Observations on the Embryonic Development of the Oriental River Prawn Macrobrachium Nipponense (De

applyparastyle "fig//caption/p[1]" parastyle "FigCapt" applyparastyle "fig" parastyle "Figure" Journal of Crustacean Biology Advance Access published 8 March 2019 Journal of Crustacean Biology The Crustacean Society Journal of Crustacean Biology 39(3) 261–266, 2019. doi:10.1093/jcbiol/ruz006

Observations on the embryonic development of the oriental river prawn Macrobrachium nipponense (De

Haan, 1849) (Decapoda: Caridea: Palaemonidae) Downloaded from https://academic.oup.com/jcb/article/39/3/261/5372354 by guest on 29 September 2021

Ke-Yi Ma, Xiao-Qing Tian, Zhi-Qiang Liu and Gao-Feng Qiu National Demonstration Center for Experimental Fisheries Science Education Shanghai Ocean University, Shanghai, 201306, China; Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, 201306, China; Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306, China; and Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, 201306, China

Correspondence: Gao-Feng Qiu; E-mail: [email protected] (Received 22 September 2018; accepted 6 February 2019)

ABSTRACT The embryonic development of the oriental freshwater prawn Macrobrachium nipponense (De Haan, 1849) is described from observations on whole-mount embryos with nuclei stained using Sytox® Green. The prawn eggs are centrolecithal, non-polar, and oval-shaped, with a diameter of 300–500 µm. The early cleavage pattern shows mixed cleavage modes. Notably, cells could shift to the interior of the embryo to form a visual blastopore, which is characteris- tic during the gastrulation stage. The putative intracellular body (ICB) could be discriminated at fertilized-egg, four-cell, eight-cell, and 16-cell stages, with volume gradually decreasing along with cell division. It is noted that the embryonic development takes a long period of approximately twenty-one days before hatching, which is quite different to what is known from penaeid shrimps. The various modes and profiles of early cleavage existing in crustaceans could imply a transition in their evolution. Keywords: blastopore, cleavage, intracellular body (ICB), Sytox® Green stain

INTRODUCTION important species for freshwater aquaculture in China, with cultured production increasing every year (Ma et al., 2012; Qiao et al., Crustaceans display various segmentation patterns and processes 2017). during early embryonic development, such as cleavage and gastru- As a useful model organism for studying reproduction and lation (Celada et al., 1991; Nazari et al., 2003; Müller et al., 2004; development among decapods (Ma et al., 2012), studies related Biffis et al., 2009; Foote et al., 2010; Grattan et al., 2013; Scholtz & to gene cloning and gene expression in M. nipponense have been Wolff, 2013; Klann & Scholtz, 2014). The diversity of this devel- increasing (Ma et al., 2012; Jin et al., 2017; Qiao et al., 2017). opment is found even among closely related species. For instance, There is nevertheless still much information to discover about the various cleavage and gastrulation modes have been described embryonic development in this species or even in Macrobrachium (Scholtz & Wolff, 2013). This diversity is also reflected in the in general (Chen et al., 2012). The development of the blastopore hatching stages or time. For example, some decapod species hatch has yet to be studied in M. nipponense. As an essential period during at the nauplius stage, whereas others could begin as zoeae or even ontogeny, a deep understanding of the embryogenesis will be of show direct development (Scholtz, 2000). great help for the study of the developmental biology, or even for Decapod crustaceans include many commercially important sexual development and sex control in the species. We examined species, including penaeid shrimps, freshwater prawns, cray- the embryogenesis of M. nipponense from the fertilized-egg to the fishes, lobsters, and crabs (Jung et al., 2011; Ma et al., 2012). The zoea stage using the Sytox® Green staining method and described oriental river prawn Macrobrachium nipponense (De Haan, 1849) the major events throughout embryonic development, including (Palaemonidae) is widely distributed in freshwaters and low-salin- the appearance of cleavage furrows, blastopore, and appendage ity estuaries in China and other Asian countries (Ma et al., 2012, anlagen. We also compared the duration of embryonic develop- 2016; Jin et al., 2017; Qiao et al., 2017). Owing to its excellent ment in M. nipponense with those of penaeid shrimps. adaptability and high commercial value, M. nipponense has been an

MATERIALS AND METHODS rinsed twice in PBST (1 × PBS, with a final concentration of 0.1% Tween 20) to minimize residues of other solutions. The nuclei Specimens of the embryos were the stained for 3 h with Sytox® solution Healthy oriental river prawns M. nipponense, with wet body weight (1:10,000 dilution). The embryos, after rinsing with PBST solu- of approximately 4–6 g each, were collected from a local aquacul- tion, were mounted between two cover slips in DABCO-glycerine ture farm in Shanghai and transferred alive to our laboratory. All (Sigma-Aldrich, St. Louis, MO, USA) to observe both sides of the adult prawns were cultured in a freshwater tank at 25 ± 2 °C the embryos separately. Selected embryos were later observed and fed a commercial diet twice a day. Female prawns were cap- using a fluorescence microscope (Olympus, Tokyo, Japan) and tured and placed in an aquarium under constant aeration. Mature photographed. male individuals were subsequently added to the aquarium to copulate with the females, with a male:female ratio of 2:1. The male prawns were taken out immediately after copulation and the RESULTS gravid females maintained until spawning. Fertilized egg to cleavage stage After spawning, the fertilized eggs of M. nipponense are adhered to Embryo sampling the pleopods of the female. Aside from the vitelline membrane Downloaded from https://academic.oup.com/jcb/article/39/3/261/5372354 by guest on 29 September 2021 Embryonic development of the oriental river prawn is a continu- packaging the egg, the outside of each egg adjoins tightly to a ous process from one-cell to zoea stages (Qiu et al., 2013). The secondary envelope, which is clearly distinguishable and mainly embryos were therefore checked at 1 h intervals under a micro- plays the role of protection and adherence of the eggs. No obvi- scope. According to their morphological characteristics as previous polarity could be observed in the fertilized eggs and their sur- ously described (Chen et al., 2012), embryos at various stages were face was smooth without any fissures. The egg is oval-shaped with collected from the pleopods of the ovigerous females and fixed the long and short axes of approximately 500 µm and 300 µm, with 4% paraformaldehyde in phosphate-buffered saline (PBS) respectively. Polar bodies could not be recognized throughout solution overnight at 4 °C. embryogenesis. Under the microscope, the undivided nucleus of a zygote also could not be distinguished owing to the rich yolk (Fig. 1A). With the first cleavage (approximately 2 h after spawning), Embryonic nucleus staining the daughter nuclei, which are most likely located in the middle of After fixation, the embryos were washed three times with 1 × the yolk-rich egg, separate along the long axis (Fig. 1B). PBS, and stored in methanol at –20 °C until use. Before stain- In the process of second nuclear division, the two nuclei divided ing with Sytox® Green (Invitrogen, Carlsbad, CA, USA), all the into four nuclei along the short axis. After completion of the embryos were gradually rehydrated using methanol, with a gradi- second nuclear division, cleavage furrows could not be observed ent of 95%, 80%, 70%, and 50%. The embryos were rehydrated clearly (Fig. 1C) compared to the subsequent cleavages (Fig. 1D).

Figure 1. Zygote to cleavage stage in M. nipponense stained with Sytox® Green: Zygote, two-cell stage, four-cell stage, eight-cell stage, 16-cell stage, 32-cell stage, 64-cell stage, and 128-cell stage, respectively (A–H). The arrows show the location of potential intracellular body (ICB), detected in cytoplasm at the zygote, four-cell, eight-cell and 16-cell stages. Scale bar = 100 µm. This figure is available in color at Journal of Crustacean Biology online.

262 EMBRYONIC DEVELOPMENT OF MACROBRACHIUM NIPPONENSE

The transition from meroblastic to holoblastic cleavage seems to of the embryo to form the blastopore (Fig. 2B). Due to appear- take place after second division. After completion of the third ance of an empty region left behind by consumption of a large division, the furrows could be clearly seen on the embryonic sur- amount of yolk (Chen et al., 2012), this phase of gastrulation could face, and seemingly divide the entire egg into eight equal-size generate vitellophages (Klann & Scholtz, 2014). A number of cells blastomeres (Fig. 1D). The nuclei gradually transfer from the subsequently begin to gather around the blastopore (Fig. 2C). The center to the surface of the embryo, leading to formation of an cell numbers are relatively reduced on the other end of the long eight-cell stage. axis (Fig. 2D). More cells concentrate around the blastopore at It seems that the fourth to seventh cleavage divisions (Fig. the later stage of gastrulation (Fig. 2E) and cell numbers on the 1E–H) are perpendicular to each other, respectively. As the num- other end of the long axis are fewer as compared to earlier stages ber of cells increases, the cleavage furrows at the seventh cleav- (Fig. 2F). Because the animal-vegetal axis could not be determined age division are no longer clearly visible as compared to those in at earlier stages, the position of the blastopore can serve as the the previous divisions (Fig. 1D–G) due to the limitations of the first marker of the vegetal pole. It took more than four days to get staining method. After completion of the seventh nuclear division, through the blastoderm and gastrulation stages. a 128-cell embryo was formed (Fig. 1H), so that the embryonic development enters into the blastoderm stage. It takes about three

Nauplius to zoea stages Downloaded from https://academic.oup.com/jcb/article/39/3/261/5372354 by guest on 29 September 2021 days from first to seventh cleavages. It is noteworthy that at the fertilized-egg, four-cell, eight-cell, and 16-cell stages, a possible The cells at the germinal area continue to aggregate as embry- RNA-enriched intracellular body (ICB) was detected in the cyto- onic development progresses. At the beginning of the nauplius plasm at the edge of a blastomere (Fig. 1A and 1C, E), which is stage, the first formations of morphogenetic structures are a pair similar to observations in a penaeid shrimp (Pawlak et al., 2010). of bilaterally symmetrical anlagen of optic lobes and a posterior growth zone (Fig. 3A), displaying a V-shaped germ band. A sym- Blastoderm and gastrulation stages metrical anlagen of antennules (the first antennae) appear in the region between the optic lobes and the posterior growth zone, fol- As shown in Figure 2A, cell division takes place along the longi- lowed by the differentiation of the second antennae and the man- tudinal axis of the embryo from 128 to 256 cells. From the blas- dibles (Fig. 3B). Parallel with this phase, the stomodaeum, which toderm stage onwards, the speed of cell division is extremely fast is the origin of the anterior intestine, could also be identified in and blastomeres become increasingly smaller, making it difficult to the center of the region of high cell concentration (Fig. 3B). All count the number of cells. structures of the nauplius stage become more pronounced in mor- At the beginning of the gastrulation stage, some cells start to phology as development progresses (Fig. 3C, D). As the cells fur- congregate at one end of the long axis to form the germinal area. ther concentrate (distinguishable through the collective brightness Meanwhile, this germinal area starts depressing into the interior of the stained nuclei), the stomodaeum invaginates and is covered

Figure 2. Development of blastoderm to gastrulation stages M. nipponense stained with Sytox® Green: blastoderm stage (A); gastrulation stage (B–G). The arrows indicate the blastopore. Scale bar = 100 µm. This figure is available in color at Journal of Crustacean Biology online.

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Figure 3. Formation of M. nipponense nauplius stained with Sytox® Green: area outside the post-naupliar region during embryonic development, of which F is a 180° rotation of E. ol, optic lobe; pg, posterior growth zone; a1, antennule; a2, antenna; md, mandible; st, stomodaeum; lb, labrum; cp, caudal papilla. Scale bar = 100 µm. This figure is available in color at Journal of Crustacean Biology online. by the newly formed labrum at this phase. Meanwhile, the cells at affects the cleavage pattern. Although both M. nipponense and the posterior growth zone are further accumulated to form a caudal penaeid shrimp Litopenaeus vannamei (Boone, 1931) belong to the papilla, which is separated from the pairs of mandibles by an obvi- group of yolk-rich and intralecithal species (Hertzler, 2005), their ous structural boundary (Fig. 3C). At this stage, the post-naupliar early cleavage patterns of eggs are quite different. The cleavage region is supposedly formed by cells which are located posterior to pattern shows mixed cleavage modes in M. nipponense whereas the caudal papilla. During further embryogenesis, anlagen of the holoblastic cleavage is found in L. vannamei. Yolk in the embryo optic lobes, antennules, and antennae stretch towards the flanks, of M. nipponense is generally concentrated in the center of the egg, and the terminal of caudal papilla dehisces to form the anlage of and the initial cleavages are only limited to the fission of nuclei, the furca (Fig. 3D). The cells outside of the post-naupliar region without cytoplasm dividing. Three to four hours after spawning, are obviously fairly sparse (Fig. 3E, F). furrows start to be established from the outside. It seems to repre- After further development, the embryo occupies more than half sent a holoblastic cleavage mode, in which blastomeres are drasti- the egg volume. The expansion speed of the optic lobes is faster cally separated by furrows, but these early cleavage furrows are than that of the anlagen of antennules, antennae, and mandibles, superficial and do not reach the central yolk mass. which had appeared at the nauplius stage (Fig. 4A). The anlagen Although orientation of the second cleavage can take on either of maxillipeds and pereiopods also symmetrically appears at the an equatorial or meridional division pattern in the freshwater thorax of the embryo (Fig. 4A). Initially, these anlagen are not shrimp Caridina multidentata (Stimpson, 1860; Klann & Scholtz, clearly visible owing to their much lower cell density. As develop- 2014), the second cleavage orientation in M. nipponense was found ment progresses, the anlagen of the optic lobes differentiate into to be strictly transverse after observing many embryos (data not the inner optic ganglions, medial eye stalks, and outer compound shown). When reaching the 256-cell stage, partial cells on both eyes (Fig. 4B). Meanwhile, the anlagen of maxillipeds and pereio- sides of the equatorial plate are divided along the long axis. Other pods become more obvious owing to increased cell density. There than those, most of the cells, especially those distributed near the is a dramatic cell proliferation in the pleonic region resulting in ends of the longitudinal axis, show irregular division perhaps the strongly elongated pleon at the zoea stage (Fig. 4C). Compared due to increasing embryonic curvature and relative cell number with cell accumulation at both ends of the egg, the number of towards the ends. “dorsal” cells, the extra-embryonic region gradually decreases The time required for embryonic development in crustaceans (Fig. 4D–F). Embryogenesis from the nauplius to zoea stages takes is not only associated with external environmental factors (Celada approximately two weeks in M. nipponense. et al., 1991), such as water temperature, but also closely related to egg size and yolk content. Palaemonids, with an embryonic diameter of 300–1,890 µm (Nazari et al., 2003; Antunes & Oshiro, DISCUSSION 2004; Müller et al., 2004), adopt a reproductive strategy of a long period of embryonic development (Nazari et al., 2003). We found The yolk not only provides all energy and nutrition for animal that in M. nipponense (embryonic diameter of 300–500 µm) embry- embryonic development, but the amount of the yolk sometimes onic development takes approximately twenty-one days before

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Figure 4. Zoea stage of M. nipponense stained with Sytox® green: D–F are the dorsal view of the development shown in A–C, respectively; ol, optic lobe; a1, antennule; a2, antenna; cp, caudal papilla; ca, carapace; ma, maxilliped; pe, pereiopods; og, optic ganglion; es, eye stalk; ce, compound eye. The red ellipses show one lateral part of the pereon; the white circles in D–F show that posterior cells accumulate at both ends of the egg in A–C, respectively. Scale bar = 100 µm. This figure is available in color at Journal of Crustacean Biology online. hatching. The penaeid shrimp Penaeus chinensis (Osbeck, 1765), distinguished the putative ICB at the fertilized-egg, four-cell, eight- however, only requires about two days for its embryonic develop- cell, and 16-cell stages, as reported during cleavage of the embryos ment (embryonic diameter less than 300 µm) (Cao et al., 1994). of the penaeid M. japonicus (Pawlak et al., 2010). Although we did The egg of P. chinensis is isolecithal, with less yolk compared to the not observe the ultrastructure of the ICB, we clearly observed centrolecithal egg of M. nipponense (Liu et al., 1994). Due to the a decreasing trend in ICB volume from the fertilized egg to the smaller volume of each fertilized egg of P. chinensis, energy allo- 16-cell stage cleavage (Fig. 1A, 1C–1E), which is similar to that in cation per embryo might be limited, resulting in no benefit from penaeid shrimps (Biffis et al., 2009; Pawlak et al., 2010). Embryonic taking a long time for its embryonic development. In contrast, genome activation generally occurs during cleavage (Graf et al., M. nipponense egg is larger and has more amount of yolk in storage. 2014; Palfy et al., 2017), promoting a dramatic reprograming of A longer period of embryonic development is therefore reason- specific genes referred to as the maternal-to-embryonic transition. able for M. nipponense. As a result, the main reason for smaller ICB could be attributed to The duration between early and later periods of embryonic the dynamic loss of material as embryogenesis progresses (Grattan development is also distributed unequally in M. nipponense. Our et al., 2013) and generation of novel transcripts by the embryo itself. results show that embryonic development during the first four It has been suggested that ICBs detected in cytoplasm can periods, from fertilized-egg to gastrulation stages, take about serve to trace the primordial germ cells (PGCs) (Biffis et al., 2009; seven days, whereas the nauplius and zoea stages take more than Pawlak et al., 2010). Previous studies coincidentally indicated that fourteen days, thus occupying two thirds of the embryonic devel- the gene vasa could be used to locate PGCs as well (Oezhan-Kizil opment. This allocation of time is expected, because the more et al., 2009; Qiu et al., 2013). In M. nipponense, the signal of vasa complex structures are mostly formed during the two later periods transcripts is concentrated on a granule region, where a single (Chen et al., 2012). Important structures such as the mandibles and blastomere represents the prawn PGCs (Qiu et al., 2013). Future compound eyes, begin to gradually form when the development ultrastructural and co-localization studies will help to reveal of the embryo enters into the nauplius stage. Furthermore, all whether the M. nipponense ICB and vasa transcripts are traced at biramous appendages have to differentiate before hatching, which the same location. is after zoea stage in M. nipponense. The longer durations of time required for the nauplius and zoea stages are not at all surprising. No intracellular body (ICB), a putative germ granule, had been ACKNOWLEDGEMENTS reported in Macrobrachium prior to this study. Previous studies had nevertheless traced the potential ICB in several penaeid species This work was supported by Shanghai Sailing Program (project (Biffis et al., 2009; Foote et al., 2010; Pawlak et al., 2010; Grattan 17YF1408000), Special Fund for the Development of Science and et al., 2013). Enriched with RNA and localized in the D lineage, Technology of Shanghai Ocean University (project A2-0203-17- the ICB is supposed to play an essential role in determination 100201), the Doctoral Scientific Research Foundation of Shanghai of germ line fate (Pawlak et al., 2010; Grattan et al., 2013). We Ocean University (A2-0203-17-100302), and the Training Scheme

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