian and Pacific Oceans (, , ). It is a fast-growing (Barón, unpublished data), highly valued species (, ), and therefore a good candidate for local aquaculture. It is targeted by artisanal fisheries using trawl and seine nets, baited traps and scuba diving (), and is suitable for processing into a diversity of consumer products (). Although there is detailed information on most stages of the O. trimaculatus life cycle (, , ), there is still a knowledge gap on the phase of embryonic development. The aim of this work is to characterize the morphological changes during embryogenesis, as well as the duration of the process and the embryonic survival. The results are expected to be useful to complement the available information on its life cycle, which will allow the comparison of evolutionary traits with other related species, and to provide important knowledge for the design of hatchery practices. MATERIALS AND METHODSTop Specimen collection and experimental design Twenty-six ovigerous O. trimaculatus females carrying embryos at the earliest stage of development (i.e. morula in the first or second cell division) were hand-collected by SCUBA diving on subtidal sand bottoms of Nuevo Gulf near the city of Puerto Madryn, Argentina (42°25′S, 64°07′W) during the reproductive season (October-December) of 2014. Specimens were transported to the Experimental Marine Aquarium of the National Patagonian Centre (CCT CONICET-CENPAT), where they were acclimated in plastics tanks containing filtered seawater with continuous aeration. The females were marked using coloured rubber bands placed around the cephalothorax between the chelipeds and the first pair of pereiopods, in such a way that these could not be removed. The maximum widths of their carapaces and abdomens were measured with a digital caliper to the nearest 0.1 millimetre at the beginning of the experiments. Seawater temperature during embryonic incubation experiments was set at 13±1°C (mean±sd), simulating not only the seawater temperature at the time of crab collection but also the average surface seawater temperature during the reproductive season (). Salinity was kept at 33±1 (mean±sd), resembling typical values from waters of Nuevo Gulf (), and photoperiod was fixed at 14 h light:10 h dark, emulating the natural light cycle at the time of experimentation. The crabs were fed fish meat once a week and 20% of the seawater volume in the tanks was renewed daily. To allow important relevant developmental changes to be observed through embryogenesis while minimizing animal stress, females were randomly sorted into three groups in which individual egg masses were sampled: 1) weekly (n=10); 2) three times per week (n=10); and 3) once, after hatching (control; n=6). The control group was used to determine the total duration of embryonic development without causing any stress. Taking into account that differences in the rate of oxygen delivery to embryos could have effects on their survival and on the duration of embryogenesis (), three ≥10 egg-samples (i.e. developing embryos) were carefully excised from the inner (i.e. deep, near the bases of the pleopods), middle (i.e. at mid-depth) and outer parts (i.e. on the external surface) of the egg mass on each sampling. Eggs were taken using thin surgical forceps, placed in seawater on excavated slides and examined “in vivo” under an Olympus stereomicroscope with total magnification of 100×. Based on changes in morphological and physiological characters such as yolk colour, yolk content (%), development of appendages, chromatophores and eye pigmentation, five stages were defined to characterize the embryonic development. Representative morphological features of each of the stages were photographed using a Nikon D3000 digital camera. Observations, measurements and calculations Since preliminary observations showed significant shrinkage and deformation of eggs preserved in fixing solutions, all measurements were made on fresh samples. Fifty eggs from each development stage, ten from each of five different females, were used for measurements. These eggs were individualized with forceps under a dissecting stereomicroscope (Olympus CH30) with total magnification of 10× and photographed using a Nikon D3000 digital camera. Resulting images were then processed using the ImageJ program to obtain three orthogonal measurements of egg diameter. Except for the earliest developmental stage, from morula to gastrula, these corresponded to the body axes of embryos: Diameter I (DI, antero-posterior axis), Diameter II (DII, ventro-dorsal axis), and Diameter III (DIII, latero-lateral axis). Based on these, several morphometric variables were calculated, including area (A), volume (V), ellipticity (e) and elongation (E) as defined by : A = π r², where r is half of the mean egg diameter; V = 1/6 π d³, where d is the mean egg diameter;

e= \frac{DI}{DII}

and

$E = \frac{DI – DII}{DI + DII}$

The standard colour of live embryos was determined following the Pantone® Colour Formula Guide (Pantone Inc., Carldtadt, USA) and was recorded as P followed by the number that represents the code of the Pantone colour scale.

The relative duration of each embryonic stage (RDE_i) was estimated as

${RDE}_{i} = \frac{{DE}_{i}}{DE} 100$ ,

where DE_i is the absolute duration of embryonic stage i and DE is the total length of embryogenesis.

Since it has been reported that O. trimaculatus suffers substantial egg loss during embryonic development (Brante et al. 2004Brante A., Cifuentes H.O.P., Wolf A., et al. 2004. Latitudinal comparisons of reproductive traits in five brachyuran species along the Chilean coast. Rev. Chil. Hist. Nat. 77: 15-27.), and considering that in many crustacean species egg cleaning results in the non-selective detachment of embryos from the egg mass (Brante et al. 2004Brante A., Cifuentes H.O.P., Wolf A., et al. 2004. Latitudinal comparisons of reproductive traits in five brachyuran species along the Chilean coast. Rev. Chil. Hist. Nat. 77: 15-27.), estimation of embryonic mortality is not straightforward for the species. Instead, based on the assumption that embryos remain attached to the egg mass during brief periods after death, “instantaneous embryonic mortality” at the end of stage i (EM_i) was estimated as

${EM}_{i} = \frac{D_{i}}{T} 100$ ,

where D_i is the number of dead embryos in a sample of the egg mass taken at the end of stage i and T is the total number of eggs in the same sample, including live and dead embryos. Embryos were considered dead when yolk had a different colour or texture to those of its neighbours. In stages II to V, when the embryo was clearly distinguishable, yolk droplets were smaller and dispersed around the embryo.

Similarly, instantaneous mortality from the end of the last stage of embryonic development to the end of hatching (EM_h) was estimated as

${EM}_{h} = \frac{D_{h}}{T} 100$ ,

where D_h is the number of dead embryos found in samples of the egg mass and T is the total number of eggs, including broken egg capsules remaining attached to the pleopods after hatching.

To measure heartbeat frequency of embryos in an advanced stage of development, the beating of 10 embryos’ hearts was counted for a minute and then averaged.

Data analysis

When normality (Kolmogorov-Smirnov test) and homoscedasticity (Fisher test) assumptions were fulfilled, differences in mean values of different morphometric variables between different stages of embryonic development were tested using one-way analysis of variance (ANOVA), followed by post hoc comparisons between stages using Tukey tests. Otherwise, non-parametric Kruscal-Wallis tests and Dunn post hoc tests were used. The Spearman correlation test was used to observe the relation between the carapace width of females and the dimensions of their eggs.

RESULTSTop

Ovigerous females used for the incubation experiments ranged from 63.4 to 99.2 mm in carapace width and 17.2 to 31.5 mm in abdomen width. Optimum egg sampling frequency to detect important changes during embryogenesis was three times a week. Survival of ovigerous females during the experiments was 100% for the control group and 90% for the groups of females whose eggs were sampled weekly and three times a week.

Five stages of embryonic development were defined for O. trimaculatus based on several morphological characteristics, as described below:

Stage I

After fertilization, eggs are spherical, 309.76±7.13 μm in diameter and intense orange-coloured (P. 123) (Fig. 1: A1-A5). Two transparent membranes, inner and outer (Fig. 1: A1), cover the egg, whose volume is completely occupied by yolk. The external egg cover is adhesive at spawning and projects into a funiculus that holds the egg attached to the female’s pleopod setae. After cell division starts (Fig. 1: A2), slender grooves delineate polygonal areas on the egg surface, and the centrolecytic zygote progressively transforms into morula (Fig. 1: A3), blastula and gastrula. In contrast, in unfertilized eggs the outer cover and the egg yolk are not in contact (Fig. 1: A1). No organ structures are observed at this stage, which lasts 9.65±1.01 days, representing an RDE of 26.76±2.58% (Table 1).

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Fig. 1. – Morphological changes in the eggs and embryos of Ovalipes trimaculatus during the embryological development stages (stage I, A1-A5; Stage II, B1-B3; stage III, C1-C4; stage IV, D1-D5; stage IV, E1-E8). Scale bar = 100 μm (A1 at D2; D4; E1; E2; E5 and E6). Scale bar = 50 μm (D3; D5; E3; E4; E6). References: antenna (a’); antennule (a); chromatophores (ct); external cover (ec); eye (e); heart formed (h); internal cover (ic); maxilla (m’); maxillipeds (mp); maxillule (m); optic globe (op); primordial structures (p); setae (s); telson (t); thoracic-abdominal buds (ta); yolk (y).

Table 1. – Kruskal-Wallis tests for differences in values of egg morphometric variables between stages of embryonic development of Ovalipes trimaculatus. RDE, relative duration of embryonic stage; DI-DIII, egg diameters; A, area; V, volume; E, elongation and e, ellipticity. Values are mean±standard deviation; n, number of eggs analysed. See detailed description of morphometric dimensions, indexes and colours in the section of Materials and Methods.

	Stage I	Stage II	Stage III	Stage IV	Stage V	Kruskal-Wallis Test significance and p values
RDE (%)	26.76±2.58	22.36±2.76	22.38±2.10	16.19±1.75	13.3±1.29	p<0.01
D I	307.26±9.10	324.26±11.97	328.8±16.92	364.76±18.11	368.36±15.96	H=176.96; p<0.01
D II	312.26±8.38	329.72±13.17	333.16±14.827	370.36±16.69	376.12±18.89	H=178.34; p<0.01
D III	311.06±7.83	325.4±11.61	330.1±15.47	361.78±19.42	368.16±16.48	H=180.42; p<0.01
A	493.98±11.31	520.48±18.181	552.82±21.52	581.97±29.21	589.70±22.95	H=183.96; p<0.01
V	165.54±3.73	174.56±5.85	175.29±6.89	195.80±8.74	197.15±6.29	H=183.96; p<0.01
E	0.008±0.016	0.008±0.017	0.004±0.017	0.007±0.013	0.010±0.016	p=0.4488
e	1.016±0.033	1.017± 0.036	1.009±0.035	1.016±0.028	1.021±0.034	p=0.4489
Colour	P123	P157	P484	P464	P469
n	50	50	50	50	50

Stage II

Eggs are spherical, 326.99±11.17 μm in diameter and orange-coloured (P. 157) (Fig. 1: B1-B3). The embryonic primordium becomes evident as a translucent bulge on the egg’s surface at the animal pole (Fig. 1: B1), which represents the ventral region of the developing embryo. Rudiments of some embryonic appendages become visible as a long pair of preoral structures emerging along the antero-posterior axis, the antennules being the first, followed by a shorter pair of antennae (Fig. 1: B2). Both appendages are uniramous and elongated. The posterior thoraco-abdominal plate has a short, wide projection with its distal extremity bifurcated into the two lobes of the forming telson (Fig. 1: B3). The yolk content represents approximately 90% of the egg volume. Stage duration is 8.10±0.94 days, which represents an RDE of 22.36±2.76% (Table 1).

Stage III

Eggs are spherical, 331.98±18.38 μm in diameter and dark-red coloured (P. 484) (Fig. 1: C1-C4). The stage begins when the first three pairs of appendages (i.e. antennule, antennae and mandible) are formed (Fig. 1: C3, C4). Developing structures represent approximately 40%-50% of the egg volume, the rest being yolk. The antennule and antenna are tube-shaped structures without setae, growing caudally parallel to each other while mandibles become comparatively smaller (Fig. 1: C3, C4). As this stage progresses, abdominal somites and elongated rudiments of the rest of the appendages form posteriorly. At the distal end of the abdomen, the telson is biforked, still without setae. Afterwards, maxillule, maxillae and first and second maxillipeds form, while crescent-shaped carapace rudiments appear on the anterior part of the cephalothorax. At the end, four short terminal setae appear on each telson lobe. The stage lasts 7.83±0.90 days, which represents an RDE of 22.38±2.10% (Table 1).

Stage IV

Eggs are spherical, 367.56±16.69 μm in diameter and brown-coloured (P. 464).). Pigmented eyes are first visible (Fig. 1: D1). The antennules are uniramous and longer than antennae, displaying two long terminal setae. The antennae are uniramous and with two terminal setae. The maxillule and maxilla are uniramous. The optic lobes are well developed, showing a pigmentation pattern consisting of a dark curved line growing from the antennule side of the compound eyes (Figs. 1: D4, D5). The first and second maxillipeds are biramous, with three terminal setae at the end of the endopods (Fig. 1: D4). The abdomen grows continuously incorporating segments. In the biforked telson, four pairs of setae are visible on each lobule (Fig. 1: D3). At the end, yolk reserves are significantly reduced to approximately 30% to 40% of the egg volume. Stage duration is 5.59±0.66 days, which represents an RDE of 16.19 ±1.75% (Table 1).

Stage V

Eggs are spherical, 372.24±16.32 μm in diameter and dark brown–grey coloured (P. 469) (Fig. 1: E1-E8). The yolk is reduced to a few droplets in the dorsal part of the embryo and its volume progressively reduces through this stage while the embryo grows (Fig. 1: E1). Heartbeat and dorsal spine become first noticeable and body motion can be observed. Dot-like pigmented chromatophores appear on the bases of the mandibles and maxillipeds and on each abdominal segment (Fig. 1: E2-E8). Pigmentation on the ocular globe expands and increases while the compound eye enlarges. A transversal pigmented line appears on the mouth. At the middle of this stage the eyes are completely formed and pigmented (Fig. 1: E2). The uniramous antennule displays two terminal setae. The biramous antenna has a short and wide endopodite with one seta and an exopodite with two setae. The mouth has a large chromatophore with horseshoe shape and spots of pigments on each side of mouth. The first maxilliped has a chromatophore on the long coxa; its endopodite and exopodite have two and four terminal setae, respectively. The second maxilliped displays a chromatophore on the coxa. The exopodite has four terminal setae. Neither pereiopods nor pleopods can be observed. The abdomen has five segments, with the sixth fused to the telson (Fig. 1: E4). All of these segments have chromatophores on each side. The biforked telson has six terminal setae on each lobe. Sporadic motions of the appendages and abdomen can be observed, becoming more frequent by the time of hatching. The cardiac frequency is approximately 180.62 per minute. This stage ends just before hatching, and lasts 4.76±0.42 days, which represents an RDE of 13.3±1.29%. (Table 1)

When embryonic development completes, females induce hatching by flapping their abdomens and rhythmically moving their pleopods. Egg hatching starts by breaking of the chorion in the anterior zone, above the cephalic part of embryos. Hatching of the entire brood occurs in a single pulse between the first night and early morning hours. Empty chorionic membranes stay adhered to pleopods until the next moult.

Egg sampling frequency had no significant effect on the total duration of embryogenesis (ANOVA: F=2.81; p=0.083; df=23). The relative position of embryos in the egg mass (inner, middle or outer) did not significantly affect the duration of development (ANOVA; F=0.14; p=0.8727; df=69), so O. trimaculatus embryogenesis is synchronous for all embryos carried by a single mother. The average total length of embryogenesis was 35.7±2.11 days (n=24) from the earliest stages of development (i.e. 2 or 4–cell morula) to hatching. To evaluate the survival of embryos, a two-way Kruskal-Wallis test for (instantaneous embryonic mortality) was applied, with relative position of embryos in the egg mass and stage of embryonic development as the explaining variables. While significant differences were found (H=50.59; p<0.01), post hoc comparisons (Dunn test) revealed that embryonic mortality was higher at hatching than at the end of each of the stages of embryonic development, and that position of eggs in the egg mass had no effects on survival of embryos at 13°C and constant aeration (Table 2).

Table 2. – Instantaneous embryonic mortality (EM) of Ovalipes trimaculatus at the end of different stages of development and hatching. H, hatching. Different letters denote significant differences of post hoc comparisons for each location in all stage of development. EM values are mean±standard deviation; n, number of samples with egg mass analysed (10 eggs per sample).

Stage of embryonic development (i)	EM_s-i (%) Inner	EM_s-i (%) middle	EM_s-i (%) outer	n
I	0.00^a	0.00^a	0.00^a	20
II	0.00^a	0.00^a	0.00^a	20
III	1.00±2.61^a	1.42±3.63^a	1.45±2.75^ab	20
IV	0.00^a	0.00^a	0.00^a	20
V	0.25±1.12^a	0.00^a	0.42±2.04^a	20
H	3.50^b	3.50^b	3.50^b	10

No significant correlation was found between the dimensions of recently extruded (ρ=0.15) or pre-hatched (ρ=0.44) eggs and the carapace width of the brooding females. Therefore, morphometric data of eggs were pooled together for comparisons between embryonic stages. As shown by the elongation (E) and ellipticity (e) indexes, eggs were spherical and preserved their shape throughout all developmental stages (Fig. 2; Table 1). Increases in egg area (A) and volume (V) were observed during development, but egg size differences were significant only in the transition from stage I to II and from stage III to IV (Table 1).

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Fig. 2. – Boxplots of three different dimensions of eggs and indexes of egg shape during successive stages of embryonic development of Ovalipes trimaculatus. Different letters denote significant differences between stages using the Dunn test. Detailed descriptions of egg dimensions and indexes are included in the Material and Methods section.

DISCUSSIONTop

Cellular differentiation in crustacean embryos starts soon after gastrulation and requires enormous energy expenditure (Soundarapandian et al. 2013Soundarapandian P., Ilavarasan N., Varadharajan D., et al. 2013. Embryonic development of commercially important Portunid crab Charybdis feriata (Linnaeus). J. Mar. Sci. Res. Dev. 3: 122.). Some authors consider that the main events of the embryonic development occur from the onset of cleavage to the formation of gastrula (Nagao et al. 1999Nagao J., Munehara H., Shimazaki K. 1999. Embryonic development of the hair crab Erimacrus isenberckii. J. Crustac. Biol. 19: 77-83., Müller et al. 2003Müller Y.M.R., Nazari E.M., Simoes-Costa M.S. 2003. Embryonic stages of the freshwater prawn Macrobrachium olfersi (Decapoda, Palaemonidae). J. Crustac. Biol. 23: 869-875.), but changes occurring during this period are difficult to observe in live eggs without special techniques. After inward blastoderm migration by cellular proliferation into the yolk mass (Adiyodi 1988Adiyodi R. 1988. Reproduction and development. In: Burggren W., McMahon B.R. (eds) Biology of the land crabs. Academic Cambridge, England Press. 139-185.), primordial structures start growing and extend ventrally, being easy to observe and describe in live eggs. Therefore, stages of embryonic development of crustaceans are generally defined based on distinctive external morphological features such as the proportion of yolk relative to total embryonic volume, appearance of the primordium, degree of development of appendages, pigmentation of the ocular globe, formation of chromatophores, and initiation of heartbeat (Nagao et al. 1999Nagao J., Munehara H., Shimazaki K. 1999. Embryonic development of the hair crab Erimacrus isenberckii. J. Crustac. Biol. 19: 77-83.). Although a more detailed description of development could have been achieved by characterizing additional stages of embryonic development, in this study external characteristics useful for rapid and easy classification during hatchery operations or population field studies were used to define developmental stages based on general criteria used for decapods (Felder et al. 1985Felder D.L., Martin J.W., Goy J.W. 1985. Patterns in early post-larval development of decapods. In: Wenner A.M. (ed.), Larval growth, 2. Crustacean issues. Balkema Press, Rotterdam, Netherlands. 163-226.).

Eye formation follows the pattern described for the crustacean vision ontogeny (Cronin and Jinks 2001Cronin T.W., Jinks R.N. 2001. Ontogeny of vision in marine crustaceans. Amer. Zool. 41: 1098-1107.). The general sequence of ontogenic changes observed during the embryogenesis (Table 3) and the increase in size and complexity of abdominal and ocular processes of O. trimaculatus are similar to those reported for other portunids. However, since only external morphological features were used to characterize stages of embryonic development of species from the group, further examination based on histology and other techniques could reveal differences in the chronology of development between species. Also, as observed in other portunid crabs (Ates et al. 2012Ates M.C.D., Quinitio G.F., Quinitio T., et al. 2012. Comparative study on the embryonic development of three mud crabs Scylla spp. Aquac. Res. 43: 215-225., Ikhwanuddin et al. 2012Ikhwanuddin M., Ara M.N., Siti-Aimuni H., et al. 2012. Fecundity, embryonic and ovarian development of blue swimming crab, Portunus pelagicus (Linnaeus, 1758) in coastal Water of Johor, Malasya. Pak. J. Biol. Sci. 15: 720-728., Soundarapandian et al. 2013Soundarapandian P., Ilavarasan N., Varadharajan D., et al. 2013. Embryonic development of commercially important Portunid crab Charybdis feriata (Linnaeus). J. Mar. Sci. Res. Dev. 3: 122.), in O. trimaculatus the embryonic development is synchronous in all eggs independently of their location in the egg mass. This pattern could reflect a homogeneous oxygen distribution to the developing embryos resulting from the ventilation of egg masses through abdominal flapping by brooding females, as reported for the species by Fernández et al. (2002)Fernández M., Pardo L.M., Baeza J.A. 2002. Patterns of oxygen supply in embryo masses of brachyuran crabs throughout development: the effect of oxygen availability and chemical cues in determining female brooding behavior. Mar. Ecol. Prog. Ser. 245: 181-190..

Table 3. – Comparison of the embryogenesis in several portunid species.

Species	Embryonic development and mean diameter				References
	Blastula-cleavage-gastrula (mean diameter)	Primordium of embryo - eye placode	Organogenesis- pigment appears	Chromatophores appear-heartbeat
Charybdis feriata	Spherical shape 0.298-0.36 mm	Spherical shape 40 mm	Elliptical shape 0.42 mm	Spherical shape 0.369-0.45 mm	Sundarapandian et al. 2013Soundarapandian P., Ilavarasan N., Varadharajan D., et al. 2013. Embryonic development of commercially important Portunid crab Charybdis feriata (Linnaeus). J. Mar. Sci. Res. Dev. 3: 122., Josileen 2011Josileen J. 2011. Captive spawning, hatching and larval development of crucifix crab, Charybdis feriatus (Linnaeus, 1758). J. Mar. Biol. Assoc. India. 53: 35-40.
Portunus pelagicus	Spheric shape 0.307±0.037 mm	Spherical shape 0.386±0.039 mm		Spherical shape 0.396±0.033 mm	Ikhwanuddin et al. 2012Ikhwanuddin M., Ara M.N., Siti-Aimuni H., et al. 2012. Fecundity, embryonic and ovarian development of blue swimming crab, Portunus pelagicus (Linnaeus, 1758) in coastal Water of Johor, Malasya. Pak. J. Biol. Sci. 15: 720-728., Ravi 2013 Ravi R., Manisseri. M.K. 2013. Alterations in size, weight and morphology of the eggs of blue swimmer crab, Portunus pelagicus Linnaeus, 1758 (Decapoda, Brachyura, Portunidae) during Incubation. Turk. J. Fish. Aquat. Sci. 13: 509-515.
P. sanguinolentus	Spherical shape 0.34 mm	Spherical shape 0.41 mm	Spherical shape 0.48 mm	Elliptical shape 0.57-0.64 mm. Posteriorly spheric shape 0.73 mm	Samuel and Soundarapandian 2009 Samuel N.J., Soundarapandian P. 2009. Embryonic development of commercially important portunid crab Portuns sanguinolentus (Herbst). J. Anim. Vet. Adv. 1: 32-38.
Scylla serrata	Spherical shape 0.34 mm 0.40 mm	Spherical shape 0.41 mm 0.52 mm	Spherical shape 0.48 mm 0.60 mm	Elliptical shape. 0.65 mm. Posteriorly spheric shape 0.73 mm	Samuel and Soundarapandian 2009Samuel N.J., Soundarapandian P. 2009. Embryonic development of commercially important portunid crab Portuns sanguinolentus (Herbst). J. Anim. Vet. Adv. 1: 32-38., Ates et al. 2012Ates M.C.D., Quinitio G.F., Quinitio T., et al. 2012. Comparative study on the embryonic development of three mud crabs Scylla spp. Aquac. Res. 43: 215-225.
S. tranquebarica and S. olivacea	Spherical shape 0.40 mm	Spherical shape 0.52 mm	Spherical shape 0.60 mm	Elliptical shape. 0.65 mm. Posteriorly spherical shape 0.73 mm	Samuel and Soundarapandian 2009Samuel N.J., Soundarapandian P. 2009. Embryonic development of commercially important portunid crab Portuns sanguinolentus (Herbst). J. Anim. Vet. Adv. 1: 32-38., Ates et al. 2012 Ates M.C.D., Quinitio G.F., Quinitio T., et al. 2012. Comparative study on the embryonic development of three mud crabs Scylla spp. Aquac. Res. 43: 215-225.
Ovalipes trimaculatus	Spherical shape 0.40 mm	Spherical shape 0.52 mm	Spherical shape 0.60 mm	Spherical shape 0.65 mm. Posteriorly 0.73 mm	Du Preez and Mcachlan 1984Du Preez, H.H., McLachlan, A. 1984. Biology of the three spot swimming crab, Ovalipes punctatus (De Haan) III. Reproduction, fecundity and egg development. Crustaceana 47(3): 285-297.
Ovalipes trimaculatus	Spherical shape 0.309-0.347 mm	Spherical shape 0.326 mm	Spherical shape 0.367 mm	Spherical shape 0.372-0.378 mm	This study

Along with fecundity measurements, estimates of embryonic mortality can be used to infer the number of brooding females necessary to obtain a given amount of larvae or to anticipate live feed requirements for a batch of larvae production in hatcheries. Numerous sources of embryonic mortality are documented for brachyuran crabs, acting either at oviposition, during embryogenesis or at hatching (Kuris 1990Kuris A.M. 1990. A review of patterns and causes of crustacean brood mortality. In: Wenner A. and Kuris A. (eds), Crustacean egg production. Crustacean issues 7: 117-141, Balkema, Rotherdam.). Among others, eggs can fail to attach to female pleopods (Choy 1988Choy S.C. 1988. Reproductive Biology of Liocarcinus puber and L. holsatus (Decapoda, Brachyura, Portunidae) from the Gower Peninsula, South Wales. Mar. Ecol. 9: 227-241.), detach due to mechanical stress (Kuhn et al. 2011Kuhn P.S., Graham S., Choi J.S. 2011. Influence of senescent algae, temperature, tides, currents, and embryo detachment on Chionoecetes opilio (Snow crab) larval release. J. Crustac. Biol. 31: 100-105.) or die during incubation due to adverse external conditions such as nitrite concentration development in culture systems, poor oxygen availability, unsuitable salinities and temperatures, embryo predation, viral, bacterial and fungal diseases, epibionts and parasites (Balasundaram and Pandian 1982Balasundaram C., Pandian T.J. 1982. Egg loss during incubation in Macrobrachium nobilii (Henderson and Mathai). J. Exp. Mar. Biol. Ecol. 59: 289-299., Harper and Talbot 1984Harper R.E., Talbot P. 1984. Analysis of the epibiotic bacteria of lobster (Homarus) eggs and their influence on the loss of eggs from the pleopods. Aquaculture 36: 9-26., Hamasaki et al. 2003Hamasaki K. 2003. Effects of temperature on the egg incubation period, survival and developmental period of larvae of the mud crab Scylla serrata (Forskal) (Brachyura: Portunidae) reared in the laboratory. Aquaculture 219: 561-572.). Also, mortality may be the result of unsuccessful fertilization of some eggs of a clutch (Choy 1988Choy S.C. 1988. Reproductive Biology of Liocarcinus puber and L. holsatus (Decapoda, Brachyura, Portunidae) from the Gower Peninsula, South Wales. Mar. Ecol. 9: 227-241.). Only accounting for mechanical stress, O. trimaculatus can lose up to 56.7% of its developing embryos (Brante et. al. 2004Brante A., Cifuentes H.O.P., Wolf A., et al. 2004. Latitudinal comparisons of reproductive traits in five brachyuran species along the Chilean coast. Rev. Chil. Hist. Nat. 77: 15-27.). Therefore, since “instantaneous mortality” estimations reported here do not account for mechanical egg loss, they should not be taken as absolute figures, but only used to compare embryonic mortality due to physiological stress between stages of development. Although a slight increase in instantaneous embryonic mortality was noted starting from stage III, the most noticeable mortality was observed during the hatching process (Table 2). This suggest that embryos should be treated gently during the final stages of development, especially at hatching, as indicated for other portunid crabs, Charybdis feriata (Sundarapandian et al. 2013Soundarapandian P., Ilavarasan N., Varadharajan D., et al. 2013. Embryonic development of commercially important Portunid crab Charybdis feriata (Linnaeus). J. Mar. Sci. Res. Dev. 3: 122.) and Scylla serrata (Davis 2004aDavis J.A., Churchill G.J., Hecht T., et al. 2004a. Spawning characteristics of the South African mud crab Scylla serrata (Forskål) in captivity. J. World Aquacult. Soc. 35: 121-131.).

Eggs of portunid species show variations in size (Wear 1974Wear R.G. 1974. Incubation in British decapod crustacean and the effects of temperature on the rate and success of embryonic development. J. Mar. Biol. Assoc. U.K. 54: 745-762.). Positive relationships between female body size and egg size have been reported for several species within the group, including Ovalipes catharus (Haddon 1994Haddon M. 1994. Size-fecundity relationships, mating behavior and larval release in the New Zealand paddle crab, Ovalipes catharus (White 1843) (Brachyura: Portunidae). New Zeal. J. Mar. Fresh. 28: 329-334.) and Portunus pelagicus (Ravi and Manisseri 2013Ravi R., Manisseri. M.K. 2013. Alterations in size, weight and morphology of the eggs of blue swimmer crab, Portunus pelagicus Linnaeus, 1758 (Decapoda, Brachyura, Portunidae) during Incubation. Turk. J. Fish. Aquat. Sci. 13: 509-515.). In contrast, in this study no significant relationship was found between these variables, as has also been observed in S. serrata (Churchill 2003Churchill G.R. 2003. An investigation into the captive spawning, egg characteristic and egg quality of the mud crab (Scylla serrata) in South Africa. Workshop of mud crab culture, ecology and fisheries, Can Tho University, Vietnam, 8-10 th January 2001.), Charybdis feriata (Soundarapandian et al. 2013Soundarapandian P., Ilavarasan N., Varadharajan D., et al. 2013. Embryonic development of commercially important Portunid crab Charybdis feriata (Linnaeus). J. Mar. Sci. Res. Dev. 3: 122.) and some species of Cancer (Hines 1991Hines A.H. 1991. Fecundity and Reproductive output in nine species of Cancer crabs (Crustacea, Brachyura, Cancridae). Can. J. Fish. Aquat. Sci. 48: 267-275.).

As the embryos of O. trimaculatus develop, egg size increases gradually, reaching maximum dimensions by the time of hatching. Increases in egg volume and area recorded in this study are similar to those reported for other portunids (Fig. 2, Table 3), but are moderate in comparison to some species, whose eggs can double their volume at the end of embryonic development (Soundarapandian et al. 2013Soundarapandian P., Ilavarasan N., Varadharajan D., et al. 2013. Embryonic development of commercially important Portunid crab Charybdis feriata (Linnaeus). J. Mar. Sci. Res. Dev. 3: 122., Ates et al. 2012Ates M.C.D., Quinitio G.F., Quinitio T., et al. 2012. Comparative study on the embryonic development of three mud crabs Scylla spp. Aquac. Res. 43: 215-225.). In general, eggs of benthic marine decapods increase their initial water content by 33-40% during embryonic development (Stella et al. 1996Stella V., Lopez L., Rodriguez E. 1996. Fecundity and brood biomass investment in the estuarine crab Chasmagnathus granulata Dana 1851. Crustaceana 69: 307-312.).
During the first stages of embryogenesis, yolk catabolism increases the amount of free metabolites providing the energy required for the formation of tissues, so more water, and consequently more volume, is required for solvation (Stella et al. 1996Stella V., Lopez L., Rodriguez E. 1996. Fecundity and brood biomass investment in the estuarine crab Chasmagnathus granulata Dana 1851. Crustaceana 69: 307-312.). Also, progressive intake and retention of water are necessary during metabolic reactions of lipid oxidation (Pandian 1967Pandian T.J. 1967. Changes in chemical composition and caloric content of developing eggs of the shrimp Crangon crangon. Helgol. Wiss. Meeresunters. 16: 216-224., Lardies and Wehrtmann 1997Lardies M.A., Wehrtmann I.S. 1997. Egg production in Betaeus emarginatus (H. Milne Edwards, 1837) (Decapoda: Alpheidae): fecundity, reproductive output and chemical composition of eggs. Ophelia 46: 165-174.). At the final stages of development, egg volume increments are more closely related to the rise of internal osmotic pressure, which generates more water intake, and to the simultaneous or independent mechanisms that allow movements of the embryos aimed at tearing the chorion and initiating hatching (Davis 1965Pandian T.J. 1967. Changes in chemical composition and caloric content of developing eggs of the shrimp Crangon crangon. Helgol. Wiss. Meeresunters. 16: 216-224., Lardies and Wehrtmann 1997Lardies M.A., Wehrtmann I.S. 1997. Egg production in Betaeus emarginatus (H. Milne Edwards, 1837) (Decapoda: Alpheidae): fecundity, reproductive output and chemical composition of eggs. Ophelia 46: 165-174., Pandian 1967Pandian T.J. 1967. Changes in chemical composition and caloric content of developing eggs of the shrimp Crangon crangon. Helgol. Wiss. Meeresunters. 16: 216-224.). In O. trimaculatus in particular, marked increments in egg dimensions were observed between stages I and II, when the thoracic-abdominal plate and the primordium of the embryo start forming appendages and grow, requiring extra space within the egg. Also, a pronounced increase in egg dimensions was observed in the transition from stages III to IV, when embryogenesis completes and formed embryos enlarge their appendages. Moreover, these differences are statistically significant (Fig. 2). In regard to shape variations, eggs of O. trimaculatus remain spherical throughout embryonic development, showing near-to-zero elongation index values in all stages of development, just like the eggs of most brachyuran crabs (Hines 1982Hines A.H. 1982. Allometric constraints and variables of reproductive effort in brachyuran crabs. Mar. Biol. 69: 309-320., Yamaguchi 2001Yamaguchi T. 2001. Incubation of eggs and embryonic development of the fiddler crab Uca lactea (Decapoda, Brachyura, Ocypodidae). Crustaceana 74: 449-458., Pinheiro and Hattori 2003Pinheiro M.A.A., Hattori G.Y. 2003. Embryology of the mangrove crab Ucides cordatus (Brachyura: Ocypodidae). J. Crustac. Biol. 23: 729-737.), including those from the closely related Ovalipes catharus (Haddon 1994Haddon M. 1994. Size-fecundity relationships, mating behavior and larval release in the New Zealand paddle crab, Ovalipes catharus (White 1843) (Brachyura: Portunidae). New Zeal. J. Mar. Fresh. 28: 329-334., Ravi and Manisseri 2013Ravi R., Manisseri. M.K. 2013. Alterations in size, weight and morphology of the eggs of blue swimmer crab, Portunus pelagicus Linnaeus, 1758 (Decapoda, Brachyura, Portunidae) during Incubation. Turk. J. Fish. Aquat. Sci. 13: 509-515.).

It is known that in marine organisms, including crustaceans, pigment content determines egg quality to a great extent due to its critical role in many metabolic processes, including egg respiration and vitamin A production, its protective action against the effects of oxygen, UV and ionizing radiation, and its function in gene expression, among others (Craik 1985Craik J.C.A. 1985. Egg quality and egg pigment content in salmonid fishes. Aquaculture 47: 61–88., Kalinina et al. 2009Kalinina M.V., Zadorozhnyib P.A., Vinnikovac N.A. 2009. Sexual Cycle Properties and Changes in Carotenoid Levels in Ovary of Japanese Mitten Crab Eriocheir japonica from Rivers of Primorye. J. Dev. Biol. 39 (3): 176-182., Wade et al. 2015Wade N.M., Gabaudan J., Glencross B.D. 2015. A review of carotenoid utilization and function in crustacean aquaculture. Rev. Aquaculture.). Egg colour progression throughout embryogenesis is common in crustaceans, and in general terms it reflects the initial consumption of typically yellow or orange-coloured yolk, superimposed on the production of red-, brown- and grey-coloured pigments on chromatophores and eyes during advanced stages (Sigana 2002Sigana D.A. 2002. Breeding cycle of Thalamita crenata (Latreille, 1829) at Gazi Creek (Maftaha Bay), Kenya. Western Indian Ocean. J. Mar. Sci. 1: 145-153., Parimalam 2001Parimalam K. 2001. Embryonic and larval development of the hermit crab, Clibanarius longitarsus (de Haan) (Crustacea: Decapoda: Anomura). MSc. thesis: Annamalai Univ. India.). Although this pattern of colour change throughout development has been reported in studies conducted on other portunid species (Ravi and Manisseri 2013Ravi R., Manisseri. M.K. 2013. Alterations in size, weight and morphology of the eggs of blue swimmer crab, Portunus pelagicus Linnaeus, 1758 (Decapoda, Brachyura, Portunidae) during Incubation. Turk. J. Fish. Aquat. Sci. 13: 509-515.), to our knowledge there are no objective colour classifications (with the Pantone® Colour Formula Guide), as reported in this study.

This work provides the first information on the embryogenesis of O. trimaculatus. Although experimental results were obtained from incubations at a single combination of temperature and salinity, they represent a necessary step preceding studies aimed at understanding the effects of environmental variables on embryogenesis. Further experimental work is necessary to optimize hatchery practices and to supplement the pre-existing knowledge on the life cycle of the species in the wild, for management purposes.

ACKNOWLEDGEMENTSTop

We thank the personnel of the nautical and aquarium services (CCT CONICET-CENPAT) for their collaboration in crab collection and setting of experimental work. This work was carried out in the framework of the projects PIP 0354/14 and PICT-2014-1498 granted by the National Council of Science and Technology (CONICET).

This study includes partial contents of the doctoral thesis of A. Martelli at the Universidad Nacional of Comahue (Argentina) and was financed with a research grant from the National Council for Scientific and Technical Research (CONICET, Argentina).

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