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Evaluating spawning induction methods for the tropical black-lip , Saccostrea echinata

Nowland, Samantha; O'Connor, Wayne; Elizur, Abigail; et.al. https://research.usc.edu.au/discovery/delivery/61USC_INST:ResearchRepository/12142219730002621?l#13142219720002621

Nowland, S., O’Connor, W., Elizur, A., & Southgate, P. (2021). Evaluating spawning induction methods for the tropical black-lip rock oyster, Saccostrea echinata. Reports, 20, 1–10. https://doi.org/10.1016/j.aqrep.2021.100676 Document Type: Published Version

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Please do not remove this page Aquaculture Reports 20 (2021) 100676

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Aquaculture Reports

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Evaluating spawning induction methods for the tropical black-lip rock oyster, Saccostrea echinata

Samantha J. Nowland a,b,*, Wayne A. O’Connor c, Abigail Elizur d, Paul C. Southgate b a Aquaculture Unit, Department of Industry, Tourism and Trade, Government, GPO Box 3000, Darwin, NT, 0801, Australia b School of Science, Technology & Engineering and Australian Centre for PacificIslands Research, University of the Sunshine Coast, 90 Sippy Downs Drive, Sippy Downs, , 4556, Australia c NSW Department of Primary Industries, Port Stephens Fisheries Institute, Taylors Beach, NSW, 2316, Australia d Genecology Research Centre, University of the Sunshine Coast, 90 Sippy Downs Drive, Sippy Downs, Queensland, 4556, Australia

ARTICLE INFO ABSTRACT

Keywords: When developing a species-specific hatchery protocol it is important to investigate the triggers for spawning, Spawning which is the foundation of the production cycle. This study evaluated multiple spawning induction techniques to Induction optimise the spawning success of black-lip rock oyster, Saccostrea echinata, broodstock. Initially, the most Oyster effective method for non-destructively opening broodstock was determined, to allow intramuscular injection of Saccostrea echinata chemical stimuli. Following this, the efficacy of seven spawning induction treatments, including two physical Salinity Muscle relaxant shock techniques, three chemical induction techniques, a combination of physical and chemical induction and strip spawning were assessed. Based on the outcomes of these trials, the combined and independent effects of reduced salinity and addition of sperm, as well as the potential of neuropeptides to trigger spawning, were evaluated. Results demonstrated that a concentration of 30 g/L of MgCl2 is an effective muscle relaxant for commercial and research application for S. echinata. It was determined that salinity reduction, rather than temperature increase, is a key factor for spawning induction. Strip spawning is a viable option for S. echinata; however, sperm motility was significantlyaffected, and fecundity and fertilisation rates were lower (although not significant,P > 0.05) when compared to other induction methods tested. A combination of physical and chemical induction, involving reduced salinity and addition of sperm, was the most successful treatment; 80 % of broodstock spawned within 19 min of salinity drop and addition of sperm. This method delivers significant production improvements for S. echinata, particularly regarding the time taken to induce spawning, and is therefore recommended for application in the hatchery.

1. Introduction year-round supply of spat (Maccacchero et al., 2005; FAO, 2011). Hatchery production has been established for Crassostrea iredalei and Oysters are a growing aquaculture commodity in tropical regions Crassostrea belcheri in Malaysia (Tan et al., 2014), Crassostrea angulata in that provide important economic and subsistence benefits in many Vietnam (O’Connor et al., 2016) and Crassostrea rhizophorae in Brazil countries (Nowland et al., 2019b; Willer and Aldridge, 2020). (Maccacchero et al., 2005). Other countries are progressing towards Commercial-scale production (>100 t per annum) occurs in Brazil, hatchery production by investing in species-specific research and northern Chile, southern China, Cuba, India, Indonesia, Malaysia, development, such as the establishment of culture techniques for Mexico, The Philippines, Senegal, Taiwan, Thailand and Vietnam Striostrea prismatica in Ecuador (Lodeiros et al., 2017). (Nezon, 1988; Huang and Lee, 2014; O’Connor et al., 2016; FAO, 2020). The black-lip rock oyster (Saccostrea echinata) (Quoy and Gaimard, Production in most of these countries, however, relies on collection of 1835) is an emerging aquaculture species that has received research wild spat as a source of culture stock, which can be unreliable (Nowland attention and investment. It occurs throughout the Indo-Pacific, from et al., 2019b). Research into hatchery production of tropical rock oysters Japan to New Caledonia, and across northern Australia, from Cone Bay is therefore increasing, stimulated by the need to guarantee a reliable, in to Bowen in Queensland (Nowland et al., 2019d).

* Corresponding author at: GPO Box 3000, Darwin, Northern Territory, 0801, Australia. E-mail addresses: [email protected] (S.J. Nowland), wayne.o’[email protected] (W.A. O’Connor), [email protected] (A. Elizur), [email protected] (P.C. Southgate). https://doi.org/10.1016/j.aqrep.2021.100676 Received 17 November 2020; Received in revised form 12 March 2021; Accepted 24 March 2021 Available online 7 April 2021 2352-5134/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/). S.J. Nowland et al. Aquaculture Reports 20 (2021) 100676

Recent research has focused on developing the potential of S. echinata as production for this developing aquaculture species. a new aquaculture commodity in tropical Australia (Cobcroft et al., 2020). Studies have investigated population genetics (McDougall, 2018; 2. Materials and methods Nowland et al., 2019d) and reproductive seasonality of wild oysters (Nowland et al., 2019c), described larval development (Southgate and 2.1. Broodstock and histology Lee, 1998; Nowland et al., 2018a) and optimised rearing conditions for larval growth and survival (Nowland et al., 2018b, a). However, there is Broodstock oysters (S. echinata) with length (dorsoventral measure­ a lack of information relating to methods for reliable spawning induc­ ment; DVM) and width (anteroposterior measurement; APM) of 100 ± tion, which is an important requirement for routine commercial hatch­ 1.99 mm and 74.49 ± 1.55 mm, respectively, were originally collected ◦ ′ ′′ ery production. Developing a hatchery protocol for a species new to from South Goulburn Island, Northern Territory, Australia (11 38 46 S ◦ ′ ′′ aquaculture usually involves trialling different methods for spawning 133 25 14 E) and held in earthen ocean filledponds near the Northern induction to identify the most effective method for the target species. Territory Government’s, Darwin Aquaculture Centre, for use when Successful spawning induction under hatchery conditions requires oys­ required. Broodstock were randomly collected from this population in ters with mature gametes (Li et al., 2010), which can be obtained December 2016 for Experiment 1 and February 2017 for Experiment 2. directly from the wild during periods of natural maturation (Nowland Some broodstock were used for both experiments, therefore, treatment et al., 2019c) or by conditioning broodstock within the hatchery numbers are provided for each experiment. Broodstock were rinsed with ´ (Chavez-Villalba et al., 2002; Maneiro et al., 2017). Spawning may be freshwater after collection and scrubbed clean of any fouling using an triggered by several endogenous and exogenous factors, and methods iodine solution; 10 mL of Providone-Iodine Antiseptic Solution into 10 L often involve chemical induction, physical shock (i.e. rapid change in an of freshwater. Broodstock length (mm), width (mm), height (mm) and environmental factor such as water temperature or salinity) or a com­ weight (g) were recorded before each experiment. After spawning in­ bination of both (Helm et al., 2004; Aji, 2011). Furthermore, physical duction trails, broodstock were held in a 7600 L cylindrical tank within removal of gametes from mature oyster gonads, or ‘strip spawning’, is labelled baskets for two weeks to assess survival before being returned to commonly employed when a high degree of fertilisation control is the holding ponds. required, such as that required in selective breeding programs (Utting Pervious work on wild S. echinata has documented a gender ratio of and Spencer, 1991; O’Connor et al., 2008). 1:1.4 (female:male) and synchronised reproductive development It has long been documented that the presence of spermatozoa in (Nowland et al., 2019c). Because broodstock are valuable and it is not culture water may trigger spawning in conspecifics, as spermatozoa possible to determine gender or reproductive condition without sacri­ contain pheromones that cause chemical stimulation (Galtsoff, 1938, ficinganimals, the exact gender ratio and reproductive condition of the 1940). These pheromones were later found to be intrinsic sperm mem­ oysters in each treatment group was unknown. Therefore, the gender brane proteins (Rice et al., 2002), which remain bioactive even after ratio was estimated to be 1:1.4 (female:male) (Nowland et al., 2019c), sperm viability has been eliminated (e.g. via microwave), and can be although in some cases the ratios were subsequently found to be used to elicit spawning while avoiding uncontrolled fertilisation (Wal­ different. The reproductive condition of broodstock was determined lace et al., 2008). Among the chemical agents used successfully to induce before and after Experiment 1 and Experiment 2, by taking histological spawning of bivalve molluscs are serotonin (Gibbons and Castagna, sections of fiverandomly selected broodstock on the firstand last day of 1984) and hydrogen peroxide (Rose, 1990). More recently, the effects of the experiments. Transverse 6-mm-thick sections of the dorsal visceral neuropeptides to induce spawning in oysters has been investigated mass, from the point where the labial palps meet the gills, and parallel (Bernay et al., 2006; Chansela et al., 2008). Bernay et al. (2006), for with the hinge line, were made for each oyster. The sections were then example, were the firstto provide experimental evidence of peptidergic placed into histological cassettes, fixedin 10 % seawater formalin for 48 control of spawning in oysters when they demonstrated that the regu­ h and then processed and embedded in paraffin.Sections of 4 mm were latory peptides, APGWamide and PIESVD, trigger increases in shell made using standard histological techniques (Luna, 1968; Raphael et al., closure frequency of mature female Crassostrea gigas. 1976) and stained with hematoxylin and eosin for histological evalua­ Physical ‘shocks’ that mimic natural environmental changes readily tion. Sections were assessed microscopically to determine gender and induce mature oysters to spawn. They include rapid change in water reproductive condition according to the Dinamani (1974) classification temperature (Utting and Spencer, 1991) or salinity (Stephen and Shetty, system described for Saccostrea glomerata. These phases of gamete 1981), the addition of microalgae (Starr et al., 1990) and emersion or development include; resting (R), early development (G1–G2), late mimicking tidal patterns (Korringa, 1947). However, academic reviews development (G3), ripe (G4), discharged (G5) and residual (Gx). When of this topic remain equivocal as to specific spawning triggers in the more than two stages coexisted in the same sample, the dominant stage natural environment (Helm et al., 2004; Aji, 2011). In the hatchery, was assigned to the specimen. change in water temperature is the main physical trigger for spawning Mean gonad index (GI) was determined for each sampling period and temperate oyster species, such as C. gigas (Helm and Millican, 1977) and was calculated following the formula described by (Seed, 1969): Crassostrea virginica (Wallace et al., 2008). However, in tropical species, M = Σ(ns)/N changing salinity may be the chief spawning stimulus (Stephen and (1) Shetty, 1981). Past research suggests that this may be the case for Where n is the number of oysters present in a given gametogenic stage, s S. echinata; Coeroli et al. (1984) reported that spawning stimulation of is the numerical ranking of the given gametogenic stage, and N is the S. echinata involved a reduction in salinity from 35 to 25 ppt for over one total number of individuals in the sample (Seed, 1969; Braley, 1982, hour. Southgate and Lee (1998) similarly noted that spawning induction 1984). The following is the numerical ranking (s) scheme for the Dina­ of S. echinata almost always required sudden reduction in salinity, and mani (1974) gametogenic classificationsystem; R = 0, G1 = 1, G2 = 2, postulated that this is likely a reflectionof heavy seasonal rainfall being G3 = 3, G4 = 4, G5 = 0 and Gx = 0. The index may vary from zero, when a spawning cue under natural conditions. Nowland et al. (2019c) all the individuals in the sample have spawned (G5) or are in the resting investigated the spawning seasonality of S. echinata in the wild and (R) phase, to four, when all the individuals within the sample are hypothesised that monsoonal rainfall and increased phytoplankton sexually ripe (G4). availability may initiate spawning activity. This study evaluated multi­ ple spawning induction techniques for S. echinata broodstock to deter­ mine the most effective method to obtain viable gametes in isolation, 2.2. MgCl2 relaxation of S. echinata broodstock without the risk of uncontrolled fertilisation. Results provide an important basis for progress towards commercial scale hatchery Because some of the spawning induction methods evaluated in this

2 S.J. Nowland et al. Aquaculture Reports 20 (2021) 100676 study required intramuscular injection, a pilot experiment was needed was reduced by the addition of DI freshwater. to determine the most effective method for non-destructively opening Spawning induction methods followed those described for S. echinata broodstock. Magnesium chloride (MgCl2) was trailed as it is a S. glomerata by O’Connor et al. (2008). Briefly,broodstock (n = 18) were successful muscle relaxant for S. glomerata (Butt et al., 2008), a closely placed into individual aquaria and left overnight without FSW (17 h). related species. Different concentrations of MgCl2 were trialled to Following this, aquaria were filled with ambient FSW and oysters determine the optimum conditions. Broodstock oysters (n = 60) were allowed to acclimate for 2 h, before heaters in the header tanks were exposed to a solution of either 30, 50 or 70 g/L of MgCl2 (MagSalt by turned on in treatment aquaria (n = 9) and flowrates were adjusted to ◦ HY-CLOR, Australia) dissolved in deionised (DI) freshwater for 6 h. For increase the seawater temperature at a rate no more than 1 C every 10 ◦ concentrations of 30 and 50 g/L, MgCl2 salinity was adjusted to 36 ± 1 min. Water temperature was increased to a maximum of 7 C above ◦ ppt using instant ocean salt (Aquarium Systems, France), the salinity of ambient (37 C) and oysters were held at this temperature for 30 min. At the 70 g/L MgCl2 concentration was recorded and controls were held in this time, DI freshwater was added to the header tanks to reduce salinity 36 ppt filtered seawater (FSW) without MgCl2 (n = 15 per treatment). from 36 to 22 ppt and oysters held in these conditions for 2 h. If oysters For further detail on methods see Butt et al. (2008). Time to complete did not spawn within this period, water temperature and salinity shock relaxation (i.e., opening) was assessed and recorded every 15 min over a were repeated a maximum of three times (three stress cycles), after 6 h (360 min) period; which was determined as the time at which oysters which the experiment was ended for any oysters that did not spawn. did not respond to gentle irritation of the mantle (O’Connor and Lawler, Water temperature and salinity were not changed in control aquaria (n ◦ 2002); the experiment was repeated twice. = 9) and remained at ambient conditions (30 ± 1 C and 36 ppt).

2.3.3. Chemical induction: addition of sperm 2.3. Experiment 1: investigating multiple spawning induction methods A sperm suspension was collected by sacrificing 17 broodstock to obtain 7 male broodstock in adequate reproductive condition. Shallow Six spawning induction methods were trialled in this study including cuts were made into the gonad and sperm washed from the incisions two physical shock techniques, three chemical induction techniques and using FSW and passed through a 160 μm nylon mesh screen. The a combination of physical and chemical induction, as well as physical resulting sperm suspension was pooled and a 1:10 serial dilution in FSW removal of gametes (see Table 1). During the experiment, broodstock was performed 4–6 times, and sperm were counted using a haemocy­ were held in individual 5 L aquaria (one oyster per aquarium) with 12 tometer. The final concentration of sperm was 26.2 × 106 /mL. The μ ± ◦ m FSW at ambient temperature and salinity (30 1 C and 36 ppt). sperm suspension was made on the day of the experiment and stored in When broodstock spawned, the number of eggs released, percent sperm the refrigerator, for a maximum of 4 h, when not in use (O’Connor et al., motility and percent fertilisation (defined by cell division 1.5 h post 2008). fertilisation; Nowland et al., 2018a) were recorded. Methods for each Broodstock (n = 18) were placed into individual aquaria and left out spawning induction experiment are detailed below. of water overnight (17 h). The following day aquaria were filled with ◦ ambient FSW (30 ± 1 C and 36 ppt) and oysters allowed to acclimate for 2.3.1. Physical shock: emersion 2 h. Following this, 2 mL of sperm suspension was added directly into = Broodstock (n 18) were placed into individual aquaria and left treatment aquaria (n = 9) and the equivalent volume of FSW added to = overnight without seawater (17 h) and control broodstock (n 18) were control aquaria (n = 9). Oysters were held in these conditions for 1 h. If placed into identical aquaria, with FSW overnight. Air and water tem­ oysters did not spawn an additional 2 mL of sperm suspension or FSW peratures were recorded every 5 min with two temperature data loggers was added and oysters held under these conditions for 1 h; this was (HOBO water temp pro v2 U22 001, United States of America), in both repeated twice. The experiment ended 3 h after the final addition of treatment and control aquaria. Following this, aquaria were filled with sperm suspension or FSW (total of 6 mL). ambient FSW and broodstock held in these conditions for up to 8 h, after which the experiment ended for any oysters that did not spawn. 2.3.4. Chemical induction: addition of sperm extract Methods for preparing sperm extract followed those described for 2.3.2. Physical shock: increased temperature and reduced salinity Pinctada maxima by Taylor et al. (2018). Briefly, sperm was stripped To allow water temperature and salinity manipulation without dis­ from three S. echinata broodstock that were in reproductive condition, = turbing broodstock, FSW was supplied to the 5 L aquaria (n 18) used to passed through a 160 μm nylon mesh screen and stored in a 15 mL falcon ◦ hold the oysters from one of six 100 L cylindrical header tanks. Each tube at 80 C. To process, the sperm sample was thawed and centri­ ◦ header tank supplied FSW to three aquaria by individual low voltage fuged at 4000 × g for 30 min at 4 C to separate sperm cells (pellet) from pumps (AQUAPRO AP200LV, Australia) and 10 mm flexible tubing. sperm-free milt (supernatant) which was gently poured off and dis­ Header tank FSW was heated using thermostatically controlled immer­ carded. The sperm pellet was held on ice and homogenised for 10 min on ± ◦ sion heaters ( 0.5 C) (EHEIM thermocontrol 25, Germany) and salinity speed 3–4 (IKATI8 basic ULTRA-TURRAX, Malaysia), before being processed by freeze-thaw three times to rupture the sperm cells. The ◦ Table 1 sample was then centrifuged for 30 min at 4000 × g at 4 C to produce a List of spawning induction techniques and respective methods trialled in sperm membrane pellet and the supernatant was removed. The sperm Experiment 1. membrane pellet was frozen using dry ice before being lyophilised. Technique Method Broodstock (n = 36) were placed into individual aquaria and left out physical shock (1) emersion of water overnight (17 h). Following this, aquaria were filled with ◦ (2) increased temperature and reduced salinity * ambient FSW (30 ± 1 C and 36 ppt) and allowed to acclimate for 2 h. chemical induction (3) addition of sperm Sperm extract aliquots of lyophilised sperm membrane (n = 18), ^ (4) addition of sperm extract equivalent to 0.5 mL of stripped sperm per dose, were made up to 5 mL (5) serotonin = physical and chemical (6) reduced salinity and addition of sperm with FSW. Aliquots of only FSW (n 18) were made for controls. Ali­ combination quots were added directly to respective aquaria and oysters were held in physical removal of gametes (7) strip spawning* these conditions for 1 h. If there was no spawning response, an addi­ * Modified from methods described by O’Connor et al. (2008) for Saccostrea tional aliquot of sperm extract or FSW was added to aquaria. The glomerata. experiment ended for oysters that did not spawn 3 h after four aliquots ^ Modified from methods described by Taylor et al. (2018) for Pinctada (total of 20 mL) had been added. maxima.

3 S.J. Nowland et al. Aquaculture Reports 20 (2021) 100676

2.3.5. Chemical induction: serotonin the presence of sperm as spawning stimuli for S. echinata. Treatments Broodstock (n = 36) were placed into individual aquaria and left out included: (a) reduced salinity and addition of sperm (n = 10); (b) of water overnight (17 h). The following day, aquaria were filledwith 30 reduced salinity only (n = 10); (c) addition of sperm only (n = 10); and g/L MgCl2 solution and oysters held for 3 h under these conditions to (d) controls (n = 10). induce muscle relaxation (see methods for 2.2 pilot experiment). Sero­ A sperm suspension was collected by stripping sperm from brood­ tonin solution was made to a concentration of 2 mM (Gibbons and stock (n = 4) and a 1:10 serial dilution in FSW was performed and sperm Castagna, 1984) by adding 0.17 g of crystalline serotonin (5-hydroxy­ were counted using a haemocytometer. The finalconcentration of sperm tryptamine creatinine sulfate complex, Sigma Chemical Co.) to 250 mL was 26.1 × 106 /mL. To allow salinity manipulation without disturbing of FSW. Once oysters (n = 18) were completely relaxed they were broodstock, 100 L cylindrical header tanks (n = 8) supplied 5 aquaria (n removed from the aquaria and injected with 0.2 mL of serotonin solution = 40) with FSW by individual low voltage pumps (AQUAPRO AP200LV, into the adductor muscle using a 25 gauge needle and 1 mL syringe, Australia) and 10 mm flexible tubing. Salinity was reduced by the while control oysters (n = 18) were injected with the equivalent volume addition of DI freshwater. of saline solution (0.9 % NaCl; AEROWASH, Australia). Oysters were Broodstock (n = 40) were placed into individual aquaria and left out ◦ then transferred to aquaria filled with ambient FSW (30 ± 1 C and 36 of water overnight (17 h). The following day, aquaria were filled with ◦ ppt). The experiment concluded for oysters that did not spawn within 4 ambient FSW (30 ± 1 C and 36 ppt) and oysters were allowed to h of intramuscular injection. acclimate for 2 h. Salinity was reduced from 36 to 22 ppt in the header tanks by adding DI freshwater (treatments a and b) and 2 mL of sperm 2.3.6. Physical and chemical combination: reduced salinity and addition of suspension was added directly into aquaria (treatments a and c). The sperm equivalent volume of FSW was added to control aquaria (treatment d) This experiment utilised the same header tank design as described and when independent effects were being investigated (treatments b and above for method two “physical shock: increased temperature and c). Oysters were held in these conditions for 1 h. If oysters did not spawn reduced salinity”, so that salinity could be manipulated without dis­ an additional 2 mL of sperm suspension or FSW was added and oysters turbing broodstock. Broodstock (n = 18) were placed into individual held in these conditions for 1 h. The experiment ended 3 h after final aquaria and left out of water overnight (17 h). The following day aquaria addition of sperm suspension or FSW (total of 4 mL). were filledwith ambient FSW and oysters allowed to acclimate for 2 h. Salinity was reduced from 36 to 22 ppt in the header tanks by adding DI 2.4.2. Chemical induction: neuropeptides freshwater and 2 mL of sperm suspension was added directly into Methods followed those described by In et al. (2016) who identified treatment aquaria (n = 9) and the equivalent volume of FSW added to neuropeptides in S. glomerata (APGWamide and buccalin) that trigger control aquaria (n = 9). See method three “chemical induction: addition spawning. Both APGWamide and buccalin were used in this study. of sperm” for details on collection and concentration of the sperm sus­ Neuropeptides were synthesized by China Peptides Co. Ltd. and within pension used. Oysters were held in these conditions for 1 h. If oysters did one peptide treatment group, 40 μg of each bioactive peptide was pooled not spawn an additional 2 mL of sperm suspension or FSW was added into 10 μL of DI water, as this was the volume designed for one injection and oysters held in these conditions for 1 h. The experiment was ended 3 (Table 2). h after the final addition of sperm suspension or FSW had been added To investigate the effects of APGWamide and buccalin as spawning (total of 4 mL). stimuli for S. echinata, four treatments were trailed: (a) APGWamide and buccalin (n = 10); (b) APGWamide only (n = 10); (c) buccalin only (n = 2.3.7. Physical removal of gametes: strip spawning 10); and (d) controls (n = 10). Broodstock (n = 40) were placed into Broodstock (n = 34) were left out of water overnight (17 h), before individual aquaria and left out of water overnight (17 h). The following being sacrificedby removing the right (top) valve. After microscopically day aquaria were filled with 30 g/L MgCl2 solution for 3 h to induce assessing gender and gamete quality (sperm motility and egg shape, size muscle relaxation (see methods for 2.2. pilot experiment). When oysters and number (O’Connor et al., 2008)) of each oyster, four males and four were completely relaxed they were removed from aquaria and injected females in good reproductive condition were selected. Gametes were with either 5 μL of both APGWamide and buccalin (treatment a), 10 μL stripped following methods modified from Helm et al. (2004). Shallow of either APGWamide or buccalin (treatments b and c), or 10 μL of saline cuts were made in the gonad and eggs or sperm were washed from the solution (0.9 % NaCl; AEROWASH, Australia; treatment d). Injections incisions using FSW. Great care was taken not to puncture the digestive were made into the adductor muscle with a Hamilton syringe (10R-GT gland beneath the layer of gametogenic tissue, new scalpels were used, syringe). During injections, peptides were kept cold on crushed ice and and utensils cleaned between each oyster, to avoid cross contamination. refrigerated between injections. After injection, oysters were placed ◦ Females were stripped first, and eggs were washed through a 160 μm back into aquaria filledwith ambient FSW (30 ± 1 C and 36 ppt). The nylon mesh screen into individual 500 mL containers with FSW and left experiment ended for oysters that did not spawn 6 h after intramuscular for 1 h to water-harden, before being washed on a 20 μm nylon mesh injection. screen. Males were then stripped and resulting gametes washed through a 160 μm nylon mesh screen into individual 500 mL containers. To assess gamete viability, each female was crossed with each male; con­ tainers (n = 16) were prepared by splitting the total volume of eggs from each female between four containers and topping up to 500 mL with Table 2 FSW and crossing with sperm from each male. Enough sperm was added Names and bioactive peptide sequences of peptides assessed for to ensure at least one sperm was visible at the periphery of each egg spawning induction potential in this study. (Utting and Spencer, 1991), visually assessed using a compound mi­ croscope, this was ~1 mL of sperm suspension for each container. Peptide name Bioactive peptide sequence RPGW-NH2 KPGW-NH 2.4. Experiment 2: further investigating two spawning induction methods APGWamide 2 SPGW-NH2 APGW-NH2 2.4.1. Physical and chemical combination: reduced salinity and addition of ALDRYSFFGGL-NH2 ALDKYGFFGGI-NH sperm Buccalin 2 Based on results from Experiment 1, this experiment further inves­ GLDRYSFMGGI-NH2 GLDRYGFAGSL-NH tigated the combined and independent effects of salinity reduction and 2

4 S.J. Nowland et al. Aquaculture Reports 20 (2021) 100676

2.5. Statistical analyses between genders; high (4) at the start of the experiment and low (0) at the end (post spawning). Pilot experiment, Experiment 1 (fecundity, sperm motility and fer­ tilisation) and Experiment 2 (fecundity, sperm motility, fertilisation and 3.3. Experiment 1: investigating seven spawning induction methods time to spawn) data were tested for homogeneity of variance. Pilot experiment (number of oysters relaxed), Experiment 1 (fecundity and Results from Experiment 1 are summarised in Table 3. Oysters in all fertilisation) and Experiment 2 (time to spawn data from method two, experimental treatments underwent emersion (17 h) prior to each neuropeptides) data were log transformed, while Experiment 2 data spawning induction attempt, where air temperature ranged from 26.6 to ◦ (fecundity) were square root transformed. All data were then analysed 31.6 C. No oysters spawned in the emersion, sperm and sperm extract with a one- or two-way ANOVA followed by post-hoc Tukey HSD test treatments (Table 3). During the increased temperature and reduced (where appropriate) to determine differences between methods and salinity treatment one male spawned 15 min after the firstsalinity drop treatments. The Pearson correlation coefficient was used to determine and the same male spawned again 8 min after the second salinity drop the presence of linear relationships between fecundity and broodstock (on the second stress cycle) (Table 3). Intramuscular serotonin injection length and weight, in Experiment 2. Pearson chi-square tests were used triggered spawning in four males (22 %) within 38 min and no further to compare the gender of spawned oysters between methods and the oysters spawned up to 4 h post-injection. The most successful method proportions of oysters that spawned between treatments. Analyses were trialled was salinity shock and sperm, where two females (22 %) and one completed and plots generated using R (R Core Team, 2017) with a male (11 %) spawned within 47 min of treatment; however, two males significance level of α = 0.05. (22 %) also spawned in the control (Table 3). Fecundity in the salinity and sperm treatment (mean number of eggs released, 7.7 × 106) was 3. Results four times higher than the average number of eggs obtained from strip spawning (mean number of eggs released, 1.9 × 106), however, differ­ 3.1. MgCl2 relaxation of S. echinata broodstock ences were not significant (F1, 4 = 4.851. P > 0.05) (Table 3). Fertil­ isation rates were also higher in the salinity and sperm treatment (100 There was a significant effect of MgCl2 concentration on time to %) compared to strip spawning (5–85 %). In the three spawning in­ complete relaxation of S. echinata broodstock (F4, 31 = 3.68, P < 0.05) duction methods where males spawned (increased temperature and and no significantdifference was recorded between experiment days (F1, reduced salinity, serotonin and reduced salinity and addition of sperm), 31 = 1.91, P > 0.05). After 1.5 h of exposure to MgCl2 more than 50 % of sperm motility was significantly(F 1, 10 = 7.568, P < 0.05) higher (50–80 broodstock were completely relaxed in the 30 g/L treatment (Fig. 1). %) compared to that obtained by strip spawning (30–60 %). In the two- After 6 h of exposure, 47 % of broodstock were completely relaxed in the week recovery period following Experiment 1, only one oyster died from 70 g/L treatment, while the 50 g/L MgCl2 treatment performed poorest the serotonin treatment. with only 20 % and 40 % (experiment days 1 and 2, respectively) of broodstock completely relaxed after 6 h of exposure (Fig. 1). Salinities 3.4. Experiment 2: further investigating two spawning induction methods were adjusted to 36 ppt for the 30 and 50 g/L treatments and were 50 ppt in the 50 g/L treatment, and water temperature ranged from 26.3 to Results from Experiment 2 are summarised in Table 4. Oysters ◦ ◦ 28.6 C and 27 to 28.6 C on experiment day one and two, respectively. spawned in all treatments, however, Pearson chi-square test showed a No oysters in the control treatment showed signs of relaxation and no significantdifference between the proportions of males and females that mortalities were recorded. spawned between methods (X2 = 8.28, P < 0.05). Significantly, more females spawned with exposure to reduced salinity and sperm compared 3.2. Baseline reproductive condition with neuropeptides, 22.5 % and 2.5 % respectively. Controls spawned across both methods, 40 % and 20 %, respectively, of these five males Histological sections prior to Experiment 1 identified three males and one female spawned (Table 4). and two females with low GI scores; zero (males) and 1.5 (females), Time to spawn was significantlydifferent between methods one and = < respectively. At the end of Experiment 1 GI values did not change, two (F1, 42 19.7, P 0.001). Within method one, time to spawn was = > however, one oyster of indeterminate gender was recorded. Histology not significantly different across treatments (F3, 18 2.83, P 0.05); sections for oysters used in Experiment 2 revealed two males and three however, post hoc Tukey HSD tests showed that there was a significant females at both sampling events (start and end) and GI was consistent difference between the time to spawn for oysters from treatment (a) salinity and sperm and (d) control (P < 0.05). Within method two, time to spawn was also not significantlydifferent between treatments (F3, 18 = 0.252, P > 0.05). Method one, salinity and sperm, had oysters from each treatment (a–d) spawn during the 2 h acclimation period; 8 oysters (20 %) (Fig. 2) of these, two oysters (5%) were female. Once treatments (a–d) were applied (Fig. 2, time zero) an additional 14 oysters (35 %) spawned within 19 min and no further oysters spawned for the remaining 4 h 41 min of the experiment (Fig. 2). Broodstock within method two did not spawn during the 2 h acclimation period and once treatments (a–d) were applied, spawning occurred from 2 min until 4 h 39 min post intramuscular injection (Fig. 3). Across both methods, Pearson chi-square tests showed significant differences between treatment and the proportions of oysters that spawned (X2 = 14.55, P < 0.05). The most successful treatment across both methods was (a) salinity and sperm, where 80 % of oysters (five males and three females) spawned within 19 min of the salinity drop and addition of sperm (Fig. 2). Among the method two (neuropeptide) Fig. 1. Cumulative percentage of Saccostrea echinata broodstock that became treatments, (a) APGWamide and buccalin performed best with 90 % of completely relaxed over time, after exposure to different concentrations of oysters spawning (8 males and one female) within 2.6 h of intramuscular MgCl2 (30, 50 and 70 g/L) repeated over two experiment days (exp1 and exp2). injection (Table 4 and Fig. 3). While treatments (b) APGWamide and (c)

5 S.J. Nowland et al. Aquaculture Reports 20 (2021) 100676

Table 3 Summary of spawning responses, fertilisation success and mortalities of oysters subject to six spawning induction treatments and strip spawning in Experiment 1.

Treatment n No. males No. females Fecundity ± SE Sperm motility ± SE Fertilisation ± SE (%; No. spawned spawned (×106; range) (%; range) range) mortalities

(1) emersion treatment 18 0 0 – – – 0 control 18 0 0 – – – 0 (2) increased temperature and treatment 9 1 0 – 80 – 0 reduced salinity control 9 0 0 – – – 0 (3) addition of sperm treatment 9 0 0 – – – 0 control 9 0 0 – – – 0 (4) addition of sperm extract treatment 18 0 0 – – – 0 control 18 0 0 – – – 0 (5) serotonin treatment 18 4 0 – 62.5 ± 6.5 (50–80) – 1 control 18 0 0 – – – 0 (6) reduced salinity and treatment 9 1 2 7.7 ± 2.4 (4.3–11.1) 80 100 0 addition of sperm control 9 2 0 – 70 – 0 (7) strip spawning treatment 34 4 4 1.9 ± 0.5 (0.1–2.8) 45 ± 7.5 (30–60) 41.5 ± 4.7 (5–85) 0

Table 4 Summary of spawning responses, fertilisation success and mortalities of oysters subject to spawning induction treatments in Experiment 2.

Method Treatment n No. males No. females Fecundity ± SE Sperm motility ± Fertilisation ± SE No. spawned spawned (×106; range) SE (%; range) (%; range) mortalities

(1) salinity and (a) reduced salinity and 10 5 3 12.9 ± 3.8 (5–21) 82 ± 2.3 (75–90) 96.7 ± 2.7 0 addition of sperm addition of sperm (90–100) (b) reduced salinity only 10 3 3 19.2 ± 6 91.3 ± 3.3 98.3 ± 1.4 0 (10.9–33.8) (85–99) (95–100) (c) addition on sperm 10 2 2 9.45 ± 6 (0.9–18) 60 ± 7.1 (50–70) 92.5 ± 5.3 0 only (85–100) (d) control 10 3 1 7.8 56.7 ± 15.2 90 0 (20–80) (2) neuropeptides (a) APGW and buccalin 10 8 1 14 71.3 ± 6.4 100 0 (40–90) (b) APGW only 10 5 0 – 72 ± 5 (50–80) – 0 (c) Buccalin only 10 6 0 – 81.7 ± (60–90) – 0 (d) control 10 2 0 – 80 ± 7.1 (70–90) – 0

Fig. 2. The effect of treatment (reduced salinity and addition of sperm, reduced Fig. 3. The effect of neuropeptide treatment (APGW and buccalin, APGW only, salinity only, addition of sperm only and controls) from Experiment 2 on time to buccalin only and controls) from Experiment 2 on time to spawn and spawning spawn and spawning percentage of Saccostrea echinata broodstock. Spawning percentage of Saccostrea echinata broodstock. Spawning percentage is cumula­ percentage is cumulative and vertical intercept line indicates the time of tive and vertical intercept line indicates the time of treatment application. treatment application. 4. Discussion buccalin showed similar spawning percentages, 50 % and 60 % of oys­ ters spawned (only males spawned). Across both the salinity and sperm, 4.1. MgCl2 relaxation of S. echinata broodstock and neuropeptides methods, no significant differences (P > 0.05) were detected between method and either fecundity, sperm motility or fer­ We have demonstrated that MgCl2 is an effective method for non- tilisation. Fecundity showed a weak, positive and non-significant cor­ destructively opening S. echinata broodstock to allow/enable intra­ = > = relation with broodstock weight (r 0.350, P 0.05) and length (r muscular injection. The concentration of 30 g/L MgCl2 was most effec­ 0.329, P > 0.05) and in the two-week recovery period no mortalities tive, with 50 % of broodstock completely relaxed after 1.5 h of exposure. were recorded. Interestingly, the 50 g/L concentration of MgCl2 recommended for the

6 S.J. Nowland et al. Aquaculture Reports 20 (2021) 100676 edible oysters, C. gigas (Suquet et al., 2009) and S. glomerata (Butt et al., salinity, rather than water temperature, was a key factor for spawning 2008), was the least effective concentration tested in this study. induction in S. echinata. This corroborates the findings of other studies Whereas, the recommended concentrations of 35 g/L for the more on S. echinata that suggest the importance of lower salinities to induce distantly related, Ostrea edulis (Culloty and Mulcahy, 1992) and 30 g/L spawning (Southgate and Lee, 1998; Nowland et al., 2019c) and for Ostrea chilensis (Alipia et al., 2014) and the commercial scallop Pecten improve larval development (Nowland et al., 2019a). Current hatchery fumatus (Heasman et al., 1995), was most effective. Both Heasman et al. practices for S. echinata are based on methods developed for S. glomerata (1995) and Alipia et al. (2014) concluded that a 30 g/L concentration of (O’Connor et al., 2008) and spawning induction for the latter is similar MgCl2 was the lowest and most cost effective concentration that induced to method two (increased temperature and reduced salinity). This ◦ rapid relaxation in broodstock without subsequent mortality. However, method involves slowly increasing seawater temperature (1 C every 10 studies with the pearl oyster, Pteria sterna, have reported that MgCl2 min), which is time consuming and can be logistically challenging. induces oxidative tissue stress and damage, without causing mortality Therefore, significant improvements could be made to hatchery pro­ (Granados-Amores et al., 2017, 2018). These studies highlight the tocols if increasing water temperature is shown to be unnecessary. importance of considering the possible stressful effects that muscle Physical removal of gametes via strip spawning is a fast and efficient relaxation methods can have in the initial phase (i.e., before relaxation), method of obtaining gametes in isolation and supports selective which is especially important to consider if stress is sufficientto elicit an breeding programs where gametes are kept separate and specific male unwanted spawning response (Arafa et al., 2007; Granados-Amores and female crosses made (Loosanoff and Davis, 1963). Although often et al., 2017). preferred in commercial hatcheries, the efficacy of this method varies In the current study, 100 % broodstock relaxation was not achieved with species; for example, success is relatively limited with S. glomerata but the most effective concentration (30 g/L on experiment day two) (O’Connor et al., 2008), while it is used routinely for C. gigas (Helm resulted in 93 % of broodstock completely relaxed after 6 h of exposure. et al., 2004). This is because the eggs of some species undergo a matu­ This is less effective than relaxation effects reported for the same con­ ration process during passage through the oviducts before they can be centration of MgCl2 when used with other species such as, 100 % successfully fertilized (Helm et al., 2004). Results from the current study relaxation in O. edulis after 90 min (Culloty and Mulcahy, 1992), 100 % demonstrate that strip spawning is a viable option for S. echinata; relaxation in S. glomerata after 6 h (Butt et al., 2008) and 96 % relaxation however, fecundity and fertilisation rates were lower and sperm motility in O. chilensis after 3 h (Alipia et al., 2014). This difference may be due to was significantlylower, when compared to induction where gametes are the use of large wild broodstock in the current study that may have a released naturally. If initial broodstock condition (GI) was higher, strip greater ability than cultured oysters to maintain closed shell valves and spawning may have proven more effective in the current study and is prevent the physiological effect of MgCl2 (Poulet et al., 2003). These worth investigating further if selective breeding programs are consid­ findings demonstrate that optimal MgCl2 concentrations for the most ered for this species. The availability of S. echinata broodstock is effective muscle relaxation in bivalves are species-specific and worth currently limited and sizeable natural populations are considered to be optimising when developing hatchery methods for a new species. rare (Nowland, 2019), so hatcheries are currently reluctant to sacrifice broodstock. 4.2. Experiment 1: investigating multiple spawning induction methods Methods for conditioning S. echinata broodstock within the hatchery are currently undeveloped and would provide benefits, including; an The number of broodstock that were induced to spawn in Experiment extended reproductive period, allowing year-round spat supply for 1 was low with only two females and 8 males spawning across all in­ farmers and elimination of the reliance and cost of collecting broodstock duction methods trialled. The GI of broodstock in the current study was from the wild (Chavez-Villalba´ et al., 2002; Lagreze-Squella, 2018). An comparatively low, however, differences were detected between understanding on the role of environmental factors such as temperature, methods and the combination of reduced salinity and addition of sperm, salinity and phytoplankton bloom events on reproduction and spawning performed best; two females and one male spawned within 47 min of of bivalve species is a critical first step to develop these methods. treatment. No oysters spawned in the emersion or sperm only methods, Research into the natural reproductive seasonality of S. echinata (Now­ and only a single male spawned in the increased temperature and land et al., 2019c) has gone some way to elucidating location specific salinity reduction method, supporting the notion that these factors act reproductive patters, however, future research should build on this synergistically. For example, Honkoop et al. (1999) found that a tem­ knowledge to develop effective conditioning protocols. For example, ◦ perature increase of 8.5 C in combination with the use of the chemical Chavez-Villalba´ et al. (2003) investigated location-specific broodstock fluoxetine triggers spawning in the marine clam, Macoma balthica, and condition and timing of gametogenesis to optimise broodstock condi­ that the temperature induction step was necessary to stimulate a suc­ tioning procedures for C. gigas in France. cessful spawning event in the laboratory. Furthermore, a combination of physical shock and chemical induction techniques are routinely used in 4.3. Experiment 2: further investigating two spawning induction methods commercial hatchery production of C. gigas (Helm and Millican, 1977), C. virginica (Wallace et al., 2008) and S. prismatica (Argüello-Guevara Broodstock were in good reproductive condition in Experiment 2; GI et al., 2013). values (4) were high and oysters spawned across the reduced salinity For temperate oyster species, the physical shock technique using and addition of sperm and neuropeptides treatments. However, brood­ water temperature is the most widely employed spawning method stock in both of the control treatments also spawned. Despite this, sig­ (Helm et al., 2004; O’Connor et al., 2008; Camacho et al., 2011). nificant differences were recorded between treatments and only one However, water temperature is less likely to play a critical role in female spawned from the control treatments. It is well documented that spawning induction of tropical oyster species, because seawater tem­ male oysters spawn more readily than females (Galtsoff, 1940; Aji, peratures are relatively stable throughout the tropics (Christo and 2011). Spawning during the acclimation period occurred in the reduced Absher, 2006; Paixao˜ et al., 2013; Nowland et al., 2019c). Salinity, on salinity and addition of sperm method but not in the neuropeptides the other hand, fluctuates seasonally due to rainfall and is therefore method, which is perhaps a result of the flow-through system design more likely to be a key physical spawning stimuli (Stephen and Shetty, compared with the static aquaria used in the subsequent method. The 1981; Aji, 2011). Rao (1950), for example, investigated the effects of flowthrough system may have provided additional physical stimulus for salinity on spawning of the Indian backwater oyster, Ostrea madrasensis, spawning, similar to the effect of tides. For example, a relationship be­ and reported spawning of wild oysters when salinity fell to within a tween spawning and spring tides has been observed for Ostrea lurida range of 20–28 ppt, which was also the optimal salinity found for em­ (Hopkins, 1936), O. edulis (Korringa, 1947) and C. gigas (Arakawa, 1990; bryonic development within the laboratory. In the current study, Bernard et al., 2016). The influence of hydrodynamic processes on

7 S.J. Nowland et al. Aquaculture Reports 20 (2021) 100676 spawning is an area for further investigation for S. echinata. 4.4. Conclusions Within the reduced salinity and addition of sperm method, the combined treatment of both stimuli was superior, with 80 % of brood­ This study demonstrated that spawning induction methods can be stock spawning within 19 min of treatment. Although the independent used to significantly improve the spawning success of S. echinata treatments of reduced salinity only and addition of sperm only resulted broodstock. The combination of physical and chemical induction tech­ in spawning, the synergistic effect, of salinity reduction and addition of nique, using a reduction in salinity and the addition of sperm, was the sperm, produced greater success. In a similar study by Argüello-Guevara most successful method investigated and has now been employed et al. (2013) addition of sperm was reported to enhance spawning suc­ routinely at the Darwin Aquaculture Centre hatchery, resulting in sig­ cess of S. prismatica compared with only physical shock treatments; nificantproduction improvements for this species, particularly the time water temperature decrease and increase and salinity reduction. This taken to induce spawning. While relaxation was not necessary for may be due to the positive spawning response of male oysters to the spawning induction, MgCl2 is a viable muscle relaxant for research ap­ presence of sperm (Aji, 2011). Under natural conditions, males respond plications with S. echinata and a concentration of 30 g/L is recom­ more readily than the females to environmental stimuli (i.e., increased mended to induce rapid muscle relaxation without subsequent temperature) and are the first to spawn, which stimulates spawning of mortality. females and other males nearby, and causes spawning to spread over an oyster bank (Galtsoff, 1930; Gosling, 2003). Based on the natural Declaration of Competing Interest reproductive patterns recorded for S. echinata in northern Australia, which display semi-continuous spawning throughout the monsoon The authors report no declarations of interest. season (October–April) (Nowland et al., 2019c), it is hypothesised that male oysters are primarily stimulated to spawn by a reduction in salinity Acknowledgements caused by a rainfall event (physical shock), which in turn stimulates females to spawn (chemical induction). This study was funded by the Australian, Northern Territory Gov­ Neuropeptides have been shown to influencethe reproductive cycle ernments, Department of Industry, Tourism and Trade and conducted of S. glomerata by accelerating gonadal maturation and triggering within the “Tropical Rock Oyster Aboriginal Economic Development spawning (In et al., 2016). Furthermore, the tetrapeptide APGWamide Program”. We thank the Warruwi community of South Goulburn Island has been demonstrated to play a role in the reproduction of C. gigas fe­ for supporting this project with the supply of broodstock. We also males, where it induces in vitro adductor muscle contraction followed recognise the significant support provided by the Darwin Aquaculture by oocyte release (Bernay et al., 2006). In the current study, intramus­ Centre staff, in particular we thank Cameron Hartley, Layla Hadden, cular injection with neuropeptides significantly improved the propor­ Paul Armstrong, Shannon Burchert and Shanika Jeewantha Arachchi for tion of oysters that spawned. The combination of both neuropeptides, their contribution during the experiments. APGWamide and buccalin, performed best, with 90 % of oysters spawning within 2.6 h of treatment. When tested independently these References neuropeptides also successfully induced spawning; however, only male oysters spawned. Compared with method one, that investigated reduced Aji, L.P., 2011. Review: spawning induction in bivalve. J. Penelitian Sains 14, 33–36. Alipia, T.T., Mae, H., Dunphy, B.J., 2014. A non-invasive anaesthetic method for salinity and addition of sperm, significantly fewer female oysters accessing the brood chamber of the Chilean flatoyster (Ostrea chilensis). N. Z. J. Mar. spawned when neuropeptides were employed. From previous work we Freshwater Res. 48 (3), 350–355. https://doi.org/10.1080/00288330.2014.909505. assumed a gender ratio of 1:1.4 (female:male) (Nowland et al., 2019c), Arafa, S., Sadok, S., Abed, A.E., 2007. Assessment of magnesium chloride as an anaesthetic for adult sea urchins (Paracentrotus lividus): incidence on mortality and however, spawning results indicated the ratio of males in this experi­ spawning. Aquacultural Research 38 (15), 1673–1678. ment was higher than this. Without knowing the gender of each oyster Arakawa, K.Y., 1990. Natural spat collecting in the pacific oyster Crassostrea gigas stocked in each method, the interpretation of these results are limited, (thunberg). Mar. Behav. Physiol. 17 (2), 95–128. https://doi.org/10.1080/ and it is likely that the combined APGWamide and buccalin method 10236249009378760. Argüello-Guevara, W., Loor, A., Sonnenholzner, S., 2013. Broodstock conditioning, would have been more effective at eliciting female spawning, if more spawning induction, and early larval development of the tropical rock oyster females at the right GI were exposed to it. Striostrea prismatica (Gray 1825). J. Shellfish Res. 32 (3), 665–670. https://doi.org/ In other molluscs, APGWamide has been shown to play a key role in 10.2983/035.032.0306. Bernard, I., Massabuau, J.C., Ciret, P., Sow, M., Sottolichio, A., Pouvreau, S., Tran, D., male reproduction through activation of genital eversion in the snails 2016. In situ spawning in a marine broadcast spawner, the Pacificoyster Crassostrea Lymnaea stagnalis (Koene, 2010) and Helix aspersa (Koene et al., 2000), gigas: timing and environmental triggers. Limnol. Oceanogr. 61 (2), 635–647. and induction of male spawning in the abalone Haliotis asinina (Chansela https://doi.org/10.1002/lno.10240. Bernay, B., Baudy-Floc’h, M., Zanuttini, B., Zatylny, C., Pouvreau, S., Henry, J., 2006. et al., 2008). Koene et al. (2000) suggested that neural control of mating Ovarian and sperm regulatory peptides regulate ovulation in the oyster Crassostrea has evolved conservatively in molluscs, which may explain why dispa­ gigas. Mol. Reprod. Dev. 73 (5), 607–616. https://doi.org/10.1002/mrd.20472. rate species display similar responses. The role of neuropeptides in Braley, R.D., 1982. Reproductive periodicity in the Indigenous oyster Saccostrea cucullatain Sasa Bay, Apra Harbor, Guam. Mar. Biol. 69, 165–173. https://doi.org/ S. echinata reproduction is still unknown, however, results from this 10.1007/BF00396896. initial investigation indicate that further research could benefithatchery Braley, R.D., 1984. Mariculture potential of introduced oysters Saccostrea cucullata production. tuberculata and Crassostrea echinata, and a histological study of reproduction of C. echinata. Aust. J. Marine Freshwater Res. 35, 129–141. https://doi.org/10.1071/ Mean fecundity per female S. echinata has been previously reported MF9840129. 6 as 21.5 × 10 oocytes (Nowland et al., 2018a). In the current study, Butt, D., O’Connor, S.J., Kuchel, R., O’Connor, W.A., Raftos, D.A., 2008. Effects of the female fecundity ranged from 0.1 to 33.8 × 106, which may suggest that muscle relaxant, magnesium chloride, on the Sydney rock oyster (Saccostrea glomerata). Aquaculture 275 (1–4), 342–346. https://doi.org/10.1016/j. some oysters were partially spawning. A semicontinuous spawning aquaculture.2007.12.004. pattern is typically of tropical bivalves and has been reported for Camacho, E.G., Dominguez, G.R., Armenta, O.O.Z., Haws, M., Supan, J., Bajo, L.J.A., C. iredalei in Malaysia (Teh et al., 2012), Crassostrea madrasensis in India Llamas, G.H., Valenzuela, J.E.V., Juarez,´ R.N.P., 2011. Developing Hatchery (Nair and Nair, 1987) and Crassostrea brasiliana in Brazil (Christo and Methods for the Mangrove Oyster, Crassostrea corteziensis for the Pacific Coast of Mexico. Final Reports: Investigations 2009–2011. Autonomous University of Absher, 2006). Enriquez-Diaz et al. (2009) reported geographic and Sinaloa, Mexico, pp. 280–288. seasonal variation in spawning intensity of C. gigas individuals within Chansela, P., Saitongdee, P., Stewart, P., Soonklang, N., Stewart, M., field sites in France. It is likely that different spawning induction Suphamungmee, W., Poomtong, T., Sobhon, P., 2008. Existence of APGWamide in the testis and its induction of spermiation in Haliotis asinina Linnaeus. Aquaculture methods applied in the hatchery may also elicit a variable response in 279 (1–4), 142–149. https://doi.org/10.1016/j.aquaculture.2008.03.058. spawning intensity. Assessing spawning intensity, therefore, may further confirm optimal spawning protocols for S. echinata.

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