Physiological Responses of Intrinsic Small Abalone Haliotis Diversicolor

Journal of Marine Science and Engineering

Article Physiological Responses of Intrinsic Small Abalone Haliotis diversicolor aquatilis under High Temperature Stress by Low Level 60CO Gamma Irradiation-Mediated Hormetic Eﬀect

Min-seok Jwa 1 and Chang-Yu Hong 2,* 1 Department of Future Strategy Research, Jeju Research Institute, Jeju 63147, Korea; [email protected] 2 Department of Urban Environmental Research, Jeju Research Institute, Jeju 63147, Korea * Correspondence: [email protected]; Tel.: +82-64-729-0526

Received: 27 September 2020; Accepted: 11 November 2020; Published: 12 November 2020

Abstract: Haliotis diversicolor aquatilis (H. diversicolor aquatilis) is one of the aquaculture abalone species in Jeju Island, South Korea. The high water temperature in the middle of the summer season frequently limits the aquaculture productivity of abalone. To cope with the problem, this study aimed to investigate viability, attachment rate, changes of immune response, and physiological properties of juvenile small abalone Haliotis diversicolor aquatilis against high water temperature stress under 60CO-gamma irradiation-mediated hormetic effects. To examine physiological changes of abalone, the gamma-ray irradiated and non-irradiated groups were distinguished by grouping a total of 70 abalones in a treated group and a control group (each group included twenty-five female and ten male abalones). The treated group was exposed to 60CO-gamma irradiation using a designed experimental apparatus, the control group was not. Our results revealed that the low level of gamma ray (20 Gy)-irradiated Haliotis diversicolor aquatilis showed a 100% survival rate during the experiment. After gamma ray exposure, all the abalones were cultured without feeding and bioactivities were measured to examine gamma ray-induced physiological responses. The results suggested the potential for selective breeding using gamma ray irradiation hormesis to manipulate the number of eggs, fertilization rate, hatching rate, and attachment rate. The shell length of juvenile abalone was significantly enhanced by a 20 Gy radiation dose. We could presume that the effect of hormesis in the gamma-ray irradiation parent shellfish also had a genetic effect on the offspring. In order to verify changes in immune response and stress tolerance of abalone under high temperature stress, lysozyme activity and survival rates were compared at a water temperature of 30 ◦C. Interestingly, the 60Co gamma ray-irradiated abalones exhibited almost a 1.65-fold enhanced survival rate along with reduced lysozyme activity after 12 h of high temperature stress. Our results speculate that low levels of 60Co gamma ray-mediated hormetic effects can be an effective strategy for shell length growth and high temperature stress tolerance.

Keywords: 60CO-gamma irradiation; Haliotis diversicolor aquatilis; abalone hormesis; mutation induction; Jeju Island

1. Introduction Hormesis can be explained as an adaptation to harsh changing environments. The bioactive mechanism of hormesis has yet to be established. Several hypotheses have been suggested based on studies of land animals and plants. The low dose radiation produces proteins related to the DNA recovery process while the DNA synthesis process is temporarily inhibited, providing sufficient time for the treated cells to recover. At the same time, resistance is increased when free radicals spread [1].

J. Mar. Sci. Eng. 2020, 8, 906; doi:10.3390/jmse8110906 www.mdpi.com/journal/jmse J. Mar. Sci. Eng. 2020, 8, 906 2 of 11

Low-dose radiation treatment technology such as gamma radiation may be used to induce hormesis in plant species including cabbage, pepper, and gourd [2,3] to stimulate growth, increase viability and productivity, stabilize food supply, and increase plant resistance to environmental stressors [4]. Hormesis and increased growth has also been reported for the marine abalone species, Haliotis discus hannai and Haliotis discus at gamma-ray doses of 30 Gy and 20 Gy respectively [5,6], however, no studies have been undertaken for Haliotis diversicolor aquatilis. The bioactive mechanism of hormesis has yet to be established, but several possibilities are being presented. The low dose radiation produces proteins related to the DNA recovery process while the DNA synthesis process is temporarily inhibited, providing sufficient time for the targeted cells to recover. At the same time, resistance is increased when free radicals spread [1]. These theories were established based on the experimental results of land animals and plants, but in the case of Haliotis diversicolor aquatilis, which is marine life, there are few studies. Haliotis diversicolor aquatilis is a species that only inhabits East Asia such as China and Jeju Island, Korea. It has quite a high industrial value. Climate change due to global warming has recently caused physiological changes such as growth, breeding, metabolic, and osmotic pressure, which have caused problems such as disease and death in abalone aquaculture. In particular, in the summer of 2006, low salinity seawater from China flowed into the northwestern coast of Jeju Island, causing the salinity to drop below 20 psu (practical salinity unit: %). With this phenomenon, fishermen encountered the massive death of abalone, resulting in economic losses of up to $5 million (six billion Korean won). In general, most abalones of Korea have a long growth period and have diverse growth variables due to environmental changes during this time. Stable abalone production requires an understanding of the factors that affect growth and survival [7,8]. Up to this day, aquaculture scientists have researched the correlation between Haliotis diversicolor aquatilis physiology and environmental changes [9]. Some researchers have shown that Haliotis diversicolor aquatilis, Haliotis discus, and Haliotis sieboldi in Jeju Island are subject to physiological changes due to stress induced by seawater temperature [9,10]. Studies regarding breeding mostly address topics of growth traits and genetic parameter estimation [11], but there is almost no research on active breed improvement. Methods used for plant breeding are widely used such as selective breeding, hybrid breeding, and mutation breeding by irradiation. In particular, there have been many studies on hormesis using radiation, but few studies using radiation for breed improvement. In this study, gamma-ray hormesis was induced to Haliotis diversicolor aquatilis, a flagship aquaculture species of Jeju Island. After gamma irradiation, basic physiological changes in juvenile small abalone such as attachment rate, growth rate, and immunoactivity change according to water temperature stress were recorded and analyzed to determine if 20 Gy-irradiated Haliotis diversicolor aquatilis exhibited increased growth rates.

2. Materials and Methods

2.1. Animals For the present study, we purchased Haliotis diversicolor aquatilis from Jeju City ﬁshing village societies on Jeju Island, Korea. Samples were acclimated to the experimental setup one week in a tank at 23 0.3 C and 32 0.2 psu (practical salinity unit: %) of salinity without feeding. In all, ± ◦ ± 100 female and 20 male Haliotis diversicolor aquatilis were used for the experiment. The average shell lengths (mean standard deviation) of Haliotis diversicolor aquatilis were 6.25 0.2 (range 6.05–6.45), ± ± and the average total wet weights were 32.5 1.5 g (range, 31–34 g). The twenty-ﬁve (25) females and ± ten (10) males were exposed to each gamma radiation dose (0 and 20 Gy).

2.2. Fabrication of the Case for Gamma-Ray Irradiation Use Haliotis diversicolor aquatilis is a marine invertebrate that dies if exposed to air for a long time. It shows characteristics of being mobile and lives by attaching to something else. To induce mutations by gamma irradiation, a case was made to minimize the mobility limitations of Haliotis diversicolor aquatilis J. Mar. Sci. Eng. 2020, 8, 906 3 of 11

J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 3 of 11 and theJ. Mar. stress Sci. Eng. during 2020, 8, thex FOR attachment PEER REVIEW and desorption of Haliotis diversicolor aquatilis. The case3 of 11 was a hollow acrylic box with a width of 10 cm, a length of 10 cm, and a height of 5 cm. The front surface surface was made detachable to facilitate the attachment and desorption of the Haliotis diversicolor was madesurface detachable was made detachable to facilitate to thefacilitate attachment the attachment and desorption and desorption of the ofHaliotis the Haliotis diversicolor diversicolor aquatilis . aquatilis. For this, grooves were formed on both sides of the acrylic box to facilitate the separation of For this,aquatilis grooves. For this, were grooves formed were on formed both sides on both of the sides acrylic of the box acrylic to facilitatebox to facilitate the separation the separation of the of front the front plate (Figure 1). platethe (Figure front1 plate). (Figure 1).

Figure 1. Case for irradiation with gamma-rays [2]. Figure 1. Case for irradiation with gamma-rays [2]. 2.3. 60CO Gamma Irradiation Figure 1. Case for irradiation with gamma-rays [2]. As2.3. abalone60CO Gamma species Irradiation have adhesive and migratory properties and perish after long-term exposure to2.3. air, 60CO we GammaAs designed abalone Irradiation species small acrylichave adhesive cases forand the migratory experiment. properties The and cases perish were after 10 long cm-term wide, exposure 10 cm long, and 5to cm air, high. we designed To facilitate small the acrylic disadhesion cases for the of abaloneexperiment. species, The cases we made were 10 the cm cases wide, with 10 cm a detachable long, acrylicAsand panel abalone 5 cm (Figurehigh. species To1 facilitate). Wehave conducted adhesive the disadhesion and gamma migratory of abalone radiation properties species, (GR) w exposuree and made perish the experiments cases after with long a at-detachableterm the exposure Institute forto air, Nuclearacrylic we designed panel Science (Fig andsmallure. Technology1). acrylic We conducted cases at Jejufor gamma the National experiment. radiation University, (GR The) exposure Koreacases were withexperiment 1060CO cmsgamma atwide, the Institute 10 irradiation cm long, 60 (10,000and 5for cm ci), Nuclear high. as shown ToScience facilitate in and Figure Technology the2. disadhesion GR was at setJeju atNationalof 0 abalone and 20University, Gyspecies, by regulating Koreawe made with the theCO irradiation cases gamma with irradiation time a detachable to 0 and (10,000 ci), as shown in Figure 2. GR was set at 0 and 20 Gy by regulating the irradiation time to 0 36acrylic min, panel respectively. (Figure.The 1). We radiation conducted level gamma was measured radiation using (GR a) exposure DE/FH40G-L experiment surveys meter at the (Eberline, Institute and 36 min, respectively. The radiation level was measured using a DE/FH40G60 -L survey meter TN,for Nuclear USA). Haliotis Science diversicolorand Technology aquatilis at Jejuwas National exposed University, to all of the Korea radiation with levelsCO gamma stated aboveirradiation and (10,000(Eberline, ci), as shown TN, USA). in FigureHaliotis 2. diversicolor GR was set aquatilis at 0 andwas exposed20 Gy by to regulating all of the radiation the irradiation levels stated time to 0 GR-exposed abalone groups were incubated under optimal conditions (23 0.3 ◦C and 32 0.2 psu of and 36above min, and respectively. GR-exposed abalone The radiation groups were level incubated was measured under optimal using conditions a DE/FH40G± (23 ± 0-L.3 °C survey± and 3 2 meter salinity)± 0.2 without psu of salinity) feeding without for six feeding weeks. for six weeks. (Eberline, TN, USA). Haliotis diversicolor aquatilis was exposed to all of the radiation levels stated above and GR-exposed abalone groups were incubated under optimal conditions (23 ± 0.3 °C and 32 ± 0.2 psu of salinity) without feeding for six weeks.

Figure 2. Gamma irradiation in Haliotis diversicolor aquatilis.

J. Mar. Sci. Eng. 2020, 8, 906 4 of 11

2.4. Seed Production of Haliotis Diversicolor Aquatilis after Gamma-Ray Irradiation Both Haliotis diversicolor aquatilis parents of the gamma irradiated and unirradiated samples were acclimatized at optimal water temperature (23 0.3 C) for a week, before conducting artiﬁcial seed ± ◦ production. After 1 h of excitation stimulation at 30 ◦C, UV seawater stimulation was performed to induce artiﬁcial spawning of samples. In gamma-ray-irradiated samples and unirradiated experimental control group samples, the mixing of the eggs with the sperm were separately performed to prevent mixing fertilized eggs from each group. The fertilized eggs were collected using 100 µm muller gauze, then the fertilized embryo population was counted.

2.5. Fertilization Rate and Hatching Rate Count The fertilization rate was counted and calculated with about 200,000 samples of fertilized eggs of the gamma irradiated group and an unirradiated experimental control group by separately transferring the fertilized eggs from a 1 mL glass beaker to a 50 mL glass beaker in triplicate. Then, after putting the fertilized eggs in a 20 L circular plastic container, the hatched larvae and the unhatched larvae were collected and moved to the 1 L glass beaker and the overall hatching rate was measured by pipetting 1 mL of the fertilized eggs in triplicate.

2.6. Larvae Management The fertilized eggs of Haliotis diversicolor aquatilis were accommodated in two 4-ton tanks of ﬁber reinforced plastic (1 m width 6 m length 70 cm height) with one water tank for each experimental × × group at 24 0.5 C. After the veliger larvae were fully formed, the larvae were raised to the surface ± ◦ using the air supply and water ﬂow systems. Seeds were collected by gathering the larvae on the shelter plate in which diatoms were cultured in advance.

2.7. Changes in Attachment Rate and Growth Rate The attachment rate was measured by counting Haliotis diversicolor aquatilis attached to the shelter plate by randomly selecting three shelter plates from the shelter plate set (30 cm width 30 cm length), × which were separated by seed collection dosage. The growth rate was measured by the size of three randomly selected Haliotis diversicolor aquatilis attached to the water tank shelter plate of each experimental group using Vernier calipers 90 days after hatching when the abalones could clearly be identiﬁed by the naked eye.

2.8. Immune-Activity Changes of Small Abalones Due to Water Temperature Stress For the immuno-activity changes, small abalones that were produced from the control and 20 Gy were bred for 48 weeks, and subsequently, 30 similar small abalones were selected from each group. Small abalones were used with an average shell length of 2.48 0.05 cm, shell width of 1.21 0.05 cm, ± ± and a total weight of 2.1 0.3 g. To determine whether the immune-activity increased, five abalone were ± collected when the sudden change of water temperature was at 0 h and 12 h, respectively, using the pre-studied temperature of 30 ◦C (100% of the dead-water temperature and lysozyme activity change was evaluated). The blood was collected from the blood vessels next to the heart of Haliotis diversicolor aquatilis using a 1 mL syringe with a needle standard of 26 GX 1/2 and diluted tenfold with a 0.9% saline solution. The lysozyme activity inside the serum was analyzed by a turbidimetric assay according to the following methods: Micrococcus lysodeikticus (0.1 mg/mL PBS, pH 6.8) suspension 90 µL, and serum 100 µL were mixed in a 96-well plate and reacted for 5 min in a 25 ◦C culture medium, then the absorbance was measured at 530 nm. Lysozyme activity was marked as a 0.001/mL decrease in absorbance values per 1 unit. J. Mar. Sci. Eng. 2020, 8, 906 5 of 11

J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 5 of 11 2.9. Statistical Tool 2.9.Statistical Statistical analysisTool was processed using a one-way analysis of variances (ANOVA) followed by post-hocStatistical t-tests by analysis using anwas IBM processed SPSS 22 using program. a one- Theway values analysis of of (p variances< 0.05) were (ANOVA) considered followed signiﬁcant, by andpost we-hoc experiments t-tests by were using conducted an IBM SPSS in triplicate. 22 program. The values of (p < 0.05) were considered significant, and we experiments were conducted in triplicate. 3. Results and Discussion 3. Results and Discussion 3.1. Survival Rate of Parent Shellﬁsh and Fertilization Rate 3.1.The Survival dam Rate (mother) of Parent samples Shellfish of and irradiated Fertilization 20 Gy Rate gamma-ray showed dull activity until the time of domesticationThe dam (mother) and egg samples collection, of irradiated but indicated 20 Gy agamma survival-ray rate showed of 100%. dull activity The control until the group time and theof dams domestication that were and exposed egg collection to irradiated, but indicat 20 Gyed of a gamma-raysurvival rate wereof 100%. used The to control create seedgroup production. and the Eachdams of that the spawnedwere exposed eggs to were irradiated separated 20 Gy and of gamma fertilized.-ray were used to create seed production. Each of theThe spawned number eggs of eggs were spawned separated in eachand fertilized. experimental group was measured by an optical microscope (NikonThe PROFILE number PROJECTOR of eggs spawned V-12B) in using each a experimental 1 mL pipette, group and the was results measured are shown by an in optical Figure 3. Themicroscope fertilized (Nikon eggs of PROFILE 2,000,000 PROJECTOR130 were foundV-12B) inusing the a control 1 mL pipette, group, and and the 1,000,000 results are110 shown fertilized in ± ± eggsFigure were 3. foundThe fertilized in the 20 eggs Gy of experimental 2,000,000 ± 1 group.30 were found in the control group, and 1,000,000 ± 110 fertilized eggs were found in the 20 Gy experimental group.

Figure 3. Number of eggs (A) and fertility (B) for each gamma-ray dose. * p < 0.05 for control. Figure 3. Number of eggs (A) and fertility (B) for each gamma-ray dose. * p < 0.05 for control. J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 6 of 11 J. Mar. Sci. Eng. 2020, 8, 906 6 of 11 In order to obtain accurate results, about 200,000 fertilized eggs were collected. The fertilized and non-fertilized eggs in each experimental group were counted, and as a result of examining the fertilizedIn order eggs to and obtain fertilization accurate results, rates accordingabout 200,000 to gammafertilized irradiation, eggs were collected. 185,000 ± The 170 fertilized samples and of fertilizednon-fertilized eggs eggswere in found each experimentalin the experimental group werecontrol counted, group, and and as 110,00 a result0 ± of 150 examining samples theof fertilized fertilized eggs and fertilization rates according to gamma irradiation, 185,000 170 samples of fertilized eggs eggs were found in the 20 Gy experimental group. The fertilization rate± was confirmed as 85% in the were found in the experimental control group, and 110,000 150 samples of fertilized eggs were found experimental control group and 55% in the 20 Gy experimental± group. In the mean water temperature ofin 24 the °C, 20 it Gy took experimental 8 h from fertilization group. The to fertilization hatching, 16 rate h until was formation confirmed of as the 85% young in the shell, experimental and 56 h untilcontrol the group seedling and 55% collection in the 20 stage. Gy experimental There was no group. significant In the mean difference water temperature between the of experimental 24 ◦C, it took groups8 h from (p fertilization< 0.05). to hatching, 16 h until formation of the young shell, and 56 h until the seedling collection stage. There was no significant difference between the experimental groups (p < 0.05). 3.2. Hatching Rate and Attachment Rate Measurement 3.2. Hatching Rate and Attachment Rate Measurement In order to measure the hatching and attachment rate, fertilized eggs were reared in the sea In order to measure the hatching and attachment rate, fertilized eggs were reared in the sea water water tank for each experimental group. The results are shown in Figure 4. The hatching rate was tank for each experimental group. The results are shown in Figure4. The hatching rate was indicated indicated as 70 ± 5% in the control and 30 ± 5% in the 20 Gy exposed group. The attachment rate was as 70 5% in the control and 30 5% in the 20 Gy exposed group. The attachment rate was revealed revealed± as 80 ± 5% in the control± and 65 ± 5% in the 20 Gy exposed group. The number of fertilized as 80 5% in the control and 65 5% in the 20 Gy exposed group. The number of fertilized eggs, eggs, fertilization± rate, hatching rate,± and attachment rate was lower in the 20 Gy experimental group fertilization rate, hatching rate, and attachment rate was lower in the 20 Gy experimental group than than in the control group (where the number of small abalone was low (p < 0.05)). in the control group (where the number of small abalone was low (p < 0.05)).

Figure 4. Hatching rate (A) and attachment rate (B) for each gamma-ray dose. * p < 0.05 for control. Figure 4. Hatching rate (A) and attachment rate (B) for each gamma-ray dose. * p < 0.05 for control. J. Mar. Sci. Eng. 2020, 8, 906 7 of 11 J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 7 of 11

3.3.3.3. Growth Growth Measurement Measurement of of Small Small Abalones Abalones ThirtyThirty randomly selected selectedHaliotis Haliotis diversicolor diversicolor aquatilis aquatiliattacheds attached to each to eachexperimental experimental group’s group shelter’s shelterplate in plate the waterin the tankwater was tank measured was measured after 90 after days, 90 as days, demonstrated as demonstrated in Figure in 5Figure. A total 5. A of total 65 shelter of 65 plates and 10 abalones per shelter plate were attached. An average of 650 70 small abalones were shelter plates and 10 abalones per shelter plate were attached. An average of± 650 ± 70 small abalones wereattached. attache Thed. averageThe average temperature temperature of the of aquaculture the aquaculture farm farm was was 25.6 25.6◦C and °C and the averagethe average salinity salinity was was32.6 32.6 psu. psu. The generalThe general natural natural condition condition of the of photoperiod the photoperiod was 10 was L:14 10 D. L:14 The D. control The groupcontrol grew group by 1.1 0.5 mm and the 20 Gy group grew by 1.6 0.5 mm (p < 0.05). grew± by 1.1 ± 0.5 mm and the 20 Gy group grew± by 1.6 ± 0.5 mm (p < 0.05).

Figure 5. Changes in growth in juvenile Haliotis diversicolor aquatilis for each gamma-ray dose.

3.4. ChangesFigure of 5. Immune-Activity Changes in growth by in Water juvenile Temperature Haliotis diversicolor Stress aquatilis for each gamma-ray dose.

3.4. ChangesTo investigate of Immune the-Activity cause ofby theWater high Temperature growth rate Stress after gamma irradiation, Haliotis diversicolor aquatilis stress was investigated under a high water temperature of 30 ◦C. The lysozyme activity change is anTo environmental investigate the stress cause factor of thatthe high is used growth as an indicatorrate after of gamma immune irradiation, function, as Haliotis shown diversicolor in Figure6. aquatilisThe lysozyme stress activitywas investigated change in the under treated a high group water of 20 temperature Gy was shown of 30to be°C. significantly The lysozyme higher activity at 0 h change is an environmental stress factor that is used as an indicator of immune function, as shown in than at 12 h under the 30 ◦C temperature water (p < 0.05). Additionally, the survival rate resulted in Figureterms of6. immune-activityThe lysozyme activity after 12 change h are described in the treated in Figure group7. Thisof 20 indicates Gy was theshown survival to be rate significantly of 34% in higherthe control at 0 h group than at and 12 56%h under in the the 20 30 Gy °C group temperature (p < 0.05). water (p < 0.05). Additionally, the survival rate resultedMany in terms studies of of immune breeding-activity through after mutation 12 h ar inductione described have in been Figure conducted 7. This focusingindicates on the agriculture survival rateand of its 34% effect in and the economiccontrol group value and [12 ,56%13]. Inin thisthe 20 study, Gy group low-dose (p < gamma-rays 0.05). were irradiated to parent shellfishMany of studiesHaliotis diversicolorof breeding aquatilis through. Their mutation growth induction was observed have through been conducted physiological focusing changes on agricultureand breeding. and The its next effect generation and economic of Haliotis value diversicolor [12,13]. In aquatilis this study,produced low-dose after gamma- irradiationrays were irradiatedwas also observed. to parent Generally, shellfish itof isH reportedaliotis diversicolor that gamma aquatilis ray irradiation. Their growth causes was oxidative observed stress through in cells physiologicalcaused by the changes water molecule’s and breeding. overproduction The next generation of hydroxyl of [ 14H].aliotis The resultsdiversicolor of this aquatilis study illustratedproduced afterthat there gamma were irradiatio no deathsn was of parent also observed. shellfish in Generally, the high concentration it is reported gamma-ray that gamma irradiation ray irradiation section causesof 20 Gy, oxidative but a phenomenon stress in cells of caused decreased by the activity water emerged. molecule’s Therefore, overproduction the dose of of hydroxyl gamma-ray [14]. set The in resultsthis study of this may study cause illustrated some stress that on there parent were shellfish, no deaths but of does parent not shellfish affect breeding, in the high so was concentration considered gammasuitable-ray for irradiation mutation induction. section of 20 Gy, but a phenomenon of decreased activity emerged. Therefore, the doseIt was of gamma pointed-ray out set that in this the hatchingstudy may rate cause of fertilizedsome stress eggs on producedparent shellfish, in the but control does group not affect was breeding,70 5% andso was the considered attachment s rateuitable was for 80 mutation5%, the induction. hatching rate of fertilized eggs produced in the ± ± 20 GyIt was exposed pointed group out was that 30 the hatching5%, and therate attachment of fertilized rate eggs was produced 65 5%. in The theexperimental control group group was 70 of ± ± ±20 5% Gy and showed the attachment a low number rate was of fertilized 80 ± 5%, eggs,the hatching fertilization rate of rate, fertilized hatching eggs rate, produced and attachment in the 20 rate,Gy exposedso it resultingly group was indicated 30 ± 5% a, and lower the number attachment of small rate abaloneswas 65 ± 5%. than The the experimental control group. group According of 20 Gy to showedKim et al. a low [15], number it is reported of fertilized that gamma-ray eggs, fertilization irradiation rate, in younger hatching abalone rate, and caused attachment stronger rate, DNA so and it resultinglyRNA damage indicated and caused a lower physiological number of disorders.small abalones The reductionthan the control of thehatching group. Acco andrding attachment to Kim rate et al. [15], it is reported that gamma-ray irradiation in younger abalone caused stronger DNA and RNA damage and caused physiological disorders. The reduction of the hatching and attachment rate at J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 8 of 11 J. Mar. Sci. Eng. 2020, 8, 906 8 of 11

J.high Mar. doses Sci. Eng. examined 2020, 8, x FOR in thisPEER study REVIEW was also presumed to be a result of concentration, presumably8 of by 11 causing gene damage to eggs existing in the body of the parent shellfish. highat high doses doses examined examined in inthis this study study was was also also pr presumedesumed to to be be a aresult result of of concentration concentration,, presumably presumably by by causingcausing gene gene damage damage to to eggs eggs existing existing in in the the body body of of the the parent parent shellfish. shellﬁsh.

Figure 6. Effect of high water-temperature stress on abalone Haliotis diversicolor aquatilis lysozyme Figure 6. Eﬀect of high water-temperature stress on abalone Haliotis diversicolor aquatilis lysozyme activity in the control or 20 Gy groups at a 0 h and 12 h incubation period. Figureactivity 6. in Effect the control of high or water 20 Gy-temperature groups at a 0 stress h and on 12 habalone incubation Haliotis period. diversicolor aquatilis lysozyme activity in the control or 20 Gy groups at a 0 h and 12 h incubation period.

Figure 7. Survival of abalone Haliotis diversicolor aquatilis reared with diﬀerent doses of radiation at Figure12 h after 7. Survival exposure of to abalone high-water Haliotis temperature diversicolor stress. aquatilis * p reared< 0.05 forwith control. different doses of radiation at 12 h after exposure to high-water temperature stress. * p < 0.05 for control. Figure 7. Survival of abalone Haliotis diversicolor aquatilis reared with different doses of radiation at 12 h after exposure to high-water temperature stress. * p < 0.05 for control. J. Mar. Sci. Eng. 2020, 8, 906 9 of 11

Meanwhile, Lee et al. [16] observed increments of germination rates in a gamma-ray irradiated group and a control group. This observation focused on the resulting hormesis effect of irradiated low-dose gamma-rays on the seeds of chili crop according to each dose. The optimal doses affecting growth were reported between 1 Gy and 20 Gy. Kim et al. [17–19] also observed that the growth and quantity of corn, fruit, and cabbage from the irradiation of low dose gamma-rays to seeds increased. Koepp and Kramer [20] reported that increments of the germination rate and growth of low-dose gamma-ray irradiated corn affected the quantitative equilibrium of plant hormones, and that the low-dose gamma-ray, which penetrated the seed skin, eventually improved the seed skin’s resistance to photosynthetic stress. Additionally, when the low dose gamma-ray irradiated Haliotis diversicolor aquatilis in this study, it showed a low hatching and attachment rate, but the growth was significantly high. This can be attributed to the gamma-ray irradiation affecting the growth and growth factors of the hormone system, which is similar to terrestrial plants. According to Kim et al. [21], water temperature and adaptability to low salinity environments were measured using F1 produced by hybridization of the Pagrus major (red seabream) and the Acanthopagrus schlegelii (black sea bream). It was reported that the fast-growing abalones are highly susceptible to variation in environmental changes. Invertebrate lysozymes are frequently used as an indicator of immune activity [22]. It has been known to play various roles in biodefense mechanisms by involving the opsonization and anti-parasitic action, antiviral action, and pollutant action, as it has cleansing and anti-inflammatory properties [23]. In this experiment, the lysozyme activity change from high water temperature stress was significantly greater in the gamma irradiation 20 Gy experimental group than in the control group at 0 h, and it was significantly lower than the control group at 12 h. Considering these results, the lysozyme activity was low in the control group without gamma-ray irradiation, while the lysozyme activity was high in the gamma-ray irradiation group. This suggests that the biodefense system against external stress was activated to remove harmful active oxygen, thereby showing a higher survival rate and rapid growth rate. As a result of measuring the activity of lysozyme in Haliotis diversicolor aquatilis by the low salt stimulation, it was reported that the lysozyme activity was signiﬁcantly increased and then decreased in other experimental groups compared to the control group immediately after administering stress (the same as in this study). These results suggest that the survival rate is improved by the increase in the initial immune-activity right after applying water temperature and salinity stress, and the abalones with strong resistance increase initial immune-activity, then stabilize rapidly.

4. Conclusions The results of this study on the physiological effects of gamma irradiation using Haliotis diversicolor aquatilis, an invertebrate marine organism, revealed that there was a high survival rate, growth rate, and immune activity response in low dose gamma irradiation, as shown in the published results of existing studies of land plants. In other words, these results revealed the potential of selective breeding using gamma ray irradiation hormesis regarding the number of eggs, fertilization rate, hatching rate, and attachment rate. It is presumed that the effect of hormesis in the gamma-ray irradiation parent shellfish also has a genetic effect on the offspring. These results are expected to suggest the possibility of introducing new breeding techniques through the induction of gamma-ray mutation in marine organisms with a high mortality rate due to high water temperature such as scallops and sea squirts. However, physiological indicators are sensitive to various environmental changes, species, and individuals. It is deemed necessary to set up the evaluating indicators through a detailed experiment in the future.

Author Contributions: M.-s.J. and C.-Y.H.provided direction for the research work and participated in the research. C.-Y.H. did the literature review and collected relevant data and M.-s.J. wrote the manuscript. Additionally, M.-s.J. searched and collected data on irradiation experiment as well as searched and collected literature and evidence. C.-Y.H. revised the paper. M.-s.J. and C.-Y.H. collected and analyzed the literature this paper needed. All authors have read and agreed to the published version of the manuscript Funding: This research received no external funding. J. Mar. Sci. Eng. 2020, 8, 906 10 of 11

Acknowledgments: We appreciate the productive suggestions from the Editors and anonymous reviewers and would like to give our thanks to them. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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