STAGES OF GONADAL DEVELOPMENT IN THE SPOTTED PINK SHRIMP
PENAEUS BRASILIENSIS
Martha E. Sandoval Quintero and Adolfo Gracia
A B S T R A C T
The stages of gonadal development for the female of the spotted pink shrimp Penaeus brasiliensis from the Mexican Caribbean Sea are characterized, based on histological analyses. Five stages
(immature, developing, almost mature, mature, and spawned) were determined, according to the
structure and arrangement of the germ cells in the gonads. Each of these stages corresponds macroscopically to a characteristic color, except for stages I and V, in which the coloring is similar and
can be distinguished only microscopically. A chromatic scale is provided to determine the stages
o f gonadal development, which ranges between a translucent aspect to an olive green color corresponding to the immature and mature stages. The size of the cells in each stage varied from an average o f 0.028 m m for stage I to an average of 0.231 mm for stage IV. Growth of cells is faster in
stages II and III and shows a greater overlapping of sizes in stages III and IV.
R E S U M E N
Se proporciona una caracterización de los estadios de desarrollo gonadal de las hembras de camarón rojo P e n a e u s brasiliensis en el Caribe Mexicano basadas en análisis histológicos. De acuerdo
a la estructura y disposición de las células germinales de las gónadas se determinaron cinco estadios (inmaduro, en desarrollo, casi maduros, maduros y desovados). Macroscópicamente cada uno
de estos estadios corresponde a colores caracter�sticos, excepto las etapas I y V cuya coloración es
similar y solo puede distinguirse microscópicamente. Se proporciona una escala cromática para la
determinación de los estadios de desarollo gonadal que van desde un aspecto translúcido hasta
color verde olivo correspondientes a las fases de inmaduro y maduro. El tamaño de las células en
cada etapa de madurez de las gónadas varió desde 0.028 m m en promedio para el estadio I, hasta
u n promedio de 0.231 mm para la fase IV. El crecimiento de las células es mas rápido de los estadios II a III y muestra una mayor sobreposición de tamaños en las etapas III y IV.
The spotted pink shrimp is caught throughout most of the year mainly northeast of Contoy Island in Quintana Roo State (ArreguinSanchez, 1981), except during the closed season, which was established in 1994 for the
months of August-November. Statistics for
the spotted pink shrimp landings in the Mexican Caribbean Sea show that maximal production was obtained between 1984 and 1987,
during which catches ranged from 3 0 9 - 4 7 4
tons of shrimp tails. After this period, a notable decrease in the production of this resource has occurred, causing great concern
regarding its exploitation. In 1995 and 1996,
the shrimp catch decreased noticeably in respect to the maximum obtained in 1985; less
than 200 tons were obtained.
Although production of Penaeus brasiliensis Latreille is not as large as that of other
species o f the genus Penaeus in the Gulf of
Mexico (Gracia et al., in press), it represents
an important regional fishing activity. Therefore, it is necessary to have adequate information on the factors involved in its produc-
tion to establish guidelines for biological
management of this resource. Among these
factors, knowing the dynamics of the reproductive process is essential, since it is fundamental for the renewal process of the population. Definition o f the developmental
stages of the gonads and determination of
practical gonadal indices represent a starting
point for the study of the reproductive activity of P. brasiliensis. Based on this knowledge, a maturation index of the shrimp may
be established. The combination of this index
with other parameters, such as the abundance
of reproductive individuals and fertility/size
index, would help to determine the reproductive patterns of the population. This in
turn would be instrumental in the establishment o f conservation measures for this resource.
The present study characterizes the stages
of gonadal development in the female of P.
brasiliensis, using a chromatic scale to determine the stages of maturity validated by
histological sections.
To determine whether a significant difference existed
among the average diameters of the cells, for each maturation stage, a one-way analysis of variance (ANOVA)
and the Tukey test for multiple comparisons of medians
(Zar, 1974) were applied.
RESULTS
Ovarian Stages and Chromatic Scale
Fig. 1 . F r e q u e n c y of cell size in female gonadal stages
of Penaeus brasiliensis.
MATERIALS AND METHODS
In order to establish a chromatic scale of the different
maturation stages in the female of P. bra.siliensis, a sample of 100 organisms was collected in 4 sampling trips
on board shrimp boats from the Puerto Juarez, Quintana
Roo, fishing fleet, during the months of March, July, and
October 1993, and April 1994.
For each organism, the total length, measured from the
tip of the rostrum to the tip of the telson, using a 30-cm
ichthyometer with an accuracy of 0.05 mm, and the color
of the fresh gonad were recorded. Afterward, the gonads
were fixed in 5% Formalin for histological sectioning and
microscopic determination of the degree of development.
The prevailing color for each maturation stage of the
gonad was compared with a color catalog, internationally used for serigraphy (Pantone, Inc., 1984) to establish basic reference points.
Histological analyses of the gonads were performed
using the paraffin-embedding method, consisting of: (a)
fixation of the gonadal tissue, (b) paraffin embedding, (c)
sectioning and mounting, and (d) hematoxylin-eosin staining (Drury and Wallington, 1980; Bell and Lightner, 1988).
Histological sections were observed under a microscope
to record the structure and arrangement of the ovarian
germ cells and to determine their developmental stage.
Measurements of 100 cells in each maturation stage
were made, recording the largest diameter. This process
was performed using a microscope provided with an ocular micrometer at a 0.05-llm precision.
The number of cells needed to establish the average
size for each stage was calculated from the equation: N
= Z�7d�. at a 0.01-mm precision (d), a confidence level
of 95% (Z 1 . 9 6 ) , and a standard deviation (S) estimated
for each cellular stage sampled. The N obtained for each
stage was: El = 3.11, Ell = 7.52, EIII = 30.11, and EIV
= 30.11. However, based on the central limit theorem, it
was decided to establish the size of the sample as 30 cells.
The number of females in each gonadal stage needed
to establish the average diameter of the cells for the corresponding stage was also analyzed. The standard deviation used was estimated from the average size of the cells
in 3 different organisms, obtaining for El, N = 0.345;
EII, N = 0.614; EIII, N = 0.96, and EIV, N = 0.03. Since
the sample represented only a fraction of the gonad, it
was decided to measure 30 cells from 5 different females
in each developmental stage, because it is best to sample
different "experimental units" and not subsamples of the
same sample (Steel and Torrie, 1988).
A total of 100 gonads from females of P.
brasiliensis in their different developmental
stages, representing the whole range of colors for each stage, was analyzed.
Based on microscopic observation of the
histological sections and on descriptions
made for other penaeid species (King, 1948;
Guitart and Hondares, 1980; Bell and Lightner, 1988; Bielsa et al., 1983; Guitart et al.,
1988; Levi and Vacchi, 1988; Quackenbush,
1991), five different stages in development
and cell arrangement in the ovary were identified. It is noteworthy to mention that the V
stage, which corresponds to a spawned gonad, starts its development toward new maturation during stage II (Fig. l A - E ) .
According to the structure and arrangement
of the different types of germ cells in the
ovary, the microscopic scale to determine the
different maturation stages is as follows:
Stage I. The most abundant cells in the
ovary are oogonia, which are part of the germinating epithelium. They are not uniformly
distributed inside the ovary, but clustered in
a well-defined area of the ovarian wall along
the gonad, known as the "zone of proliferation." Small basophilic oocytes were found
clustered on the periphery o f the ovary.
Stage II. The ovary is organized in chambers with oocysts containing the basophilic
oocytes at the periphery, separated by stromatic tissue and surrounded by follicle cells.
The central region of the chambers contains
oogonia.
Stage III. The ovary shows the same organization, but the oocytes are now acidophilic
and each is surrounded by follicle cells in
charge of providing nutrients. Oocytes are
still on the periphery of the chambers and
seem to be compressed due to their increased
size. Oogonia are located in the central region of the chamber, with basophilic oocytes
among them.
,
Stage IV. Acidophilic oocytes develop peripheral bodies. Oogonia and basophilic oocytes occupy the same position inside the
chambers.
Fig. 2. P e n a e u s brasiliensis. A, female gonadal Stage I. zp = germinal cell layer of oogonia "zone of proliferation", p = cysts, cf = follicle cells (40x). B, female gonadal Stage II. Cysts (p) well defined and surrounded by follicle cells (cf). Size of cell nucleus (n) and nucleoli (n') greater than cytoplasmic size (40x). C, female gonadal
Stage III. Nucleoli (n') located on periphery of cell nucleus (n). Cytoplasm (c) increasing its size in comparison to
previous stage (40x). D, female gonadal Stage IV. Main difference from stage III presence of "droplike peripheral
bodies" (cp). Nucleoli (n') in nuclear border of ova (n) (40x). E, spent ovary, gonadal Stage V. Remaining follicle
cells (cf) dispersed in gonad after ova extruded. Some cells in stage I (EI and EII) observed (40x). F, practical color
scale of ovarian developmental stages. Gonadal consistency and color shown for each ovarian stage.
Stage V. The ovarian organization disappears, leaving holes left by the released
oocytes, as well as remains of disintegrating
mature oocytes or those in the process of reabsorption, vitellin granules together with
oogonia, and basophilic oocytes disseminated
throughout the entire ovary.
Macroscopically four developmental stages
can be identified, according to four characteristic colors. Intermediate shades are considered as beginning or advanced stages,
which can be determined only by microscopic
observation of the structure and arrangement
of the ovarian cells. During the first two de-
Table 1. Mean size, range, and statistical results of stages of gonadal cells of P e n a e u s brasiliensis. *Significant
differences.
velopmental stages, the pigmentation of the
gonads varies from a translucent color to a
creamy light green. With advancing maturation, stages III and IV, the green intensifies.
The texture of the gonadal tissue and the space
occupied by the gonads are also characteristic for each developmental stage.
Based on the color and microscopic and
macroscopic observations, a practical scale to
define the degree of gonadal development is
proposed, which presents the prevailing color
for each stage (color name and code were assigned according to the Pantone catalog,
1984) (Fig. IF).
The proposed stages are:
I. Immature. Undeveloped ovary, filiform,
and flaccid consistency. Egg mean size 0.028
mm. Translucent.
II. Developing. Ovary starting maturation,
thickening of the gonad, turgid consistency.
Egg mean size 0.078 mm. Creamy color (Catalog: Lamb's wool, 12-0910).
III. Early ripe. Ovary in advanced maturation. Gonad thickening more and occupying
more space along the body, turgid consistency. Egg mean size 0.188 mm. Light green
color (Catalog: Beechnut, 14-0425).
IV. Ripe. Completely mature ovary. Anterior part of gonad occupying entire cavity of
cephalothorax, and medial and posterior portions extending along abdomen. Turgid consistency. Egg mean size 0.231 mm. Olive
green color (Catalog: Lizard 18-0629).
V. Spent. Defined through microscopic observation of gonadal sections. Not included
in the chromatic scale, since, based on its cellular organization, it is at the start of stage II
of gonadal development. Color observed in
this stage whitish, not translucent.
Cell Size
The size of the germ cells recorded for each
maturation stage varied from 0.028 m m for
stage I (range 0 . 0 2 4 - 0 . 0 3 2 ) to 0.231 mm
(range 0 . 2 2 7 - 0 . 2 3 5 ) for stage IV. Figure 2
shows that the size of the cells is greater with
advancing maturation stages, except for
stages III and IV, in which the size is very
similar. In these two stages, noticeable overlapping of sizes can be observed, since the increase in size is relatively smaller as compared to the transition from stage II to III,
when the gonadal cells present the most accelerated growth rate (Table 1).
Variance analysis applied to the set of cellular diameters of each stage revealed a statistically significant difference among them
(P = 0.05). In the same way, the Tukey test
applied to the data revealed a statistically significant difference (P = 0.05) among the average diameters of the cells in each stage of
gonadal development.
Despite the statistically significant differences among the cell diameters, the median
value of the cell diameter for each stage and
its standard deviation allow us to group the
maturation stages in two pairs, according to
cellular size, i.e., (1) stages I and II and
(2) stages III and IV, which would correspond
to immature and mature females, respectively.
DISCUSSION
Ovarian Stages and Chromatic Scale
External characteristics of the ovary, such
as color, size, and texture of the tissue are
closely related to the development and internal organization of the germ cells. Therefore,
it is possible to establish a practical scale to
determine the maturation stage of fresh gonads. The scale presented in this paper, to
determine the degree of development of females of P. brasiliensi.s, was constructed by
correlating the macroscopic and microscopic
characteristics of the gonads for the four main
developmental stages. Another external indicator of gonadal maturation is whether the
gonad can be observed through the exoskeleton. Castille and Lawrence (1991) reported that an immature gonad (stage I) cannot be distinguished through the exoskeleton,
and even the diameter of gonadal lobules is
smaller than that of the intestine. A spawned
gonad, due to its reduced size (microscopic
stage V), is equally difficult to distinguish.
From what these authors call early maturation (stage II), the gonad becomes more visible through the exoskeleton, until being
clearly distinguishable in the mature ovary
(stage IV).
These indicators can cause confusion in
field sampling. For example, in the case of a
spawned female, the direct observation of the
gonad would reveal a filiform appearance,
leading to the conclusion that it is in stage I.
However, the color is not translucent, but
whitish, which would suggest that the organism is rather in stage II. To establish the degree of maturation of a female with these
characteristics, it is important to consider the
microscopic observations that would confirm,
in this case, that it is indeed a female in stage
II of gonadal development.
Another source of confusion may arise between determining stages III and IV, since
the gonad occupies practically the whole
cephalothoracic cavity, and different shades
of green can be observed. However, from microscopic observations of histological sections, a female is defined in stage IV when
the gonad shows an intense green color
(Lizard 1 8 - 0 6 2 9 , Pantone Inc., 1984). The
peripheral bodies of the cells develop at this
time, which is the main characteristic of this
stage. Early ripe (Stage III) and ripe (Stage
IV) females can be reliably separated only by
histological gonad examination.
This type of scale, despite a certain degree
of subjectivity in the color definition, is a useful tool for field sampling on board ships for
carrying out population studies aimed at determining the status of the resource and its reproduction dynamics. Another practical importance for the use of the chromatic scale is
to be able to recognize when the female is
mature. This is useful for collecting reproductive shrimp for aquacultural purposes.
Cell Size
The size of the oocytes of P. brasiliensis
is very similar to that recorded for other pe-
naeid species (Guitart and Quintana, 1978;
Ramos and Torras, 1986; Guitart et al., 1988).
The increase in size of an oocyte from stage
I to II is not substantially different. Whereas
the development in size of a basophilic oocyte
(stage II) to an acidophilic one (stage III) is
very drastic, since the process of protein synthesis is accomplished during this time.
In this regard, Quackenbush (1991) reported that the protein production pattern in
the crustacean "egg" is divided in two stages:
(1) primary vitellogenesis, characterized by
a small increase in the diameter of oocytes,
but already in the process of developing cytological characteristics to prepare the cell for
protein synthesis, and (2) secondary vitellogenesis, during which a massive increase in
size and weight of the cells occurs and the
precursors for the cortical granules that surround the fertilized egg develop.
In the transition from stage III to stage IV,
a wide overlapping of the range of cell diameters and a small difference in size can be
observed, suggesting that the oocyte has almost completed its development. No records
exist about the time needed for development
of stage III to stage IV for spotted pink
shrimp. Crocos and Kerr (1983) pointed out
that ovarian development through stage III
and stage IV occurs during one intermolt period. For P. esculentus de Man (see Crocos,
1984), it occupies approximately 65% (mean
duration of 19 days) of the intermolt period.
These observations can be important from a
practical point of view, since the exact determination between these two stages can be
accomplished only by histological analysis
and since the visual examination of gonads
during field work based on color could lead
to errors in the differentiation of stages. It becomes necessary to establish the duration of
these stages for P. brasiliensis to avoid any
under or overestimate of the actual level of
spawning activity. The time needed for the
spotted pink shrimp gonad to achieve complete maturation after stage III is apparently
small. Both stages can be considered reproductive without biases during population
studies, provided that sampling frequency is
not less than one month. Thus, a color scale
represents a useful field tool for population
studies. Such studies should also consider information about abundance of spawners,
spawning period, and fecundity/size indices.
Information obtained from spotted pink
shrimp in the area (Sandoval Quintero, 1996)
shows that spawning occurs throughout the
year with a main peak during spring
(March-April). Knowledge of population fecundity and its seasonal fluctuations would
allow a better understanding of the reproductive dynamics of P. brasiliensis.
Color, size, and texture of the gonads of the
female shrimp are closely related to the cellular development of the ovaries. Therefore,
the chromatic scale is representative of different stages of development and is a valuable practical tool for studies on the reproductive dynamics of the spotted pink shrimp
P. brasiliensis.
ACKNOWLEDGEMENTS
Thanks are due to the Centro Regional de Investigacion Pesquera de Puerto Morelos del Instituto Nacional
de la Pesca for supporting this study. Appreciation is extended to fishermen of the Shrimp Cooperatives of Quintana Roo State for facilitating the collection of shrimp.
The authors are also grateful to Raul Sima and Gregory
Arjona for their assistance in histological analysis.
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RECEIVED: 5 September 1997.
ACCEPTED: 11 February 1998.
Addresses: (MESQ) Centro Regional de Investigación
Pesquera de Puerto Moralos, Instituto Nacional de la
Pesca, Apartado Postal 580, Cancún, Quintana Roo,
77501 México; (AG) Instituto de Ciencias del Mar y Limnologίa, U N A M , Apartado Postal 70-305, Mexico D.F.
04510 México. (e-mail: [email protected])