Specimen collection and environmental variables (surface sea temperature and chlorophyll) measure site geopositions.
During two annual periods, the reproductive cycle of the gastropod Hexaplex princeps from Puerto Ángel, Oaxaca, Mexico was studied through gonadal histology. Sex proportion for the collected individuals was not statistically different from parity although most of the time, the number of males was slightly larger than that of females, which only outnumbered males during the spawning season. The maturity stages established for females were: (1) initial oogenesis, (2) previtellogenic maturity, (3) vitellogenic maturity, (4) maturity, (5) spawning, and (6) resting; and for males: (1) initial spermatogenesis, (2) maturity, (3) spawning, (4) onset of the rest, and (5) resting. Monthly variations of maturation stages showed that H. princeps has an annual reproductive cycle with a long period of gonadal activity. The spawning season comprised from November (females) and December (males) to March, with activity peaks in January. From March to October (females) and from May to June (males), reproduction resting occurred. Spawning was related to high chlorophyll concentrations due to the upwelling processes resulting from the winds and to the cooler sea surface temperatures occurring from November to March. This study provides baseline information that may serve to establish measures for sustainable exploitation strategies and for future aquaculture implementation of this species.
- Hexaplex princeps
- reproductive cycle
- sexual proportion
- histological analysis
- maturity scales
Marine mollusks constitute one of the more important world fisheries representing around 10% of the total value and quantity . These invertebrates have been exploited since ancient times. Recently, it has been reported that omega fatty acids, including docosahexaenoic acid (DHA) are key to brain health and most likely helped to drive the evolution of the modern human brain, when hominin ancestors consumed rich DHA marine shellfish . In the world, approximately 720 gastropod species are exploited [3, 4]. In Mexico, the gastropod catch in 2013 has the 19th place with 6011 ton .
In the world, artisanal fisheries are in continuous expansion due to the growing demand and increasing value of appreciated species, and thus, the fisheries effort is augmenting . Under this scenario, it is advisable (if not indispensable) to gather baseline biological information that may be used to propose management measures promoting long-term sustainable resource exploitation .
The study of the reproductive processes in marine organisms is a fundamental biological aspect, which permits to understand their population dynamics . The reproductive season is a crucial life history trait and the proper timing of breeding may be important to provide the offspring with favorable environmental conditions and to influence parental fitness [14, 15]. The analysis of the reproductive cycle of organisms permits to know the adequate moment and intensity of the capture to avoid the population depletion.
The reproduction of Muricidae members (at which
Thus, this study is aimed to investigate the reproductive cycle of
2. Materials and methods
The organisms were obtained from the artisanal fishery with (as possible) monthly periodicity during two annual periods from January 2014 to November 2015. The organisms were caught with the help of two local free divers and the captain of an 8 m length vessel with a 40 HP outboard motor at depths from 5 to 15 m in rocky coast localities at the vicinity of Puerto Ángel, Oaxaca, Mexico, between 9:00 and 12:00 h local time (Table 1, Figure 1). The collecting sites were determined each date according to the atmospheric and sea conditions as well as the diver’s knowledge on the species availability in the zone. Our aim was to have a representative number of specimens from the region to gather the histological information from reproductive organs and tissues.
|Site||Latitude (N)||Longitude (W)|
|Punta Cometa (PC)||15° 39′ 35.4″||96° 33′ 16.5″|
|San Agustinillo (SA)||15° 39′ 48.6″||96° 33′ 01.0″|
|Playa Panteón (PP)||15° 39′ 56.1″||96° 29′ 27.1″|
|Aragón (Ar)||15° 39′ 38.2″||96° 31′ 46.8″|
|Estacahuite (Es)||15° 40′ 04.7″||96° 28′ 54.5″|
|Bajos de Aceite (BA)||15° 40′ 10.6″||96° 28′ 29.6″|
|La Mina (LM)||15° 40′ 26.7″||96° 28′ 35.7″|
|La Boquilla (LB)||15° 40′ 48.3″||96° 27′ 58.4″|
|Secretario (Sr)||15° 41′ 02.3″||96° 27′ 00.5″|
|Tijera (Tj)||15° 41′ 20.2″||96° 26′ 26.3″|
|Dominguillo (Dm)||15° 41′ 35.0″||96° 26′ 02.2″|
|Playita, Tembo (PT)||15° 41′ 36.1″||96° 25′ 54.3″|
|Temperature (SST) and chlorophyll (CL)||15° 38′ 44.9″||96° 28′ 45.0″|
From the caught organisms, 10–15 individuals in the interval from 8 to 12 cm in length (interval that contained more than 90% of the lengths, we collected since 2012) were separated and their shell broken to extract the soft parts, which were fixed in formalin 10% prepared with seawater . Once fixed, the specimens were transported to the Biometry and Fisheries Biology Laboratory of the Facultad de Estudios Superiores Zaragoza, UNAM, where after 48 h were washed with tap water and preserved in 70% ethylic alcohol. As there are no external characters to distinguish sex, the specimens were dissected to examine and search for the presence or absence of penis.
The histological sections were carried out at the Histology Laboratory from the Morphology Department at the Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. The alcohol-preserved specimens were dehydrated following the usual alcohol series (70–100%) and cleared in xylol before being included in paraplax and paraffin. The embedded tissues were sliced into sections of 5 μm thickness using a microtome and mounted over glass slides. The preparations were stained with the Hematoxylin-Eosin method  to facilitate the determination of the gonad development stages. The sections were fixed with Entalan and covered with glass slips. Finally, the preparations were observed and photographed by means of an optical microscope with attached camera.
The sea surface temperature (SST) and chlorophyll
The comparisons of the resulting time series data were performed by means of the cross-correlation analysis [34, 35, 33] between the percentages of maturity stages against the temperature and chlorophyll values. Additional cross-correlation analyses among maturity stages were made.
3.1 Reproductive cycle
In total, 232 males and 214 females were captured. The sexual proportions throughout the study are included in Table 2. From the 446 individuals, 250 were analyzed for recognition and characterization of the gonad stages considering the degree of development besides the occurrence and abundance of gametes. The different gonad development stages were classified as follows: for females, six stages were established (Table 3, Figure 2). Stage 1 (S1) initial oogenesis, stage 2 (S2) previtellogenic maturity, stage 3 (S3) vitellogenic maturity, stage 4 (S4) maturity, stage 5 (S5) spawning, and finally, stage 6 (S6) resting. For males, five stages were recognized (Table 4, Figure 3). Stage 1 (S1) initial spermatogenesis, stage 2 (S2) maturity, stage 3 (S3) expulsion, stage 4 (S4) onset of rest, and stage 5 (S5) resting.
|Collecting data and site (main)||Total||Sexo||Male:female proportion|
|Stage 1 initial oogenesis (S1)||Occurrence of developing ovogonia and oocytes, thick follicle walls|
|Stage 2 previtellogenic maturity (S2)||Oocytes full of yolk granules; in some oocytes, the nucleus and nucleolus are observed; follicles completely mature full of oocytes|
|Stage 3 vitellogenic maturity (S3)||Follicles with thin walls and developing oocytes; yolk granules are observed and yolk platelets appear|
|Stage 4 maturity (S4)||Follicles full of yolk granules and platelets; thin follicle walls|
|Stage 5 spawning (S5)||Light in the follicles is observed; follicles partially empty; follicle walls thin with some remnant oocytes|
|Stage 6 resting (S6)||Some resting follicles besides cells or phagocytes in thick follicle walls; conspicuous conjunctive tissue|
Spawning (S5) females of
aconcentration and gonad cycle
In 2014, the highest chlorophyll
In 2015, the high chlorophyll concentrations were observed in January (1.62 mg m−3), February (1.23 mg m−3), March (1.08 mg m−3), and (exceptionally) April (11.89 mg m−3); except for April, in all other months, the stage with larger frequency was spawning (S5 and S3 for females and males, respectively) (Figures 4 and 5). In the same year, the lower chlorophyll concentrations occurred in July (0.18 mg m−3), October (0.18 mg m−3), and November (0.19 mg m−3). These chlorophyll concentration values were related to the resting stage of females and males (S6 and S5, respectively) (Figures 4 and 5).
It is possible that the April 2015, notably high (11.89 mg m−3), chlorophyll concentration originated a different pattern, in comparison with that from the same month of the previous year. The resting phase of females (S6) and males (S5) occurred with less frequency and the spawning gonad stage (S5 females and S3 males) extended to June, July, and August (Figures 4 and 5).
3.3 Temperature and gonad cycle
The lowest registered temperatures occurred in January (27.48°C), February (27.74°C), and December (27.96°C). In these months, it was observed that the spawning females (S6) and males (S3) were those with the highest frequency (Figures 4 and 5). The months with the highest temperatures were May (30.70°C), June (31.14°C), and August (30.83°C), which were related to the larger frequency of the resting stages of females (S6) and males (S5) (Figures 4 and 5).
The lowest temperatures for 2015 were registered in January (28.04°C), February (27.44°C), and March (28.11°C), which corresponded with the highest frequencies of female and male in the spawning stage (S5 and S3, respectively). The months with the larger temperature values were August (31.19°C) and September (31.24°C). In these months, female stage 4 (mature), 5 (spawning), and 6 (resting) were observed with 25, 25, and 50%, respectively; the male stages were 3 (spawning) and 4 (end of spawning, onset of the rest) (50 and 30%, respectively), and in September, 35% of stage 2 (maturity), 25% of phase 3 (spawning), and 40% of stage 5 (resting) were observed.
3.4 Smoothing and cross-correlation
To describe in more detail the variation and relationships of the reproduction stages and the environmental variables, sea surface temperature (SST) and chlorophyll
The males seem to mature (S2) early (July) but the trend is clear from September to January (Figure 7). The spawning males (S3) occur from October to February. Similarly than females, males attain the reproductive rest stage (S5) from April to August, during the warmer months.
The cross-correlograms show a direct relationship lagged 2 months between S2 and S3 (maturity, spawning stage) and a 6 month lagged high cross-correlation between spawning (S3) and resting (S5) males, corroborating significantly the above statements.
The spawning (S5) females showed a clear opposite (negative) correlation with the sea surface temperature values with a lag of 6 months, and concordant (positive) cross-correlation with chlorophyll
The sex proportion of
The histological examination of the gonads of
|Stage 1 initial spermatogenesis (S1)||Follicles active, developed with immature cells; small separated follicles with numerous immature cells (spermatogonia and spermatocytes), thick follicle walls|
|Stage 2 maturity (S2)||Follicles utterly full with a greater quantity of spermatozoids, spermatogonia, spermatocytes, and spermatids|
|Stage 3 spawning (S3)||Mature spermatozoids in expulsion, ciliated cylindric epithelium with foldings|
|Stage 4 onset of rest (S4)||Some follicles in expulsion; empty and resting follicles are observed|
|Stage 5 resting (S5)||Empty follicle lumen; resting follicles due to the expulsion of spermatozoids are observed; conspicuous conjunctive tissue|
Comparing both sexes, spawning and expulsion occurred in January, April, May, October, and November, and the larger frequency of resting individuals was registered in June and July. In this way, it is possible to recognize a period of spawning and expulsion from November to March with pikes in January and February. The resting period of females occurred from March to October, with peaks in July (2014) and April–July (2015), and the males presented high resting frequency values in June (2014) and May (2015). This does not corresponds to the reproduction times reported for
Chlorophyll concentrations have a direct relationship with the development of gonads as this reproductive process demands high energetic quantities that must be obtained from the eaten food extracted from the environment or from reserves previously accumulated or from both [6, 38].
In relation to temperature is worth to mention that along the period of study, the water temperature differences between surface-bottom lectures were not detected. The direct explanation for this finding is that the rocky coast localities where the individuals of
In this study, the predominance of reproductive stages occurred during the winter months under relatively colder temperatures. As noted before, the reproduction of the related species
From October to April but mainly from November to February, the blowing winds, known as the “Tehuanos,” originate upwelling and water vertical mixing causing an increase in chlorophyll concentrations [39, 40] by phytoplanktonic blooming. This water mixing process promotes spawning, breeding, and feeding of the aquatic species . Thus, it is possible that the food availability is the main factor affecting the onset of reproduction of
On the other side, we would like to mention that the smoothing technique applied to the maturity stage frequencies allow distinguishing in a clearer way the subjacent pattern of the reproductive cycle. With the availability of long-term data records, it is possible to use time series analysis statistical techniques, making it possible to assess the significance of the observed behaviors. From this, it can be stated that
This is the first study on the reproductive cycle of
The reproductive cycle of
The sexual proportion was not statistically different from parity, although most of the times, the number of males was slightly larger than the number of females. Only during the spawning season, females were more frequent than males.
The histological analysis permitted to establish maturity stages.
Females (six stages): (1) initial oogenesis, (2) previtellogenic maturity, (3) vitellogenic maturity, (4) maturity, (5) spawning, and (6) resting.
Males (five stages): (1) initial spermatogenesis, (2) maturity, (3) spawning, (4) onset of the rest, and (5) resting.
Monthly variations of maturation stages showed that
Reproductive events were related to high chlorophyll
We express our thankfulness to the Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México (UNAM), and the Consejo Nacional de Ciencia y Tecnología (CONACyT) for their support. Without the help of the local divers Manolo Jarquín, Primitivo Herrera Ordóñez and Captain “Beto” (Abraham Reyes López), the collection of specimens would not have been possible. We are grateful to Dr. Erika Rosales Cruz for her help with the histological photographs and to the Central de Instrumentación de Microscopía, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. This research received support from Dirección del Personal Académico, UNAM under the programs PAPIME (PE206213, PE207417) and (partially) PAPIIT (IG201215). Besides, we received support from the Carrera de Biología, Facultad de Estudios Superiores (FES) Zaragoza, UNAM. Lastly, we would thank all the students and colleagues of the Laboratorio de Biometría y Biología Pesquera, FES Zaragoza, UNAM. This research report is part of first author’s PhD thesis.
Conflict of interest
We state that there is no “conflict of interest.”