INTRODUCTION
⌅The abalone (Haliotis spp.) fishery is one of the oldest in Mexico ( Cox 1962 Cox K.W. 1962. California Abalones, Family Haliotidae. Fish Bull. 118: 1-131.). Currently, abalone products are important commodities in national and international markets, and their exploitation is sustained by several socio-economic activities conducted along the Pacific coast of the Baja California peninsula ( Searcy-Bernal et al. 2010 Searcy-Bernal R., Ramade-Villanueva M.R., Altamira B. 2010. Current Status of abalone fisheries and culture in Mexico. J. Shellfish Res. 29: 573-576. https://doi.org/10.2983/035.029.0304 , Cook 2014 Cook P.A. 2014. The Worldwide Abalone Industry. Mod. Econ. 5: 1181-1186. https://doi.org/10.4236/me.2014.513110 ). The high demand for abalone has led to intense fishing pressure and dramatic declines in wild populations ( Morales-Bojórquez et al. 2008 Morales-Bojórquez E., Muciño-Díaz M.O., Vélez-Barajas J.A. 2008. Analysis of the decline of the abalone fishery (Haliotis fulgens and H. corrugata) along the westcentral coast of the Baja California peninsula, Mexico. J. Shellfish Res. 27: 865-870. https://doi.org/10.2983/0730-8000(2008)27[865:AOTDOT]2.0.CO;2 ). To compensate for these declines, Mexican abalone aquaculture has rapidly increased ( Lafarga-De la Cruz and Gallardo-Escárate 2011 Lafarga-De la Cruz F., Gallardo-Escárate C. 2011. Intraspecies and interspecies hybrids in Haliotis: Natural and experimental evidence and its impact on abalone aquaculture. Rev. Aquac. 3: 74-99. https://doi.org/10.1111/j.1753-5131.2011.01045.x ). However, several challenges continue to hamper final production outcomes.
Worldwide, a notable bottleneck in abalone aquaculture arises due to the difficulties associated with maintaining individuals through the weaning stage, which encompasses the transition from post-settlement diatom-fed abalone to the juvenile stage macroalgae-fed abalone. The reasons for high abalone mortality during the weaning stage are still poorly understood, although they have been related to ontogenetic development in the morphology of the feeding apparatus (radula development mainly), in addition to the types of digestive enzymes present and their activities ( Johnston et al. 2005 Johnston D., Moltschaniwskyj N., Wells J. 2005. Development of the radula and digestive system of juvenile blacklip abalone (Haliotis rubra): Potential factors responsible for variable weaning success on artificial diets. Aquaculture 250: 341-355. https://doi.org/10.1016/j.aquaculture.2005.03.012 ). Moreover, food consumption in postlarvae abalone initially increases exponentially (up to a maximum point) as they grow. Thus, inadequate diets or an inability of the postlarvae to ingest and assimilate nutrients may result in delayed metamorphosis, starvation, slow growth, and ultimately death ( Takami et al. 2002 Takami H., Kawamura T., Yamashita Y. et al. 2002. Effects of delayed metamorphosis on larval competence, and postlarval survival and growth of abalone Haliotis discus hannai. Aquaculture 213: 311-322. https://doi.org/10.1016/S0044-8486(02)00338-1 , Johnston et al. 2005 Johnston D., Moltschaniwskyj N., Wells J. 2005. Development of the radula and digestive system of juvenile blacklip abalone (Haliotis rubra): Potential factors responsible for variable weaning success on artificial diets. Aquaculture 250: 341-355. https://doi.org/10.1016/j.aquaculture.2005.03.012 , Dyck et al. 2010 Dyck M., Roberts R., Jeffs A. 2010. Use of algal diets to aid early weaning in the abalone Haliotis iris. J. Shellfish Res. 29: 613-620. https://doi.org/10.2983/035.029.0309 ). To increase abalone survival during the weaning stage, farmers around the world have directed their efforts towards the use of (i) mixed diets to meet the feeding and energetic necessities of developing postlarvae ( Correa-Reyes et al. 2001 Correa-Reyes J.G., Sánchez-Saavedra M. del P., Siqueiros-Beltrones D.A., Flores-Acevedo N. 2001. Isolation and growth of eight strains of benthic diatoms, cultured under two light conditions. J. Shellfish Res. 20: 603-610., Parker et al. 2007 Parker F., Davidson M., Freeman K., et al. 2007. Investigation of optimal temperature and light conditions for three benthic diatoms and their suitability to commercial scale nursery culture of abalone (Haliotis laevigata). J. Shellfish Res. 26: 751-761. https://doi.org/10.2983/0730-8000(2007)26[751:IOOTAL]2.0.CO;2 , Hernández et al. 2009 Hernández J., Uriarte I., Viana M.T., et al. 2009. Growth performance of weaning red abalone (Haliotis rufescens) fed with Macrocystis pyrifera plantlets and Porphyra columbina compared with a formulated diet. Aquac. Res. 40: 1694-1702. https://doi.org/10.1111/j.1365-2109.2009.02267.x ) and (ii) selective breeding and abalone hybridization to generate more resistant lineages ( Lafarga-de la Cruz and Gallardo-Escárate 2011 Lafarga-De la Cruz F., Gallardo-Escárate C. 2011. Intraspecies and interspecies hybrids in Haliotis: Natural and experimental evidence and its impact on abalone aquaculture. Rev. Aquac. 3: 74-99. https://doi.org/10.1111/j.1753-5131.2011.01045.x ).
Under culture conditions, seaweeds and benthic diatoms play pivotal roles in abalone maintenance by inducing larval settlement and serving as the main nourishment sources during the early postlarvae stage. Moreover, postlarval growth rates appear to be modulated by the nutritional value, size, availability and digestibility of the supplied diet ( Carbajal-Miranda et al. 2005 Carbajal-Miranda M.J., Sánchez-Saavedra M.D.P., Simental J.A. 2005. Effect of monospecific and mixed benthic diatom cultures on the growth of red abalone postlarvae Haliotis rufescens (Swainson 1822). J. Shellfish Res. 24: 401-405. https://doi.org/10.2983/0730-8000(2005)24[401:EOMAMB]2.0.CO;2 , Correa-Reyes et al. 2009 Correa-Reyes J.G., Sánchez-Saavedra M. del P., Viana M.T et al. 2009. Effect of eight benthic diatoms as feed on the growth of red abalone (Haliotis rufescens) postlarvae. J. Appl. Phycol. 21: 387-393. https://doi.org/10.1007/s10811-008-9381-x ). Therefore, the diets used in farmed abalone should be composed of different algae to fulfil the nutritional needs of each developmental stage ( Hernández et al. 2009 Hernández J., Uriarte I., Viana M.T., et al. 2009. Growth performance of weaning red abalone (Haliotis rufescens) fed with Macrocystis pyrifera plantlets and Porphyra columbina compared with a formulated diet. Aquac. Res. 40: 1694-1702. https://doi.org/10.1111/j.1365-2109.2009.02267.x ). The use of mixed diets has not yet been universally adopted in abalone aquaculture; for example, farms continue to rely on mono-diets consisting of the macroalgae Macrocystis pyrifera because of its availability, ease of harvest and low cost. However, the poor biochemical composition of M. pyrifera (5%-12% proteins, 0.5%-1% lipids, and 46%-50% carbohydrates) may be insufficient to meet the nutritional needs of weaning abalone ( Simental et al. 2004 Simental J.A., Sanchez-Saavedra M.D., Flores-Acevedo N. 2004. Growth and survival of juvenile red abalone (Haliotis rufescens) fed with macroalgae enriched with a benthic diatom film. J. Shellfish Res. 23: 995-999.). In addition, M. pyrifera can become scarce or unavailable during winter months and in response to certain environmental changes and conditions such as those associated with the El Niño-Southern Oscillation (ENSO; Edwards 2019 Edwards M.S. 2019. Comparing the impacts of four ENSO events on giant kelp (Macrocystis pyrifera) in the northeast Pacific Ocean. Algae 34: 141-151. https://doi.org/10.4490/algae.2019.34.5.4 ).
Moreover, the use of both diatoms (e.g. Navicula spp.) and macroalgae (e.g. Ulva spp.) in abalone aquaculture systems has generated promising results by promoting the growth and survival of postlarvae ( Simental et al. 2004 Simental J.A., Sanchez-Saavedra M.D., Flores-Acevedo N. 2004. Growth and survival of juvenile red abalone (Haliotis rufescens) fed with macroalgae enriched with a benthic diatom film. J. Shellfish Res. 23: 995-999., Strain et al. 2006 Strain L.W.S., Borowitzka M.A., Daume S. 2006. Growth and survival of juvenile greenlip abalone (Haliotis laevigata) feeding on germlings of the macroalgae Ulva sp. J. Shellfish Res. 25: 239-247. https://doi.org/10.2983/0730-8000(2006)25[239:GASOJG]2.0.CO;2 , Correa-Reyes et al. 2009 Correa-Reyes J.G., Sánchez-Saavedra M. del P., Viana M.T et al. 2009. Effect of eight benthic diatoms as feed on the growth of red abalone (Haliotis rufescens) postlarvae. J. Appl. Phycol. 21: 387-393. https://doi.org/10.1007/s10811-008-9381-x ). Specifically, Ulva sp. has been shown to reduce postlarval settlement, although it increases the survival rates of farmed abalone ( Muñoz et al. 2012 Muñoz P., Ambler R., Bulboa C. 2012. Settlement, Survival, and post-larval growth of red abalone, Haliotis rufescens, on polycarbonate plates treated with germlings of Ulva sp. J. World Aquac. Soc. 43: 890-895. https://doi.org/10.1111/j.1749-7345.2012.00615.x ), so it is often supplied along with an easily digestible diatom ( Daume et al. 2004 Daume S., Huchette S., Ryan S., Day R.W. 2004. Nursery culture of Haliotis rubra: the effect of cultured algae and larval density on settlement and juvenile production. Aquaculture 236: 221-239. https://doi.org/10.1016/j.aquaculture.2003.09.035 , Daume 2006 Daume S. 2006. The roles of bacteria and micro and macro algae in abalone aquaculture: A review. J. Shellfish Res. 25: 151-157. https://doi.org/10.2983/0730-8000(2006)25[151:TROBAM]2.0.CO;2 ). A potential alternative commercial nutritional source for postlarvae abalone is Navicula spp. These benthic diatoms are among those most used in the production of algal films for abalone culture ( Siqueiros-Beltrones and Domenico 2000 Siqueiros-Beltrones D.A., Domenico V. 2000. Grazing selectivity of red abalone Haliotis rufescens postlarvae on benthic diatom films under culture conditions. J. World Aquac. Soc. 31: 239-246. https://doi.org/10.1111/j.1749-7345.2000.tb00359.x ). Moreover, in controlled feeding systems of red abalone (Haliotis rufescens), monospecific cultures of Navicula incerta have generally been shown to increase both the growth and survival rates of postlarvae ( Correa-Reyes et al. 2001 Correa-Reyes J.G., Sánchez-Saavedra M. del P., Siqueiros-Beltrones D.A., Flores-Acevedo N. 2001. Isolation and growth of eight strains of benthic diatoms, cultured under two light conditions. J. Shellfish Res. 20: 603-610.). Thus, as proposed by Carbajal-Miranda et al. (2005) Carbajal-Miranda M.J., Sánchez-Saavedra M.D.P., Simental J.A. 2005. Effect of monospecific and mixed benthic diatom cultures on the growth of red abalone postlarvae Haliotis rufescens (Swainson 1822). J. Shellfish Res. 24: 401-405. https://doi.org/10.2983/0730-8000(2005)24[401:EOMAMB]2.0.CO;2 , the use of mixed diets composed of these algal species could induce higher growth rates in postlarvae abalone.
Hybridization has recently been proved to be a simple and effective way to increase the yields of commercially important molluscs, even after only a few generations ( de Melo et al. 2016 de Melo C.M.R., Durland E., Langdon C. 2016. Improvements in desirable traits of the Pacific oyster, Crassostrea gigas, as a result of five generations of selection on the West Coast, USA. Aquaculture 460: 105-115. https://doi.org/10.1016/j.aquaculture.2016.04.017 , Li et al. 2018 Li J., Wang M., Fang J., et al. 2018. A comparison of offspring growth and survival among a wild and a selected strain of the Pacific abalone (Haliotis discus hannai) and their hybrids. 495: 721-725. https://doi.org/10.1016/j.aquaculture.2018.06.071 ). Currently, hybridization is a widely used strategy to improve the growth, behaviour and flavour of many commercially important plant and animal species, in addition to their reproductive and processing characteristics ( Hamilton et al. 2009 Hamilton M., Kube P., Elliott N., et al. 2009. Development of a breeding strategy for hybrid abalone. Proc. Assoc. Adv. Anim. Breed. Genet. 18: 350-353.). Although hybridization has been successfully applied in abalone breeding, the physiological and molecular mechanisms underlying abalone hybrid superiority remain mostly unknown. Possible explanations of hybrid advantages over pure species may include (i) heterosis, (ii) complementarity and (iii) greater allelic diversity through recombination ( Hamilton et al. 2009 Hamilton M., Kube P., Elliott N., et al. 2009. Development of a breeding strategy for hybrid abalone. Proc. Assoc. Adv. Anim. Breed. Genet. 18: 350-353.). Notably, the acquisition of new traits in hybrid offspring relies upon both the morphological and physiological characteristics of the paternal species based on the possible combinations of male and female gametes, with higher genetic similarity to maternal traits generally observed in the offspring ( Lafarga-De la Cruz and Gallardo-Escárate 2011 Lafarga-De la Cruz F., Gallardo-Escárate C. 2011. Intraspecies and interspecies hybrids in Haliotis: Natural and experimental evidence and its impact on abalone aquaculture. Rev. Aquac. 3: 74-99. https://doi.org/10.1111/j.1753-5131.2011.01045.x , Liang et al. 2014 Liang S., Luo X., You W., Luo L., Ke C. 2014. The role of hybridization in improving the immune response and thermal tolerance of abalone. Fish Shellfish Immunol. 39: 69-77. https://doi.org/10.1016/j.fsi.2014.04.014 ).
Red abalone is known for its relatively rapid growth ( Valenzuela-Miranda et al. 2015 Valenzuela-Miranda D., Del Río-Portilla M.A., Gallardo-Escárate C. 2015. Characterization of the growth-related transcriptome in California red abalone (Haliotis rufescens) through RNA-Seq analysis. Mar. Genomics 24: 199-202. https://doi.org/10.1016/j.margen.2015.05.009 ), and green abalone (H. fulgens) is particularly resistant to low nutrient or starvation conditions ( Durazo-Beltrán et al. 2003 Durazo-Beltrán E., D’Abramo L.R., Toro-Vazquez J.F., et al. 2003. Effect of triacylglycerols in formulated diets on growth and fatty acid composition in tissue of green abalone (Haliotis fulgens). Aquaculture 224: 257-270. https://doi.org/10.1016/S0044-8486(03)00223-0 ). Indeed, during both the juvenile and adult stages, green abalone may survive for several weeks under restricted food regimes by successively metabolizing stored carbohydrates first, followed by lipids and finally proteins ( Durazo-Beltrán et al. 2004 Durazo-Beltrán E., Viana M.T., D’Abramo L.R., Toro-Vazquez J.F. 2004. Effects of starvation and dietary lipid on the lipid and fatty acid composition of muscle tissue of juvenile green abalone (Haliotis fulgens). Aquaculture 238: 329-341. https://doi.org/10.1016/j.aquaculture.2004.03.025 , Viana et al. 2007 Viana M.T., D’Abramo L.R., Gonzalez M.A., et al. 2007. Energy and nutrient utilization of juvenile green abalone (Haliotis fulgens) during starvation. Aquaculture 264: 323-329. https://doi.org/10.1016/j.aquaculture.2007.01.004 ). In this study, we evaluated abalone growth in terms of shell length (SL) and the survival rates of weaning abalone supplied with six different diets. In addition, physiological performance was compared between pure red abalone (RR) and a hybrid obtained by crossing female red abalone and male green abalone gametes (RG). This combination was chosen to generate a new hybrid cross characterized by a high growth rate that is more suitable for captive maintenance.
MATERIALS AND METHODS
⌅Abalone rearing and maintenance
⌅Our experimental design addressed two main scientific questions: (i) to what extent does each diet affect the growth and survival rates of postlarvae abalone during the weaning phase (<5 mm SL) and (ii) does the growth and survival rates of a hybrid (H. rufescens [♀] × H. fulgens [♂]; RG) exceed those of pure red abalone (H. rufescens [♀] × H. rufescens [♂]; RR).
To answer these questions, pure and hybrid crosses were produced at the Aquaculture Department of the Center for Research and Higher Education of Ensenada (CICESE). Competent larvae of pure and hybrid abalone were produced by crossing 12 female red abalone and three red and three green male abalone. Initially, adult spawn was stimulated with the TRIS-H2O2 method described by Morse et al. (1977) Morse D.E., Duncan H., Hooker N., Morse A. 1977. Hydrogen peroxide induces spawning in mollusks, with activation of prostaglandin endoperoxide synthetase. Science 196: 298-300. https://doi.org/10.1126/science.403609 . After 4 h post-fertilization, the oocytes were randomly sampled and collected by sieving to corroborate the presence of fertilized oocytes. For this, each oocyte was observed in a Sedgewick-Rafter counting chamber with the help of a Nikon ECLIPSE E200 optical microscope. Fertilization was confirmed through the presence of oocytes in the first and second mitotic divisions. Pure and hybrid postlarvae abalone were initially fed with a diet of fresh N. incerta ad libitum until they reached 3±0.5 mm in SL (~ 7 months). A total of 1440 postlarvae were randomly selected and included in the experiments (up to 13 weeks). After a seven-day acclimation period, 120 abalone from each cross (RR and RG) were randomly selected and collected by sieving and then transferred to three individual 6 L plastic tanks (40 abalone per replicate) with a fine bubble aeration stone under static conditions (no water flow). Tanks were filled using 1 µm filtered and UV-sterilized seawater at 17±0.5°C. A total water change was performed once a week, and feed was provided after each water change. Faeces were removed twice a week, and the water was also refilled (up to 20% exchange). Seawater was maintained at 17±0.5°C by setting the laboratory air conditions and monitored daily with HOBO® data loggers (Onset Corp., Bourne, MA, USA). Oxygen and pH were measured with an HI 98193 oximeter (Hanna Instruments, Smithfield, RI, USA) and an HI 98127 pH meter (Hanna Instruments). The experimental systems were maintained with the same photoperiod conditions (12 h light and 12 h darkness), which were regulated through a controller installed inside the laboratory. Light was maintained at an average lux of ~2.2 mE m-2 s-1 for the tanks. Finally, alkalinity and the concentrations of ammonium, nitrites and nitrates were monitoring using API colorimetric kits, following the instructions of the manufacturer.
Diet composition and algae cultures
⌅Basic diets consisted of two fresh macroalgae of M. pyrifera (M) and Ulva ohnoi (U) and four mixed diets prepared using different combinations of these macroalgae in equal proportions in terms of weight. The microalgae N. incerta (N) was used in a constant volume of 60 mL (105-106 cells mL-1). The mixed diets were named MU, MN, UN and MUN based on their algal combination.
Macrocystis pyrifera
⌅Fresh fronds of M. pyrifera were collected weekly by the Abulones Cultivados Company from an intertidal zone near Ensenada (Baja California, Mexico; 31°17’33.00”N; 116°24’34.45”W) and maintained under optimal controlled conditions before being delivered to CICESE. Once in the laboratory, the fronds were rinsed with freshwater to remove fouling to ensure clean fresh fronds for the diets.
Ulva ohnoi
⌅The green algae Ulva ohnoi was provided by Dr. José Zertuche from the Instituto de Investigaciones of the Universidad Autónoma de Baja California (IIO-UABC). Ulva strains were collected in Bahía San Quintín, Baja California, Mexico, and two different foliar strains of Ulva ohnoi were selected to develop its commercial culture ( Zertuche-González et al. 2021 Zertuche-González J.A., Sandoval-Gil J.M., Rangel-Mendoza L.K., et al. 2021. Seasonal and interannual production of sea lettuce (Ulva sp.) in outdoor cultures based on commercial size ponds. J. World Aquac. Soc. 52: 1047-1058. https://doi.org/10.1111/jwas.12773 ). The Ulva ohnoi used in this study was produced under controlled conditions with a cultivation method consisted of “tumble culture” with aeration from the bottom of the pond ( Revilla-Lovano et al. 2021 Revilla-Lovano S., Sandoval-Gil J.M., Zertuche-Gonzalez J.A., et al. 2021. Physiological responses and productivity of the seaweed Ulva ohnoi (Chlorophyta) under changing cultivation conditions in pilot large land-based ponds. Algal Research, 56: 102316. https://doi.org/10.1016/j.algal.2021.102316 ), and fresh and clean fronds were provided once a week. Once in the laboratory of CICESE, the algae were rinsed with freshwater several times and kept in seawater for a few hours prior to being fed to the abalone.
Navicula incerta
⌅The strains of the diatom Navicula incerta, a benthonic microalga, used in this study were provided from the microalgal collection of the algae repository of CICESE. The microalgae strain was obtained in a volume of 15 mL and scaled to 150 mL non-axenic cultures via a seven-day culture using F/2 Medium (reactive grade) with sodium metasilicate solution ( Guillard 1975 Guillard R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith W.L. and Chantey M.H. (eds), Cult. Mar. Invertebr. Anim. Plenum Publ. New York 29-60. https://doi.org/10.1007/978-1-4615-8714-9_3 ) and irradiance, temperature and salinity conditions of 100 µE m-2 s-1, 17±1°C and 34 ppt, respectively. Once the strain production was stable, the diatom cultures used to feed the abalone were maintained in 150 mL for 4 days. Thereafter, a total volume of 150 mL was inoculated in 15 L of fresh 1 µm filtered (cartridge filters) and UV-sterilized seawater (25-L plastic trays) with F/2 commercial medium (ProLine F/2 Algae Food, Pentair) and grown out for four days before being harvested and fed to the abalone. A volume of 60 mL of N. incerta (105-106 cells mL-1) that had been sonicated for 2 min was added once a week to each experimental tank with a mixed diet (MN, UN, and MUN) to feed the abalone. Aeration was used to distribute the diatoms throughout the tank for 20 min and then discontinued for 1 hour to enable the diatoms to settle on the tank and macroalgae surfaces depending on the treatment.
Growth and survival
⌅During the 13-week experimental period, we evaluated the relative growth rate (RGR) of postlarvae abalone in terms of the increase in SL from the initial conditions. With the aim of reducing the amount of disturbance to all animals, as handling and/or additional stress are known to negatively affect abalone growth and their overall conditions ( Cunningham et al. 2016 Cunningham S.C., Smith A.M., Lamare M.D. 2016. The effects of elevated pCO2 on growth, shell production and metabolism of cultured juvenile abalone, Haliotis iris. Aquac. Res. 47: 2375-2392. https://doi.org/10.1111/are.12684 ), SL measurements (mm) were obtained from a random subset of abalone (n=14-30) from each replicated tank. Abalone were placed on a gridded surface, and digital photographs were taken with a stereo microscope. The images were evaluated with ImageJ v.1.47 (available at https://imagej.nih.gov/ij/ ) with a vertical and horizontal calibration of ±0.5 mm ( Hopkins 1992 Hopkins K.D. 1992. Reporting fish growth: A review of the basics. J. World Aquac. Soc. 23: 173-179. https://doi.org/10.1111/j.1749-7345.1992.tb00766.x ). To avoid confounding effects due to differences in initial SL between abalone batches, SL increases were compared using the RGR calculated for each experimental unit with Eq. (1):
where SL i is the SL at time i, and SL 0 is the initial SL. SL 0 was defined as the mean SL for each replicate tank at week 0 (Hopkins 1992).
The survival of postlarvae abalone was checked each day. Dead individuals were immediately removed from the tanks. Survival was assessed with Kaplan-Meier cumulative survival curves with the survival package in R. Statistical differences in survival curves between diets and abalone crosses were evaluated with the survdiff function from the survival package, followed by pairwise comparison with the pairwise_survdiff function from the survminer package in R.
Statistical analysis
⌅Statistically significant differences between means with regard to the RGR and abalone survival at the end of the 13-week experimental period were analysed using a two-way analysis of variance (ANOVA), with “Diet” and “Cross” (RR and RG) included as fixed factors. A Tukey honest significant difference post-hoc test was used to identify the mean differences between diet treatments. Normality and homogeneity of variance for RGR were tested using Shapiro-Wilk and Levene tests, respectively. To consider the time effect on the RGR during the whole experiment, a linear model was fitted using “Diet,” “Cross,” and “Week” as fixed factors. Thereafter, slopes were contrasted with the emtrends function from the R package emmeans. All statistical analyses were conducted in R (v. 4.0.0; R Core Team).
RESULTS
⌅Shell growth
⌅All diets successfully weaned abalone, and continuous increases in SL were maintained throughout the 13-week experiment ( Fig. 1 ). At the 13th week, the ANOVA indicated a significant effect of diet (F(5,633)=13.0; P<0.0001), a non-significant effect of abalone cross (F(1,633)=1.73; P=0.18) and a significant interaction (F(5,633)=4.8; P=0.002). The lowest increase in mean SL for both abalone crosses was obtained with the diet consisting of only M. pyrifera. Although there was no statistically significant difference between the RGR values of both crosses with this diet (RRRGR=70.90%±45.06 sd; RGRGR=48.12%±44.70 sd; Tukey>0.05; Table I ), the slopes of the models that considered the weekly values were significantly different, with RR having a steeper slope (slope for RR=6.1; slope for RG=4.4; Tukey P<0.02).
| Diet | Cross | Mean shell length at week 1 (mm±sd) | Mean shell length at week 13 (mm±sd) | Relative growth rate at week 13 (% from initial SL±sd)* |
|---|---|---|---|---|
| M | RR | 3.78 ± 1.08 | 6.45 ± 1.69 | 70.70 ± 45.07bc |
| M | RG | 4.29 ± 0.71 | 6.35 ± 1.88 | 48.12 ± 44.71c |
| MN | RR | 3.90 ± 1.09 | 8.07 ± 1.68 | 113.02 ± 59.58a |
| MN | RG | 3.78 ± 0.98 | 7.60 ± 1.75 | 104.30 ± 57.05ab |
| MU | RR | 3.78 ± 0.99 | 6.99 ± 1.75 | 87.09 ± 54.18ab |
| MU | RG | 4.11 ± 1.10 | 7.29 ± 2.16 | 78.57 ± 53.61bc |
| MUN | RR | 3.96 ± 1.14 | 8.45 ± 2.49 | 115.45 ± 67.65a |
| MUN | RG | 3.92 ± 0.88 | 7.77 ± 2.10 | 98.75 ± 55.28ab |
| U | RR | 3.70 ± 1.10 | 7.40 ± 2.05 | 99.89 ± 54.56ab |
| U | RG | 4.27 ± 0.97 | 7.81 ± 1.74 | 84.74 ± 46.55ab |
| UN | RR | 4.57 ± 1.31 | 7.94 ± 1.96 | 76.15 ± 48.96bc |
| UN | RG | 3.98 ± 0.90 | 8.58 ± 1.93 | 115.35 ± 48.22a |
The highest increase in the RGR for RR was obtained with the mixed diet MUN (RRRGR=115.45%±67.6 sd; Table I ), while the highest RGR value for the hybrid abalone was obtained with the UN diet (RGRGR=115.34%±48.2 sd; Table I ). However, it is important to highlight that there were no significant differences in the final RGR values for the MUN diet (RRRGR=115.45%±67.6; RGRGR=98.7%±55.2 sd; Tukey, P>0.05; Table I ) or in the slopes of weekly growth (slope for RR=8.7; slope for RG=8.8; Tukey, P=0.88; Fig. 1 ) between the two crosses, indicating similar beneficial effects of the MUN diet for the pure and hybrid abalone. By contrast, the beneficial effect of the UN diet occurred only in the hybrid RG, with lower RGR values (RRRGR=76.15%±48.96 sd; RGRGR=115.35%±48.22 sd; Tukey, P<0.05; Table I) and a lower slope reflecting growth in RR (slope for RR=6.4; slope for RG=8.4; Tukey, P=0.01; Fig. 1 ). The remaining diets showed no significant differences between the final RGR values ( Table I ) or in the weekly increases in RGR for the two abalone crosses ( Supplementary Material Table S1 ).
Survival between pure and hybrid crosses
⌅The Kaplan-Meier cumulative survival curves demonstrated significant differences between groups at the end of the 13-week trial ( Fig 2 ). Overall, a higher survival rate for RR was observed with the MUN diet (89.16%), whereas the lowest rate was observed with the U diet (60%). On the other hand, higher survival for RG was observed with the MN and U diets (86.66% for both diet treatments), while the lowest survival rate for RG was observed with the M diet (65%). The chi square test performed with all pairwise comparisons for all diets revealed significant differences between abalone crosses with only the MUN and U diets ( Fig. 2 ). The MUN diet induced a significantly higher survival rate in RR (P<0.001), whereas the U diet induced a significantly higher survival in RG (P<0.0001). The results from all the pairwise comparisons are shown in Supplementary Material Table S2 .
DISCUSSION
⌅Previous studies have evaluated the nutritional value of monospecific cultures of benthic diatoms as food sources for red and blacklip (H. rubra) abalone postlarvae. In both cases, higher growth rates were obtained with abalone supplied with Navicula spp. and other diatoms ( Correa-Reyes et al. 2001 Correa-Reyes J.G., Sánchez-Saavedra M. del P., Siqueiros-Beltrones D.A., Flores-Acevedo N. 2001. Isolation and growth of eight strains of benthic diatoms, cultured under two light conditions. J. Shellfish Res. 20: 603-610., 2009 Correa-Reyes J.G., Sánchez-Saavedra M. del P., Viana M.T et al. 2009. Effect of eight benthic diatoms as feed on the growth of red abalone (Haliotis rufescens) postlarvae. J. Appl. Phycol. 21: 387-393. https://doi.org/10.1007/s10811-008-9381-x ). Our findings support this observation and suggest that mixed diets including N. incerta may increase the growth and survival rates of both the pure RR abalone and RG hybrid cross. Moreover, the use of M. pyrifera should be discouraged during this stage due to the nutritional content of this macroalgae, which may not fulfil the nutritional needs of weaning abalone, and the inability of postlarvae abalone (3-5 mm) to properly ingest and assimilate this alga. This was evident in the present study, as the M diet induced the lowest growth increases for both abalone crosses (RR and RG) and also induced the lowest cumulative survival in the hybrid cross (RG).
The highest growth and survival rate for RR abalone was observed with the MUN diet. For the hybrid abalone RG, the highest growth rate was observed with the UN diet, whereas the highest survival was observed with the MN and U diets. However, it is important to note that there were no significant differences between the survival curves with the UN diet (chi square P>0.05; Supplementary Material Table S2 ), although the highest survival values for RG were observed with the MN and U diets. Therefore, we can conclude that the UN diet induced a higher growth rate and high survival rates in RG.
Accordingly, as previously reported, N. incerta should be considered one of the most appropriate nourishment sources for recently settled abalone ( Martínez-Ponce and Searcy-Bernal 1998 Martínez-Ponce D.R., Searcy-Bernal R. 1998. Grazing rates of red abalone (Haliotis rufescens) postlarvae feeding on the benthic diatom Navicula incerta. J. Shellfish Res. 17: 627-630., Simental et al. 2004 Simental J.A., Sanchez-Saavedra M.D., Flores-Acevedo N. 2004. Growth and survival of juvenile red abalone (Haliotis rufescens) fed with macroalgae enriched with a benthic diatom film. J. Shellfish Res. 23: 995-999., Carbajal-Miranda et al. 2005 Carbajal-Miranda M.J., Sánchez-Saavedra M.D.P., Simental J.A. 2005. Effect of monospecific and mixed benthic diatom cultures on the growth of red abalone postlarvae Haliotis rufescens (Swainson 1822). J. Shellfish Res. 24: 401-405. https://doi.org/10.2983/0730-8000(2005)24[401:EOMAMB]2.0.CO;2 ). Moreover, the incorporation of this diatom into abalone diets should be encouraged in farmed production scenarios, as it may reduce abalone losses during weaning, which is the most critical stage during abalone cultivation ( Viana et al. 1993 Viana M.T., López L.M., Salas A. 1993. Diet development for juvenile abalone Haliotis fulgens Evaluation of two artificial diets and macroalgae. Aquaculture 117: 149-156. https://doi.org/10.1016/0044-8486(93)90131-H , Carbajal-Miranda et al. 2005 Carbajal-Miranda M.J., Sánchez-Saavedra M.D.P., Simental J.A. 2005. Effect of monospecific and mixed benthic diatom cultures on the growth of red abalone postlarvae Haliotis rufescens (Swainson 1822). J. Shellfish Res. 24: 401-405. https://doi.org/10.2983/0730-8000(2005)24[401:EOMAMB]2.0.CO;2 , Hernández et al. 2009 Hernández J., Uriarte I., Viana M.T., et al. 2009. Growth performance of weaning red abalone (Haliotis rufescens) fed with Macrocystis pyrifera plantlets and Porphyra columbina compared with a formulated diet. Aquac. Res. 40: 1694-1702. https://doi.org/10.1111/j.1365-2109.2009.02267.x ). The benefits of N. incerta likely reflect its protein and lipid content (up to 30%; Simental-Trinidad et al. 2001 Simental-Trinidad J.A., Sánchez-Saavedra M.P., Correa-Reyes J.G. 2001. Biochemical composition of benthic marine diatoms using as culture medium a common agricultural fertilizer. J. Shellfish Res. 20: 611-617., Hernández et al. 2009 Hernández J., Uriarte I., Viana M.T., et al. 2009. Growth performance of weaning red abalone (Haliotis rufescens) fed with Macrocystis pyrifera plantlets and Porphyra columbina compared with a formulated diet. Aquac. Res. 40: 1694-1702. https://doi.org/10.1111/j.1365-2109.2009.02267.x , Ortiz et al. 2009 Ortiz J., Uquiche E., Robert P., et al. 2009. Functional and nutritional value of the Chilean seaweeds Codium fragile, Gracilaria chilensis and Macrocystis pyrifera. Eur. J. Lipid Sci. Technol. 111: 320-327. https://doi.org/10.1002/ejlt.200800140 ).
The higher growth rates obtained with N. incerta-based diets may also be related to the particular morphologies and digestive enzymes present in postlarvae abalone. Between 80 and 102 days post-settlement, early teeth appear on the radula, while the digestive gland increases in both complexity and enzyme production, suggesting that the abalone is preparing to feed on macroalgae ( Johnston et al. 2005 Johnston D., Moltschaniwskyj N., Wells J. 2005. Development of the radula and digestive system of juvenile blacklip abalone (Haliotis rubra): Potential factors responsible for variable weaning success on artificial diets. Aquaculture 250: 341-355. https://doi.org/10.1016/j.aquaculture.2005.03.012 ). Kawamura et al. (1995) Kawamura T., Saido T., Takami H., Yamashita Y. 1995. Dietary value of benthic diatoms for the growth of post-larval abalone Haliotis discus hannai. J. Exp. Mar. Bio. Ecol. 194: 189-199. https://doi.org/10.1016/0022-0981(95)00099-2 reported that both the radula structure and the ability to digest and assimilate seaweeds are fully developed in H. discus hannai measuring 2-4 mm in SL ( Kawamura et al. 1995 Kawamura T., Saido T., Takami H., Yamashita Y. 1995. Dietary value of benthic diatoms for the growth of post-larval abalone Haliotis discus hannai. J. Exp. Mar. Bio. Ecol. 194: 189-199. https://doi.org/10.1016/0022-0981(95)00099-2 ). Thus, the postlarvae abalone used in this experiment (>3 mm) were likely able to feed on seaweed. However, our observations suggest that at this time the reduced cell size of diatoms may still increase the efficiency at which the radula passes food into the mouth; hence, small cells are preferred by immature abalone, as they are easily handled and ingested ( Carbajal-Miranda et al. 2005 Carbajal-Miranda M.J., Sánchez-Saavedra M.D.P., Simental J.A. 2005. Effect of monospecific and mixed benthic diatom cultures on the growth of red abalone postlarvae Haliotis rufescens (Swainson 1822). J. Shellfish Res. 24: 401-405. https://doi.org/10.2983/0730-8000(2005)24[401:EOMAMB]2.0.CO;2 ).
The cumulative survival observed during the experimental period (13 weeks) generally ranged from 60% to 89%. These results are particularly encouraging, as survival rates lower than 70% are usually reported in weaning abalone ( Takami et al. 2002 Takami H., Kawamura T., Yamashita Y. et al. 2002. Effects of delayed metamorphosis on larval competence, and postlarval survival and growth of abalone Haliotis discus hannai. Aquaculture 213: 311-322. https://doi.org/10.1016/S0044-8486(02)00338-1 , Hernández et al. 2009 Hernández J., Uriarte I., Viana M.T., et al. 2009. Growth performance of weaning red abalone (Haliotis rufescens) fed with Macrocystis pyrifera plantlets and Porphyra columbina compared with a formulated diet. Aquac. Res. 40: 1694-1702. https://doi.org/10.1111/j.1365-2109.2009.02267.x ). Moreover, the registered growth rate met the standard growth of commercial postlarvae Haliotis rufescens in Mexican farms estimated to be ~2.0 mm/month ( Searcy-Bernal et al. 2007 Searcy-Bernal R., Pérez-Sánchez E., Anguiano-Beltrán C., Flores-Aguilar R. 2007. Metamorphosis and postlarval growth of abalone Haliotis rufescens in a Mexican commercial hatchery. J. Shellfish Res. 26: 783-787. https://doi.org/10.2983/0730-8000(2007)26[783:MAPGOA]2.0.CO;2 ). Both observations suggest that the use of mixed diets together with the rearing conditions used in this study should be considered for future experimental studies and farmed production.
Marine mollusc hybrids generally show better growth than their parents ( Alter et al. 2017 Alter K., Andrewartha S.J., Morash A.J., et al. 2017. Hybrid abalone are more robust to multi-stressor environments than pure parental species. Aquaculture 478: 25-34. https://doi.org/10.1016/j.aquaculture.2017.04.035 ); however, in this study, similar growth rates were obtained between the two crosses. The simplest explanation for such similar growth rates may be the experimental design, as both crosses were maintained within the optimum temperature conditions for red abalone (17-18°C). However, an alternative or complementary possibility may be proposed. It has been reported that the acquisition of new traits does not rely exclusively on the crossed species but also on the combination of male and female gametes ( Cai et al. 2010 Cai M., Wang Z., Ke C., et al. 2010. Allogyogenetic progeny are produced from a hybrid abalone cross of female Haliotis diversicolor and male Haliotis discus discus. J. Shellfish Res. 29: 725-729. https://doi.org/10.2983/035.029.0325 ). Accordingly, the growth rates of the green abalone [♀] × red abalone [♂] combination, the GR hybrid, should be also evaluated.
Finally, previous studies have indicated that hybrid progeny usually share high genetic similarity with their maternal parent ( Cai et al. 2010 Cai M., Wang Z., Ke C., et al. 2010. Allogyogenetic progeny are produced from a hybrid abalone cross of female Haliotis diversicolor and male Haliotis discus discus. J. Shellfish Res. 29: 725-729. https://doi.org/10.2983/035.029.0325 ). Hence, in this study, the red abalone [♀] × green abalone [♂] cross was preferred when generating hybrid progeny with the ability to grow in relatively short periods of time, which is a characteristic trait of red abalone ( Valenzuela-Miranda et al. 2015 Valenzuela-Miranda D., Del Río-Portilla M.A., Gallardo-Escárate C. 2015. Characterization of the growth-related transcriptome in California red abalone (Haliotis rufescens) through RNA-Seq analysis. Mar. Genomics 24: 199-202. https://doi.org/10.1016/j.margen.2015.05.009 ). However, the growth and survival of other abalone species and gamete combinations should be evaluated. Notably, although no significant differences between growth rates were obtained between crosses, heterosis was observed in terms of survival rates, suggesting that hybrid abalone may be better able to withstand culture conditions. Similar results have also been reported with an H. discus hannai hybrid, with the metamorphosis, growth, and survival rates of the hybrids being superior to those of the pure lineage ( Li et al. 2018 Li J., Wang M., Fang J., et al. 2018. A comparison of offspring growth and survival among a wild and a selected strain of the Pacific abalone (Haliotis discus hannai) and their hybrids. 495: 721-725. https://doi.org/10.1016/j.aquaculture.2018.06.071 ). Together, these observations should encourage hybrid farming to meet the demands of culture production while limiting abalone losses.
CONCLUSIONS
⌅In conclusion, we assessed the growth and survival rates of postlarvae (4±0.22 mm SL) of pure red abalone and a red abalone [♀] × green abalone [♂] hybrid under culture conditions. Both the hybrid and pure-line abalone were supplied with six different diets. Notably, hybrid abalone did not outperform pure red abalone in terms of survival or growth, as the highest growth rate and survival values were similar for both crosses, but a cross-specific beneficial effect of diet was evident. Overall, a mixture of M. pyrifera, U. ohnoi, and N. incerta induced the highest growth rate and survival in pure red abalone, whereas the mixture of U. ohnoi and N. incerta induced the highest growth rate and optimum survival in the hybrid abalone. Although the hybrid abalone did not show higher growth rates than those of the pure red abalone, this study sheds light on the different nutritional requirements of hybrid crosses when compared with those of pure lineages. Additionally, it is important to consider that the animals were grown at the optimum temperature of red abalone, so culturing hybrid RG at its specific optimum temperature with its specific diet may result in better outcomes for this cross.