Advances in pre-operative techniques for pearl production in the lions-paw scallop Nodipecten subnodosus: Relaxation and mantle excision

Aquaculture 356–357 (2012) 279–283

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Short communication

Advances in pre-operative techniques for pearl production in the lions-paw scallop Nodipecten subnodosus: Relaxation and mantle excision José A. Torres-Martínez a, Pedro E. Saucedo b, Carlos Rangel-Dávalos a, Héctor Acosta-Salmón b,⁎ a b

Universidad Autónoma de Baja California Sur (UABCS), La Paz, B.C.S., 23080, Mexico Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico

a r t i c l e

i n f o

Article history: Received 9 November 2011 Received in revised form 2 May 2012 Accepted 3 May 2012 Available online 11 May 2012 Keywords: Mantle tissue Nodipecten subnodosus Pearl production Relaxation

a b s t r a c t Relaxation and excision of mantle tissue from live lions-paw scallops can assist in the optimum development of pre-operative techniques for pearl production in this species. To determine the feasibility of relaxing the scallop, five scallops were exposed to each of nine relaxant treatments previously used for pearl seeding operations. Relaxation and suitable conditions for seeding operations were obtained in scallops exposed to 30 g L − 1 magnesium chloride and to 1 mL L− 1 2-phenoxyethanol. To determine the healing capacity and ability to sustain tissue excision, a section of mantle tissue was removed from 15 relaxed scallops. Survival 30 days after excision was 87% and all scallops showed signs of regeneration. Microscopic examination confirmed the elongation of the mantle epithelia and growth of new connective tissue. This relaxation technique is an important step in developing basic technology for pearl production, as previously accomplished with other species. This is an important tool that will improve successful production of lions-paw scallop cultured pearls. This can add significant value to the scallop-cultivating industry and the pearl industry. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In recent years, there has been an increased interest in pearls from non-Pinctada species, such as quahog clams (Veneridae), Melo melo (Volutidae), scallops (Pectinidae), abalone (Haliotidae), and even nautilus (Strack, 2011). In particular, pearls from the lions-paw scallop Nodipecten subnodosus (G.B. Sowerby I, 1835) show tints ranging from white to pink to purple. Some of these curiosities have reached extraordinary values for non-nacreous pearls (Bari et al., 2010). This scallop is one of the most important commercial shellfish species of the Baja California Peninsula and it meets the criteria (Mann, 1984) of fast growth and high value for a candidate species for aquaculture (Beltran-Lugo et al., 2008; Maguiño-Napurí et al., 2011; Osuna-Garcia et al., 2008). Developing pearl culturing methods for the lions-paw scallop will add value to this incipient industry. Relaxants have gained widespread acceptance in aquaculture for a number of processes. Fish are relaxed to reduce handling or transport stress (Ross and Ross, 1999); abalone are easily detached from cultivation tanks (White et al., 1996); and other mollusks are also relaxed to facilitate internal inspections and seeding operations for pearl production (Culloty and Mulcahy, 1992; Heasman et al., 1995; Norton et al., 1996, 2000). Developing methods for pearl production in alternative species would be more efficient if relaxation methods are used to facilitate access to internal organs, where

⁎ Corresponding author. Tel.: + 52 612 128 3434. E-mail address: [email protected] (H. Acosta-Salmón). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.05.004

grafting is conducted (Acosta-Salmón and Davis, 2007; Aquilina and Roberts, 2000). Reducing pre-operative stress can improve post-surgery survival and retention of nuclei (Norton et al., 2000). Many chemicals have been used to relax animals for pearl production, including 2-phenoxyethanol (Mamangkey et al., 2009), propylene phenoxetol (Norton et al., 1996), benzocaine (Acosta-Salmón et al., 2005; Ruiz-Rubio et al., 2006), clove oil (Norton et al., 1996), MS-222 (Saucedo et al., 2001), and magnesium chloride (Acosta-Salmón and Davis, 2007). These chemicals are effective, safe to use, affordable, and easy to obtain from specialized suppliers. Of these chemicals, magnesium chloride has been used with success to relax scallops (Pecten fumatus: Heasman et al., 1995). In the pearl oyster industry, mantle tissue is essential for inducing the formation of cultured pearls and is obtained from sacrificed donors and then grafted into recipient oysters to grow a cultured pearl. Also, mantle tissue can be excised from relaxed live donors and used for pearl production (Acosta-Salmón and Southgate, 2005; Acosta-Salmón et al., 2004; Mamangkey, 2009). Since the quality of a pearl is greatly determined by the quality of the mantle donor (McGinty et al., 2010; Taylor, 2002), keeping high quality mantle donors alive provides significant benefits to the pearl industry (Acosta-Salmón et al., 2004). These include the use of donors as future broodstock to improve the quality of future mantle donors and increase the value of the pearl harvest. Relaxed animals are able to survive the excision of a large piece of mantle tissue, heal the wound, and completely regenerate this part of the mantle and all its internal structures in less than three months (Acosta-Salmón and Southgate, 2005, 2006; Mamangkey and Southgate, 2009). Relaxation

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and excision of mantle tissue from live lions-paw scallops can assist developing optimal techniques for pearl production in this species. To assess this scallop for pearl cultivation, this study was conducted to determine the feasibility of safely relaxing lions-paw scallops and observing the process of healing and regeneration after excising mantle tissue. 2. Materials and methods Seventy adult lions-paw scallops, Nodipecten subnodosus, were collected in Laguna Guerrero Negro, Baja California Sur, Mexico (28°01′N, 114°11′W); their mean (± SD) shell height, shell length, and live weight was 126.6 mm (±4.4 mm), 132.9 mm (± 5.5 mm), and 562.3 g (±67.5 g), respectively. Scallops were transported in insulated coolers to maintain temperature (17–20 °C) to ensure survival. At the laboratory, they were cleaned and acclimated to experimental conditions for 10 days in a flow-through system that provided gently-aerated, 1 μm, filtered seawater (18 °C) and food consisting of a 1:1:1 mix of the microalgae Isochrysis galbana, Chaetoceros calcitrans, and Pavlova lutheri. Holding tanks were cleaned and refilled with clean seawater daily. 2.1. Relaxation To evaluate the effects of relaxant treatments, 50 of the collected scallops were divided into ten groups of five scallops each. Nine groups were exposed to the treatments described in Table 1. Five scallops were used as the control to determine the effect of handling on survival. Magnesium chloride was added directly to the seawater and stirred until dissolved. Eugenol, 2-phenoxyethanol, and propylene phenoxetol were shaken with seawater in small containers to disperse the chemicals into small droplets before adding the chemicals to the units holding the scallops (Mamangkey et al., 2009; Norton et al., 1996). To prepare the 250 and 500 mg L − 1 concentrations of benzocaine, a 1:4 w/v working solution of benzocaine-methanol was made and the desired amount of this solution was poured into hot water (92–96 °C) to ensure that the benzocaine dissolved (Acosta-Salmón et al., 2005). This solution was then poured into the chambers with seawater to reach the desired concentrations. Only magnesium chloride increased salinity (56 psu); all the other chemicals did not increase this parameter. For each relaxant, five 5 L units (replicates) were used, each containing one scallop. A plastic wedge was used to maintain the valves of each scallop open to allow immediate and simultaneous exposure to the relaxant after immersion. The experiment was conducted in a chamber set at 18 °C. Table 1 Chemicals and concentrations used to induce relaxation in the lions-paw scallop, Nodipecten subnodosus. Treatment 30 g L

−1

of magnesium chloride

0.5 mL L− 1 eugenol (clove oil) 250 mg L− 1 benzocaine 500 mg L− 1 benzocaine 0.25 g L− 1 menthol crystals 1 mL L− 1 propylene phenoxetol 2.5 mL L− 1 propylene phenoxetol

1 mL L− 1 2-phenoxyethanol 3 mL L− 1 2-phenoxyethanol

Use and reference* Ostrea edulisa, Strombus gigasb, Pecten fumatusc Pinctada albinad, P. maxima (modified from sourcee) Pteria sternaf, P. margaritiferag S. gigasb, P. maximae, P. margaritiferag, Pinctada albinad, This study Pinctada albinad, P. margaritiferad,g, P. maximae and Haliotis irish This study P. maximae, Haliotis irish

*Unless stated otherwise, these treatments have been used to induce relaxation in different species of mollusks in previous studies. aCulloty and Mulcahy, 1992; bAcostaSalmón and Davis, 2007; cHeasman et al., 1995; dNorton et al., 1996; eMamangkey et al., 2009; fRuiz-Rubio et al., 2006; gAcosta-Salmón et al., 2005; Aquilina and Roberts, 2000.

After exposure to the relaxants, scallops were continuously observed for up to one hour for signs of relaxation or for signs of unsuitability for seeding operations, such as mucus production, damaged gills, or mantle or body collapse (Acosta-Salmón et al., 2005; Mamangkey et al., 2009; O'Connor and Lawler, 2003). To determine whether a scallop was relaxed, the mantle was lightly touched with a flat metal spatula. If a strong muscular contraction was observed, the scallop was left undisturbed. If only a slight muscular response or no response was observed, the scallop was considered relaxed and the time until relaxation occurred was recorded. When relaxed, scallops were constantly observed for up to 30 min to determine the condition of the mantle and gills. At each observation, scallops were classified as either: relaxed (scallops showed signs of suitability for seeding operations) or unsuitable for seeding (scallops showed any of the previously mentioned responses). All unsuitable scallops were placed in clean seawater for recovery. After 30 min of observation, relaxed scallops were returned to clean seawater for recovery. Following these trials, scallops were returned to cultivating conditions at a depth of 10 m in a bottom-culture trestle system at El Merito, Baja California Sur, Mexico. Survival was recorded one month later. The Mann–Whitney U test (P b 0.05) was used to determine whether there were differences between the mean time required for scallops to relax in the two successful treatments (30 g L− 1 of magnesium chloride and 1 mL L − 1 2-phenoxyethanol). 2.2. Experimental wounding and tissue regeneration Fifteen scallops were used to determine if mantle excision from live scallops was feasible. Scallops were relaxed with 30 g L − 1 magnesium chloride, as described above, since this treatment proved to be effective in the relaxation trials. Once relaxed, a section of mantle tissue (50 mm × 10 mm) was cut from the ventral part. These scallops were returned to clean seawater and were maintained under cultivating conditions, as previously mentioned for 30 days, to determine survival. To determine the degree of regeneration of mantle tissue, all scallops were sacrificed and the regenerating mantle samples were sectioned for standard histological examination (Acosta-Salmón and Southgate, 2005; Acosta-Salmón et al., 2004); briefly, samples were preserved in Davidson's solution for 48 h, dehydrated, embedded in Paraplast XT, thin-sectioned to 5 μm, and stained with H–E (Howard and Smith, 1983). Finished slides were studied under a compound microscope at 10 × and 20×. 3. Results 3.1. Relaxation Four scallops exposed to 30 g L − 1 magnesium chloride relaxed in 20 to 30 min (Fig. 1). Scallops remained relaxed for 30 min, and only one of five scallops showed damage to the gills; however, the five scallops recovered after 20 min in clean seawater. Scallops exposed to 1 mL L− 1 2-phenoxyethanol relaxed between 32 and 42 min after exposure and remained in this condition for 30 min (Fig. 1). Recovery occurred in clean water after 25 min. Scallops relaxed faster (Mann–Whitney U test P = 0.013) with 30 g L− 1 magnesium chloride than with 1 mL L − 1 2-phenoxyethanol. Of scallops exposed to 3 mL L − 1 2-phenoxyethanol, their mantles collapsed within 23 min of exposure (Fig. 2). Scallops exposed to 0.5 mL L− 1 eugenol (Fig. 2) relaxed within 15 min. The mantle collapsed in four scallops after exposure for 25 min. The fifth scallop did not show a negative response after 30 min. Recovery time was not determined. Scallops exposed to 250 mg L − 1 benzocaine did not relax. Within 15 min, the mantle of three scallops collapsed and the other two had

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number of scallops

1 mL L-1 2-phenoxyethanol 5 4 3 2 1 0 1

2

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10 11 12 13 14 15 16 17

30 g L-1 magnesium chloride number of scallops

5 4 3 2 1

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wide, gaping valves, but retained muscular tone and strength for 1 h. Of scallops exposed to 500 mg L − 1 benzocaine, the mantle of the five scallops collapsed within 11 min (Fig. 2). The treatment of 0.25 g L− 1 menthol crystals was ineffective; menthol solidified immediately after contact with cold (18 °C) water. Relaxation was not observed in scallops exposed to this treatment. Three scallops exposed to 1 mL L − 1 propylene phenoxetol relaxed within 18 min. Of these, two remained relaxed for 30 min and the third scallop showed mantle collapse before 25 min exposure. The remaining two scallops revealed signs of mantle collapse before 20 min of exposure (Fig. 2). Of scallops exposed to 2.5 mL L − 1 propylene phenoxetol, three relaxed within 6 min, but their mantles collapsed after 20 min. The other two did not relax and their mantles collapsed after 10 min (Fig. 2). No significant differences in the survival of scallops 30 days after exposure to the treatments were observed between treatments. Those exposed to 30 g L − 1 magnesium chloride had 100% survival. At the other extreme, the lowest survival (60%) occurred in scallops exposed to 0.5 mL L − 1 eugenol, and 0.25 g L − 1 menthol crystals.

0 1

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3.2. Experimental wounding and tissue regeneration

time (min) Fig. 1. Number of lions-paw scallops that: did not relax ( ), relaxed (■), and were unsuitable ( ) for operation, and number of relaxed scallops returned to clean water for recovery (□) at different times after exposure to the two most successful relaxant treatments. Unsuitable scallops were returned to clean water for recovery.

Survival of scallops 30 d after excision was 87%. Mantle regeneration occurred in all scallops. Histological sections showed details of the regenerated mantle (Fig. 3). A hemocyte plug and growth of connective tissue was present, as well as several large hemolymph

2.5 mL L-1 propylene phenoxetol

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1 mL L-1 propylene phenoxetol 5

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500 mg L-1 benzocaine

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0.5 mL L-1 eugenol number of scallops

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Fig. 2. Number of lions-paw scallops that: did not relax ( ), relaxed (■), and were unsuitable ( ) for operation at different times after exposure to six relaxant treatments. Scallops exposed to 0.25 g L− 1 menthol crystals did not relax and are not shown. Unsuitable scallops were returned to clean water for recovery.

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Fig. 3. Histological view of regenerated mantle tissue in Nodipecten subnodosus 30 days after mantle excision. ct = connective tissue, ee = external epithelium, hp = hemocyte plug, hv = hemolymph vessel, ie = internal epithelium, lm = longitudinal muscles, p = periostracum secretion, rm = radial muscles.

vessels. The rudiments of a mantle lobe and periostracum secretion were also observed. 4. Discussion Similar to other species used for pearl production, scallops were easily relaxed and were able to survive the excision of a significant section of mantle tissue, with very low or no mortality. Scallops were successfully relaxed with 30 g L − 1 magnesium chloride or 1 mL L − 1 2-phenoxyethanol. This concentration of magnesium chloride was used to induce relaxation in other mollusks, including the scallop P. fumatus (Heasman et al., 1995). This chemical increases salinity and some studies have used this chemical dissolved in distilled water to compensate for the increase in salinity (Butt et al., 2008). However, similar to our results, the increased salinity did not have harmful effects on the queen conch also used during the development of similar techniques to assist pearl production (Acosta-Salmón and Davis, 2007). While 2-phenoxyethanol at 3 mL L− 1 has been used with success in pearl oysters and abalone (Mamangkey et al., 2009; White et al., 1996), this treatment did not relax scallops and provoked mantle collapse after 23 min exposure. A lower concentration of 2-phenoxyethanol (1 mL L− 1) was successful for relaxing lions-paw scallops. The 0.5 mL L− 1 eugenol treatment relaxed the scallops, but provoked mantle collapse after 25 min exposure. Two of the scallops spawned after recovery. Some relaxants reduce the incidence of unplanned spawning (Heasman et al., 1995). In this trial, scallops were collected in March, when gametogenesis starts. This species spawns mainly in May and in October through November every year (Arellano-Martínez et al., 2004); therefore, spawning may be attributed to the treatment. Different results have been obtained in other bivalves exposed to eugenol. In the gold-lipped pearl oyster (Pinctada maxima) for example, excess of mucus production and mortality was observed after exposure to 1.5 mL L− 1 clove oil (Mamangkey et al., 2009). However, the pearl oyster (Pinctada albina) relaxed and did not produce mucus when exposed to 0.5 and 1.5 mL L− 1 clove oil (Norton et al., 1996), which is consistent with our findings with lions-paw scallops. Benzocaine was unsuccessful at inducing relaxation in the lions-paw scallop, as was also reported for the queen conch Strombus gigas (Acosta-Salmón and Davis, 2007) and the blackfoot paua abalone Haliotis iris (Aquilina and Roberts, 2000). Scallops exposed to the low concentration (250 mg L − 1) did not relax and those exposed to the high concentration (500 mg L − 1) had collapsed mantle tissue within 11 min. It seems that lions-paw scallops tolerate very narrow concentrations of relaxants or short times of exposure to relaxants. Regardless, mantle tissue was the organ that first showed the side effects of the relaxant treatments.

The most common side effect in the scallops exposed to relaxant treatments was collapse of the mantle. No excessive production of mucus or body collapse was observed and only one individual showed gill damage (30 g L − 1 magnesium chloride). The damage to the gills was presumably repaired quickly because all scallops undergoing this treatment survived. Mantle collapse has been widely documented (Acosta-Salmón et al., 2005; Mamangkey et al., 2009; Norton et al., 1996; O'Connor and Lawler, 2003) as a side effect to relaxant treatments. Mantle collapse of scallops in our study was considered an unsuitable response for pearl seeding, since the mantle loses its tone and falls into the soft tissues, which makes our attempts for exploration or operation difficult. Additionally, scallops with collapsed mantles should not be used as mantle donors, since healing and developmental capacity of collapsed mantle is yet unknown (Acosta-Salmón et al., 2005). Mantle tissue removal from relaxed scallops was successfully completed and most survived the excision. Although the scallops were much slower to regenerate than reports for other species (Acosta-Salmón and Southgate, 2005, 2006), epithelial cells proliferated in the mantle tissue of lions-paw scallops. Similar findings were previously reported for three pearl oysters—Pinctada fucata, P. margaritifera, and P. maxima (Acosta-Salmón and Southgate, 2005, 2006; Mamangkey and Southgate, 2009). The pallial artery could not be observed in our mantle samples, but will likely evolve from the large hemolymph vessels present in the regenerated section, since elongation of epithelia and growth of new connective tissue had taken place. However, a hemocyte plug that disappears a few days after wounding the pearl oysters (Acosta-Salmón and Southgate, 2006), was still present in our scallops after 30 days. Mantle regeneration after excision was incipient, compared to what occurs in pearl oysters (Acosta-Salmón and Southgate, 2005, 2006). After 30 days, the extent of regeneration of mantle in this trial was equivalent to mantle that regenerated in 6 to 15 days in the Akoya pearl oyster P. fucata (Acosta-Salmón and Southgate, 2005). The mantle tissue in scallops shows a higher degree of complexity, since this organ assists the scallop during swimming (Stephens, 1978) and also provides highly developed sensory functions (ocelles) that are absent in Pinctada pearl oysters. Presumably, these structures require a longer period to regenerate. The usefulness of relaxation treatment in scallops has been described. Estimating reproductive stages, identification of possible diseases, and induction of spawning, using intragonadal or intramuscular injections are some activities that are simplified with the use of appropriate relaxation techniques in scallops (Heasman et al., 1995). Although scallops tend to gape after air exposure, a wedge is required to maintain the valves open, which in non-relaxed scallops can lead to physical injury of mantle tissue, tearing of the adductor muscle, or rupture of the hinge ligament (Heasman et al., 1995). With the potential for pearl

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production in the lions-paw scallop, the usefulness of a relaxation technique is increased significantly. 5. Conclusion This study showed that the lions-paw scallop N. subnodosus can be relaxed in 20–40 min by using 30 g L − 1 magnesium chloride or 1 mL L − 1 of 2-phenoxyethanol. Also, we demonstrated that scallops are able to survive excision of a section of mantle tissue. During relaxation, scallops showed an extended mantle and a slow response to physical manipulation. After recovery, scallops showed very high survival rates. The relaxation technique is an important step in developing basic technology for producing pearls in this species, as has been done with other species. This important tool will improve the potential for successful production of cultured pearls in lions-paw scallops. In turn, this can add significant value to the scallop industry and the pearl industry. Acknowledgements The authors thank Philippe Danigo (Marimex del Pacífico) for donating the scallops. We also thank the following staff at CIBNOR: Mario Osuna-García, José D. Barajas-Frias, and Pablo Ormart-Castro for technical support maintaining the scallops; Carmen RodríguezJaramillo for processing of the mantle tissue samples for histological examination; Enrique Calvillo-Espinoza, Jorge Angulo-Calvillo, and Juan José Ramirez-Rosas for field support; and Ira Fogel for editorial improvements. This study was part of a thesis funded by SEP-CONACYT Project 81249 (“Evaluación de la calidad gonádica de la almeja mano de león, Nodipecten subnodosus y su influencia en la viabilidad larvaria”). References Acosta-Salmón, H., Davis, M., 2007. Inducing relaxation in the queen conch Strombus gigas (L.) for cultured pearl production. Aquaculture 262, 73–77. Acosta-Salmón, H., Southgate, P.C., 2005. Mantle regeneration in the pearl oysters Pinctada fucata and Pinctada margaritifera. Aquaculture 246, 447–453. Acosta-Salmón, H., Southgate, P.C., 2006. Wound healing after excision of mantle tissue from the Akoya pearl oyster, Pinctada fucata (Gould). Comparative Biochemistry and Physiology Part A 143, 264–268. Acosta-Salmón, H., Martínez-Fernández, E., Southgate, P.C., 2004. A new approach to pearl oyster broodstock selection: can saibo donors be used as future broodstock? Aquaculture 231, 205–214. Acosta-Salmón, H., Martínez-Fernández, E., Southgate, P.C., 2005. Use of relaxants to obtain saibo tissue from the blacklip pearl oyster Pinctada margaritifera and the Akoya pearl oyster Pinctada fucata. Aquaculture 246, 167–172. Aquilina, B., Roberts, R., 2000. A method for inducing muscle relaxation in the abalone, Haliotis iris. Aquaculture 190, 403–408. Arellano-Martínez, M., Ceballos-Vázquez, B.P., Villalejo-Fuerte, M., García-Domínguez, F., Elorduy-Garay, J.F., Esliman-Salgado, A., Racotta, I.S., 2004. Reproduction of the lion's paw scallop Nodipecten subnodosus Sowerby, 1835 (Bivalvia: Pectinidae) from Laguna Ojo de Liebre, B.C.S., México. The Journal of Shellfish Research 23, 723–729. Bari, H., Lam, D., Fried, K., 2010. Pearls. Rizzoli, New York. 336 pp.

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