Jenynsia multidentata, (Jenyns, 1842) (Jenyns, 1842)

López-Rodríguez, Nathalia C., de Barros, Cíntia M. & Petry, Ana Cristina, 2017, A macroscopic classification of the embryonic development of the one-sided livebearer Jenynsia multidentata (Teleostei: Anablepidae), Neotropical Ichthyology 15 (4), No. e 160170, pp. 1-10 : 3-7

Results

In total, 155 females that were deprived of males for 60 days and raised up to over 30 days after a mating attempt survived and were used for histological processing. Their total length ranged between 34.47 and 68.48 mm (mean ± S.D.: 47.66 ± 6.95 mm) and they showed a high variation in the degree of ovary development. Ovaries were in general small-sized, and resembled the virgin or resting (n = 77) stage, whereas others harbored embryos on early (n = 27) and later stages of development (n = 51), which were visible due to their pigmented eyes. According to the chronological ordering of the females throughout the 58 days, no sequence of advancing embryonic development was recorded. Despite recording the mating and harassments of females by males, it was not possible to determine whether females were fertilized if they presented ovaries in folliculogenesis, or even if they would be able to become pregnant under captive conditions. Additionally, spermatozoa were not detected in any of the 80 histological slides analyzed.

Characterization of the embryonic development stages. According to the morphological analysis of the embryos, it was possible to discriminate eight stages of development, beginning from Stage 1, the fertilized egg (ovum) through Stage 8, in which the embryos are ready to birth ( Tab. 1 ; Fig. 1 ).

Stage 1: Fertilized egg. Fertilized eggs measured approximately 0.3 mm in diameter, with yolk granules and a well-defined perivitelinic zone. Although the eggs were found free inside the ovary, they were also seen grouped, forming sets on the cephalic region, from where they seemed to migrate along the ovary wall until the central cavity to continue the development ( Figs. 1 a-b).

Stage 2: Outline of the optic vesicle. The embryo was unpigmented and the body curved with a diameter of approximately 0.9 mm. The YSL was abundant. It was possible to distinguish the following three regions of the body: cephalic, corporal, and terminal. The outline of the optic vesicle was evident, and the somites had a pointed look, indicating its future division in dorsal and ventral muscle mass (epiaxial and hypoaxial, sensu Kimmel et al., 1995) ( Figs. 1 c-d).

Stage 3: Beginning of eye pigmentation. The embryo was still curved with a diameter of approximately 1.5 mm. The YSL was present in most embryos. The greater development of the cephalic region, which represented approximately 30% of body size, was evident. The eye pigmentation begins in the layers of the retina. Pigmentation on the back of the head was first detected in few embryos. The optic nerve, three to five crests of branchial arches with hyaline cartilaginous tissue and neural tube were identified. Moreover, the liver occupying a large part of the abdominal cavity, and the portions of the intestine were observed. From this stage, it was possible to count the embryos, which could be differentiated based on the pigmentation of the retina and/ or iris ( Figs. 1 e-f).

Stage 4: Sprout of the caudal fin. The body of the embryo began to distend, was thin, translucent and had little muscle mass. The low frequency of embryos at this stage and the greater variability in their size made it difficult to estimate their total body length. The head accounted for 25% of the body size. The mouth opening and the early development of the caudal fin were observed. The retinas were pigmented and the first punctate melanophores were identified in the dorsal region. Remnants of the YSL were still detected ( Figs. 1 g-h).

Stage 5: Sprouts of the dorsal and anal fins. The head was more developed. The arrangement of the dorsally punctiform melanophores resembled a heart. The body gets thicker, and in most embryos, the trunk is fully distended. The total length was approximately of 5 mm. Punctiform melanophores were scattered along the flanks. The remnants of YSL were still visible in some embryos. The operculum and the dorsal and anal fins were visible ( Figs. 1 i-j).

Stage 6: Intrusion of ovarian folds. The embryo was fully distended and cephalic and corporal regions of the body attained greater proportionality. The pigmentation was the most conspicuous feature for the identification of this phase. It was possible to identify three lines of punctiform melanophores along the dorsum, one in each sideline and one at the base of the dorsal and anal fins, and the concentration of large punctate chromatophores on the back of the head. The odd fins were much more developed and were observed as rays on the rounded caudal fin. The remnants of the YSL were not more evident and ovarian folds were inserted through the gills of all embryos to constitute the branchial pseudoplacenta ( Figs. 1 k-l).

Stage 7: Occurrence of dendritic melanophores. The embryo increased in thickness and total length (which ranged between 6.9 and 9.0 mm) and remained connected to the ovarian tissue through its folds that resembled a cord. The muscles were more developed. Dendritic melanophores were detectable, and the pigmentation was more intense in the mandibular region and the dorsal and lateral sides of the head, advancing to the caudal direction. The upper mouth was evident, and in some embryos, the ovarian folds were inserted through the mouth ( Figs. 1 m-n).

Stage 8: Mature embryo. The body was robust; the caudal peduncle was thicker compared to the previous phase, and TL varied between 11.6 and 14.3 mm. The pigmentation was spread throughout the body – around the mouth and eyes. In the dorsal region of the head, there was a cluster of punctiform melanophores resembling a heart. In the dorsal region and the upper sideline, the grouped melanophores (with greater intensity) resembling spots showed the following two patterns: dendrite (or branched/ramified/starshaped) and staining (punctate melanophores together). The pigmentation of all retinal layers was completed. Through the thin ovarian membrane, it was possible to identify embryos that maintain their cord connection with the mother ( Figs. 1 o-p).

Structure connecting female-embryo. The first detectable maternal-embryonic connection was a cord like structure, which was first recorded in almost elongated and highly pigmented embryos from Stage 6 onwards. This connection lay on the ovarian tissue folds (villus) and extended throughout the gills either via the oral (less frequently recorded) or pharyngeal cavity (most frequently recorded) of the embryo ( Figs. 2 a-b). In a single batch, all embryos were connected to the ovarian tissue (villus) through a cordlike structure, which was maintained until the final stages of development, when embryos were mature and ready to birth ( Fig. 2c ). Histological analyses of villus revealed the occurrence of blood vessels located adjacent to epithelium in the connective tissue ( Fig. 2d ).

Variation in the weight of batches and reproductive tissues through embryonic development. According to the dry weight recorded in 15-18 batches of each developmental stage, significant increases occurred in the total developing embryos despite minor changes in maternal reproductive tissues. On an average, the batches of embryos in the initial stages of development showed substantially smaller weight than those that are close to maturity or mature ( Tab. 2 ). While the total dry weight of these mature embryos (Stage 8) increased steeply with the developmental stage (an increment of 66-folds, Tab. 2 ), it increased only slightly in the other reproductive tissues. The significant interaction term between predictor variables ( Tab. 3 ) indicated an effect on the stage of the development of the relation between the dry weight of the batch and the other reproductive tissues.

After controlling the effect of reproductive tissues, almost 90% of the variation in the total dry weight of the batch was explained by the developmental stages (ANCOVA, F = 62.424, d.f. = 11;81, r 2 = 0.880, P <0.001) ( Tab. 2 ). According to the comparison of the adjusted means, total dry weight of developing embryos from the Stage 6 onwards was significantly heavier than those in Stages 3-4 ( Fig. 3 ). Macroscopic records of the ovaries and embryos revealed that a thin layer (ovarian membrane) covers the batches of mature embryos. However, other maternal-embryonic structures (i.e., the cord-like structure) may override the losses represented by the thinning of the ovarian wall in the later developmental stages on the weight in maternal reproductive tissues.

Figures

Fig. 1. Stereoscopic (a, c, e, g, i, k, m, o) and light microscopy (b, d, f, h, j, l, n, p) images stained with Hematoxylin and Eosin of the stages of embryonic development of the one-sided livebearer Jenynsia multidentata. Y: yolk; PS: perivitelinic space; YG: yolk granule; Oov: outline optic vesicle; YSL: syncytial layer of the yolk; R: retine; Arrow head: somites; Nt: neural tube; L: liver; I: intestine; On: optic nerve; Cr: crest of branchial arch; Mo: mouth opening; *: miomers; B: brain; H: heart; Sb: swimbladder; Op: operculum; Baf: bud of the anal fin; Bdf: bud of the dorsal fin; Sc: spinal cord; Df: dorsal fin; Af: anal fin; Cf: caudal fin; Ra: rays of caudal fin; Bpp: branchial Pseudoplacenta; Vr: vertebra. Scale bars of 0.5 mm (a, c, e, g, i, k, m, o) and of 0.2 mm (b, d, f, h, j, l, n, p).

Fig. 2. Morphology of maternal-embryonic connections from the one-sided livebearer Jenynsia multidentata, based on macroscopic embryo and maternal-embryo structure (a); histological section of the embryo and maternal-embryonic (b); cystovarian with villus forming a maternal-embryonic connection (c); and blood vessels from the maternal-embryonic connection (d). Arrow: maternal-embryonic connection; Arrow-head: blood vessels; OC: oral cavity; Emb: embryo; V: villus; E: epithelium; CT: connective tissue. Scale bars of 0.5 mm (a), 200 Mm (b, c) and 20 Mm (d).

Fig. 3. Variation in dry weight of batches of developing embryos (adjusted mean ± 95% confidence interval) of the one-sided livebearer Jenynsia multidentata through the progressive stage of development, after controlling the effect of the dry weight of reproductive tissues. The values of the x-axis range from the beginning of eye pigmentation (Stage 3) to the mature embryo (Stage 8), according to the stages proposed by this study. Different letters (A-D) identify averages that differed significantly between stages of development according to Tukey’s test.

Tables

Table 1. Characterization of the embryos of the one-sided livebearer Jenynsia multidentata at the proposed eight stages of development. Scale bar of 0.5 mm.

Stage	Individual dry weight (mg) ± S.D.	Sketches	Morphological features
1	0.007 ± 0.006		Fertilized egg – Eggs presenting yolk granules.
2			Outline of optic vesicle – Curved unpigmented embryo. Cephalic, corporal and terminal regions of relative similar size. Detectable outline of the optic vesicle and somites.
	0.032 ± 0.032
3				Beginning of the eye pigmentation – Curved embryo with a greater
	0.069 ± 0.028		development of the cephalic region, almost 30% of the body size. Pigmentation of the layers of retina and of the top of the head. Detectable
			optic nerve, three to five gill archs and intestine.
4			Sprout of the caudal fin – The body is thin and translucent. Detectable mouth
	0.093 ± 0.023		opening and the early development of the caudal fin.The retinas are pigmented
			and the first punctate melanophores are located in the dorsal region.
5			Sprouts of dorsal and anal fins – The body is thicker and the trunk is fully
	0.193 ± 0.074		distended. Punctiform melanophores on the top of the head resemble a heart
			and others scattered along the flanks. Detectable opercle, dorsal and anal fins.
6			Intrusion of the ovary folds – The embryo is fully distended. Three lines of
	0.746 ± 0.326		punctiform melanophores along the dorsum. Large punctate chromatophores on the top of the head. Fully developed odd fins. The ovarian folds are inserted through the gills.
7			Occurrence of dendritic melanophores – The embryo increases in length
			and thickness and remains connected to the ovarian tissue through its folds.
	1.869 ± 0.710		Pigmentation is more intense in the mandibular region, dorsal and lateral sides
			of the head. The upper mouth is evident and ovarian folds are inserted both
			through the gills and the mouth.
8			Mature embryo – The body is robust. The pigmentation is spread throughout
			the body: around the mouth and eyes, in the dorsal region and upper the sideline
	4.243 ± 1.524		grouped melanophores resembled dendritic and stained spots. Pigmentation of
			all layers of the retina. Embryos may maintain connection with the mother
			throughout the ovarian folds.

Table 2. Variation in dry weight of the batch of developing embryos (n) and reproductive tissues (mean ± S.D.) and their ratio across the developmental Stages 3 and 8 of the one-sided livebearer Jenynsia multidentata inhabiting coastal lagoons of the northern of Rio de Janeiro State, Brazil.

Stage	n per Lagoon			Dry weight (mg)		Ratio (BDE/RT)
	Pitanga	Catingosa	Garça	Batches of developing embryos (BDE)	Reproductive tissues (RT)
3	0	6	9	0.6 ± 0.3	4.0 ± 2.0	0.1500
4	2	9	4	1.1 ± 0.6	3.7 ± 1.5	0.2973
5	10	3	5	4.2 ± 2.5	5.1 ± 2.4	0.8235
6	5	3	7	11.8 ± 5.6	5.8 ± 1.7	2.0345
7	5	3	7	34.3 ± 24.9	5.8 ± 2.4	5.9138
8	2	6	7	54.6 ± 19.7	5.5 ± 1.6	9.9273