Gametogenesis, sex ratio and energy metabolism in Ostrea angasi: implications for the reproductive strategy of spermcasting marine bivalves

Gametogenesis, sex ratio and energy metabolism in Ostrea angasi: implications for the... Abstract Spermcasting is an unusual reproductive strategy in some aquatic species in which functional males release gametes to fertilize eggs inside the body cavity of functional females. As some spermcasting bivalves have high ecological and economical importance, the understanding of their reproductive strategy is crucial for aquaculture, fisheries and conservation. This study investigates gametogenesis, sex ratio and energy metabolism in Ostrea angasi to elucidate the reproductive strategy of spermcasting bivalves. Gonad histology indicated asynchronous development of spermatozeugmata, but synchronous development of oocytes within an individual. The hermaphroditic individuals released spermatozeugmata before egg ovulation. The population of 2–3 year old oysters comprised 46.7% hermaphrodites and showed a highly skewed male to female ratio of 7:1. This species primarily metabolized glycogen as the energy source for gametogenesis, with an overlapping period of energy storage and utilization. This pattern of gametogenesis suggests multiple production of spermatozeugmata in a reproductive season in male and hermaphroditic oysters, but a single episode of egg ovulation in female and hermaphroditic oysters. The dynamics of energy metabolism indicate that O. angasi follows a strategy for energy metabolism that is intermediate between those of conservative and opportunistic species. This study suggests that unsynchronized gamete development, skewed sex ratio and intermediate energy metabolism are adaptive strategies in the reproduction of spermcasting bivalve species. INTRODUCTION Most marine animals release gametes directly into the water column, where fertilization occurs. However, there are a few species in which males release sperm in this way and females then inhale sperm into their body cavity for egg fertilization (Bishop & Pemberton, 2006; Barazandeh et al., 2013). This reproductive mode has been found in a variety of taxonomic groups including hydroids, corals, polychaetes, bivalves, tunicates, entoprocts, brachiopods, bryozoans and pterobranchs (Pemberton et al., 2003). This mode of fertilization inside the body by inhaling sperm from the water column has been described as spermcast mating (Pemberton et al., 2003), for which synonymous terms are larviparity (Buroker, 1985), egg brooding (Phillippi, Hamann & Yund, 2004) and egg brooding free spawning (Johnson & Yund, 2004). To avoid confusion with other variants of internal fertilization, we use the term spermcasting for the species that fertilize eggs inside the body by acquiring sperm from the surrounding water. The reproductive modes of oysters in the family Ostreidae vary between genera. For example, species of Ostrea perform spermcasting, whereas Crassostrea species are broadcasting. In Ostrea, a male or a hermaphrodite releases sperm as spermatozeugmata, which are inhaled by a female or a hermaphrodite to fertilize the eggs in the mantle cavity (Ó Foighil, 1989). The subsequent larval development takes place inside the mantle cavity, protected by the maternal parent (Chaparro, Navarrete & Thompson, 2006). In Crassostrea, on the other hand, both types of gametes are released into the water column where fertilization and embryo development occur. Compared with broadcasting and internal spawners, very little information exists regarding the reproductive strategy of spermcasting species. Gametogenesis is the process in which gametocytes undergo cell division and differentiation to form haploid gametes. Asynchrony in maturation of the male and female gametes during gametogenesis leads to synchronous spawning, whereas discrepancy in gamete development leads to asynchronous spawning (Styan & Butler, 2003; Chaves-Fonnegra et al., 2016). Since spermcasting bivalves are incapable of movement after settlement, acquisition of functional spermatozeugmata by a female is necessary for successful reproduction. The development patterns of spermatozeugmata and eggs could provide insight for understanding their role in the reproduction of spermcasting species (Falese, Russell & Dollahon, 2011). The sex ratio is a fundamental indicator for reproduction success in dioecious species. Nearly equal numbers of males and females are produced in most gonochoristic species, giving a balanced sex ratio (Fisher, 1930), whereas the sex ratio can skew towards one sex in hermaphroditic species. A highly male-biased sex ratio has been found in spermcasting Ostrea edulis, but the reported proportion of simultaneous hermaphrodites differs among studies (Kamphausen, Jensen & Hawkins, 2011; Acarli et al., 2015). The family Ostreidae includes species of both the broadcasting and spermcasting modes, but our understanding of the reproductive strategies of the latter is far less than of the former. This duality of reproductive mode makes oyster species a suitable model to study the relationship between sex ratio and reproductive mode. Energy metabolism in marine bivalves is closely associated with reproductive activities. Energy is stored as glycogen, protein and lipid in various tissues and mobilized for physiological and reproductive activities (Berthelin, Kellner & Mathieu, 2000; Benomar et al., 2010). The dynamics of the biochemical composition in storage tissues and the body condition index reveal the energy metabolism strategy of a species. As gametogenesis is a highly energy-demanding process, marine bivalves use either an opportunistic or a conservative strategy to control energy expenditure during gametogenesis (Bayne, 1976). Opportunistic species obtain and store energy prior to the process of gametogenesis, whereas conservative species mainly rely on energy obtained from exogenous feeding during the process of gametogenesis. In a temperate region, temperature and food supply are seasonally variable and environmental variation regulates the reproductive clock (Garrido & Barber, 2001; Enríquez-Díaz et al., 2009); consequently, most species follow the conservative energy-metabolism strategy (Darriba, San Juan & Guerra, 2005; Li et al., 2006; Karray et al., 2015). However, the energy metabolism strategy needs to be evaluated for a species which such as Ostrea angasi, which has a protracted spawning period and grows in an area with low food supply, in order to understand how energy is supplied for a long spawning season without much calorific intake. In this study, the Australian flat oyster O. angasi G.B. Sowerby II, 1871 was used as a representative of spermcasting bivalves. This species was abundant in Australia before the arrival of European settlers, but almost perished from its natural habitat in the late 18th and early 19th century due to overfishing (Nell, 2001; Alleway & Connell, 2015). Over the last decade, the production of O. angasi has increased and it has become an emerging species for aquaculture in Australia (Heasman et al., 2004; O’Connor & Dove, 2009). However, the poor understanding of the reproductive strategy of marine spermcasting bivalves hinders their aquaculture, fishery management and conservation. The objective of this study is to elucidate the reproductive strategy of spermcasting marine bivalves by examining the patterns of gametogenesis, sex ratio and energy metabolism in O. angasi. MATERIAL AND METHODS Oyster and sea water sampling Flat oysters and sea water were collected at the leases of the Pristine Oyster Farm in Coffin Bay, South Australia, from April 2014 to May 2015. Oysters were transported within 24 h in a chilled Styrofoam box to the laboratory at Flinders University. The oysters were then cleaned with a brush and blotted using paper towel before taking length and weight measurements. The age of oysters in this study was 2–3 years (mean ± SD = 76.8 ± 5.2 mm shell length and 72.0 ± 10.9 g wet weight, n = 840) (Fig. 1). Figure 1. View largeDownload slide Ostrea angasi, showing the main internal organs. Abbreviations: AM, adductor muscle; G, gonad; M, mantle; S, shell. Figure 1. View largeDownload slide Ostrea angasi, showing the main internal organs. Abbreviations: AM, adductor muscle; G, gonad; M, mantle; S, shell. Environmental parameters Monthly measurement of environmental variables at the sampling site included water temperature, salinity and chlorophyll a. After filtering out debris and zooplankton through a 300-μm screen, phytoplankton was collected on a 0.45-μm Millipore filter, the Millipore filter then wrapped with aluminium foil and stored at –20 °C for up to 7 days before analysis. Chlorophyll a was extracted by dissolving the filter paper in 90% acetone and stored at 4 °C for 24 h. The determination of chlorophyll a concentration was on a microplate reader (CLARIOstar, BMG Labtech) at the wavelengths of 647 nm and 664 nm, using the formula: Chlorophyll a (mg l–1) = 11.87A664−1.786A647 for calculations (Ritchie, 2006), where A664 and A647 represent absorbance at 664 nm and 647 nm, respectively. Histological observation on gonad tissues The procedures for histological preparation of Ostrea angasi gonad/visceral tissues followed that described by Hassan, Qin & Li (2016). The protocol included: fixing 3-mm pieces of tissue in Davidson’s fixative, submerging in different grades of alcohol and xylene, embedding in paraffin wax, cutting sections at 5 μm, mounting a microscope slide, staining with haematoxylin and counter-staining with eosin. The histological slides were photographed on an inverted microscope (Nikon Eclipse TS100-F). Gonad development of O. angasi was categorized in five stages: (1) resting gonad, (2) early gametogenesis, (3) advanced gametogenesis, (4) mature gonad and (5) spawned gonad, based on the criteria used for O. edulis (Da Silva, Fuentes & Villalba, 2009). Larval occurrence and sex ratio A total of 60 oysters were opened each month to count those bearing larvae. Oyster larvae were classified as white-sic, grey-sic and black-sic following Carson (2010). Identification of sex category (male, female, hermaphrodite or undifferentiated) was based on gonad histology and microscopic observation and was recorded for 840 oysters from April 2014 to May 2015. Unless spermatozeugmata were present, the larvae-bearing oysters were considered female. Biochemical composition The combined gonad-visceral tissues were used for analyses of biochemical composition, because the gonad and digestive system are anatomically intermingled. Tissues from 10 live oysters were pooled and used for analysis of glycogen, protein and lipid content. Tissues were freeze-dried for 48 h and stored at −80 °C for no more than 3 months prior to analysis. Glycogen, protein and lipid content were determined according to the modified Anthrone technique (Roe & Dailey, 1966), Coomassie Bradford assay (Bradford, 1976) and modified Bligh Dyer method (Folch, Lees & Sloane-Stanley, 1957), respectively. Condition index Prior to condition index (CI) analysis, oysters were cleaned by scrubbing off barnacles and debris, blotted dry, the valves were opened, shells and soft tissues were freeze-dried for 48 h and weights were measured using an analytical scale (0.01 g). The condition index (CI) was calculated according to the formula: CI=dry flesh weightdry shell weight×100. Data calculation and statistical analysis To calculate the gonad maturity index (GMI), oysters were ranked from 1 to 4 based on the gonad developmental stage, ranked as follows: 1, inactive resting or spent stage; 2, early gametogenesis; 3, advanced gametogenesis and 4, mature stage. The GMI was calculated according to the formula: GMI=Ʃ(n×F)N (Vaschenko, Hsieh & Radashevsky, 2013), where n is the number of oysters at a certain gonad stage; F is the gonad-ranking score (1–4) and N is the total number of oysters in the sample. Pearson correlation was applied to measure the relationships of (1) GMI vs biochemical compositions and CI, and (2) temperature vs chlorophyll a. The data on chlorophyll a, biochemical compositions and CI were tested for normality and homogeneity using Shapiro-Wilk and Levene’s tests. Due to the violation of normality assumption, data on CI were square-root transformed prior to analysis. One-way ANOVA was used to determine the difference between months in chlorophyll a, biochemical compositions and CI. Tukey’s post hoc test was used when significant differences were found. The data were considered statistically significant at P < 0.05. All the data were analysed using SPSS v. 23 (IBM, Armonk, NY). RESULTS Environmental parameters Seawater temperature gradually decreased from 19.3 °C in April to 11.2 °C in July 2014 and increased from 12.0 °C in August 2014 to 24.2 °C in February 2015 (Fig. 2A). Salinity was relatively stable, but decreased from 41.7 ppt in April to 36.5 ppt in July 2014 and then increased from 36.8 ppt in August 2014 to 41.3 ppt in April 2015. The monthly differences in chlorophyll a content were significant (F = 6.79, df = 13, P < 0.05). Chlorophyll a decreased from 1.27 mg l–1 in April to a minimum of 0.92 mg l–1 in July 2014 and then increased to a maximum of 1.95 mg l–1 in January 2015 (Fig. 2B). Seawater temperature and chlorophyll a were positively correlated (r = 0.74, n = 42, i.e. 3 replicates per month over 14 months, P < 0.05). Figure 2. View largeDownload slide Monthly variations of environmental parameters in Coffin Bay, South Australia from April 2014 to May 2015. A. Seawater temperature and salinity. B. Chlorophyll a. Different letters indicate significant differences (P < 0.05) among the monthly values. Each bar represents mean ± SE of three replicates. Figure 2. View largeDownload slide Monthly variations of environmental parameters in Coffin Bay, South Australia from April 2014 to May 2015. A. Seawater temperature and salinity. B. Chlorophyll a. Different letters indicate significant differences (P < 0.05) among the monthly values. Each bar represents mean ± SE of three replicates. Gametogenesis In the resting stage, gonad follicles were empty and the sex category was not distinct (Fig. 3A). With the progress of spermatogenesis, encapsulated spermatozeugmata were formed in gonad follicles at the advanced stage. The same oysters carried different developmental stages of spermatozeugmata, i.e. presence of spermatocytes and spermatids at the advanced spermatogenesis stage (Fig. 3B). At the start of oogenesis, gonad follicles consisted of only a few oogonia, but the oogonial cells gradually aggregated and became larger at the advanced stage (Fig 3C). Within each individual, the oocytes were of similar size and the oocytes matured at a similar time. Vitellogenesis, yolk deposition in the ooplasm, started at stage 3 and continued until full maturation of the eggs. In an hermaphroditic gonad, both male and female gametes were in a similar developmental stage (Fig. 3D), but the gonad follicles of partially spawned individuals indicated that spermatozeugmata were released before ovulation (Fig. 3E). Subsequently, empty inflated gonad follicles indicated complete absorption of residual gametes after spawning. Gravid oysters carried larvae in the pallial cavity (Fig. 3F). Figure 3. View largeDownload slide Histological sections of Ostrea angasi during gametogenesis. A. Resting gonad. Sex category is difficult to distinguish at this stage. Arrows indicate developing gonad follicles between mantle and digestive gland. B–D. Mature gonads of male, female and hermaphrodite. Single and double arrows indicate spermatocytes and spermatids, respectively, in male. Arrows indicate mature oocytes in female. White and black arrows indicate spermatids and mature eggs, respectively, in hermaphrodite. E. Partially spawned gonad in hermaphrodite. Single white, double and black arrows indicate residual spermatids, eggs and phagocytes, respectively. F. Arrows indicate larvae in pallial cavity in a gravid oyster. Abbreviations: CT, connective tissue; DD, digestive diverticula; GF, gonad follicle; M, mantle; PC, pallial cavity. Scale bar = 100 μm. Figure 3. View largeDownload slide Histological sections of Ostrea angasi during gametogenesis. A. Resting gonad. Sex category is difficult to distinguish at this stage. Arrows indicate developing gonad follicles between mantle and digestive gland. B–D. Mature gonads of male, female and hermaphrodite. Single and double arrows indicate spermatocytes and spermatids, respectively, in male. Arrows indicate mature oocytes in female. White and black arrows indicate spermatids and mature eggs, respectively, in hermaphrodite. E. Partially spawned gonad in hermaphrodite. Single white, double and black arrows indicate residual spermatids, eggs and phagocytes, respectively. F. Arrows indicate larvae in pallial cavity in a gravid oyster. Abbreviations: CT, connective tissue; DD, digestive diverticula; GF, gonad follicle; M, mantle; PC, pallial cavity. Scale bar = 100 μm. Spawning periodicity Gonad histology revealed active gametogenesis throughout the year (Fig. 4A). However, both gonad histology and larvae-bearing oysters indicated a spawning period from May to December, with a relatively higher spawning intensity from September to December (Fig. 4A, B). Figure 4. View largeDownload slide Monthly variations of (A) gonad development stages and (B) frequency of larvae-bearing Ostrea angasi collected from Coffin Bay, South Australia from April 2014 to May 2015. Figure 4. View largeDownload slide Monthly variations of (A) gonad development stages and (B) frequency of larvae-bearing Ostrea angasi collected from Coffin Bay, South Australia from April 2014 to May 2015. Occurrence of larvae Oysters carried larvae in the mantle cavity from May to December. The percentage of larvae-bearing oysters gradually increased from 2.5% in May to a maximum of 10% in October and then decreased to 3.3% in December (Fig. 4B). Sex ratio Based on the monthly sex-ratio data, no pattern of sex-ratio change was evident within this study period. The monthly sex-ratio data confirmed the occurrence of simultaneous hermaphroditism in this species (Fig. 5). A relatively higher percentage of undifferentiated gonads indicated that the postspawning gonadal stage lasted from October to February. The percentages of males, females, hermaphrodites and undifferentiated individuals were 41.3%, 5.8%, 46.7% and 6.2%, respectively, among the 840 oysters observed. The male to female ratio was 7:1. In hermaphroditic gonads, proportions of male and female gametes ranged from highly skewed to one sex to similar proportions of both sexes. Figure 5. View largeDownload slide Percentage of sex categories of Ostrea angasi collected from Coffin Bay, South Australia from April 2014 to May 2015. Sixty oysters were observed each month to determine sex categories. Figure 5. View largeDownload slide Percentage of sex categories of Ostrea angasi collected from Coffin Bay, South Australia from April 2014 to May 2015. Sixty oysters were observed each month to determine sex categories. Dynamics of biochemical composition The monthly differences in glycogen (F = 20.09, df = 13, P < 0.05), protein (F = 3.96, df = 13, P < 0.05) and lipid (F = 15.29, df = 13, P < 0.05) content were significant. The glycogen content increased from 171.7 mg g–1 in April to 217.5 mg g–1 in August and subsequently decreased to a minimum of 106.9 mg g−1 in December. The glycogen content again increased from 140.9 mg g−1 in January to a maximum of 252.7 mg g−1 in May. The protein content increased from 320.7 mg g−1 in April to a maximum of 338.8 mg g−1 in August and decreased to a minimum of 258.5 mg g−1 in February. In the following months, the protein content further increased up to 324.3 mg g−1 in May. The lipid content was relatively stable, but increased from 136.5 mg g−1 in April to a maximum of 146.2 mg g−1 in August and subsequently decreased to a minimum of 113.5 mg g−1 in December (Fig. 6A). The GMI was positively correlated with glycogen (r = 0.70, n = 14, P < 0.05), protein (r = 0.63, n = 14, P < 0.05) and lipid (r = 0.91, n = 14, P < 0.05) content. Chlorophyll a was negatively correlated with protein (r = -0.51, n = 42, P < 0.05) and lipid (r = –0.55, n = 42, P < 0.05) content, but the correlation between chlorophyll a and glycogen was not significant (r = 0.063, n = 42, P > 0.05). Figure 6. View largeDownload slide Monthly variations of energy storage compounds and condition index of Ostrea angasi from April 2014 to May 2015. A. Glycogen, protein and lipid content (mg g−1 dry tissue) in gonad-visceral tissues. Each bar represents mean ± SE of 3 replicates. Different letters within each row indicate significant differences among monthly values of glycogen (middle row; P < 0.05) protein (top row, P < 0.05) and lipid (bottom row; P < 0.05) content. B. Condition index. Each bar represents mean ± SE of 20 individuals. Different letters indicate significant monthly differences in condition index (P < 0.05). Figure 6. View largeDownload slide Monthly variations of energy storage compounds and condition index of Ostrea angasi from April 2014 to May 2015. A. Glycogen, protein and lipid content (mg g−1 dry tissue) in gonad-visceral tissues. Each bar represents mean ± SE of 3 replicates. Different letters within each row indicate significant differences among monthly values of glycogen (middle row; P < 0.05) protein (top row, P < 0.05) and lipid (bottom row; P < 0.05) content. B. Condition index. Each bar represents mean ± SE of 20 individuals. Different letters indicate significant monthly differences in condition index (P < 0.05). Dynamics of condition index The monthly variations in CI were significant (F = 8.39, df = 13, P < 0.05). CI increased from 3.8 in April to a maximum of 4.7 in July and decreased to a minimum of 2.8 in December (Fig. 6B). In the following months, CI gradually increased up to 4.0 in May. CI was positively correlated with GMI (r = 0.87, n = 14, P < 0.05), glycogen (r = 0.54, n = 14, P < 0.05), protein (r = 0.65, n = 14, P < 0.05) and lipid content (r = 0.82, n = 14, P < 0.05). DISCUSSION This study has elucidated the reproductive strategy of the spermcasting Ostrea angasi from the perspectives of gametogenesis, sex ratio and energy metabolism. The asynchronous development of spermatozeugmata in pure males and synchronous maturation of eggs in pure females imply multiple releases of sperm and single ovulation of eggs. In hermaphroditic individuals, although the male and female gametes could develop at a similar pace, spermatozeugmata would normally be released before egg ovulation. As hermaphrodites can take both male and female roles, the high percentage of hermaphrodites in the population may compensate for the limited representation of females during mating. Similar to many other bivalves, glycogen is the main energy reserve for gametogenesis in O. angasi (Dridi, Romdhane & Elcafsi, 2007; Ke & Li, 2013) and this species adopts an energy metabolism strategy that is intermediate between conservative and opportunistic species, since it uses energy from both tissue storage and instantaneous food intake during gametogenesis. The gonad-maturation stages of O. angasi varied greatly among individuals and a small proportion of the population was mature at any time within the spawning season. This gametogenesis pattern indicates that only a small proportion of the population spawns at a given time. In a population with a very small number of spawning individuals, synchronization of release of spermatozeugmata and eggs is an important determinant of fertilization success (Styan & Butler, 2003). Since the acquisition of male gametes by a female is constrained by the physical distance between male and female in sedentary bivalves (Styan, 1999), production of spermatozeugmata in multiple batches would enhance fertilization success when one batch of spermatozeugmata fails to reach females. Interestingly, the impact of location on egg fertilization is minimized in O. puelchana by the attachment of dwarf males to the shells of females (Pascual, 1997). In O. angasi, spermatozeugmata maintain functionality for up to 24 h after their release in sea water (Hassan et al., 2016) and successful fertilization should be possible if a female or a hermaphrodite acquires spermatozeugmata within this period. The duration of the gametogenesis and spawning periods varies among spermcasting bivalves. In O. angasi, all the five gonad maturation stages were found throughout the whole year, but larvae-bearing oysters were found from May to December. Individuals of O. nomades with different gonad maturation stages were also found throughout the year (Siddiqui & Ahmed, 2002), whereas gametogenesis has been reported to take place seasonally in O. edulis (Da Silva et al., 2009). Individuals brooding larvae were found during an 8-month period in O. chilensis (Brown et al., 2010), but for 4 months in O. puelchana (Castaños et al., 2005). This variation in the temporal pattern suggests that different spermcasting bivalve species adapt to local environmental condition for gametogenesis and spawning. In this study, the sex ratio of O. angasi was highly skewed towards males and a large proportion of the population was of simultaneous hermaphrodites. Whereas broadcasting oysters first mature as a male and change sex to female at an older age (Mazón‐Suástegui et al., 2011), spermcasting oysters may not follow a similar pattern in sex change. The high proportion of male to female (sex ratio 7:1) in O. angasi might be due to the use of relatively small-sized oysters (77 mm; 2–3 years old) in this study. However, no pattern of sex change was clear within the 14-month study period. Spermcasting O. edulis also have a highly skewed male to female ratio, 6:1 at a shell length of 50–70 mm (Kamphausen et al., 2011; Acarli et al., 2015), suggesting a general trend of male dominance in spermcasting oysters in a similar size range. A review of the sex ratio of two ostreid genera (Crassostrea for broadcasting oysters and Ostrea for spermcasting oysters) reveals that the sex ratios of broadcast and spermcast spawners are different (Table 1). The percentage of hermaphroditic individuals is about 1% in broadcasting spawners (Steele & Mulcahy, 1999; Enríquez-Díaz et al., 2009; Castilho-Westphal, Magnani & Ostrensky, 2015), but is over 35% in spermcasting spawners (Acarli et al., 2015; this study) at a similar size range. These contrasting sex ratios of oysters in different taxonomic and spawning groups, and their regulation, require further study in relation to the spawning strategies of broadcasting and spermcasting species. Table 1. Comparison of sex ratio between two ostreid genera, Crassostrea and Ostrea, which represent broadcast and spermcast spawners, respectively. Reproductive strategy  Species  Geographical location  Oyster age and size  Male to female ratio  Hermaphrodite occurrence  References  Broadcast spawning  Crassostrea gigas  Dungarvan and Cork Harbour, Ireland  2 years; 9.2 cm  1:1  <1%  Steele & Mulcahy (1999)  N Patagonia, Argentina  >4 cm  1:1  None  Castaños, Pascual & Camacho (2009)    English Channel and Bay of Biscay, France  1–2 years  1.69:1 and 1.22:1  <1%  Enríquez-Díaz et al. (2009)  Gulf of Tunis and Bizert lagoon, Tunisia  8–10 cm  1.4:1  <1%  Dridi, Romdhane & Elcafsi (2014)  C. angulata  Western coast of Taiwan  10–15 cm  1:0.9  4.2%  Vaschenko et al. (2013)  C. corteziensis  Coastal lagoon in NW Mexico  8–10.3 cm  1:3  None  Rodríguez-Jaramillo et al. (2008)  C. brasiliana  Paraná, Brazil  1.1–9.4 cm  1:2.65  1%  Castilho-Westphal et al. (2015)  Spermcast spawning  Ostrea edulis  Solent, UK  4–6 years; 5–7 cm  6:1  2%  Kamphausen et al. (2011)  Izmir Bay, Turkey  >5 cm  19.3:1  37%  Acarli et al. (2015)  O. angasi  Coffin Bay, South Australia  2–3 years; 6.4–8.9 cm  7:1  46.7%  This study  Reproductive strategy  Species  Geographical location  Oyster age and size  Male to female ratio  Hermaphrodite occurrence  References  Broadcast spawning  Crassostrea gigas  Dungarvan and Cork Harbour, Ireland  2 years; 9.2 cm  1:1  <1%  Steele & Mulcahy (1999)  N Patagonia, Argentina  >4 cm  1:1  None  Castaños, Pascual & Camacho (2009)    English Channel and Bay of Biscay, France  1–2 years  1.69:1 and 1.22:1  <1%  Enríquez-Díaz et al. (2009)  Gulf of Tunis and Bizert lagoon, Tunisia  8–10 cm  1.4:1  <1%  Dridi, Romdhane & Elcafsi (2014)  C. angulata  Western coast of Taiwan  10–15 cm  1:0.9  4.2%  Vaschenko et al. (2013)  C. corteziensis  Coastal lagoon in NW Mexico  8–10.3 cm  1:3  None  Rodríguez-Jaramillo et al. (2008)  C. brasiliana  Paraná, Brazil  1.1–9.4 cm  1:2.65  1%  Castilho-Westphal et al. (2015)  Spermcast spawning  Ostrea edulis  Solent, UK  4–6 years; 5–7 cm  6:1  2%  Kamphausen et al. (2011)  Izmir Bay, Turkey  >5 cm  19.3:1  37%  Acarli et al. (2015)  O. angasi  Coffin Bay, South Australia  2–3 years; 6.4–8.9 cm  7:1  46.7%  This study  Gametogenesis affects energy storage status and body condition (Vite-García & Saucedo, 2008; Karray et al., 2015), which is supported by this study as the GMI was positively correlated with glycogen, protein and lipid content, and with the CI. However, the negative correlations of protein and lipid with chlorophyll a involve a more complex interaction. The food availability and spawning intensity are positively related to water temperature, but increased spawning activity can reduce energy stores in oysters (Newell & Branch, 1980), explaining the negative correlations of protein and lipid with chlorophyll a in O. angasi. Marine bivalves allocate energy to gametogenesis by metabolizing compounds such as glycogen, protein and lipid, but the strategy for metabolizing these compounds is species-specific. Glycogen serves as the main energy reserve in most bivalves, but protein and lipid are also used as an additional or alternative energy source (Mathieu & Lubet, 1993). The large variation in glycogen content among prespawning, spawning and postspawning oysters suggests that glycogen serves as the main energy source for gametogenesis in O. angasi. Moreover, the dynamics of protein content suggest that O. angasi could use protein as an additional energy source, especially when the level of the glycogen reserve becomes low. Similarly, both glycogen and protein are metabolized to supply energy for gametogenesis in C. gigas (Dridi et al., 2007), Mactra veneriformis (Ke & Li, 2013), Perna picta (Shafee, 1989) and Atrina japonica (Lee et al., 2015). The consistent lipid content throughout the year indicates low lipid metabolism for gametogenesis in O. angasi, although lipid is the main energy source in spermcasting O. edulis (Ruiz et al., 1992). The energy required for gametogenesis in O. angasi was derived from energy stored in tissues and from instantaneous food intake. The periods of energy storage and energy utilization for gametogenesis overlapped from March to September; therefore, this species uses an energy metabolism strategy that is intermediate between those of conservative and opportunistic species. An intermediate energy metabolism strategy is also adopted by P. perna (Benomar et al., 2010), M. veneriformis (Ke & Li, 2013) and Pteria sterna (Vite-García & Saucedo, 2008), although none of these species has a protracted spawning period like O. angasi. In contrast, spermcasting O. edulis uses an opportunistic energy metabolism strategy, in which energy supply for gametogenesis is predominantly derived from instantaneous food intake (Ruiz et al., 1992). This contrast in energy metabolism between the spermcasting congeners O. edulis and O. angasi in different geographical locations suggests that the strategy for energy metabolism depends on species and on environmental conditions. Interestingly, energy provision from degenerated eggs is another energy-retrieval strategy for the protracted spawner Pecten fumatus during a period of low food availability (Mendo et al., 2016). The empty inflated gonad follicles observed in O. angasi suggest that this oyster could resorb unspawned gametes, but future study is needed to understand the pathways of energy allocation from degenerated gametes towards the next round of gametogenesis. In conclusion, the reproduction in O. angasi is characterized by asynchronous gamete development and a highly skewed male to female sex ratio. This species follows an energy metabolism pattern intermediate between conservative and opportunistic species. The particular reproductive traits of O. angasi may suggest their more general adaptive significance in organisms with spermcasting reproduction. ACKNOWLEDGEMENTS We are grateful to Brendan Guidera of the Pristine Oyster Farm, Coffin Bay for supplying flat oysters. We thank Michelle Norman for histological preparation and Sophie Leterme for providing microscopic imaging facilities. Algae and Biofuels Facility of National Collaborative Research Infrastructure Strategy (NCRIS) at Aquatic Sciences, South Australian Research and Development Institute, provided analytical services. Comments from two anonymous reviewers and from Associate Editor Yoichi Yusa substantially improved the quality of this manuscript. This work was financially supported by the research fund of the South Australian Oyster Research Council and a Flinders International Postgraduate Research scholarship from Flinders University to the first author (FIPRS No. 33004553). REFERENCES Acarli, S., Lök, A., Kirtik, A., Acarli, D., Serdar, S., Kucukdermenci, A., Yigitkurt, S., Yildiz, H. & Saltan, A.N. 2015. Seasonal variation in reproductive activity and biochemical composition of flat oyster (Ostrea edulis) in the Homa Lagoon, Izmir Bay, Turkey. Scientia Marina , 79: 487– 495. Google Scholar CrossRef Search ADS   Alleway, H.K. & Connell, S.D. 2015. 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Gametogenesis, sex ratio and energy metabolism in Ostrea angasi: implications for the reproductive strategy of spermcasting marine bivalves

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Abstract

Abstract Spermcasting is an unusual reproductive strategy in some aquatic species in which functional males release gametes to fertilize eggs inside the body cavity of functional females. As some spermcasting bivalves have high ecological and economical importance, the understanding of their reproductive strategy is crucial for aquaculture, fisheries and conservation. This study investigates gametogenesis, sex ratio and energy metabolism in Ostrea angasi to elucidate the reproductive strategy of spermcasting bivalves. Gonad histology indicated asynchronous development of spermatozeugmata, but synchronous development of oocytes within an individual. The hermaphroditic individuals released spermatozeugmata before egg ovulation. The population of 2–3 year old oysters comprised 46.7% hermaphrodites and showed a highly skewed male to female ratio of 7:1. This species primarily metabolized glycogen as the energy source for gametogenesis, with an overlapping period of energy storage and utilization. This pattern of gametogenesis suggests multiple production of spermatozeugmata in a reproductive season in male and hermaphroditic oysters, but a single episode of egg ovulation in female and hermaphroditic oysters. The dynamics of energy metabolism indicate that O. angasi follows a strategy for energy metabolism that is intermediate between those of conservative and opportunistic species. This study suggests that unsynchronized gamete development, skewed sex ratio and intermediate energy metabolism are adaptive strategies in the reproduction of spermcasting bivalve species. INTRODUCTION Most marine animals release gametes directly into the water column, where fertilization occurs. However, there are a few species in which males release sperm in this way and females then inhale sperm into their body cavity for egg fertilization (Bishop & Pemberton, 2006; Barazandeh et al., 2013). This reproductive mode has been found in a variety of taxonomic groups including hydroids, corals, polychaetes, bivalves, tunicates, entoprocts, brachiopods, bryozoans and pterobranchs (Pemberton et al., 2003). This mode of fertilization inside the body by inhaling sperm from the water column has been described as spermcast mating (Pemberton et al., 2003), for which synonymous terms are larviparity (Buroker, 1985), egg brooding (Phillippi, Hamann & Yund, 2004) and egg brooding free spawning (Johnson & Yund, 2004). To avoid confusion with other variants of internal fertilization, we use the term spermcasting for the species that fertilize eggs inside the body by acquiring sperm from the surrounding water. The reproductive modes of oysters in the family Ostreidae vary between genera. For example, species of Ostrea perform spermcasting, whereas Crassostrea species are broadcasting. In Ostrea, a male or a hermaphrodite releases sperm as spermatozeugmata, which are inhaled by a female or a hermaphrodite to fertilize the eggs in the mantle cavity (Ó Foighil, 1989). The subsequent larval development takes place inside the mantle cavity, protected by the maternal parent (Chaparro, Navarrete & Thompson, 2006). In Crassostrea, on the other hand, both types of gametes are released into the water column where fertilization and embryo development occur. Compared with broadcasting and internal spawners, very little information exists regarding the reproductive strategy of spermcasting species. Gametogenesis is the process in which gametocytes undergo cell division and differentiation to form haploid gametes. Asynchrony in maturation of the male and female gametes during gametogenesis leads to synchronous spawning, whereas discrepancy in gamete development leads to asynchronous spawning (Styan & Butler, 2003; Chaves-Fonnegra et al., 2016). Since spermcasting bivalves are incapable of movement after settlement, acquisition of functional spermatozeugmata by a female is necessary for successful reproduction. The development patterns of spermatozeugmata and eggs could provide insight for understanding their role in the reproduction of spermcasting species (Falese, Russell & Dollahon, 2011). The sex ratio is a fundamental indicator for reproduction success in dioecious species. Nearly equal numbers of males and females are produced in most gonochoristic species, giving a balanced sex ratio (Fisher, 1930), whereas the sex ratio can skew towards one sex in hermaphroditic species. A highly male-biased sex ratio has been found in spermcasting Ostrea edulis, but the reported proportion of simultaneous hermaphrodites differs among studies (Kamphausen, Jensen & Hawkins, 2011; Acarli et al., 2015). The family Ostreidae includes species of both the broadcasting and spermcasting modes, but our understanding of the reproductive strategies of the latter is far less than of the former. This duality of reproductive mode makes oyster species a suitable model to study the relationship between sex ratio and reproductive mode. Energy metabolism in marine bivalves is closely associated with reproductive activities. Energy is stored as glycogen, protein and lipid in various tissues and mobilized for physiological and reproductive activities (Berthelin, Kellner & Mathieu, 2000; Benomar et al., 2010). The dynamics of the biochemical composition in storage tissues and the body condition index reveal the energy metabolism strategy of a species. As gametogenesis is a highly energy-demanding process, marine bivalves use either an opportunistic or a conservative strategy to control energy expenditure during gametogenesis (Bayne, 1976). Opportunistic species obtain and store energy prior to the process of gametogenesis, whereas conservative species mainly rely on energy obtained from exogenous feeding during the process of gametogenesis. In a temperate region, temperature and food supply are seasonally variable and environmental variation regulates the reproductive clock (Garrido & Barber, 2001; Enríquez-Díaz et al., 2009); consequently, most species follow the conservative energy-metabolism strategy (Darriba, San Juan & Guerra, 2005; Li et al., 2006; Karray et al., 2015). However, the energy metabolism strategy needs to be evaluated for a species which such as Ostrea angasi, which has a protracted spawning period and grows in an area with low food supply, in order to understand how energy is supplied for a long spawning season without much calorific intake. In this study, the Australian flat oyster O. angasi G.B. Sowerby II, 1871 was used as a representative of spermcasting bivalves. This species was abundant in Australia before the arrival of European settlers, but almost perished from its natural habitat in the late 18th and early 19th century due to overfishing (Nell, 2001; Alleway & Connell, 2015). Over the last decade, the production of O. angasi has increased and it has become an emerging species for aquaculture in Australia (Heasman et al., 2004; O’Connor & Dove, 2009). However, the poor understanding of the reproductive strategy of marine spermcasting bivalves hinders their aquaculture, fishery management and conservation. The objective of this study is to elucidate the reproductive strategy of spermcasting marine bivalves by examining the patterns of gametogenesis, sex ratio and energy metabolism in O. angasi. MATERIAL AND METHODS Oyster and sea water sampling Flat oysters and sea water were collected at the leases of the Pristine Oyster Farm in Coffin Bay, South Australia, from April 2014 to May 2015. Oysters were transported within 24 h in a chilled Styrofoam box to the laboratory at Flinders University. The oysters were then cleaned with a brush and blotted using paper towel before taking length and weight measurements. The age of oysters in this study was 2–3 years (mean ± SD = 76.8 ± 5.2 mm shell length and 72.0 ± 10.9 g wet weight, n = 840) (Fig. 1). Figure 1. View largeDownload slide Ostrea angasi, showing the main internal organs. Abbreviations: AM, adductor muscle; G, gonad; M, mantle; S, shell. Figure 1. View largeDownload slide Ostrea angasi, showing the main internal organs. Abbreviations: AM, adductor muscle; G, gonad; M, mantle; S, shell. Environmental parameters Monthly measurement of environmental variables at the sampling site included water temperature, salinity and chlorophyll a. After filtering out debris and zooplankton through a 300-μm screen, phytoplankton was collected on a 0.45-μm Millipore filter, the Millipore filter then wrapped with aluminium foil and stored at –20 °C for up to 7 days before analysis. Chlorophyll a was extracted by dissolving the filter paper in 90% acetone and stored at 4 °C for 24 h. The determination of chlorophyll a concentration was on a microplate reader (CLARIOstar, BMG Labtech) at the wavelengths of 647 nm and 664 nm, using the formula: Chlorophyll a (mg l–1) = 11.87A664−1.786A647 for calculations (Ritchie, 2006), where A664 and A647 represent absorbance at 664 nm and 647 nm, respectively. Histological observation on gonad tissues The procedures for histological preparation of Ostrea angasi gonad/visceral tissues followed that described by Hassan, Qin & Li (2016). The protocol included: fixing 3-mm pieces of tissue in Davidson’s fixative, submerging in different grades of alcohol and xylene, embedding in paraffin wax, cutting sections at 5 μm, mounting a microscope slide, staining with haematoxylin and counter-staining with eosin. The histological slides were photographed on an inverted microscope (Nikon Eclipse TS100-F). Gonad development of O. angasi was categorized in five stages: (1) resting gonad, (2) early gametogenesis, (3) advanced gametogenesis, (4) mature gonad and (5) spawned gonad, based on the criteria used for O. edulis (Da Silva, Fuentes & Villalba, 2009). Larval occurrence and sex ratio A total of 60 oysters were opened each month to count those bearing larvae. Oyster larvae were classified as white-sic, grey-sic and black-sic following Carson (2010). Identification of sex category (male, female, hermaphrodite or undifferentiated) was based on gonad histology and microscopic observation and was recorded for 840 oysters from April 2014 to May 2015. Unless spermatozeugmata were present, the larvae-bearing oysters were considered female. Biochemical composition The combined gonad-visceral tissues were used for analyses of biochemical composition, because the gonad and digestive system are anatomically intermingled. Tissues from 10 live oysters were pooled and used for analysis of glycogen, protein and lipid content. Tissues were freeze-dried for 48 h and stored at −80 °C for no more than 3 months prior to analysis. Glycogen, protein and lipid content were determined according to the modified Anthrone technique (Roe & Dailey, 1966), Coomassie Bradford assay (Bradford, 1976) and modified Bligh Dyer method (Folch, Lees & Sloane-Stanley, 1957), respectively. Condition index Prior to condition index (CI) analysis, oysters were cleaned by scrubbing off barnacles and debris, blotted dry, the valves were opened, shells and soft tissues were freeze-dried for 48 h and weights were measured using an analytical scale (0.01 g). The condition index (CI) was calculated according to the formula: CI=dry flesh weightdry shell weight×100. Data calculation and statistical analysis To calculate the gonad maturity index (GMI), oysters were ranked from 1 to 4 based on the gonad developmental stage, ranked as follows: 1, inactive resting or spent stage; 2, early gametogenesis; 3, advanced gametogenesis and 4, mature stage. The GMI was calculated according to the formula: GMI=Ʃ(n×F)N (Vaschenko, Hsieh & Radashevsky, 2013), where n is the number of oysters at a certain gonad stage; F is the gonad-ranking score (1–4) and N is the total number of oysters in the sample. Pearson correlation was applied to measure the relationships of (1) GMI vs biochemical compositions and CI, and (2) temperature vs chlorophyll a. The data on chlorophyll a, biochemical compositions and CI were tested for normality and homogeneity using Shapiro-Wilk and Levene’s tests. Due to the violation of normality assumption, data on CI were square-root transformed prior to analysis. One-way ANOVA was used to determine the difference between months in chlorophyll a, biochemical compositions and CI. Tukey’s post hoc test was used when significant differences were found. The data were considered statistically significant at P < 0.05. All the data were analysed using SPSS v. 23 (IBM, Armonk, NY). RESULTS Environmental parameters Seawater temperature gradually decreased from 19.3 °C in April to 11.2 °C in July 2014 and increased from 12.0 °C in August 2014 to 24.2 °C in February 2015 (Fig. 2A). Salinity was relatively stable, but decreased from 41.7 ppt in April to 36.5 ppt in July 2014 and then increased from 36.8 ppt in August 2014 to 41.3 ppt in April 2015. The monthly differences in chlorophyll a content were significant (F = 6.79, df = 13, P < 0.05). Chlorophyll a decreased from 1.27 mg l–1 in April to a minimum of 0.92 mg l–1 in July 2014 and then increased to a maximum of 1.95 mg l–1 in January 2015 (Fig. 2B). Seawater temperature and chlorophyll a were positively correlated (r = 0.74, n = 42, i.e. 3 replicates per month over 14 months, P < 0.05). Figure 2. View largeDownload slide Monthly variations of environmental parameters in Coffin Bay, South Australia from April 2014 to May 2015. A. Seawater temperature and salinity. B. Chlorophyll a. Different letters indicate significant differences (P < 0.05) among the monthly values. Each bar represents mean ± SE of three replicates. Figure 2. View largeDownload slide Monthly variations of environmental parameters in Coffin Bay, South Australia from April 2014 to May 2015. A. Seawater temperature and salinity. B. Chlorophyll a. Different letters indicate significant differences (P < 0.05) among the monthly values. Each bar represents mean ± SE of three replicates. Gametogenesis In the resting stage, gonad follicles were empty and the sex category was not distinct (Fig. 3A). With the progress of spermatogenesis, encapsulated spermatozeugmata were formed in gonad follicles at the advanced stage. The same oysters carried different developmental stages of spermatozeugmata, i.e. presence of spermatocytes and spermatids at the advanced spermatogenesis stage (Fig. 3B). At the start of oogenesis, gonad follicles consisted of only a few oogonia, but the oogonial cells gradually aggregated and became larger at the advanced stage (Fig 3C). Within each individual, the oocytes were of similar size and the oocytes matured at a similar time. Vitellogenesis, yolk deposition in the ooplasm, started at stage 3 and continued until full maturation of the eggs. In an hermaphroditic gonad, both male and female gametes were in a similar developmental stage (Fig. 3D), but the gonad follicles of partially spawned individuals indicated that spermatozeugmata were released before ovulation (Fig. 3E). Subsequently, empty inflated gonad follicles indicated complete absorption of residual gametes after spawning. Gravid oysters carried larvae in the pallial cavity (Fig. 3F). Figure 3. View largeDownload slide Histological sections of Ostrea angasi during gametogenesis. A. Resting gonad. Sex category is difficult to distinguish at this stage. Arrows indicate developing gonad follicles between mantle and digestive gland. B–D. Mature gonads of male, female and hermaphrodite. Single and double arrows indicate spermatocytes and spermatids, respectively, in male. Arrows indicate mature oocytes in female. White and black arrows indicate spermatids and mature eggs, respectively, in hermaphrodite. E. Partially spawned gonad in hermaphrodite. Single white, double and black arrows indicate residual spermatids, eggs and phagocytes, respectively. F. Arrows indicate larvae in pallial cavity in a gravid oyster. Abbreviations: CT, connective tissue; DD, digestive diverticula; GF, gonad follicle; M, mantle; PC, pallial cavity. Scale bar = 100 μm. Figure 3. View largeDownload slide Histological sections of Ostrea angasi during gametogenesis. A. Resting gonad. Sex category is difficult to distinguish at this stage. Arrows indicate developing gonad follicles between mantle and digestive gland. B–D. Mature gonads of male, female and hermaphrodite. Single and double arrows indicate spermatocytes and spermatids, respectively, in male. Arrows indicate mature oocytes in female. White and black arrows indicate spermatids and mature eggs, respectively, in hermaphrodite. E. Partially spawned gonad in hermaphrodite. Single white, double and black arrows indicate residual spermatids, eggs and phagocytes, respectively. F. Arrows indicate larvae in pallial cavity in a gravid oyster. Abbreviations: CT, connective tissue; DD, digestive diverticula; GF, gonad follicle; M, mantle; PC, pallial cavity. Scale bar = 100 μm. Spawning periodicity Gonad histology revealed active gametogenesis throughout the year (Fig. 4A). However, both gonad histology and larvae-bearing oysters indicated a spawning period from May to December, with a relatively higher spawning intensity from September to December (Fig. 4A, B). Figure 4. View largeDownload slide Monthly variations of (A) gonad development stages and (B) frequency of larvae-bearing Ostrea angasi collected from Coffin Bay, South Australia from April 2014 to May 2015. Figure 4. View largeDownload slide Monthly variations of (A) gonad development stages and (B) frequency of larvae-bearing Ostrea angasi collected from Coffin Bay, South Australia from April 2014 to May 2015. Occurrence of larvae Oysters carried larvae in the mantle cavity from May to December. The percentage of larvae-bearing oysters gradually increased from 2.5% in May to a maximum of 10% in October and then decreased to 3.3% in December (Fig. 4B). Sex ratio Based on the monthly sex-ratio data, no pattern of sex-ratio change was evident within this study period. The monthly sex-ratio data confirmed the occurrence of simultaneous hermaphroditism in this species (Fig. 5). A relatively higher percentage of undifferentiated gonads indicated that the postspawning gonadal stage lasted from October to February. The percentages of males, females, hermaphrodites and undifferentiated individuals were 41.3%, 5.8%, 46.7% and 6.2%, respectively, among the 840 oysters observed. The male to female ratio was 7:1. In hermaphroditic gonads, proportions of male and female gametes ranged from highly skewed to one sex to similar proportions of both sexes. Figure 5. View largeDownload slide Percentage of sex categories of Ostrea angasi collected from Coffin Bay, South Australia from April 2014 to May 2015. Sixty oysters were observed each month to determine sex categories. Figure 5. View largeDownload slide Percentage of sex categories of Ostrea angasi collected from Coffin Bay, South Australia from April 2014 to May 2015. Sixty oysters were observed each month to determine sex categories. Dynamics of biochemical composition The monthly differences in glycogen (F = 20.09, df = 13, P < 0.05), protein (F = 3.96, df = 13, P < 0.05) and lipid (F = 15.29, df = 13, P < 0.05) content were significant. The glycogen content increased from 171.7 mg g–1 in April to 217.5 mg g–1 in August and subsequently decreased to a minimum of 106.9 mg g−1 in December. The glycogen content again increased from 140.9 mg g−1 in January to a maximum of 252.7 mg g−1 in May. The protein content increased from 320.7 mg g−1 in April to a maximum of 338.8 mg g−1 in August and decreased to a minimum of 258.5 mg g−1 in February. In the following months, the protein content further increased up to 324.3 mg g−1 in May. The lipid content was relatively stable, but increased from 136.5 mg g−1 in April to a maximum of 146.2 mg g−1 in August and subsequently decreased to a minimum of 113.5 mg g−1 in December (Fig. 6A). The GMI was positively correlated with glycogen (r = 0.70, n = 14, P < 0.05), protein (r = 0.63, n = 14, P < 0.05) and lipid (r = 0.91, n = 14, P < 0.05) content. Chlorophyll a was negatively correlated with protein (r = -0.51, n = 42, P < 0.05) and lipid (r = –0.55, n = 42, P < 0.05) content, but the correlation between chlorophyll a and glycogen was not significant (r = 0.063, n = 42, P > 0.05). Figure 6. View largeDownload slide Monthly variations of energy storage compounds and condition index of Ostrea angasi from April 2014 to May 2015. A. Glycogen, protein and lipid content (mg g−1 dry tissue) in gonad-visceral tissues. Each bar represents mean ± SE of 3 replicates. Different letters within each row indicate significant differences among monthly values of glycogen (middle row; P < 0.05) protein (top row, P < 0.05) and lipid (bottom row; P < 0.05) content. B. Condition index. Each bar represents mean ± SE of 20 individuals. Different letters indicate significant monthly differences in condition index (P < 0.05). Figure 6. View largeDownload slide Monthly variations of energy storage compounds and condition index of Ostrea angasi from April 2014 to May 2015. A. Glycogen, protein and lipid content (mg g−1 dry tissue) in gonad-visceral tissues. Each bar represents mean ± SE of 3 replicates. Different letters within each row indicate significant differences among monthly values of glycogen (middle row; P < 0.05) protein (top row, P < 0.05) and lipid (bottom row; P < 0.05) content. B. Condition index. Each bar represents mean ± SE of 20 individuals. Different letters indicate significant monthly differences in condition index (P < 0.05). Dynamics of condition index The monthly variations in CI were significant (F = 8.39, df = 13, P < 0.05). CI increased from 3.8 in April to a maximum of 4.7 in July and decreased to a minimum of 2.8 in December (Fig. 6B). In the following months, CI gradually increased up to 4.0 in May. CI was positively correlated with GMI (r = 0.87, n = 14, P < 0.05), glycogen (r = 0.54, n = 14, P < 0.05), protein (r = 0.65, n = 14, P < 0.05) and lipid content (r = 0.82, n = 14, P < 0.05). DISCUSSION This study has elucidated the reproductive strategy of the spermcasting Ostrea angasi from the perspectives of gametogenesis, sex ratio and energy metabolism. The asynchronous development of spermatozeugmata in pure males and synchronous maturation of eggs in pure females imply multiple releases of sperm and single ovulation of eggs. In hermaphroditic individuals, although the male and female gametes could develop at a similar pace, spermatozeugmata would normally be released before egg ovulation. As hermaphrodites can take both male and female roles, the high percentage of hermaphrodites in the population may compensate for the limited representation of females during mating. Similar to many other bivalves, glycogen is the main energy reserve for gametogenesis in O. angasi (Dridi, Romdhane & Elcafsi, 2007; Ke & Li, 2013) and this species adopts an energy metabolism strategy that is intermediate between conservative and opportunistic species, since it uses energy from both tissue storage and instantaneous food intake during gametogenesis. The gonad-maturation stages of O. angasi varied greatly among individuals and a small proportion of the population was mature at any time within the spawning season. This gametogenesis pattern indicates that only a small proportion of the population spawns at a given time. In a population with a very small number of spawning individuals, synchronization of release of spermatozeugmata and eggs is an important determinant of fertilization success (Styan & Butler, 2003). Since the acquisition of male gametes by a female is constrained by the physical distance between male and female in sedentary bivalves (Styan, 1999), production of spermatozeugmata in multiple batches would enhance fertilization success when one batch of spermatozeugmata fails to reach females. Interestingly, the impact of location on egg fertilization is minimized in O. puelchana by the attachment of dwarf males to the shells of females (Pascual, 1997). In O. angasi, spermatozeugmata maintain functionality for up to 24 h after their release in sea water (Hassan et al., 2016) and successful fertilization should be possible if a female or a hermaphrodite acquires spermatozeugmata within this period. The duration of the gametogenesis and spawning periods varies among spermcasting bivalves. In O. angasi, all the five gonad maturation stages were found throughout the whole year, but larvae-bearing oysters were found from May to December. Individuals of O. nomades with different gonad maturation stages were also found throughout the year (Siddiqui & Ahmed, 2002), whereas gametogenesis has been reported to take place seasonally in O. edulis (Da Silva et al., 2009). Individuals brooding larvae were found during an 8-month period in O. chilensis (Brown et al., 2010), but for 4 months in O. puelchana (Castaños et al., 2005). This variation in the temporal pattern suggests that different spermcasting bivalve species adapt to local environmental condition for gametogenesis and spawning. In this study, the sex ratio of O. angasi was highly skewed towards males and a large proportion of the population was of simultaneous hermaphrodites. Whereas broadcasting oysters first mature as a male and change sex to female at an older age (Mazón‐Suástegui et al., 2011), spermcasting oysters may not follow a similar pattern in sex change. The high proportion of male to female (sex ratio 7:1) in O. angasi might be due to the use of relatively small-sized oysters (77 mm; 2–3 years old) in this study. However, no pattern of sex change was clear within the 14-month study period. Spermcasting O. edulis also have a highly skewed male to female ratio, 6:1 at a shell length of 50–70 mm (Kamphausen et al., 2011; Acarli et al., 2015), suggesting a general trend of male dominance in spermcasting oysters in a similar size range. A review of the sex ratio of two ostreid genera (Crassostrea for broadcasting oysters and Ostrea for spermcasting oysters) reveals that the sex ratios of broadcast and spermcast spawners are different (Table 1). The percentage of hermaphroditic individuals is about 1% in broadcasting spawners (Steele & Mulcahy, 1999; Enríquez-Díaz et al., 2009; Castilho-Westphal, Magnani & Ostrensky, 2015), but is over 35% in spermcasting spawners (Acarli et al., 2015; this study) at a similar size range. These contrasting sex ratios of oysters in different taxonomic and spawning groups, and their regulation, require further study in relation to the spawning strategies of broadcasting and spermcasting species. Table 1. Comparison of sex ratio between two ostreid genera, Crassostrea and Ostrea, which represent broadcast and spermcast spawners, respectively. Reproductive strategy  Species  Geographical location  Oyster age and size  Male to female ratio  Hermaphrodite occurrence  References  Broadcast spawning  Crassostrea gigas  Dungarvan and Cork Harbour, Ireland  2 years; 9.2 cm  1:1  <1%  Steele & Mulcahy (1999)  N Patagonia, Argentina  >4 cm  1:1  None  Castaños, Pascual & Camacho (2009)    English Channel and Bay of Biscay, France  1–2 years  1.69:1 and 1.22:1  <1%  Enríquez-Díaz et al. (2009)  Gulf of Tunis and Bizert lagoon, Tunisia  8–10 cm  1.4:1  <1%  Dridi, Romdhane & Elcafsi (2014)  C. angulata  Western coast of Taiwan  10–15 cm  1:0.9  4.2%  Vaschenko et al. (2013)  C. corteziensis  Coastal lagoon in NW Mexico  8–10.3 cm  1:3  None  Rodríguez-Jaramillo et al. (2008)  C. brasiliana  Paraná, Brazil  1.1–9.4 cm  1:2.65  1%  Castilho-Westphal et al. (2015)  Spermcast spawning  Ostrea edulis  Solent, UK  4–6 years; 5–7 cm  6:1  2%  Kamphausen et al. (2011)  Izmir Bay, Turkey  >5 cm  19.3:1  37%  Acarli et al. (2015)  O. angasi  Coffin Bay, South Australia  2–3 years; 6.4–8.9 cm  7:1  46.7%  This study  Reproductive strategy  Species  Geographical location  Oyster age and size  Male to female ratio  Hermaphrodite occurrence  References  Broadcast spawning  Crassostrea gigas  Dungarvan and Cork Harbour, Ireland  2 years; 9.2 cm  1:1  <1%  Steele & Mulcahy (1999)  N Patagonia, Argentina  >4 cm  1:1  None  Castaños, Pascual & Camacho (2009)    English Channel and Bay of Biscay, France  1–2 years  1.69:1 and 1.22:1  <1%  Enríquez-Díaz et al. (2009)  Gulf of Tunis and Bizert lagoon, Tunisia  8–10 cm  1.4:1  <1%  Dridi, Romdhane & Elcafsi (2014)  C. angulata  Western coast of Taiwan  10–15 cm  1:0.9  4.2%  Vaschenko et al. (2013)  C. corteziensis  Coastal lagoon in NW Mexico  8–10.3 cm  1:3  None  Rodríguez-Jaramillo et al. (2008)  C. brasiliana  Paraná, Brazil  1.1–9.4 cm  1:2.65  1%  Castilho-Westphal et al. (2015)  Spermcast spawning  Ostrea edulis  Solent, UK  4–6 years; 5–7 cm  6:1  2%  Kamphausen et al. (2011)  Izmir Bay, Turkey  >5 cm  19.3:1  37%  Acarli et al. (2015)  O. angasi  Coffin Bay, South Australia  2–3 years; 6.4–8.9 cm  7:1  46.7%  This study  Gametogenesis affects energy storage status and body condition (Vite-García & Saucedo, 2008; Karray et al., 2015), which is supported by this study as the GMI was positively correlated with glycogen, protein and lipid content, and with the CI. However, the negative correlations of protein and lipid with chlorophyll a involve a more complex interaction. The food availability and spawning intensity are positively related to water temperature, but increased spawning activity can reduce energy stores in oysters (Newell & Branch, 1980), explaining the negative correlations of protein and lipid with chlorophyll a in O. angasi. Marine bivalves allocate energy to gametogenesis by metabolizing compounds such as glycogen, protein and lipid, but the strategy for metabolizing these compounds is species-specific. Glycogen serves as the main energy reserve in most bivalves, but protein and lipid are also used as an additional or alternative energy source (Mathieu & Lubet, 1993). The large variation in glycogen content among prespawning, spawning and postspawning oysters suggests that glycogen serves as the main energy source for gametogenesis in O. angasi. Moreover, the dynamics of protein content suggest that O. angasi could use protein as an additional energy source, especially when the level of the glycogen reserve becomes low. Similarly, both glycogen and protein are metabolized to supply energy for gametogenesis in C. gigas (Dridi et al., 2007), Mactra veneriformis (Ke & Li, 2013), Perna picta (Shafee, 1989) and Atrina japonica (Lee et al., 2015). The consistent lipid content throughout the year indicates low lipid metabolism for gametogenesis in O. angasi, although lipid is the main energy source in spermcasting O. edulis (Ruiz et al., 1992). The energy required for gametogenesis in O. angasi was derived from energy stored in tissues and from instantaneous food intake. The periods of energy storage and energy utilization for gametogenesis overlapped from March to September; therefore, this species uses an energy metabolism strategy that is intermediate between those of conservative and opportunistic species. An intermediate energy metabolism strategy is also adopted by P. perna (Benomar et al., 2010), M. veneriformis (Ke & Li, 2013) and Pteria sterna (Vite-García & Saucedo, 2008), although none of these species has a protracted spawning period like O. angasi. In contrast, spermcasting O. edulis uses an opportunistic energy metabolism strategy, in which energy supply for gametogenesis is predominantly derived from instantaneous food intake (Ruiz et al., 1992). This contrast in energy metabolism between the spermcasting congeners O. edulis and O. angasi in different geographical locations suggests that the strategy for energy metabolism depends on species and on environmental conditions. Interestingly, energy provision from degenerated eggs is another energy-retrieval strategy for the protracted spawner Pecten fumatus during a period of low food availability (Mendo et al., 2016). The empty inflated gonad follicles observed in O. angasi suggest that this oyster could resorb unspawned gametes, but future study is needed to understand the pathways of energy allocation from degenerated gametes towards the next round of gametogenesis. In conclusion, the reproduction in O. angasi is characterized by asynchronous gamete development and a highly skewed male to female sex ratio. This species follows an energy metabolism pattern intermediate between conservative and opportunistic species. The particular reproductive traits of O. angasi may suggest their more general adaptive significance in organisms with spermcasting reproduction. ACKNOWLEDGEMENTS We are grateful to Brendan Guidera of the Pristine Oyster Farm, Coffin Bay for supplying flat oysters. We thank Michelle Norman for histological preparation and Sophie Leterme for providing microscopic imaging facilities. Algae and Biofuels Facility of National Collaborative Research Infrastructure Strategy (NCRIS) at Aquatic Sciences, South Australian Research and Development Institute, provided analytical services. Comments from two anonymous reviewers and from Associate Editor Yoichi Yusa substantially improved the quality of this manuscript. This work was financially supported by the research fund of the South Australian Oyster Research Council and a Flinders International Postgraduate Research scholarship from Flinders University to the first author (FIPRS No. 33004553). REFERENCES Acarli, S., Lök, A., Kirtik, A., Acarli, D., Serdar, S., Kucukdermenci, A., Yigitkurt, S., Yildiz, H. & Saltan, A.N. 2015. Seasonal variation in reproductive activity and biochemical composition of flat oyster (Ostrea edulis) in the Homa Lagoon, Izmir Bay, Turkey. Scientia Marina , 79: 487– 495. Google Scholar CrossRef Search ADS   Alleway, H.K. & Connell, S.D. 2015. 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Journal of Molluscan StudiesOxford University Press

Published: Feb 1, 2018

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