Journal of Shellfish Research, Vol. 23, No. 1, 205–209, 2004.

KARYOTYPE OF PACIFIC RED ABALONE HALIOTIS RUFESCENS (ARCHAEOGASTROPODA: HALIOTIDAE) USING IMAGE ANALYSIS

CRISTIAN GALLARDO-ESCÁRATE,1,2 JOSUÉ ÁLVAREZ-BORREGO,2,* MIGUEL ÁNGEL DEL RÍO PORTILLA,1 AND VITALY KOBER3 1Departamento de Acuicultura. División de Oceanología, 2Departamento de Óptica, 3Departamento de Ciencias de la Computación, División de Física Aplicada, Centro de Investigación Científica y de Educación Superior de Ensenada. Km 107 Carretera Tijuana – Ensenada, Código Postal 22860. Ensenada, B.C. México

ABSTRACT This report describes a karyotypic analysis in the Pacific red abalone Haliotis rufescens using image analysis. This is the first karyotype reported for this species. Chromosome number and karyotype are the basic information of a genome and important for ploidy manipulation, genomic analysis, and our understanding about chromosomal evolution. In this study we found that the diploid number of chromosomes in the red abalone was 36. Using image analysis by rank-order and digital morphologic filters, it was possible to determine total length of chromosomes and relative arm length in digitally enhanced image, elimination of noise and improving the contrast for the measurements. The karyotype consisted of eight pairs of metacentric chromosomes, eight pairs of submetacentric, one pair submetacentric/metacentric, and one pair of subtelocentric chromosomes. The black abalone, Haliotis cracherodii, also with 36 chromosomes and with a similar geographic distribution, has eight pairs of metacentric, eight pairs of submetacentric, and two pairs subtelocentric. This study contributes with new information about the karyology in the family Haliotidae found in California Coast waters and gives some support the Thetys’ model about biogeographical origin, from the Mediterranean Sea to the East Pacific Ocean.

KEY WORDS: karyotype, Haliotis refescens, chromosome number, image analysis, red abalone

INTRODUCTION In recent years, great attention has been given to image pro- cessing by the use of rank-order statistics. Rank-order and mor- Abalones are economically important marine gastropods that phologic digital filters can be applied to computer and optical reach moderate-to-high prices in the world market (Oakes & Ponte research (Kober et al. 2001a). These digital filters are very effi- 1996). To date, only eight species of abalone, Haliotis corrugata, cient in removing additive and impulsive noise, as well as in image H. cracherodii, H. fulgens, H. kamtschatkana. kamtschatkana, H. enhancing and restoring the microscopical images. The reason for k assimilis, H. sorenseni, H. walallensis, and H. rufescens, have their success in image processing is that they can suppress noise been reported in the further East Pacific (Geiger & Poppe 2000). without destroying important image details, such as edges and The Pacific red abalone, H. rufescens, is found from southern fines lines. The aim of this work is to describe the karyotype of Oregon to northern California in the United States and to the Baja Pacific red abalone Haliotis rufescens by using rank-order and California Peninsula in Mexico (Lindberg 1992). Aquaculture pro- morphologic filters for suppression of noise and enhancing con- duction of this species began during the last decade in Mexico, trast of the chromosomal images. where the whole life cycle is managed under controlled conditions. In addition to the importance on conservation, taxonomic work MATERIALS AND METHODS and karyological analyzes are also useful tools in providing fun- damental information for animal breeding programs such as inter- Gametes specific hybridization (Miyaki et al. 1997) and chromosome-set manipulation (Arai et al. 1986, Fujino et al. 1987,Kudo et al. 1991, Pacific red abalone Haliotis rufescens were obtained from the Zhang et al. 1998). A review about the biogeographical origin of broodstock kept in the commercial farm “Abulones Cultivados the Family Haliotidae reported by Geiger & Groves (1999) using S.A” at Eréndira, Baja California, México. The abalones were chromosome data in abalone showed that the abalones could be maintained in a 250-L tank with a recirculating sea water system classified in three groups according to their chromosomes number. until the spawning induction. Gametes were obtained through in- inhabits the European–Mediterranean duction spawning according to Morse et al. (1977). Eggs were ,(28 ס The first group (2n ␮ inhabits the Indigo–Pacific region, and collected in a 25- m sieve and resuspended into 2-L container (32 ס area. The second (2n ␮ represents the abalones of the North with seawater filtered (0.45 m) and irradiated with ultraviolet (36 ס the third group (2n Pacific region. Geiger & Groves (1999) suggest that the biogeo- light. Fertilization was conducted by adding sperm to the eggs graphical origin of abalone was in the European–Mediterranean suspension at a final density of 50 sperms per egg. is a relict species. Most (28 ס area because H. tuberculata (2n Chromosome Obtaining probably this area could have been the starting point of the radia- tion, extending the distribution of the abalone forward to East from Twenty to 24 hours after fertilization, swimming trochophore the Indo–Pacific to the North Pacific Ocean. This theory has been larvae of pacific red abalone were placed in a 0.005% colchicine called the Tethys’ model. In the California Cost, however, karyo- solution for 3–4 h. After the antimitotic treatment, the samples logical data have been performed only for the black abalone H. were subjected to 45 min in a hypotonic solution (50% sea water cracherodii (Minkler, 1977). Thus, more studies are necessary to and distilled water). The larvae were fixed in Carnoy solution (3:1, support the Tethys’ model and to know the karyology of abalone methanol: acetic acid) by rinsing the samples three times in the species from California Coast. fixative at intervals of 10 min. Fixed cells were stored at 5°C overnight in a refrigerator. The sample of cellular suspension was *Corresponding author. E-mail: [email protected] made by cell dissociation in 50% acetic acid and strong agitation

205 206 GALLARDO-ESCÁRATE ET AL. with a Pasteur pipette. The cellular suspension was then dropped consists of two steps. Outliers and small details are first detected onto a heated microscope slides (45°C) and air dried. using spatial relationships between the color image components. Then, the detected pixels are replaced with the output of the vector Chromosome Observation via Computer median filter over a local spatially connected area excluding the outliers, while desired detail and noise-free pixels are left unal- The chromosomal images were obtained from unstained tered. The size of a moving window is 7 × 7 elements. The spa- metaphases. The best 10 images were digitalized with a 24-bit tially connected neighborhood called as a CEV neighborhood is video color frame grabber (model CG-7, Scion Corp.) using a defined as a subset of pixels (v ) of the moving window, which phase-contrast microscopy (Zeizz Axiolab) equipped with a RGB n,m are spatially connected with the central pixel v , and whose values color video camera (model 1040 COHU). To remove impulse k,l deviate from the value of the central pixel at most by predeter- noise as well as small undesirable details in chromosomal image, mined quantities. The rank filter is given by: we use rank-order filters with spatially connected neighborhoods (Kober et al. 2001a, 2001b). These filters use spatial and rank v ,ifSIZE͑CEV͓v ͔͒ՆThrd information of spatially connected areas of the input image within = ͭ n,m n,m vˆn,m ͑ ͓ ͔ − ͓ ͔͒ a moving window to produce the output. The used rank algorithm MED S vn,m CEV vn,m , otherwise

Figure 1. A, Digital image of metaphase plate from larvae of Haliotis rufenscens. B, Karyotype of Pacific red abalone Haliotis rufenscens. KAROTYPE OF PACIFIC RED ABALONE 207 where Thrd is a threshold value of either outlier or undesired detail sis, the maximum length of the chromosomes was 5.67 ± 0.56 ␮m detection, “-” denotes the set difference operation; S is a small and the minimum was 3.75 ± 0.071 ␮m (Table 1). The ratio of the subset of the pixels of the moving window. The pixels associated chromosomes arms plotted in the karyo-ideogram indicated that with outliers after the detection are excluded from the S neighbor- this species possessed eight metacentric pairs (chromosome Nos. hood. MED and SIZE denote the median operation and the size of 1, 3, 6, 7, 14, 15, 17, and 18), eight submetacentric pair (nos. 2, 4, a set, respectively. 5, 8, 9, 10, 11 and 13), one pair (no. 12) was classified as sub- Identification of homologue chromosomes was performed by metacentric/metacentric chromosome and one pair of subtelocen- digital correlation. After identification of homologue chromo- tric chromosome (no. 16). Chromosome pairs 14 and 17 were somes, determinations of chromosome arm lengths (short and difficult to identify using only such morphologic characteristics as large) were measured with an Image Pro-Plus software version 4.0 the relative length, because of their mutually overlapping standard (Media Cybernetics). The measurements were used for determiner deviations (Fig. 2). relative lengths of chromosomes and centromeric index (100 × length of short arm/total chromosome length). The relative lengths of the chromosome arm was plotted the karyo-ideogram according DISCUSSION Spotorno (1985). Karyotype was arranged by decreasing size and classified according to Levan et al. (1964). To our knowledge, this is the first report of ploidy level and chromosome number in the Pacific red abalone Haliotis rufescens. RESULTS Karyological studies of abalone species belonging to the North Oriental Pacific are scarce; in fact, only the karyotype of the black ס All chromosomal images in this study were processed using abalone H. cracherodii is described with 2n 36 (Minkler, 1977), image analysis. The originals digitals images were obtained with since unknown the karyology of Pacific red abalone H. rufescens the CCD camera, in this images were preset noise and contrast in the California Coast at present. In contrast, karyological studies low, however, after digital image processing it is observed a good in abalones species have been reported in North Occidental Pacific performance on image edge, enhanced contrast of the chromo- (Arai et al. 1982, Nakamura 1986, Okumura et al. 1999). The somes and allowing mensurations morphologic more exact. Figure morphologic comparison between H. rufescens and H. cracherodii 1A, shows the images enhanced of unstained metaphase plate by showed that both species comprised 36 chromosomes. However, the algorithm. they are only similar in the metacentric chromosomes number The karyological observations on larvae of Pacific red abalone equal to eight pairs. The differences are that red abalone has nine showed a diploid number equal to 36 chromosomes (Fig. 1A). The submetacentric pairs (chromosome no. 12, which is submetacen- distribution of relative chromosome lengths observed was derived tric/metacentric) and one subtelocentric pair, whereas the back to perform the karyotype of Pacific red abalone Haliotis rufen- abalone has eight submetacentric pairs and two subtelocentric scens (Fig. 1B). The mean values and standard deviations of the pairs. This conformation of the karyotype in H. cracherodii sug- relative lengths and total lengths of 18 chromosomes pairs were gest a deletion process of the short arm in one of submetacentric estimated from arm length measurements in well-spread unstained chromosome pairs, originating an extra subtelocentric pair. It is metaphase plates from the larvae using chromosomal image analy- possible that H. cracherodii was divergent from the one species with more number of submetacentric chromosomes. This suggests that H. cracherodii has a higher genetic difference than H. rufe- TABLE 1. scens. According to Owen et al. (1971) and Leighton (2000), rela- tively few cases of hybridization in mollusks have been reported, Relative lengths (LR), total lengths (TL), and centromeric index (CI) however, in Haliotis, with the exception of H. cracherodii, each of chromosomes of Haliotis rufescens southern Californian species of abalones was found to have hy- bridized with at least other species. The impossibility the H. crach- Chromosome LR (%) TL (µm) CI Type erodii for hybridize with other species of abalones proved genetic 1 6.52 ± 0.56 5.67 ± 0.19 43.95 ± 0.15 M distantly related. 2 6.42 ± 1.03 5.58 ± 0.07 32.53 ± 0.18 SM Phylogenetic relationships studies by Meyer (1967) among 3 6.20 ± 0.55 5.39 ± 0.06 46.91 ± 0.11 M California species were analyzed by immunochemical comparing 4 6.09 ± 0.78 5.30 ± 0.10 31.30 ± 0.13 SM of the hemocyanins and showed that phylogenetically close spe- 5 6.03 ± 0.91 5.24 ± 0.01 29.91 ± 0.09 SM cies were distinguished from those more distantly related. Two 6 5.97 ± 0.26 5.20 ± 0.03 44.66 ± 0.15 M relatively closely related groups were found; 1) H. rufescens, H. 7 5.84 ± 0.44 5.08 ± 0.13 41.68 ± 0.03 M sorenseni, and H. kamtschatkana assimilis, and 2) H. corrugata, 8 5.83 ± 0.68 5.07 ± 0.07 31.72 ± 0.29 SM H. fulgens, and H. walallensis appeared moderately distinct from 9 5.74 ± 1.01 4.99 ± 0.07 26.38 ± 0.15 SM 10 5.34 ± 0.80 4.65 ± 0.04 28.97 ± 0.13 SM these groups, whereas H. cracherodii was decidedly different from 11 5.32 ± 0.70 4.63 ± 0.09 29.70 ± 0.13 SM the other species. The amino acid sequences of lysin proteins of 12 5.26 ± 0.52 4.58 ± 0.02 34.67 ± 0.08 SM/M seven California abalone species were deduced from the cDNA 13 5.17 ± 0.78 4.50 ± 0.04 27.59 ± 0.17 SM sequences (Lee & Vacquier 1992, 1995). These studies are in 14 5.10 ± 0.17 4.44 ± 0.07 46.34 ± 0.21 M general agreement with the immunochemical comparison by 15 4.96 ± 0.31 4.32 ± 0.05 42.74 ± 0.20 M Meyer (1967), but H. cracherodii was placed closer to H. corru- 16 4.95 ± 1.63 4.31 ± 0.05 16.31 ± 0.14 ST gata, leaving H. fulgens in a more distant relationship to the other 17 4.45 ± 0.27 4.46 ± 0.06 45.56 ± 0.15 M California species. 18 4.31 ± 0.34 3.75 ± 0.07 44.48 ± 0.19 M Morphometric comparison of chromosomes between the other M, metacentric; SM, submetacentric; ST, subtelocentric. studied abalone species showed variations of diploid number 208 GALLARDO-ESCÁRATE ET AL.

Figure 2. Karyo-ideogram of Haliotis rufenscens. M, metacentric; SM, submetacentric; ST, subtelocentric; T, telocentric.

.characteristic, except for H 32 ס among 28 to 36 chromosomes (Jarayabhand et al. 1998) (Table 2). 28, Indo–Pacific region with 2n The review of diploid chromosome number of the existing karyo- aquatilis with diploid number equal to 34 chromosomes, south and finally Pacific North area with a 32 ס types abalone according to Geiger & Groves (1999), showed that Japan area with 2n .chromosome number of 36 ס the increment begins in European Mediterranean area with 2n

TABLE 2. Review of diploid chromosome number of abalone.

Haliotis Diploid Number Geographic Occurrencea Reference

H. tuberculata 28 European–Mediterranean Arai & Wilkins 1986 H. lamellose 32 European–Mediterranean Colombera & Tagliaferri 1983 H. diversicolor aquatilis 32 Indo–Pacific Nakamura 1985 H. aquatilis 34 Indo–Pacific Nakamura 1985 H. diversicolor 32 Indo–Pacific Arai et al. 1988 H. planata 32 Indo–Pacific Arai et al. 1988 H. varia 32 Indo–Pacific Jarayabhand et al. 1998 H. asinina 32 Indo–pacific Jarayabhand et al. 1998 H. ovina 32 Indo–Pacific Jarayabhand et al. 1998 H. exigua 32 South Japan Arai et al. 1988 H. discus discus 36 North Pacific Arai et al. 1982 H. discus hannai 36 North Pacific Arai et al. 1982, Okumura et al. 1999 H. modaka 36 North Pacific Nakamura 1986 H. cracherodii 36 North Pacific Winkler 1977 H. rufenscens 36 North Pacific This study a Geographical areas according Geiger & Groves 1999. KAROTYPE OF PACIFIC RED ABALONE 209

This study contributed with new information about the kar- ACKNOWLEDGMENTS ology in the family Haliotidae found in California Cost waters and supports the Thetys’ model about biogeographical origin This study was supported by the Mexican National Council of discussed by Geiger & Groves (1999) and Geiger & Poppe Science and Technology (Project 36075-B). The authors thank to (2000). C. Paniagua for manuscript review and for technical assistance.

LITERATURE CITED Arai, K. & N. Wilkins. 1986. Chromosomes of Haliotis tuberculata L. Leighton, D. 2000. The biology and culture of the California abalones. Aquaculture 58:305–308. Pittsburgh, PA: Dorrance Publishing Co., Inc. Arai, K., K. Fujino & M. Kudo. 1988. Karyotypes and zymogram differ- Levan, A., K. Gredga & A. Sandberg. 1964. Nomenclature for centromere ences among three species of the abalones Haliotis planate, H. varia position on chromosome. Hereditas 52:201–220. and H. diversicolor diversicolor. Bull. Jpn. Soc. Sci. Fisheries 54: Lindberg, D. 1992. Evolution, distribution and systematics of Haliotidae. 2055–2064. In: S. Shepherd, M. Tegner & S. Guzmán, editors. Abalone of the word, Arai, K., H. Tsubaki, Y. Ishitani & K. Fujino. 1982. Chromosome of biology, fisheries and cultured. 3 – 18. Oxford: Fishing News Books. Haliotis discus hannai Ino and H. discus Reeve. Bull. Jpn. Soc. Sci. Meyer, R. 1967. Hemocyanins and the systematics of California Haliotis. Fisheries 48:1689–1691. Ph.D. Dissert. Stanford Univ. 92 pp. Arai, K., F. Naito & K. Fujino. 1986. Triploidization of the pacific abalone Minkler, J. 1977. Chromosomes of the black abalone (Haliotis crachero- with temperature and pressure treatments. Bull. Jpn. Soc. Sci. Fisheries dii). Experientia 33:1143. 52:417–422. Miyaki, K., O. Tabeta & H. Kayano. 1997. Karyotypes of the two species Colombera, D. & F. Tagliaferri. 1983. Chromosomes of Haliotis tubercu- of abalone Nordotis discus and N. gigantean. Fisheries Sci. 63:179– lata and H. lamellose. Caryologia 36:231–234. 180. Fujino, K., S. Okumura & H. Inayoshi. 1987. Temperature tolerance dif- Morse, D., H. Duncan, N. Hooker & A. Morse. 1977. Hydrogen peroxide ference among normal diploid and triploid Pacific abalones. Nippon induces spawning in mollusks, with activation of prostaglandin endo- Suisan Gakkishi 53:12–21. peroxide synthetase. Science 196:298–300. Nakamura, H. 1985. The chromosomes of Haliotis diversicolor aquatilis Geiger, D. & L. Groves. 1999. Review of fossil abalone (Gastropoda: (Archaeogastropoda: Haliotidae). Mol. Rev. 18:113–114. Vestigastropoda: Haliotidae) with comparison to recent species. J. Pa- Nakamura, H. 1986. Chromosomes of Archaeogastropoda (Mollusca: leontol. 73:872–885. Prosobranchia), with some remarks on their citotaxonomy and phylog- Geiger, D. & G. Poppe. 2000. A conchological iconography. The Family eny. Pub. Seto Marine Biol. Lab. 31:191–297. Haliotidae. New York: ConchBooks, pp.327. Oakes, F. & R. Ponte. 1996. The abalone market: opportunities for cultured Jarayabhand, P., R. Yom-La & A. Popongviwat. 1998. Karyotypes of abalone. Aquaculture 140:187–195. J. Shellfish marine mollusks in the family Haliotidae found in Thailand. Okumura, S., S. Kinugawa, A. Fujimaki, W. Kawai, H. Maehata, K. Res. 17:761–764. Yoshioka, R. Yoneda & K. Yamamori. 1999. Analysis of karyotype, Kober, V., M, Mozerov, J., Alvarez-Borrego & I. Ovseyevich. 2001a. Rank chromosome banding, and nucleolus organizer region of pacific aba- image processing using spatially adaptive neighborhoods. Pattern Rec- lone, Haliotis discus hannai (Archaeogastropoda: Haliotidae). J. Shell- ognition Image Analysis 11:542–552. fish Res. 18:605–609. Kober, V., M, Mozerov & J. Alvarez-Borrego. J. 2001b. Nonlinear filters Owen, B., J. Mclean & R. Meyer. 1971. Hybridization in the Eastern with spatially-connected neighborhoods. Optical Eng. 40:971–983. Pacific abalones (Haliotis). Bull. Los Angeles County Museum Natural Kudo, M., K. Arai & K. Fujino. 1991. Triploidization of Halliotis diver- History Sci. 9:1–37. sicolor diversicolor. Nippon Suisan Gakkishi 57:1263–1267. Spotorno, A. 1985. Conceptos y métodos en cariología descriptiva y com- Lee, Y. & V. Vacquier. 1992. The divergence of species-specific abalone parada. In: Fernández-Dononso R., editor. El núcleo, los Cromosomas sperm lysins is promoted by positive Darwinian selection. Biol. Bull. y la Evolución. UNESCO, pp.135–163. 182:97–104. Zhang, G., Z. Wang, Y. Chang, Y. Song, Y. Ding, Y. Wang & R. Wang. Lee, Y. & V. Vacquier. 1995. Evolution and systematics in Haliotidae 1998. Triploid induction in the pacific abalone Haliotis discus hannai (Mollusca: Gastropoda): inferences from DNA sequences of sperm Ino by 6-dimethylaminopurine and the performance of triploid juve- lysin. Marine Biol. 124:267–278. niles. J. Shellfish Res. 17:783–788.