Gametogenic Cycle of the Oysters Pinctada capensis (Sowerby III, 1890) and Saccostrea cucullata (Born, 1778) (Class Bivalvia) in Inhaca Island, Southern Mozambique: A Subsidy for Bivalve Culture in the Region

Abstract

This study describes reproductive aspects of the Pinctada capensis (pearl oyster) and Saccostrea cucullata (rocky shore oyster) in Inhaca Island, southern Mozambique (Western Indian Ocean). Adult oysters were collected monthly over two years within seagrass banks for P. capensis and rocky shore habitats for S. cucullata. The animals were evaluated using biometric and histological analyses of the gonads. Of the total population, females were predominant among larger individuals (>55 mm) and males were more dominant among smaller individuals (<55 mm) for both species. The sex ratio was (1 M–1.5 F) for Pinctada capensis and (1 M–1.6 F) for Saccostrea cucullata. Five gonad maturation stages were identified: indifferent, developing I, developing II, ripe and spent. The size at first maturity was mm and 26.2 mm for pearl oysters females and males, respectively, whereas for rocky shore oysters was 32.8 and 28.3 mm for females and males, respectively. We conclude that the reproduction of S. cucullata and P. capensis occurs mainly in summer, with a short resting period in winter, when many oysters are in the indifferent stage. These results provide valuable information to fisheries for management of both species in the area.

1. Introduction

Oysters are key species for the ecology of coastal and estuarine areas [1]. They provide important ecological services such as improving water quality, helping to stabilize the coastline and protecting erosion by building their reefs, and they are also an important product of fisheries and aquaculture in many countries [2]. These invertebrates are widely distributed around the world. In tropical regions, oysters occur in rivers and coastal areas, and most species occur in narrow bands or form dense banks at a tidal range where desiccation, fouling and predation are minimized [3].

Although most of the biological aspects of tropical oysters are comparable to that of temperate oysters, the effects of environmental factors on reproductive activity are not well documented. The sex ratio in oysters may vary from species to species due to factors such as organ age and genetic differences, and the gametogenic cycle could be influenced by environmental parameters [4,5]. The development of the gametes is a process that consumes a lot of energy depending on the nutrients obtained in the recent feeding or the reserves accumulated during the period of reproductive rest [3].

The reproduction activity of oysters can occur at any time of the year, greatly influenced by the changes in water temperature and the availability of food [6]. Spawning is related to water temperature fluctuations (25 to 30 °C), reaching a peak when temperatures are very high [7]. Additionally, spawning cycles in bivalves are related to food availability, which is often measured as seasonal variations in chlorophyll a or suspended particulate matter (SPM) [8].

The most reliable method for evaluating the development of the reproductive cycle of oysters is based on the histology of the gonads [9]. Histological methods categorize the reproductive cycle of the oysters according to the characteristics of tissue cells observed at different stages of gonadal development [10]. However, this type of analysis tends to be subjective and should be used in conjunction with quantitative methods, such as the condition index (CI), for a better assessment of the reproductive aspects [7,10].

Studies of the reproductive cycle, spawning periods, and occurrence of larvae of oyster species are essential in understanding the population dynamic of wild stocks, a basic requirement for the management of natural banks and application of conservation and/or exploitation of commercial marine/food species [11]. Moreover, a better understanding of the reproductive cycle of the native oysters and its relation to the environment is needed for farming [12], as this will permit the development of more effective and efficient techniques for the maintenance of the oyster and breeding conditions in the laboratory and for the successful production of seed.

Among the oysters, the pearls oyster Pinctada capensis (G.B. Sowerby, 1890) inhabits tropical and sub-tropical waters. This species is abundant along the western coast of Africa, where it occurs associated with seagrasses beds in sheltered lagoons, channel, intertidal reef platforms and other habitats [13]. Saccostrea cucullata (Born, 1778) has a wide Indo-Pacific distribution, ranging longitudinally from East and South Africa [14], to the Pacific Islands, and latitudinal from Japan [15] to Australia [16] and New Zealand [17]. This species is abundant in the eastern African coast, where it forms a distinct band in the mid to upper balanoid zone. Spawning has been reported to be continuous except for the monsoon time in India, and continuous but peaked during the rainy season in East Africa [18].

In Mozambique, P. capensis is found in the Bazaruto Archipelago and Maputo Bay. It thrives in shallow and relatively clear waters, generally fixed in substrates within seagrass beds, at depths from 5 to 30 m [4]. S. cucullata is found in Pemba Bay, Palma and Mocímboa da praia (northern Mozambique), Bazaruto Archipelago [19], Xai-Xai beach as well as in Maputo Bay [20]. These species stand out due to the important role they play as a protein source for local communities as well as in the use of shells in art craft. In Inhaca Island, P. capensis is more important for the local community than S. cucullata, because its harvest is higher as it is collected at more accessible sites [21].

Information on the reproductive activity of P. capensis and S. cucullata is not available in Mozambique despite their biological and socioeconomic importance as a component of benthic fauna in Inhaca Island. This paper is part of research on the population dynamics of these oyster species in Maputo Bay aimed at providing information for possible use in the development of oyster culture in the region. Therefore, the present study aimed to report baseline information on their reproductive biology by investigating the reproductive activity, sex ratio and its relation to size and size at first maturity.

2. Materials and Methods

This study was carried out in Inhaca island (latitude 26°07′ S, longitude 32°56′ E), which is located 32 km in front of Maputo City, Mozambique (Figure 1). The Island has a total area of approximately 42 km² and is part of the Ponta de Ouro Marine Park. The area mediates the shallow Maputo Bay and the open waters of the Indian Ocean, a transition zone from a tropical to a sub-tropical climate with a rich diversity of both terrestrial and marine ecosystems [22].

There are two distinct seasons—the hot and rainy season (from November to April) and the cold and dry season (from May to October)—with an average air temperature of 23 °C and a sea water temperature varying from 18 °C (July) to 32 °C (January) [18].

The eastern part of the Island is characterized by strong currents and waves while the western part is more sheltered [23]. The tides are semidiurnal and have maximum amplitudes of approximately 3.1 m in high spring tides [22]. During low tide, a large stretch of beach is exposed, making it an interesting site for the collection of many invertebrates, including oysters [23,24].

Two sampling sites were established in Inhaca Island based on occurrence and accessibility: site I (Bangua) for P. capensis and site II (Ponta Torres) for S. cucullata (Figure 1). Sampling was conducted monthly during spring tide, over a period of 2 years (November 2016–December 2017 and January to October 2019) within seagrass banks for pearl oyster P. capensis exposed during low tides (around 3 m deep in high waters) and rocky shore habitats for S. cucullata. Data on seawater temperature throughout the year were obtained from existing records (Asia Pacific Research Data Center Home—http://apdrc.soest.hawaii.edu/data/data.php accessed on 27 March 2020). Fifty specimens of oysters from each species were randomly collected in the two defined sampling sites, they were transported to the Ecology Laboratory, Department of Biological Sciences at Eduardo Mondlane University and were kept in a freezer at 4 °C for further processing. In the Lab, the oysters were cleaned, and shell length (SL) and shell width (SWi) were measured using a digital caliper (precision of 0.001 mm), while total wet weight (TW), tissue weight (TiW) and shell weight were weighed using a digital scale (precision of 0.001 g) [25].

The animals were classified histologically, according to the type of germ cells present in their gonads, into males, females, hermaphrodites (in which both oocytes and spermatozoa were found in the same individuals) and in sexual resting (in which no germ cells were found in the gonads, making sex determination impossible), according to [26].

For all individuals, soft tissues were carefully separated from shells and washed in distilled water. Both soft tissue and shells were put in an oven at 80 °C for 24 h and then weighed. The condition index (CI) was calculated individually as the ratio of dry weight of soft tissues divided by the dry weight of shell × 100 using the following formula [27]

$$CI=100*\frac{Wsoft{ }_{tissue}}{W_{total}}$$

The reproductive condition of the oysters was verified on the basis of degree of development and numerical density of the gametes, being determined by the examination of the macroscopic appearance of the gonads and the microscopic examination of gonads tissue smears. Due to a lack of external dimorphism, the shell valves were parted, and a subjective estimation of gonad volume made. Gonad smears were examined at 100× magnification. Each specimen was classified based on the presence or absence of mature or immature germ cells according to the scale modified from [28] (

Table 1. Stages of gonadal development in oyster.

Stage	Characteristics
Indifferent	No gonad visible. This has two possible explanations—adults with recovering gonads after spawning or immature juveniles
Developing I	Gonad tissue visible, but it is very difficult to distinguish sex
Developing II	Gonad tissue are evident and sexes can be distinguished. Gametes are abundant, but the majority of the spermatozoids are hardly moving and pedunculated oocytes are present
Ripe	Gonad with rapid moving spermatozoids or spherical oocytes
Spent	Gonads are empty and thin. Coexistence of cells being reabsorbed and mature cells

). The sex ratio (expressed as number of females per number of males F:M) was determined.

To estimate the minimal size of first maturation, the relative frequencies of juvenile (immature) and adults (mature) were distributed by length classes (4.5 mm interval) of individuals in both sexes, analyzed separately. The minimum size at first maturation corresponds to the size at which 50% of individuals are sexually active [21].

The data were adjusted to a dose–response model to determine the size at which at least 50% of the individuals in the population can reproduce (L50). The data obtained were adjusted to the following logistical curve:

$$P=\frac{1}{1+\exp \left[-r\left(L-L_{50}\right)\right]}$$

where P is the proportion of mature specimens in each class of shell length (SL), r is the slope of the curve and L₅₀ is the length of the first maturation [29].

Data Analysis

Data were grouped into size classes (4.5 mm SL) to detect variations in the sexual proportion as a function of specimen size. Sex ratio data were pooled for the whole study period. The chi-square test (X²) was used to determine differences in sexual proportions. In order to identify seasonal trends in gonad development, each gonad stage (i.e., immature, developing, ripe/mature and spent) was reported by size in proportion to the total catch of oysters. Sizes at sexual maturity (SW₅₀) were estimated separately using the proportion of specimens in each 4.5 mm SL size classes with mature gonads and only oysters considered mature (the last developing stage) were used.

Data analysis was performed using SPSS software [30]. Normal distribution of the data was tested using the Shapiro–Wilks test. The variance homogeneity was evaluated using Cochran’s test.

Monthly averages of CI were calculated for each species. ANOVA, followed by Tukey’s post hoc test [31], was used to confirm monthly critical differences in the reproductive variables (CI). The results are presented as the means (±standard error) and the significance level used for the tests was 5%. In addition, simple Pearson correlation analyses were used to test whether monthly average CI could be related to variability in temperature.

3. Results

3.1. Sex Ratio

A total of 2400 individuals were collected during the study period, 1200 specimens of P. capensis and 1200 specimens of S. cucullata. From all P. capensis sampled, 374 (31.2%) specimens were males, 558 (46.5%) were females, 247 (20.6%) were indeterminate, while 21 (1.8%) had both sperm and eggs present in the gonad (Figure 2A). Bisexuality was not common for this species. Additionally, these specimens showed no evidence that both gonads were functional (i.e., intersexes). The sex ratio (male:females) for this species was 1:1.5. Statistically significant differences were found in the proportions of the overall sex ratio (p < 0.05). For S. cucullata, 405 (33.8%) were males, 643 (53.6%) were females, 70 (5.8%) undetermined and 82 (6.8%) had both sperm and eggs (Figure 2B). There was a greater predominance of females than males for the overall samples during the study period. The sex ratio (M:F) was 1:1.6 (1 M–1.6 F). Significant differences were found in the proportions of the sex ratio (p < 0.001).

The pear oyster sex ratio showed a clear dominance change from male to females with increasing size classes. The majority of individuals <45 mm SL were males and female were more common from 55–60 mm to upper size of 90–95 mm (Figure 3A). For rocky shore oysters, the majority of individuals <40 mm SL were males. Above this size, the proportion of females increased progressively, reaching 100% at the upper size classes (Figure 3B).

3.2. Temperature and the Condition Index

Water temperature in this study area ranged between 21.5 and 23.6 °C in winter and between 24.4 and 27.8 °C in summer. The highest value (27.9 °C) was recorded in January 2019, whereas the lowest value (21.5 °C) was recorded in July 2017 (Figure 4).

Changes on condition index (CI) of the oyster P. capensis and S. cucullata are illustrated in Figure 4. Condition index varied over the study period for both species. The major peaks occurred in December 2016 and January 2017 and 2019 while a minor peak occurred in July 2017 and 2019 for pear oyster. For the rocky shore oyster, a major peak was recorded in January 2017 and February 2019 and the minor peak were recorded in May 2017 and August 2019. Statistical analysis showed significant change in CI between the months over the study period for P. capensis (ANOVA, F = 7.12 p < 0.05) and for S. cucullata (ANOVA, F = 6.31 p < 0.05). Additional correlation analyses, showed that for both species, there was a strong correlation between monthly average temperature and average CI (Figure 5). Temperature explained 72% (p < 0.001) and 63% (p < 0.001) of the variability in CI for S. cucculata and P. capensis respectively. This indicates that, despite some variability in temperature and CI among years, the condition of both species is tightly linked to season and temperature.

Overall, results obtained in this study indicate that a large proportion of the population spawns in summer season with a peak from January to March and a minor peak from October to December. The remaining period of the year is characterized by lower proportions of population spawns for both species (Figure 6 and Figure 7). During the winter season from May to July, more s are in the indifferent stages; and from August, the oysters begin to develop their gonad into developing stage 1. In the early warm season, when the water temperature starts to increase, more oysters of both species are in developing stage 2.

3.3. Minimum Size at First Maturity

The relationship between the percentage of mature P. capensis and total shell length (SL) for females and males is presented in Figure 8. Size at first maturation was 27 mm SL and 26.52 mm SL for female and males, respectively. For S. cucullata, the minimum size at first maturation was estimated as a size of 32.8 mm SL and 28.3 mm SL for females and males, respectively (Figure 9).

4. Discussion

This study provides new and extensive information about several life-history characteristics, in particular reproductive aspects of two common and important oyster species, pear oyster Pinctada capensis and rocky shore oyster Saccostrea cucullata, in Inhaca Island in southern Mozambique. This information can help contribute to a more sustainable use of these valuable species in the area but may also be put in the perspective of similar species in tropical and sub-tropical areas.

For example, in the sex ratios observed for both oyster species in this study, with a predominance of females over males and the occurrence of hermaphrodites in natural populations is consistent with what has been found in many other studies on oysters. This includes a study [32] on Crassostrea gigas from Korea, a study [33] on Crassostrea rhiziphorae from Guaratuba Bay, a study [34] on Crassostrea brasiliana from Guaratuba Bay, Brazil, a study [35] on Pinctada radiata from northern Karkennah Island, Tunisia, a study [36] on P. radiate and P. margaritifera from southwestern and a a study [37] on Saccostrea cucullata from Maharashtra, India.

Similarly, females were larger than males, being more common in upper size classes of shell length. The authors of [38] found that 39% of oysters of the species P. imbricata less than 71 mm shell height were females but this ratio for oysters bigger than 70 mm was reversed (66% females). This coincides with our results in which females outnumbered males for pear oyster above 55 mm and rocky shore oyster above 50 mm in shell length, respectively. The authors of [2] used diocious and hermaphroditic C. gigas individuals to determine the genetic control of the coexistence of protandric sex change and observed different ratios of females. The ratio according to the authors was related to the age of the animal: a significant proportion of the oysters matured first as males and changed to females in later years (i.e., prontandrous hermaphroditism). Several authors suggested that these sexual changes are known to be potentially related to food availability. The authors of [8] suggested that good conditions will favor females, whereas bad conditions, or stress, favor males.

The presence of gonads with both male and female gametes may indicate a pathological finding. In this case, the animals were termed “intersex” (i.e., both reproductive systems were not functional) by [32], a phenomenon that, according to the authors, was induced by aquatic pollutants and chemical endocrine disruptors. These authors also suggested that the increase in intersex individuals within a population could serve as a bioindicator of environmental quality.

The lower proportion of males compared with females for both P. capensis and S. cucullata poses no risk to population maintenance within the environment because, according to [25], males may release gametes more frequently than females due to faster gonadal recovery.

In the present study, values of the condition index (CI) for both oyster species varied during the study period and over the year. This index, showed a tendency of increase with rise of temperature and decrease in period of low temperature. Values of the Condition Index (CI) can be indicative of the reproductive period or nutritional status of individuals [39]. In the summer (October–March), the increase in temperature increase the metabolic rate of many invertebrates including oysters and consequently increasing their consumption of microalgae and suspended particulate matter, contributing to the storage of energy in form of glycogen, lipids and proteins that will be used for reproduction [40].

A high condition index in warmer seasons can be justified by the greater availability of food that is obtained in this period. The authors of [41], in a study on the seasonal cycle of planktonic communities on the Island of Inhaca, found that the concentration of nutrients is higher in the warmer months (summer), a time when rain brings a greater flow of nutrients through the rivers flowing into Maputo Bay. Following the peak of nutrients, the authors of [41] also registered maximum values of chlorophyll-a in March, with a tendency to decrease in the colder months. Many studies have attempted to explain the effect of environmental parameters on gonadal development and reproduction of different species of bivalve mollusks. Various factors such as temperature, salinity, and food availability in the environment can affect the gametogenic cycle of bivalves [42].

Seasonal spawning activity observed in this study also correlates with observations of recruitment of other species in other tropical and sub-tropical regions which peaks in the summer, from February to April [43]. The reproductive activity of the oysters is affected by the seasons and, therefore, by increases in seawater temperature. The observations of temperature and histological data showed that oysters at the early development stage first were found during the months when the temperature of seawater was close to 21 °C. Animals in the late growing stage began to appear during October, when the average temperature rose above 24 °C. As the temperature continued to rise from November through March, oysters reached the mature and spawning stages. These events occurred at the beginning and the end of this period, respectively. This information is consistent with previous findings that the periods of gametogenesis of the same species may vary among geographical areas [44]. Studies performed in different locations showed that the time required for gametogenesis and for maturation of the sexual cells increase with latitude [45].

The results here obtained on the size at sexual maturity revealed that males of P. capensis mature earlier at 26.5 mm SL compared to females at 27 mm SL and males of S. cucullata also mature earlier at 28.3 mm SL compared to females at 32.8 mm SL, as is generally the case for most oyster species [8]. Early maturity in males may confer advantages in competition in egg fertilization from highly fecund females. This might be the biological factor to ensure that all eggs are fertilized because of the protandrous situation experienced by these species.

The results of the histological analysis demonstrated that oyster gametogenesis is not continuous during the year and tends to be more intense in the summer periods when seawater temperature rises and lower or absent during the winter when seawater temperature drops. Here, we provided valuable baseline information in a wide plan to decrease exploitation and introduce aquaculture practices, towards the sustainable management and conservation of P. capensis and S. cucullata, and ultimately to ameliorate people’s livelihoods.