MATERIALS AND METHODS

Ten adult males and 40 females Macrobrachium tenellum prawns were collected in the Colotepec River (15°48'19″N, 97°01'09″W) Oaxaca, Mexico. Prawns were caught with hand nets along with the vegetation of the river bank. They were transported in 250-L cylindrical containers filled with water from the river and with continuous aeration. Once in the laboratory (Laboratorio de Acuacultura, CIIDIR-Oaxaca-IPN), all prawns were acclimated for 24 h. After that, two males and 10 females were maintained in 1000-L tanks with fresh water at 28°C, 5.0 mg O₂ L^-1, 12:12 light:dark, and fed with 35% protein shrimp pellet (Camaronina^® Ciudad Obregon, Mexico). None of the females was berried initially; they were maintained in freshwater since salty water is proper for larval development only in Macrobrachium prawns. Once they were fed and placed with males, females started to molt and became berried. Over a week, several females lay eggs, approximately four or five per week, but this was irregular. Only some days, a berried female was obtained. After three weeks, a total of 23 berried females were utilized for the study. When a female was detected as berried, it was separated and placed with the other females in the same condition. Those females were transported to the Biochemistry Laboratory of the UNAM-UABJO School of Medicine to work with the eggs in sterile conditions. Embryonic stages from egg masses were observed and classified.

The eggs were taken from the ovigerous masses and rinsed with distilled water before removing them with dissection pincers for analysis. Embryological stages were classified according to color in agreement with ^{Guzmán et al. (1981)}. Then, half of the eggs were rinsed again with distilled water (2x) and kept in 1.5 mL microtubes filled with a PBS solution 1x (NaCl, kH2PO4, KCl, pH 7.2) and stored at −80°C until their analysis. The other half was preserved in 4% PFH (4% paraformaldehyde in PBS 1x) for three hours in dark conditions. They were then rinsed with a saline solution (0.85% NaCl) and stored at 4°C until analysis.

Morphological changes in the embryos were observed by placing the eggs under an optical microscope (Zeiss-PrimoStar, USA) at 4 and 10x. Pictures were taken with a digital camera (Axiocam-ERC- 5s, Germany) and the ZEN program (Zeiss, Germany) using the manual extended focus and measurement annotation module. Distinctive features at every stage of development were registered and analyzed both in life and from pictures.

The biochemical analysis was executed on the frozen eggs in PBS 1x (-80°C). The homogenate was obtained by chopping the eggs with a mortar (sterilized and cooled to −80°C), adding 300 μL of RIPA buffer with protease inhibitors (Sigma Chemicals, cat. S8220) at −4°C. This homogenate was then centrifuged at 12,000 rpm for 10 min at 0°C (2x), and the supernatant separated and frozen at −80°C until use. The quantification of proteins in the supernatant was measured with the BCA micro method (Bicincolinic Acid, Pierce, USA) using bovine albumin serum as standard (^{Dort et al. 2016}). A 25 μL sample was placed in triplicate in a microplate, and 200 μL of BCA reagent was added to each. Then, the plate was incubated at 37°C for 30 min, and the absorbance was recorded at 570 nm. Total protein values were calculated in mg mL^-1. Protein extract determinations were performed according to ^{Mexía-Salazar et al. (2008)}. SDS-PAGE (electrophoresis in polyacrylamide gels under reducing and denaturing conditions) using a 5% concentrator gel (acrylamide/BIS, water, 0.5 M Tris, pH 6.8, 10% SDS, ammonium persulfate, TEMED) and another 12.5% separator (acrylamide/BIS, water, 1.5 M Tris, pH 8.8, 10% SDS, ammonium persulfate, TEMED). Samples were diluted 1:1 with Laemmli 2x buffer to obtain a concentration of 50 μg mL^-1 of total protein. Then, the samples were incubated at 98°C for 10 min, and electrophoresis was run at 100 V. After electrophoresis, the gel was stained with Coomassie blue (BioRad, USA) and after 12 h, destained with a decolorizing solution (10% acetic acid, 50% methanol, 50% distilled water).

Proteomic analyses were executed in agreement with ^{Hernández-Pérez et al. (2019)} that includes only stage E eggs. Protein extracts were lyophilized and sent for analysis to Creative Proteomics (Shirley, NY, USA). The analysis performed was as follows, using the Nano LC-MS/ MS platform: 5 mg of dissolved protein was placed in 200 μL of 50 mM ammonium bicarbonate. The solution was transferred to Microcon YM-10 (Millipore) devices. The device was centrifuged at 12,000 rpm at 4°C for 10 min. Subsequently, 200 μL of 50 mM ammonium bicarbonate was added to the concentrate and centrifuged again and reduced to 10 mM DTT at 56°C for 1 h and alkalized with 20 mM IAA at ambient temperature in the dark for 1 h. Then, the solution was centrifuged at 12,000 rpm g at 4°C for 10 min and rinsed with 50 mM ammonium bicarbonate. One hundred microliters of 50 mM ammonium bicarbonate and free trypsin were added to the protein solution at a 1:50 ratio, and the solution was incubated at 37°C for 12 h. Finally, the solution was centrifuged at 12,000 rpm at 4°C for 10 min. Ten hundred microliters of 50 mM ammonium bicarbonate were added to the solution and centrifuged again. The extracted peptides were lyophilized, and peptides were resuspended in 2 to 20 μL of 0.1% formic acid before LC-MS/MS analysis. Once the analysis was finished, a search on the Macrobrachium proteins database (Crustacea, Decapoda) was performed using Proteome Discoverer 2.0 (ThermoFisher Scientific). The parameters were established as follows: the protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable). The enzymatic specificity was adjusted to trypsin; the maximum fractures lost were fixed at 2; the precursor ion masses' tolerance was set at 10 ppm, and the MS/MS tolerance was 0.6 Da.

From these analyses, only peptides identified with high confidence were selected for protein identification. Data from biochemical samples were analyzed in triplicate using the Minitab Statistical Software, version 17.

RESULTS

Six different embryonic developmental stages were recognized (n = 50). Figure 1 depicts the morphological changes per stage observed in the embryos, emphasizing the appearance of new structures for each stage. Sizes and color changes in the eggs (n = 50), ranging from 481 to 692 μm length and 4.38 to 469 μm width are shown (Table 1). A continuous increase in volume along developmental time is observed.

Figure 1 Morphological changes in the embryos of Macrobrachium tenellum along with embryonic development. os: ocular spot, ce: cephalon, pr: pereion, pl: pleon, tl: telson, v: yolk. a) New eggs: spherical, no cells or structures are observed, translucent yolk, b) stage A: the blastomeres are observed, and the yolk is denser and more homogeneous, c) stage B: the cephalic structures and the tagmas that correspond to the prymordia of the pereion and the pleon appears, d) stage C: the embryo has a “C” shape. It is located at one side of the egg, and the pleon begins to split into segments. There is pigmentation of the ocular spot, and yolk occupies more than half the egg, e) stage D: the oval ocular spot with reddish pigmentation is forming, and the segmentation of the pleon is more evident. The telson is starting, and the yolk occupies less than half the egg, f) stage E: the embryo occupies most of the egg, eyes totally pigmented, pereion, pleon, telson, and uropods well-defined. The embryo is almost fully developed. 

Concerning biochemical changes, the concentration of soluble protein varied depending on the stage (R² = 0.95): new eggs: 5.04 mg mL^-1; eggs in stage A and stage B with 24.35 and 4.23 mg mL^-1, respectively; eggs in stage C with 13.3 mg mL O^-1; and eggs in stages D and E with 0.75 and 1.17 mg mL^-1, respectively. The molecular weight of the proteins found in every stage is shown (Fig. 2). Protein bands at 250-kDa molecular weight were observed in all stages, but with overexpression only in stage E. Proteins with a weight of 20, 250, 50, 37, 25 kDa were present in all stages. Protein bands with medium and small molecular weight varied depending on the stage, and that was the case of new eggs, where only protein bands with a molecular weight of 150 and 100 kDa were observed. In stage C, just the protein band of 2 kDa was found. In stages A and D, only the 10 kDa protein band was present. In the D and E stages, protein bands were current with 42, 41, 39, 27, and 23 kDa. Also, eight proteins with at least 1000 amino acids were identified in stage D eggs, mostly related to vitellogenin (Q95P34) and vitellogenin (A0A0E3JBN6) with a molecular weight of 285 and 286 kDa, respectively (Table 2).

Figure 2 According to the stage of embryonic development and molecular weights, protein bands along with Macrobrachium tenellum embryonic development. a) New hatch, b) eggs in stage A, c) eggs in stage B, d) eggs in stage C, e) eggs in stage D, f) eggs in stage E. 

DISCUSSION

The embryonic development encountered in the present work is equivalent to that proposed by Muller et al. (2004) for Macrobrachium potiuna and M. olfersi. Four stages have been recognized: new eggs, prenaupliar, naupliar, and postnaupliar. It is very similar to the five stages identified by ^{Graziani et al. (1993)} for M. carcinus or the nine stages proposed by ^{García-Guerrero & Hendrickx (2009)}. All these different species might have the same developmental process, differing only in developmental time and the description criteria. Different approaches or criteria describe a part of a life cycle comparable in all embryonic stages of other crustaceans and almost equal in any Macrobrachium species. From the observations made in the eggs of the present work and aimed at giving a synthesized classification, at least three development stages can be proposed: pre-embryonic stage (stages A and B), during which segmentation and gastrulation occur. Then, the embryonic stage equivalent to stages C and D implies the development of primary cephalic structures. Stage E, which is the most extended and complex, begins with developing the ocular and abdominal structures and agrees with the pre-naupliar, naupliar, and post-naupliar stages, as defined by ^{Müller et al. (2003)} and ^{García-Guerrero & Hendrickx (2009)}.

There are different criteria for characterizing embryonic development stages (^{Nazari et al. 2000}), but the development seems always to follow a common pattern in all Palaemonid prawns. However, its duration and success depend on several external factors. For M. tenellum, the time from new eggs to hatching at 27°C is 17 ± 1 d (^{Espinosa-Chaurand et al. 2011}), less if compared with M. carcinus and M. americanum, in which the development time is 20 ± 1 d at 24 and 25°C, respectively (^{Graziani et al. 1993}, ^{García-Guerrero & Hendrickx 2009}, ^{García-Guerrero et al. 2013}). Also, ^{Müller et al. (2003)} worked with some of the smaller species of the genus (M. olfersi), in which embryonic development may last 14 ± 1 d at 25°C and have the shortest period recorded. It is also important to consider that incubation time could be species-dependent but is influenced by water physico-chemistry, being temperature the main factor (^{Habashy et al. 2012}). The temperature might make the developmental time of embryonic structures slower or faster, making the classification into stages, based on time, a non-reliable criterion, leading to arbitrary stages. To avoid this problem, the present results classified the development into percentages, as initially proposed by Sandman & Sandman (1991) and later by ^{Tinikul et al. (2016)}: stage A = 20%, B = 40%, C = 60%, D = 80% and E = 100%. This criterion allows a comprehensible distribution of the stages and permits the comparison of embryo-genesis among species in a more accurate way (^{Odinetz-Collart & Rabelo 1996}).

In Macrobrachium species, particularly M. tenellum and M. americanum, all structures appear and grow in a similar sequence, following matching patterns (^{García-Guerrero et al. 2015}). Morphological features of M. tenellum observed in the present study are equivalent to those described for other Macrobrachium prawns, such as M. olfersii (^{Müller et al. 2003}), M. acanthurus (^{Müller et al. 2007}), and M. americanum (^{García-Guerrero & Hendrickx 2009}). Indeed, ^{Espinosa-Chaurand et al. (2011)} indicate that M. tenellum in its early stages of development begins with a holoblastic segmentation at approximately 4 h after fertilization that ends at 32 h. It seems to be equivalent to stage A of the present study (Fig. 1). The first structures of the embryo begin to differentiate starting on the third day and, on the fourth day, the appendices are formed, equivalent to stage B. The optical lobes develop during the seventh day, the pigment of the eyes at the end of the eighth day, and on the 10th day, the chromatophores appear, and the heart is formed and beating, which is equivalent in this work to stages C and D, respectively. The embryo is well-formed on the 12^th day, similarly to those obtained in the observation of E stage eggs of the present work. In M. americanum, this method even allows observing major embryonic structures' development without cytological techniques (^{García-Guerrero & Hendrickx 2009}). This technique seems to be proper because the external structures are few and well-differentiated. Most of them are easily observed through the chorion. A partial match with other Decapoda such as Palaemonetes argentinus (^{Müller et al. 2003}) is also observed. These authors mention that this method is precise and easy to apply for decapod embryology, and the present work confirms that embryonic development depicts a similar matching pattern. The staging method based on percentages has been useful to characterize the embryonic development of M. americanum (^{García-Guerrero & Hendrickx 2009}). As in the present work, this criterion was also useful to describe and divide the embryonic development in percentages based on morphological changes observed in the egg. It is clear that the method could be useful for any Decapoda with lecithotropic centrolecithal eggs such as Caridea and Astacidea (^{Sandeman & Sandeman 1991}, ^{García-Guerrero et al. 2003}), Brachyura (^{García-Guerrero & Hendrickx 2004}), and Anomura (^{García-Guerrero & Hendrickx 2006}).

The morphogenesis of the prawn eggs registered in the present work (Table 1) agrees with ^{Müller et al. (2004)}, establishing that an egg increases along with embryonic development. M. tenellum eggs are smaller in comparison with other cogeneric species. For example, M. amazonicum (^{Meireles et al. 2013}), M. carcinus (^{Graziani et al. 1993}), or even M. rosenbergii (^{García-Ulloa et al. 2004}) are reported to have larger eggs. However, differences in size among the same genus species are probably due to variations in the total amount of water and yolk inside the egg, which can vary not only among species but also among the same species' eggs. Many factors, including female fitness and feeding during maturation and female size, the embryonic stage, or water physico-chemistry, produce a subtle effect on egg size (^{García-Guerrero & Hernández-Sandoval 2012}). From these results and previous works, it is clear that egg size is related to the species' reproductive strategy and not to the species' average or maximum size.

On the other hand, many previous works with Macrobrachium prawns that proteins in prawn eggs are the main source for tissue synthesis and an additional energy production source and development. ^{García-Guerrero (2009)} and ^{García-Guerrero & Hernández-Sandoval (2012)} state that proteins are regularly the most abundant component in the eggs, as reported for M. americanum and M. occidentale. From these works, it has been inferred that proteins lower their concentration significantly along with the development. However, as observed in the present work, soluble proteins increase later and continue until ending egg development. This phenomenon occurred with differences among stages; from the initial stage A to B, there was a drastic decrease in total proteins' concentration from 24.35 to 4.23 mg mL^-1. It seems that this is due to an intensive tissue synthesis during gastrulation. Instead of what occurs in further stages C, D, and E, where an increase in the total concentration of protein occurred, another decrease is observed from 13.3 to 0.75 and 1.17 mg mL^-1, respectively. This trend is similar to that observed by ^{Dinakaran et al. (2013)} for Macrobrachium idella idella, in which the concentration of dissolved protein in the egg gradually decreased from stage I (69.32%) to stage IV (54.56%); these authors established that in zoea I stage the concentration of total protein decreases as hatching approaches, reaching 52.42%.

Morphological and size changes come with modifications in the concentration of biochemical composition. Proteins continuously change in type and amount in the egg depending on stage. The molecular protein weight can be subtracted from the protein band's migration distance to observe such variations, in agreement with ^{Maldonado-Alconada & Jorrin-Novo (2011)}. It is observed that depending on the developmental stage, proteins vary in molecular weight, but there are some bands (Fig. 2) that are equivalent in their kDa for different stages of development. Hence, their presence is continuous along with the development. It seems that it is the same protein at different stages and with varying expression levels. The same protein kind could be required along with the whole embryonic development for other tasks. In M. tenellum, all samples of embryonic stages had protein bands with a molecular weight of 250 kDa, highlighted in Figure 2, whereas in other species, such as Macrobrachium borelli, the most abundant proteins are expressed in bands at 94 and 112 kDa. A possible explanation could be the presence of many high molecular weight proteins that cause DNA and protein overexpression (^{García et al. 2006}).

Proteins are regularly the major constituent in crustacean eggs since they are essential for tissue synthesis and energy source fuel (^{García-Guerrero et al. 2003}, ^{Luo et al. 2004}). Those previous works also recognized that morphological changes occurring in the eggs at different stages are mainly due to continuous protein synthesis and degradation, which constantly produce variations in egg biochemical composition (^{Habashy et al. 2012}). Proteins are essential for most metabolic functions (^{Qiao et al. 2017}). Their exact function in M. tenellum has not been determined yet; they are likely used for species' same functions. ^{Jaramillo et al. (2017)}, working with M. olfersii, indicate that the analysis of protein expression with genes allows establishing the link of the physiological mechanisms at the molecular level, occurring along with the ontogenetic development of prawns.

Also, protein types reported in this work are similar to those described for the larval and post-larval stages of M. olfersii and M. rosenbergii (^{Barman et al. 2012}, ^{Priyadarshi et al. 2015}, ^{Jaramillo et al. 2017}). Those works suggest that vitellogenin (vg) had the highest gene expression levels in protein isolated from eggs. Other studies indicate that it is also possible to find similarities among proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L8 (RpL8), ribosomal protein L18 (RpL18), β-actin (β-act), and elongation factor −1α (EF-1α). Still, these were not quantified in the present work, so comparisons cannot be offered. From those proteins, GAPDH is the most common after vitellin-type proteins. Vitellin-originated proteins have the highest sequence of peptides (Table 2) because they are synthesized in the ovary before and during egg formation and are channeled to the eggs as a lipid carrier (^{García et al. 2008}). For example, ^{Lafontaine et al. (2017)} stated that vg is abundant in immature M. rosenbergii females. The same occurs in other Palaemonidae, such as Palaemon elegans (^{Sanders et al. 2005}, ^{Bai et al. 2016}). The latter authors support that vg is the extra-ovarian precursor of yolk proteins or vitelline, which provides nutrients and energy and the whole development (^{Matozzo et al. 2008}). Regarding M. tenellum, it was observed that variations in the amount of proteins, such as vitellin, seem to be due to changes in the total concentration of soluble proteins from stage to stage, which is a consequence of embryonic developmental changes.

This work helps to describe that embryogenesis in crustaceans exhibits a common pattern in both morphological changes and protein profile and development. Structures forming inside the egg are quite similar among species, whereas subtle differences in the genetic expression of proteins and development are found in close species. Those subtle differences among species at different stages of embryonic development can be tracked or determined in the eggs, explaining, perhaps, slight differences in requirements. A better understanding of the use of vg-originated proteins along the embryonic development of crustacean eggs could be reached with specific studies, at the molecular level, including comparisons of this process among species. ^{Jaramillo et al. (2016)} suggested that molecular sequences from Macrobrachium prawn eggs can help to understand differences in developmental models among close species with a bio-molecular approach.