The identification of genetic resistance to amyotrophia in Japanese abalone, Haliotis discus discus.

ABSTRACT This experiment was designed to evaluate the genetic resistance to amyotrophia of Japanese abalone (Haliotis discus discus) under a mixed rearing environment. Two selected families that had previously shown resistance to abalone amyotrophia as candidates, two nonselected families as controls that had previously not shown resistance, and two hybrid families were used. Individuals from these families were fertilized and mixed immediately after hatching, and then they were raised in a mixed rearing tank at an abalone hatchery during the period of frequent spontaneous occurrences of abalone amyotrophia. Furthermore, we have isolated seven new microsatellite DNA loci for identifying families, and so the six offspring families in the mixed rearing tank could be discriminated unambiguously. The survival ratio of the two selected families was 87% and 93% after the occurrence of mass-mortality caused by abalone amyotrophia, whereas that of the four other families ranged from 0% to 37%. Survival performance among the offspring families was significantly different under the mixed rearing environment. This result suggests strongly that resistance to abalone amyotrophia of juveniles is related to the genetic characters of the spawners. Therefore, this experiment shows that resistance to the disease is a heritable trait, because the two selected families produced offspring with a high survival ratio after the occurrence of abalone amyotrophia.

KEY WORDS: abalone amyotrophia, Japanese abalone (Haliotis discus discus), microsatellite, parentage analysis, resistance

INTRODUCTION

Japanese abalones include four species that are economically important for commercial fisheries and as aquaculture resources in Japan, because of their high market value and their high volume of catch. However, the volume of abalone catch has been continuously decreasing for about 30 y (Anon. 2001). Therefore to increase and stabilize the commercial catch of abalone, techniques for mass production of hatchery-produced abalone seed have been developed over the last 20 y and the use of these techniques has been spread throughout the many prefectural hatcheries in Japan. In particular, the mass production of hatchery juveniles of 2 species, Haliotis discus hannai and Haliotis discus discus that support significant local fisheries, has been well studied. The juvenile production of H. d. hannai has increased smoothly year by year, and more than 20 million juvenile abalone were produced in 1999. However, the hatchery juvenile production of H. d. discus has been stagnant because mass mortalities of juvenile abalone age 0 y have frequently occurred in many abalone hatcheries, especially during the early summer when the rise in temperature is a serious problem. One of the causes of mass mortality was elucidated as an infectious disease characterized clinically with muscular atrophy and histologically with abnormal cell masses originating from nerve tissue (Nakatsugawa 1990, Momoyama et al. 1999). The disease, due to the amyotrophy of the foot muscle, has been tentatively called abalone amyotrophia. Recently, virus particles from juvenile abalone affected with amyotrophia was isolated (Otsu & Sasaki 1997, Nakatsugawa et al. 1999), however the pathogenicity of the virus has not been proved yet. Furthermore, it was reported that the mortality of juveniles spawned from one pair of parents was significantly lower than that of several other pairs during a spontaneous occurrence of abalone amyotrophia leading to mass mortality (Okada et al. 1999). This fact suggests that the establishment of abalone families resistant to abalone amyotrophia may be possible using selective breeding.

Mixed rearing is regarded as one of the most effective methods for evaluating genetic traits, because it can minimize confounding genetic traits and environmental factors. In separate tanks, even after careful environmental standardization, environmental effects of an unknown nature are often observed to cause phenotype variation among tanks (Bagley et al. 1994, Herbinger et al. 1999). Microsatellite DNA are well known as hyper-variable genetic markers with a high discriminating power, and are regarded as an invaluable tool in the investigation of parentage analysis of hatchery populations (Herbinger et al. 1995, Blouin et al. 1996, O'Reilly et al. 1998, Perez-Enriquez et al. 1999, Hara & Sekino 2003). Discrimination of families using microsatellite DNA typing makes it possible to evaluate the genetic properties of juveniles from a mixed rearing tank.

We have developed several microsatellite DNA markers for parentage analysis in abalone to establish an evaluation method for genetic traits in mixed rearing tanks. The objective of this study is to ascertain the ability to discriminate experimental families using the developed microsatellite DNA markers and to evaluate the survival performance of the selected families resistant to abalone amyotrophia, after the spontaneous occurrence of mass mortality caused by abalone amyotrophia in the abalone hatchery.

MATERIALS AND METHODS

Six abalone families, H. d. discus, were fertilized artificially by pairing of three mature dams and three mature sires (Table 1). Of the six parents used, 3 parents (SD#1, SD#2, and SS#1) were the offspring from two families that had a remarkably higher survival rate than other four families among the six experimental families during an outbreak of abalone amyotrophia at Mie prefectural fish farming center in 1995 (Okada et al. 1999). Moreover, the relationship between dam SD#1 and sire SS#1 was full sib, and that of dams SD#2 and SS#1 was half sib. These three parents were used for producing the candidate families resistant to abalone amyotrophia. One dam (ND1#) and 2 sires (NS#1 and NS#2) were nonselected, that is, these abalone parents were drawn randomly from the wild population. There were no relationship among ND#1, NS#1 and NS#2. The family #1 and #2, the family #3 and #4, and the family #5 and #6 were respectively called the selected, hybrid and nonselected families (Table 1). The larval offspring of the six families were mixed immediately after hatching, and were allowed to settle on plastic plates for collecting larvae in one tank. The mixed offspring (49,500 at the beginning of this experiment) were raised from November 1999 to April 2000 at an abalone hatchery of the Misaki Fisheries Co-operative Association, located in Ehime Prefecture of Japan.

To investigate the survival of abalone juveniles of the six mixed families during a spontaneous occurrence of mass mortality caused by abalone amyotrophia, 49,500 juveniles from 6-15 mm in shell length were subsequently reared from April to September 2000 in the experimental 10-ton tank of the Misaki hatchery, where mass mortality due to abalone amyotrophia has continuously occurred for several years. During the experimental rearing period, dead and moribund juveniles were removed almost every day while being observed in the experimental tank; the water temperature was measured and the number of surviving juveniles was calculated. Sampled offspring for parentage analysis were drawn randomly from the live juvenile individuals in May and August, before and after the period of mass mortality respectively. Twenty moribund juveniles were examined histologically during the occurrence of mass mortality, using a light microscope, to confirm if the mortality was caused by abalone amyotrophia.

Genomic DNA was extracted from the foot muscle tissue of the 6 parents and the sampled offspring. We have developed new hyper-variable microsatellite DNA markers for parentage analysis according to the method described in Sekino et al. (2000). Detailed information about the polymerase chain reaction (PCR) amplification protocols are found in Sekino and Hara (2001). Allele sizes were determined relative to a molecular size marker (ALFexpress Sizer 50-500, Amersham Pharmacia Biotech) in combination with the Allelelinks computer software (Amersham Pharmacia Biotech), and alleles were designated according to PCR product size. The relatedness between parents and offspring was examined by matching genotypes at each locus. Survival ratio were estimated from the percentage compositions of the families and total number of offspring reared in experimental tank.

Differences in the composition of families between before and after during mass mortality, and multiple comparisons among each family were done by [chi square] analysis, using the package Statistica (StatSoft Inc, Tokyo).

RESULTS

The estimated number of reared juvenile abalone during the experimental period is shown in Figure 1. Dead juveniles were rarely found in the experimental mixed rearing tank from April to May. After that, mass mortality occurred from June to July, and bad almost ceased in August. The total estimated number of juveniles during the period from May to August decreased from 49,500 to 21,500. Decrease in feed consumption, increase in individuals with reduced activity and the appearance of abnormal growth in shell edge were observed in the experimental tank from June to July. However, these symptoms disappeared in August, and it appeared that the abnormal shell edges of individuals had recovered. During the experimental period, the temperature of the mixed rearing tank was 12.9[degrees]C to 17.5[degrees]C from April to the middle of May and was over 18[degrees]C in the end of May. After that, the temperature was continually increased reaching 23.9[degrees]C by the end of July, and staying above 24[degrees]C in August and September (Fig. 2). Furthermore, abnormal cell masses that were originating from nerve tissue and characteristic in abalone amyotrophia were histologically observed in the toot muscle section of all twenty moribund juvenile abalone, using a light microscope (Fig. 3).

[FIGURES 1-3 OMITTED]

We developed 7 new microsatellite DNA loci, Hd63, Hd201, Hd306, Hd3105, Hd535, Hd562, and Hd584 from the abalone genome DNA for this experiment, and the PCR primers pertaining to them are given in Table 2. All 7 loci were successfully amplified for 6 abalone parents, and the genotypes of the 6 abalone parents for the 7 microsatellite DNA loci and the number of alleles per locus are listed in Table 3. The number of alleles per locus ranged from 6 to 8 with an average of 6.7, and alleles unique to one parent were found at all loci. Further, all the parents except SD#1 had unique alleles. These microsatellite DNA loci were hyper-variable and stable to analysis.

The parents of individual offspring could be discriminated unambiguously by observations of the unique alleles and/or by comparison of the multi loci genotype for the 7 microsatellite DNA. The parentage inferred from more than two loci agreed perfectly for all analyzed individuals. The families of all offspring were determined precisely by the relatedness between parents and offspring. The percentages and individual number of offspring related to 6 families in May and those of August are shown in Table 4.

The estimated numbers of the selected families (family #1 and #2) were 38 and 22 of the 236 analyzed individuals, that is 16.1% and 9.3% respectively of the total mixed families in May, and the percentage compositions of these families had proportionally increased to 34.6% and 18.7% respectively in August. However, the percentage composition of the two hybrid and two nonselected families (family #3, #4, #5, and #6) decreased from May to August. Especially, offspring of the families #6, which (15.3%) was 36 of the 236 analyzed individuals, were not found at all in August. The percentage composition of the 6 families in May was significantly different compared with that in August (P < 0.01). Furthermore, on the multiple comparisons among individual number of each family in May and August, significant differences were recognized between the two selected families (family #1 and 2) and the other four families and between the family #6 and the other families (P < 0.05). The estimated survival ratio of the selected two families (family #1 and #2), which was calculated based on the total number of offspring reared in the tank and the percentage composition of each family, was more than 80%. However, the estimated survival ratios of the other families were less than 40%.

DISCUSSION

Mass mortality occurred spontaneously from June to July in the experimental mixed rearing tank at the abalone hatchery, and more than half of the juvenile abalone died. This period of mass mortality coincided with the season of mass mortalities reported previously (Nakatsugawa 1990, Momoyama et al. 1999, Okada et al. 1999). Momoyama et al. (1999) histologically examined individual abalone from mass mortalities that occurred in different hatcheries during this period from 1988 to 1996, and they reported that most of mass mortalities were attributable to abalone amyotrophia. The characteristic relation between the occurrence of abalone amyotrophia and ambient water temperature has been reported as follows: the progress of abalone amyotrophia occurs when the water temperature rises to 18[degrees]C or higher and is suppressed at more than 23[degrees]C (Nakatsugawa 1990, Okada et al. 1999). The range of water temperature of this experimental mixed rearing tank during this mass mortality was consistent with the typical temperature range of mass mortality caused by amyotrophia disease. Unhealthy individual juveniles and with shell edge abnormalities appeared in the experimental tank during this mass mortality period. These symptoms coincide with the characteristics of abalone amyotrophia. Furthermore, abnormal cell masses that were previously reported as characters of abalone amyotrophia (Nakatsugawa 1990, Momoyama et al. 1999) could be observed from all individuals examined histologically. These findings strongly suggest that the mass mortality during this experimental rearing period was caused by abalone amyotrophia.

The usefulness of microsatellite DNA markers for parentage determination in hatchery populations has been previously reported (Herbinger et al. 1995, Perez-Enriquez et al. 1999, Hara & Sekino 2003). In this experiment, the parent-offspring relationships could be unequivocally identified for all abalone offspring mix-reared from the larval stage onward using the seven new microsatellite DNA loci, and the families of the offspring were accurately discriminated. As a result, the change of the percentage composition of the six families from May to August suggests that the survival of the 2 selected families (family #1 and #2) were significantly better than that of the other 4 families (family #3, #4, #5, and #6) after the occurrence of mass mortality caused by abalone amyotrophia. Juvenile individuals of the six families were reared in the same tank, thus minimizing environmental effects immediately after hatching; therefore their survival performances among families are considered to have been strongly influenced by heritability.

The two selected families were produced from the families that had higher survival rates than the other families after an occurrence of mass mortality caused by abalone amyotrophia, and in this experiment their offspring bad survived higher rates than the other families examined. This result suggests that resistance to abalone amyotrophia of the selected families is a heritable trait. However, the estimated survival ratios of the 2 hybrid families (family #3 and #4) that were produced by the dam of the candidate families tolerant to the disease did not differ from that of the one nonselected family (family #5). The family of spawners used as tolerant to abalone amyotrophia in this experiment might not be perfectly fixed genetically. Differences between families for survival ratios in NS#1 and NS#2 of nonselected sires indicate the existence of various degrees of tolerance to abalone amyotrophia in wild abalone populations.

The results of this experiment strongly suggested that resistances to abalone amyotrophia is due to genetic effects. Establishment of a method of experimental infection by isolation of the pathogen of abalone amyotrophia and development of high performance families resistant to the disease, using selective inbreeding techniques, would likely help the problem of mass mortality caused by amyotrophia disease for juvenile production of Japanese abalone, Haliotis discus discus.

TABLE 1.

Pairing of dams and sires in the six families. Relation of the dam,
SD#1 and the sire, SS#1 is full-sib, and that of the dam, SD#2 and
SS#1 is half sibe. There is no relationship among the dam, ND#1,
the sires NS#1 and NS#2.

            Selected         Hybrid       Non-selective

Family     #1      #2      #3      #4      #5      #6

Dam       SD#1    SD#2    SD#2    SD#2    ND#1    ND#1
Sire      SS#1    SS#1    NS#1    NS#2    NS#1    NS#2

TABLE 2.

Core microsatellite sequences, primer sequences, and annealing
temperatures (Tm) for seven microsatellite DNA loci of Japanese abalone
Haliotis discus discus used in this experiment.

Locus    Core Repeat Sequence (5'-3')      Primer Sequence (5'-3')

Hd63     [(CA).sub.16]                     F-CACTATATAAATGCGGCATAAG
                                           R-GCTTTGTAAGTGCAGTAATC
Hd201    [(CA).sub.5][(CG).sub.2]          F-CTTTTATGAATATGCGATTTCCTGA
         [(CGCA).sub.3][(CG).sub.2]        R-TCACTCTGTGAAGGTTCATACTCCA
         [(CGCA).sub.6]CT[(GCACC).sub.3]
         GCACG[(CA).sub.8]
Hd306    [(CA).sub.5]CAC[(CA).sub.7]       F-GGAACAGTTTACAAGGTGGGAGCA
         [(CCA).sub.2][(CA).sub.19]C       R-GGTTTGTTTACAGGCCGCCATCGC
         (CA)3C[X.sub.4]
         [(AC).sub.4]G[(CA).sub.4]X
         [(CA).sub.6]
Hd3105   [(CA).sub.5][(CACG).sub.2]TA      F-GTTGTAATGGTGAATCGGAC
         [(CGCA).sub.3][(CG).sub.2]C       R-CACTAACGTAGTGAGGTGCA
         [(CACG).sub.6][(CATG).sub.2]
         [(CA).sub.6]
Hd535    [(CACT).sub.14]                   F-TTTAACTCTACATGCCGAAG
                                           R-TACTGTCAGTCCACATAGGAT
Hd562    [(TG).sub.2][(TTG).sub.3]         F-TGGTTGTGGCCTTGTCTGTTTC
         [(TCG).sub.2][(TTG).sub.7]        R-TATAGCTGGAATGCTCAGTGCG
Hd584    [(ACTC).sub.17]                   F-TATGACGGGAATATTGCTAA
                                           R-CAAAATGTGGTTAACATAGATAT

Locus    Tm ([degrees]C)    GenBank Accession No. *

Hd63           50                  AB025376
Hd201          61                  AB085642
Hd306          64                  AB085643
Hd3105         50                  AB085644
Hd535          50                  AB085645
Hd562          63                  AB085646
Hd584          51                  AB085647

* The nucleotide sequence data will appear in the DDJB/EMBL/GenBank
nucleotide databases with the accession numbers.

TABLE 3.

Genotypes of the 6 parents at the 7 microsatellite DNA loci in Pacific
abalone, Haliotis discus discus. Block characters indicate unique
alleles.

                     Dam                          Sire

Locus     SD#1      SD#2      ND#1      SS#1      NS#1      NS#2

Hd63     176/176   170/172   178/180   172/176   170/184   172/172
Hd201    200/222   190/222   196/218   200/222   196/224   174/212
Hd306    256/296   270/270   226/226   256/296   226/226   194/242
Hd3105   218/252   218/226   216/224   218/252   220/220   266/266
Hd535    175/211   183/195   175/175   187/211   175/199   175/195
Hd562    163/180   163/169   159/169   163/173   171/187   169/173
Hd584    131/131   131/131   135/139   131/131   115/143   115/119

             Numbers of          Numbers of
Locus    Alleles in Parents    Unique Alleles

Hd63             6                   3
Hd201            8                   5
Hd306            6                   3
Hd3105           7                   4
Hd535            6                   3
Hd562            8                   3
Hd584            6                   3

TABLE 4.

Estimated number of offspring and survival ratio of six families reared
in tank during the occurrence of mass mortality from May to August.
Numbers of offspring (Nt) are estimated based on the genetic markers of
microsatellite DNA. Number in parentheses are the percentage of
offspring of each family. N(M) and N(A) means the estimated actual
number of offspring of families in the tank in May and August based on
the percentage respectively.

                                                Estimated Number of
                                                     Offspring

                                                   May
         Family ([female] x [male])               Nt (%)      N (M)

#1 (SD#1 x SS#1)                                 38 (16.1)     7970
#2 (SD#2 x SS#1)                                 22 (9.3)      4604
#3 (SD#2 x NS#1)                                 56 (23.7)    11732
#4 (SD#2 x NS#2)                                 27 (11.4)     5643
#5 (ND#1 x NS#1)                                 57 (24.2)    11979
#6 (ND#1 x NS#2)                                 36 (15.3)     7574
Total number of offspring analyzed              236 (100)

Total number of offspring reared in the tank                  49500

                                                Estimated Number of
                                                     Offspring

                                                  August
         Family ([female] x [male])               Nt (%)      N (A)

#1 (SD#1 x SS#1)                                 37 (34.5)     7418
#2 (SD#2 x SS#1)                                 20 (18.7)     4021
#3 (SD#2 x NS#1)                                 22 (20.6)     4429
#4 (SD#2 x NS#2)                                  6 (5.6)      1204
#5 (ND#1 x NS#1)                                 22 (20.6)     4429
#6 (ND#1 x NS#2)                                  0 (0)           0
Total number of offspring analyzed              107 (100)

Total number of offspring reared in the tank                  21500

                                                Estimated Survival
                                                  Ratio (%) * 1
         Family ([female] x [male])             (N(A)/N(M) x 100)

#1 (SD#1 x SS#1)                                    [93.sup.a]
#2 (SD#2 x SS#1)                                    [87.sup.b]
#3 (SD#2 x NS#1)                                    [38.sup.c]
#4 (SD#2 x NS#2)                                    [21.sup.d]
#5 (ND#1 x NS#1)                                    [37.sup.c]
#6 (ND#1 x NS#2)                                     [0.sup.e]
Total number of offspring analyzed

Total number of offspring reared in the tank            43

* 1 Values with different superscripts indicate significant
differences (P < 0.01).

ACKNOWLEDGMENT

The authors thank Dr. Katsuhiko Wada, National Research Institute of Fisheries Science and Dr. Takuji Okumura, National Research Institute of Aquaculture for helpful suggestions and advice of statistical analysis. The authors also thank Dr. Mamoru Nishimura, Mie Prefectural Fisheries Experimental Station for the donation of the candidate abalone resistant to abalone amyotrophia and Mr. Shogo Kikuchi and Mr. Kondo, Misaki Fisheries Cooperative Association for rearing the abalone seed and supplying the samples. This study was supported in part by a grant-in-aid (Development fundamental technologies for effective genetic improvement of aquatic organisms program) from the Ministry of Agriculture, Forestry and Fisheries, Japan.

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MOTOYUKI HARA, (1), * MASASHI SEKINO, (2) AKIRA KUMAGAI, (3) AND TOMOYOSHI YOSHINAGA (4)

(1) National Research Institute of Aquaculture, Nansei, Mie, 516-0193, Japan; (2) Tohoku National Fisheries Research Institute, Shiogama, Miyagi, 985-0001, Japan; (3) Miyagi Prefectural Freshwater Fisheries Research Station, Daiwa, Miyagi, 981-3625, Japan; (4) University of Tokyo Faculty of Agriculture, Bunkyo, 113-8657, Tokyo, Japan

* Corresponding author. E-mail: [e-mail]