Introduction

Freshwater shrimp species belonging to Macrobrachium genus constitute one of the most diversified and abundant crustacean groups. They are mainly spread in tropical and subtropical regions, and include more than 200 described species (Murphy and Austin 2005). In African lagoon sectors, there are about ten shrimp species belonging to Macrobrachium genus (Powell 1980). Among the seven Macrobrachium species discovered in Benin, Macrobrachium vollenhovenii (Herklots, 1857) and Macrobrachium macrobrachion (Herklots, 1851) are very important in shrimp fishery with a relative abundance of 44.75% and 51.50%, respectively (Kouton 2004; Agadjihouèdé 2006). Shrimps are one of crustaceans groups, important for continental aquatic farm activities due to their value in commercial exchanges worldwide (FAO/OMS 2011). In Benin, freshwater shrimps have a high economical and commercial value. They contribute substantially to commercial fisheries and local incomes (Sachi et al. 2016). However, statistical data showed that shrimp quantity exported from Benin decreased from 703 tons in 2002 to 32 tons in 2006 (OMC/FAO/UEMOA 2007). According to Sohou and Djiman (2011), this decrease was due to overexploitation of available species. Thus, it is important to study the biology of shrimps involved in fishery in order to undertake their breeding and avoiding their natural population decrease.

Although some studies focused on diversity, biology, morphology, ecology (Lalèyè et al. 2005; Adite et al. 2013) and on rearing and feeding (Gangbe et al. 2016) were carried out in the Macrobrachium species, their embryology remains to be explored. Therefore, the current study aimed to identify the embryonic development stages of M. vollenhovenii in order to master the reproduction conditions of this species in captivity and provide basic data on its ontogeny.

Material and methods

Techniques of capture, identification, selection and transport of the biological material

The specimens used in the current study were captured by fishermen using nets in the lagoon of Grand-Popo between Hêvê and Gbècon, villages located at about 06°16′57.81“N and 01°50’30.84”E. Specimens’ identification was carried out according to the method of Bile et al. (2011). For females, ovaries maturation was detected by sight according to the technique of Browdy and Samocha (1985). Selected shrimps were then transported to the laboratory, waterless early in the morning from 5 to 7 a.m. by using aerated baskets.

Condition of shrimp’s reproduction

In order to reduce spawning duration, twenty females and ten dominant males sexually mature were selected and distributed, two females for one male, in ten circular ponds of 1.76 m3 volume containing 0.3 m3 of fresh water (salinity varying from 0.06 to 0.17 g L−1). Pipe (PVC 100) and water hyacinth (Eichhornia crassipes) were put in these ponds to favour spawning. The upper sides of the shrimps’dactyls were removed to avoid cannibalism. Shrimps were subject to natural photoperiod and fed commercial pelleted feed. The water physico-chemical parameters were measured daily at 7 a.m. and 4 p.m. by using multimeter “calypso ORCHIDIS SN-ODEOA 2138”. Therefore, begetters breeding was carried out at 27.51 ± 2.5 °C, pH 6.2 ± 1 and dissolved oxygen 4.8 ± 1.5 mg L−1.

Spawning and eggs hatching

Spawning was checked in advanced mature females every four hours. The first spawning occurred the seventh day of rearing. Thus, eleven gravid females, among which four presented a very bad fecundity, were recorded. One of the females with a good fecundity and spawing was chosen for the eggs incubation trial. It was transferred to the hatchery in a 28 L tank. Spawning date and hour were noticed. This female was carefully monitored in order to avoid eventual claws and paws loss and then assess correctly the total weight of eggs by calculating the difference between the gravid female weight and its weight after egg hatching. The hatchery water salinity varied from 2 ppt to 8 ppt for ten days and was adjusted to 12 ppt till hatching (Doume Doume et al. 2013). The different ranges of salinity were adjusted by mixing sea water with fresh water. Eggs incubation was performed at 28 ± 1.5 °C. Dissolved oxygen rate was 6 ± 0.5 mg L−1 with pH 7 ± 0.3.

Counting of eggs at the spawning

To estimate eggs number, ten sub-samples of different masses (0.001 g; 0.002 g; 0.003 g; 0.004 g; 0.005 g; 0.006 g; 0.007 g; 0.008 g; 0.009 g and 0.010 g) were taken by using an electronic scale « OHAUS Pioneer » with 0.001 g precision. The eggs of each sub-sample were then counted in triplicate. This enabled to realize a linear regression calculation: y = ax + b (with “x” eggs mass and “y” sampled eggs number). Moreover, three samples of thirty eggs were removed from ovigerous female and observed to estimate the average fecundity rate.

Eggs observation, drawing and measuring during the embryonic development period

During the first three days of incubation period, thirty eggs samples were taken every two hours from the gravid female for microscopic observation. The following days, samplings were carried out daily. Eggs were observed in photonic microscope (BIMICRO/WF 10×/20) in order to describe their morphology and identify their embryogeny stages. The microscopic observations enabled to identify fertilized and unfertilized eggs. Photos were taken at ×100 magnification. Major stages of embryonic development were hand drawn in lateral face. The nomenclature used for the embryonic development description followed prior studies (Anderson 1982; Müller et al. 2004; García-Guerrero and Hendrickx 2009; Sudhakar et al. 2014). The method adopted focused on the different major events, aggregated into seven big successive stages without fixed duration. Furthermore, the eggs mean length was assessed by measurement of linear axes of fifty eggs each 48 h by using Photoshop software version 7.0. from axes measurement. Egg volume (mm3) was calculated according to the following formula: V = π/6 × (Ls)2 × Lb; with Lb = length of the big axe (mm) and Ls = length of the small axe of egg (mm); π = 3.14 (Vadrucci et al. 2007). Moreover, three samples of 0.007 g of eggs were counted each 48 h to determine egg mean weight in relation to time.

Data analysis

STATISTICA version 6 software was used to realize a correlation test among eggs growth parameters. The regression curves between eggs number and mass were drawn by using Excel. Slopes provoked by eggs growth were determined using the following formula:

$$ b=\frac{\sum \left(x-\overline{x}\right)\left(y-\overline{y}\right)}{\sum {\left(x-\overline{x}\right)}^2} $$

With “b” representing the slope, “x” the sampling period and “y” the mean measurement of samples/period.

Results

Fecundity of females

After spawning, gravid females incubate eggs in their pleopods untill hatching. Egg clusters were maintained due to slim and transparent adhesive substance. The total weight of eggs spawned by the chosen female (weighing 60.51 g and measuring 153 mm) was x = 7.29 g. According to the aforementioned regression equation, total eggs number was estimated to 83,323 (Fig. 1), with an average fecundity rate of 81.6%.

Fig. 1
figure 1

Assessment of Macrobrachium vollenhovenii eggs mean number in sub-samples by regression analysis

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Eggs characteristics and embryonic development

Microscopic observation of non-fertilized eggs showed non bright grey vitellin grains randomly disposed or condensed in egg cytoplasm. A lack of veritable nucleus was also noticed. Non-fertilized egg inside was yellow-dark (Fig. 2a, c) or grey (Fig. 2b), and presented sometimes a bright perivitellin space (Fig. 2c).

Fig. 2
figure 2

Microscopic observation of non-fertilized Macrobrachium vollenhovenii eggs (10 × 10). a, c egg inside yellow-dark, b  egg inside grey 

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Fertilized eggs clusters changed colour simultaneously with embryos growth. Thus, during the embryonic development, fertilized eggs colour changed from orange to yellow after eight days and then brown after ten days and finally grey on the thirteenth day prior to hatching (Fig. 3, Table 1).

Fig. 3
figure 3

Colouration of Macrobrachium vollenhovenii eggs during the embryogeny

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Table 1 Variation of colour, form, mean value ± SD of dimensions, weight and volume of fertilized eggs of Macrobrachium vollenhovenii, during the embryonic development period
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Fertilized eggs growth in relation to time

During the embryonic development, eggs height increased considerably in major axes inducing consequently weight and volume increase, and then eggs form variation (Table 1). On the other hand, positive significant correlations were noticed among eggs growth parameters since growth also provoked an increase of eggs weight and volume (Table 1). However, a disproportioned growth among eggs weight, volume and axes length was noticed during embryonic development period (Fig. 4). Thus, the growth of egg big axe length increased with 0.0307 linear slope while that of small axe was 0.0121. Eggs volume and weight increased similarly with a slope of 0.0068 and 0.0069, respectively.

Fig. 4
figure 4

Macrobrachium vollenhovenii eggs growth in weight, height and volume in relation to time

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Embryonic development of Macrobrachium vollenhovenii

Embryonic development process is divided in seven great main stages: fertilization, primitive mitosis, morula, blastula, gastrula, nauplius and larvae (Fig. 5). Drawn version of these embryonic stages is presented in Fig. 6.

  • Stage I: Fertilization (00–02 h)

Fig. 5
figure 5

Embryonic development stages of Macrobrachium vollenhovenii (10 × 10). a fertilization start; b making up of a cell (protoplaste) called blastomere; c two cells stage; d four cells stage; e morula eight cells stage; f morula sixteen cells stage; g morula thirty two cells stage; h blastula sixty four cells stage; i blastula one hundred twenty eight cells stage; j end of blastula stage with irregular mitosis succession; k gastrula (nuclear regression); l gastrula (nuclear regression); m gastrula with flats mesoblaste-endoblaste-ectoblaste appearance; n pre-nauplius with red-dark traces (occular pigments) on thee nuclear mass; o mid-nauplius in progress with posterior somites growth and eye pigmentation; p nauplius with eyes making up; q nauplius with advanced eyes condensation; r end nauplius stage with eyes making up and end of segmentation; s larvae start; t larvae ready to hatch

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Fig. 6
figure 6

Drawing of Macrobrachium vollenhovenii embryonic development stages. ad- abdomen; al- Antenna; ap- appendix; as- abdominal segment; bt- blastomere; ch- chorion; em- external membrane; ey- eye; fr- furrow; ht- heart; lb- labre; lo- orange liquid; lp- optical lobe; lv- lecithotroph vesicles; nc- nucleus; nm- nuclear mass; op- ocular pigment; sp- perivitellin space; str- stria; ts- telson

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At the beginning (0 h), eggs presented a bright and transparent membrane. It was spherical and filled with a uniform orange coloured liquid. There was no evidence of tissues or cells presence.

From 0 to 02 h, the whole orange bright liquid in the egg started fragmenting into small oil droplet. A primordial cell marked by a black spot was made up in the central side of the egg: it was the central nucleus or protoplast.

  • Stage II: Primitive mitosis: “two first cell divisions” (02–06 h)

From 02 to 04 h, the first mitosis took place by protoplast division. This led to two blastomeres each characterized by a central black nucleus, visible through the small oily droplet. At this stage, no binary division furrow was observed.

From 04 to 06 h, four blastomeres were formed each containing a black central nucleus. Nucleuses were almost equidistant. These blastomeres originating from the division of those previously obtained, were separated by narrow furrows.

  • Stage III: Morula (06–18 h)

From 06 to 10 h, a third division led to eight blastomeres occupying the entire egg inside instead of the small oily droplets. Each of these cells contained a grey central nucleus.

From 10 to 14 h, there were 16 new blastomeres coming from the division of eight cells blastomeres previously observed. These cells were small and without nucleus. Furrows separating cells were entirely visible. At this stage, nucleus mass delimitated by the chorion, a thin membrane surrounding by a visible perivitellin. This marked the fourth mitotic division similar to the third.

From 14 to 18 h, 32 small blastomeres appeared and were localized in the central side of egg. Thus, egg’s nuclear volume decrease was noticed. This was the fifth mitotic division similar to the fourth.

  • Stage IV: Blastula (18–96 h)

From 18 to 24 h, 64 blastomeres were formed by irregular cell divisions with overlapping. Nucleus volume increased tending to fill the egg inside.

From 24 to 36 h, nucleus volume increased and filled completely the egg inside. Irregular divisions continued producing irregular height and high blastomeres concentrated in a central circular area of the egg.

From 36 to 96 h, irregular divisions occurred, characterized by the flattening of blastodermic cells. At this stage the circular region sheltering the high blastomere density seemed to merge to the big cells.

  • Stage V: Gastrula “depression of the nuclear region” (96–168 h)

From 96 to 120 h, there was a decrease of the nuclear mass enabling the building of a light easily discernible area in pole of the egg. This area expanded longitudinally forming a light liquid.

From 120 to 144 h, nuclear mass still decreased inducing an increase of the light area volume through internal periphery of the egg. This liquid became translucent. Some dark traces appeared in the nuclear region. At this stage, the egg previously spherical changed to oval form.

From 144 to 168 h, the translucent liquid divided in abdominal segments inducing the setting up of three germ layers, namely ectoderm, mesoderm and endoderm, according to polarity axe. These constitute the trunk of the developing primitive embryo. Moreover, some red-dark spots appeared in the nuclear mass.

  • Stage VI: Nauplius (168–288 h)

From 168 to 192 h, some small red-dark traces were mainly localized in both lateral parts of the nuclear mass.

From 192 to 216 h, red-dark traces blended and formed two small eyes disposed in each lateral side of the nuclear mass.

From 216 to 240 h, eyes were more visible. Some small red-dark spots were still on the nuclear mass. At this stage, the structure of the embryo abdomen was visible.

From 240 to 264 h, the abdomen was well developed and occupied about 2/3 of the embryo mass. Ocular spots increased and took an oval form.

From 264 to 288 h, the abdomen segmentation ended. Eyes were entirely pigmented. Chromatophores appearedred on the abdominal segments. The heart of embryo presented a translucent structure, spread dorsally in cardiac tube and started beating. At this stage, contractions were non rhythmical and lacked coordination. This cardiac rhythm was noticed by lecithotroph vesicles formed under the dorsal part of the embryo.

  • Stage VII: Larvae (288–336 h)

From 288 to 312 h, appendices were formed and visible. The abdomen of the embryo was folded on its body with the telson oriented to the labre. Embryo had therefore a well-defined « C » like form. At this stage, the embryo was of clear-grey colour.

From 312 to 336 h, structures surrounding the heart were more differentiated. The heart beat continuously with a rhythmical movement equal to 13 beats per minute approximately. Certain red-dark traces were visible in the embryo body. The abdomen was entirely segmented. Yellow lecithotroph vesicles became translucent and occupied the major part of the embryo head. Eyes volume increased and was surrounded with striations. Jawbone, labre, mandibles and paws were well developed. The egg was therefore ready to hatch. First larvae were obtained 339 h after spawning.

Discussion

According to the results, females of M. vollenhovenii incubated eggs in their abdomen close to pleopods until hatching thanks to an adhesive substance. At this stage, gravid females cleaned eggs constantly by pleopods movement. This incubation mode was also noticed with many other decapods like Macrobrachium idae (Heller, 1862) by Sudhakar et al. (2014) while it differs from those of Penaeidae species which directly release eggs into water (François 2003). For most of crustaceans, eggs incubation system might be one of the causes of the group success. Therefore, hatchery mode implemented by females of M. vollenhovenii insures a great survival rate by protecting them against predators as well as disfavored environmental conditions (Ching and Velez 1985).

Microscopic observation of M. vollenhovenii eggs in this study enabled to follow embryo structure without performing cytological analysis. This method is adapted to the embryogeny of decapods due not only to the low complexity of egg internal structure but also to the superficial position of egg embryo (Sudhakar et al. 2014). Meroblastical and holoblastical division favoured a slow embryonic development in M. vollenhovenii species. Thus, after the different mitotic stages (meroblastical division), eyes spot pigments appeared the second time, following successively by the formation of appendix, abdomen and cephalothoraxes outline (holoblastical division). This process is almost standard for majority of decapods (Scholtz 2000). The same trend was noticed for most of malacostraceans leading to zoes after hatching (Anderson 1982). However, holoblastical or total division happened in eggs containing few quantity of yolk «oligolecith egg» (Hertzler 2002). So, the morphological development of the embryo was rapid and led to free larvae with three pairs of phanera. This trend was noticed with most of branchiopods, maxillopods and peneids (Hertzler and Clark 1992). Thus, the quantity of the yolk in eggs of different crustacean species is closely tied to embryonic development models (Anderson 1982). In M. vollenhovenii, the chronology of appearance of all embryonic structures was similar to that noticed by Müller et al. (2004) for species belonging to Palaemonidae family, namely Macrobrachium olfersii (Wiegmann, 1836), Macrobrachium potiuna (Müller, 1880), Palaemon pandaliformis (Stimpson, 1871) and Palaemonetes argentinus (Nobili, 1901). Nevertheless, there are differences among the respective embryogeny periods. The duration of embryonic development recorded in M. vollenhovenii was 14 days at 28 ± 1.5 °C. This result was similar to that (13 to 14 days at 28.2 to 30.4 °C) recorded by Willführ-Nast et al. (1993) for this species. Similar embryogeny durations were also recorded for M. idea by Sudhakar et al. (2014). However, eggs height might influence the embryonic development duration. Therefore, according to Müller et al. (2004), small eggs of M. olfersii, and Palaemonetes argentinus (Nobili, 1901) had an embryonic development duration equal to 14 and 12 days, respectively, while for big eggs of M. potiuna, this duration was 21 days at the same temperature 26 ± 2 °C. Likewise, embryogeny duration may also vary for few days according to the tropical species and from one geographical area to the other (Celada et al. 1991).

Furthermore, during M. vollenhovenii embryonic development, a concomitant variation of eggs colour was noticed. Thus, fertilized eggs colour changed from orange to yellow then brown and grey before hatching. These colour changes were similar to those recorded for Macrobrachium heterochirus (Wiegmann, 1836) and Macrobrachium ohione (Smith, 1874) by Truesdale and Mermilliod (1979) and for Macrobrachium rosenbergii (De Man, 1879) by Ling (1969). In return, it differed from those noticed for Macrobrachium idella idella (Hilgendorf, 1898) and M. idae eggs that changed from green opaque to clear green, yellow brownish and white successively before hatching (Ajith Kumar 1990; Sudhakar et al. 2014). Besides, M. vollenhovenii eggs height increased mainly in the big axes during the embryonic development. This was recorded for other crustacean species like most of malacostraceans (Kobayashi and Matsuura 1995).

Conclusions

The current study focused on the embryonic development of the freshwater shrimp M. vollenhovenii, enable to know more about reproduction and survival conditions of the species as well as about its embryonic development cycle. Different nuances noticed for eggs cluster colour (orange, yellow, brown and grey) corresponded to the successive stages of embryonic development. This latter is characterized by two types of division such as partial or meroblastical cleavage and holoblastical or total cleavage. At the end of the embryonic development, muscular tissues differentiated early in the abdomen. Some cardiac movements were observed shortly before hatching. The nuclear division process noticed enabled a deeper characterization of a particular development model of Palaemonidae. M. vollenhovenii larvae has a form comparable to that of the adult.

Further to this study, the mastery of M. vollenhovenii larval rearing would be an important trump for the whole ontogenetic development description and in addition, for the success of the shrimp breeding.