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Karyotypes of diploid and triploid Mercenaria mercenaria (Linnaeus).

ABSTRACT Northern quahogs (=hard clam; Mercenaria mercenaria) is a commercially important bivalve naturally distributed along the American Atlantic coast. Because the hard clam was first introduced into China as a candidate for aquaculture in 1997, research has been conducted on its ecologic habits, ecological physiology, metabolism, growth, reproduction, artificial propagation, and seed culture. Little information about their chromosomes and karyotype is available. The present study is designed to study the karyotype of the diploids and triploids to provide a reference to genetic research on introduced M. mercenaria. Triploids were produced by inhibiting the release of second polar body (PBII) with a treatment of 450 gmol/mL 6-deimethylaminopurine (6-DMAP), with untreated fertilized eggs developing as normal diploid controls. Chromosome preparations were made from trochophore larvae with drop-splash method. Chromosomes were measured and classified according to Leven criteria. Statistical analysis of relative length and arm ratio revealed that, this species had a normal diploid chromosome number of 2n = 38, and the karyotype consists of 15 pairs of metacentric (M) and 4 pairs of submetacentric (SM) chromosomes, NF = 76. The triploid karyotype formula was 45 M + 12 SM, NF = 114. The heterochromosomes and satellite chromosomes were not observed in all chromosomes in the complement.

KEY WORDS: Mercenaria mercenaria, quahog, hard clam, karyotype, chromosome, diploid, triploid

INTRODUCTION

Classic techniques for chromosome analysis generally have gained accurate assessment of chromosome numbers and morphology in a wide variety of aquatic invertebrate species (Thiriot-Quievreux 1994); yet the number of species investigated from the cytogenetic point of view is scarce, only about 160 of approximately 15,000 species in the Bivalvia class (Zheng et al. 2000). Hard clam (Mercenaria mercenaria) is a commercially important bivalve, belonging to the family of Veneridae. It naturally distributes from the Gulf of St. Lawrence to the Gulf of Mexico and has been introduced to California in the United States. Hard clam is one of major shellfish favored by United States consumers. Because of its high price in market, rapid growth rate and wide tolerance range of temperature, salinity, and dissolved oxygen, hard clam has been a major aquaculture species in the United States (Menzel 1970, Manzi 1985, Kraeuter & Castagna 2001). Because the hard clam were first introduced into China in 1997, research has been conducted on its ecologic habits, ecological physiology, metabolism, growth, reproduction, artificial propagation, and seeds culture (Chang et al. 2002, Lin et al. 2002, Lin et al. 2005, Zhang et al. 2003). Little information about their chromosomes and karyotype is available. The present study was conducted to document the karyotype of the diploid and triploid of hard clams to provide a reference for the future genetic breeding and phylogenetic studies.

MATERIAL AND METHODS

All experiments were conducted at the Field Station of Zhejiang Mariculture Research Institute (ZMRI), located in Yueqing Bay, Zhejiang Province, southern China (lat. 28[degrees]N). Mercenaria mercenaria seeds were originally introduced from the Haskin Shellfish Research Laboratory, Rutgers University, New Jersey in 1999, and planted in an earth pond in the Field Station of ZMRI. The broodstock were 3 y old and 6 to 8 cm in shell length. These animals were conditioned for 3 wk, with supplement feeding of a mix of cultured microalgae and daily water changes. Spawning was induced by 12-h air-dry followed by flowing water.

Description of Triploid Inducing

Artificial fertilization was conducted at 25[degrees]C in 25 ppt seawater with a pH of 8.1. After 30 min of fertilization, when the 50% of the eggs released the first polar body, triploid was induced by inhibiting the release of the polar body II (PBII) with 450 [micro]mol/mL 6-deimethylaminopurine (6-DMAP) for 20 min. The untreated eggs were allowed to develop as normal diploid controls. Trochophores were collected for chromosome preparations at 14 to 16 h posthatching.

Chromosome Preparations

The drop-splash method was used according to the following protocol: a colchicine (0.05%) treatment of a pool of trochophores in sea water for 2 h, mechanical disruption of cells, including centrifugation at 1000 rpm/10 min, hypotonic treatment in distilled seawater (1:1) for 30 min and then in 0.075 mol/L KC1 30 min, washing to eliminate hypotonic solution (centrifuging at 800 rmp for 10 min), fixation involved three changes (20 min each) in freshly prepared solution of absolute methanol-acetic acid (3:1) and washing (centrifuging at 800 rmp for 10 min). The pellet was resuspended in 0.5 mL of cold fixative solution methanol-acetic acid (3:1), and a drop was splashed onto prewarmed slides. After drying, cells were stained with 0.03% Leishman solution made in phosphate buffer (pH 6.8). The clear metaphases with well-spread chromosomes were selected and photographs were taken with a Nikon E600 U-III photomicroscopic system.

Chromosomes of the best spread were cut out of the photographs and paired on the basis of size and centromere position for karyotype analysis according to Levan criteria (1964). Relative length (R1) was expressed as 100 times the individual chromosome length divided by the total length of the haploid complement. Centromeric index (Ci) was calculated by dividing 100 times the length of the short arm by the total chromosome length. Arm ratio (Ar), was calculated (length of long arm divided by the length of the short arm) to allow comparison with previous studies. Mean and standard deviation (SD) of relative length, arm ratio, and centromeric index were calculated for each pair.

RESULTS

We counted chromosomes in 83 metaphases from diploids and 84 metaphases from triploids. Our results showed that metaphases with 38 chromosomes dominated in diploids and that with 57 chromosomes dominated in triploids, accounting for 78.31% and 75% of all metaphases, respectively (Table 1). So the diploid number of chromosomes of M. mercenaria was 2n = 38, and the triploid number was 3n = 57.

Data on relative length, arm ratio, centromeric index and the chromosome classification for diploids are given in Table 2, and these for triploids are given in Table 3. The relative length decreased progressively from pair 1 to pair 19 and showed basically the same overall appearance in diploids and triploids. The heterochromosomes and satellite chromosomes were not observed in diploids or triploids (Fig. 1). The diploids had 15 metacentric chromosome pairs and 4 submetacentric chromosome pairs (no. 5, 9, 11, 17), and the total chromosome arm number (NF) was 76. The triploids had 45 metacentric chromosomes and 12 submetacentric chromosomes, and the total chromosome arm number (NF) was 114.

The karyological data of M. mercenaria can be summarized as the following formula:

2n = 38 = 30m + 8 sm, NF = 76; and 3n = 57 = 45 in + 12 sm, NF = 114.

DISCUSSION

Our study demonstrates that the diploid chromosome number of M. mercenaria is 38, which is in agreement with what have been reported for this species (Menzel & Menzel 1965, Wang & Guo 2001, Wang & Guo 2007). According to Nakamura (1985), 2n = 38 is the most frequent chromosome number in the class Bivalvia (40% of the reported 125 species). The majority of about 89 species reported in the class Bivalvia, exception for Pectinidae and Teredinidae, had mostly metacentric and submetacentric chromosomes. In the present study, the chromosome number and karyotype of hard clam are similar to the most members of Veneridae, such as Ruditapes philippinarum, R. aureus, and R. decussates (Borsa & Thiriot-Quievreux 1990, Corni & Trentini 1990) and different from other family of Veneroida, such as Solen strictus (Wang et al. 1998a), Sinonovacula constricta (Wang et al. 1998b), Hiatula chinensis (Que et al. 1999), which is in general agreement with phylogenetic classification. Although chromosomes from diploid and triploid karyotypes are similar in relative length, variation exists in arm ratio. The karyotype formula reported here are quite different from that of Wang and Guo (2007), probably reflecting variations in chromosome condensation and/or errors in pairing and measurement.

[FIGURE 1 OMITTED]

Comparison on the Karyotype of Diploid and Triploid M. mercenaria

The chromosome composition of triploid individuals induced by inhibiting the release of the second polar body released is based on the addition of a set of haploid chromosome to the diploid genome, so that the chromosome types of triploids should theoretically be identical to that of diploids. Yan et al. (1999) reported that all chromosome types of triploid Pinctada martensis was unanimous to diploids. Zheng et al. (2000) also reported that all chromosome of triploids and diploids of the Pacific oyster Crassostrea gigas were meta-centric. Our findings are in general agreement with previous studies. The karyotypes of diploids and triploids have the same formula, and the relative length of homologous chromosomes from diploid and triploid M. mercenaria was roughly the same with the less than 2% of variation range. On the other hand, some variation existed in arm ratio. As shown as Table 2, 3, one of 4 pairs of SM chromosomes between diploid and triploid M. mercenaria does not match. This could be attributed by the following three factors. First of all, it may be caused by measurement error, because the chromosomes are highly condensed. Secondly, different metaphases or cells had different reactions to same chromosome preparation. Thirdly, 6-DMAP may have some harmful effects on chromosomes through unknown mechanisms. For chromosome classification, we consider that it is strongly subjective if simply using arm ratio as only standard. For instance, chromosome 16 had no significant variation in arm ratio between diploids and triploids, 1.63 [+ or -] 0.13 (SD) in diploids and 1.75 [+ or -] 0.04 (SD) in triploids, according to nomenclature of Levan et al. (1964) where an arm ratio of 1.7 serves as a dividing point between metacentric and submetacentric chromosomes, therefore, the No.16 chromosome was classified to metacentric type in diploids and submetacentric type in triploids. In fact, such minute errors of measurement are common, so that the chromosome classification based on such criteria should be used as reference only.

Analysis on the Methods of Chromosome Preparation

At present, chromosome spreads of bivalves are usually prepared with three methods using different tissues: gonad, embryos, and adult gills. We did comparative experiments on chromosome preparation from the three tissue-specific methods. Our results showed that gonad chromosome preparation, especially with mature eggs, didn't produce clear metaphase plates, and chromosome arms are tangled probably because of meiotic pairing. There were few well-spread chromosomes, so it's difficult to do karyotypic analysis. For adult gill chromosome preparation in M. mercenaria, we got more mitotic metaphase plates than other species, such as Meretrix meretrix (Lu et al. 2003)and Tegillarca granosa (Zheng et al. 1996), and better chromosomal morphologies. But all chromosomes were too small to be measured accurately for karyotypic analysis. Only chromosome preparation with trochophore larvae of M. mercenaria could produce clear mitotic metaphases and well-spread chromosomes in suitable sizes.

In the present study, we took successive samples to search for the best sampling time. Our results indicate that initial trochophore larvae produce the most mitotic metaphase plates with the best chromosomal morphology. This finding may be of reference value for chromosome preparation in other molluscan species.

The hypotonic treatment plays an important role in chromosome preparation. We got good results with 20% diluted sea water and 0.075 mol/L KCI solution as two hypotonic treatments. In comparison, the hypotonic treatment with 20% diluted sea water produced more mitotic metaphase plates and well-spread chromosomes, whereas the 0.075 mol/L solution yielded good chromosome morphology and didn't exhibit the appearance of "dirty" chromosomes. Drying was also an important factor in chromosome preparation. We found that the longer the slides were dried, the better the staining results.

ACKNOWLEDGMENTS

The authors thank Dr. Ximing Guo and Dr. John N. Kraeuter for suggestions on this research. The authors also particularly thank Dr. Huayong Que for comments on this paper. This study was financially supported by Zhejiang Provincial Science & Technology Program (No. 0111021014).

LITERATURE CITED

Borsa, P. & C. Thiriot-Quievreux. 1990. Karyological and allozymic characterization of Ruditapes Philippinarum, R. aureus and R. decussates (Bivalvia, Veneridae). Aquaculture 90:209-227.

Chang, Y., J. Song, G. Wang, J. Ding, S. Yah & X. Wang. 2002. Seeds breeding of hard clam Mercenaria mercenaria. J. Fish. Sci. China 9:43-47. (in Chinese with English abstract)

Corni, M. G. & M. Trentini. 1990. The chromosomes of Venerupes surea and Ruditapes philippinarum of the Northern Adiriatic Sea (Bivalvia, Hetrodonta, Veneridae). Venus 49:258-261.

Kraeuter, J. & M. Castagna. 2001. Biology of the hard clam [M]. Amsterdam: Elsevier Science B V. pp. 221-255.

Levan, A., K. Fredga & A. Sandberg. 1964. Nomenclature for centiomeric position on chromosome. Hereditas 52:201-220.

Lin, Z. H., X. Chai, J. Fang, J. Zhang & Q. Xie. 2002. An aptitude test on the environmental habits of Mercenaria mercenaria. J. Ningbo University. 15:19-22. (in Chinese with English abstract)

Lin, Z. H., L. Shan, X. Chai, X. Ying, J. Fang, J. Zhang & Y. Zhang. 2005. The reproductive biology of hard clam Mercenaria mercenaria (Linnaeus, 1758). Oceanologia Et Limnologia Sinica. 36:430-436. (in Chinese with English abstract)

Lu, Z., X. Chai, B. Liu, J. Fang, J. Zhang, Z. Lin & T. Li. 2003. Studies on the karyotypes of diploid and triploid of Meretrix meretrix. J. Fish. Sci. China 10:519-522. (in Chinese with English abstract)

Manzi, J. 1985. Clam aquaculture. In: J. V. Hunter & E. E. Brown. editors. Crustacean and mollusk aquaculture in the United States [M]. Westport, CT: The AVI Publishing Company Inc. pp. 275-304.

Menzel, R. 1970. The species and distribution of quahog clams Mercenaria. Proc. Natl. Shellfish Assoc. 50:8. (abstract)

Menzel, R. & M. Menzel. 1965. Studies on chromosomes to two species of quahog clams and their hybrids. Biol. Bull. 129:181-188.

Nakamura, H. 1985. A review of molluscan cytogenetic information based on the CISMOCH--Commputerized index system for molluscan chromosomes. Bivalvia, polyplacophora and cephalopoda. Venus 44:193-225.

Que. H. Y., Q. Qi & w. Qiu. 1999. Karyotype of four species in Veneroida (Lamellibranchia) [M]. Trans. Chinese Soc. Malacol. VIII. pp. 75-83. (in Chinese with English abstract)

Thiriot-Quievreux, C. 1994. Advances in cytogenetics of aquatic organisms. In: A. R. Beaumont, editor. [M]. Genetics and evolution of aquatic organisms. Landon: Chapman & Hall. pp. 369-388.

Wang, J. X., X. Zhao, L. Zhou & J. Xiang. 1998a. Karyotypes of three species of marine bivalve mollusks. ACTA Oceanologia Sinica 20:102-107. (in Chinese with English abstract)

Wang, J. X., X. Zhao, L. Zhou & J. Xiang. 1998b. Chromosome study of Sinonovacula constricta (Bivalvia). Oceanologia Et Limnologia Sinica 29:191-196. (in Chinese with English abstract)

Wang, Y. & X. Guo. 2001. Chromosomal mapping of the vertebrate telomeric sequence (TTAGGG)n in four bivalve molluscs by fluorescence in situ hybridization. J. Shellfish Res. 20:1187-1190.

Wang, Y. & X. Guo. 2007. Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization. Mar. Biol. 150:1183-1189.

Yan, B., A. Wang, L. Ye & Q. Su. 1999. The karyotype of the diploid and triploid Pinctada martensii. Guangxi Sciences 6:219-221. (in Chinese with English abstract)

Zhang, T., H. Yang, B. Liu, B. Li, S. Liu, H. Wen, Y. He & F. Zhang. 2003. Effects of environmental factors on the survival and growth of juvenile hard clam Mercenaria mercenaria (Linnaeus, 1758). Oceanologia Et Limnologia Sinica 34:142-149. (in Chinese with English abstract)

Zheng, X. D., R. Wang, Z. Wang & H. Zhang. 2000. Studies on karyotypes of diploid and triploid of Crassostrea gigas. J. Fish. Sci. China 7:96-97. (in Chinese with English abstract)

Zheng, J. S., M. Wang, D. Guo, X. Xu & Q. Gao. 1996. Studies on karyotypes of Scapharca subcrenate. S. broughtonii and Tegillarca granosa of Arcidae. ACTA Oceanologica Sinica 18:78-85. (in Chinese with English abstract)

LIN ZHI-HUA, (1,2) * LU ZHEN-MING, (3) CHAI XUE-LIANG, (2) FANG JUN (2) AND ZHANG JIONG-MING (2)

(1) Fisheries College, Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, P.R.C:

(2) Zhejiang Mariculture Research Institute, 6-1 Hetongqiao, Wenzhou, Zhejiang 325005, P.R.C; (3) Marine Science College, Zhejiang Ocean University, 105 Wenhua Road, Zhoushan, Zhejiang 316000, P.R.C

* Corresponding author. E-mail: zhihua9988@126.com
TABLE 1.
The chromosomes number of diploid
and triploid M. mercenaria.

 Diploid (2n)

 No. No. Percentage
Chromosome Cells (%)

 [less 4 4.82
 than or
 equal
 to] 34
 35 3 3.62
 36 3 3.62
 37 6 7.23
 38 65 78.31
 39 1 1.20
 40 1 1.20

 Triploid (3n)

 No. No. Percentage
Chromosome Cells (%)

 [less 5 5.95
 than or
 equal
 to] 54
 55 2 2.38
 56 4 4.76
 57 63 75.0
 58 5 5.95
 59 4 4.76
 60 1 1.20

TABLE 2.
The karyotype analysis in 10 mitotic metaphases
of diploid M. mercenaria.

 Relative
 No. of Length Arm Ratio
Chromosome (X [+ or -] SD) (X [+ or -] SD)

 1 7.05 [+ or -] 0.27 1.22 [+ or -] 0.07
 2 6.60 [+ or -] 0.16 1.39 [+ or -] 0.12
 3 6.00 [+ or -] 0.22 1.28 [+ or -] 0.06
 4 5.89 [+ or -] 0.13 1.43 [+ or -] 0.11
 5 5.73 [+ or -] 0.11 1.94 [+ or -] 0.18
 6 5.57 [+ or -] 0.09 1.37 [+ or -] 0.08
 7 5.52 [+ or -] 0.08 1.39 [+ or -] 0.12
 8 5.41 [+ or -] 0.07 1.54 [+ or -] 0.22
 9 5.32 [+ or -] 0.05 1.86 [+ or -] 0.18
 10 5.20 [+ or -] 0.11 1.44 [+ or -] 0.14
 11 5.18 [+ or -] 0.07 1.76 [+ or -] 0.16
 12 5.12 [+ or -] 0.05 1.37 [+ or -] 0.12
 13 4.99 [+ or -] 0.08 1.29 [+ or -] 0.10
 14 4.85 [+ or -] 0.10 1.25 [+ or -] 0.09
 15 4.71 [+ or -] 0.10 1.55 [+ or -] 0.18
 16 4.66 [+ or -] 0.12 1.63 [+ or -] 0.13
 17 4.50 [+ or -] 0.15 1.73 [+ or -] 0.21
 18 4.20 [+ or -] 0.15 1.33 [+ or -] 0.14
 19 3.50 [+ or -] 0.26 1.22 [+ or -] 0.06

 No. of Centromere Type of
Chromosome Index Chromosome

 1 45.05 [+ or -] 1.42 m
 2 41.84 [+ or -] 2.10 m
 3 43.86 [+ or -] 1.15 m
 4 41.15 [+ or -] 1.86 m
 5 34.01 [+ or -] 2.09 sm
 6 42.19 [+ or -] 1.43 m
 7 41.84 [+ or -] 2.10 m
 8 39.37 [+ or -] 3.38 m
 9 34.96 [+ or -] 2.21 sm
 10 40.98 [+ or -] 2.36 m
 11 36.23 [+ or -] 2.10 sm
 12 42.19 [+ or -] 2.14 m
 13 43.67 [+ or -] 1.91 m
 14 44.44 [+ or -] 1.78 m
 15 39.21 [+ or -] 2.78 m
 16 38.02 [+ or -] 1.38 m
 17 36.90 [+ or -] 2.87 sm
 18 42.92 [+ or -] 2.58 m
 19 45.05 [+ or -] 1.22 m

m, metacentric; sm, submetacentric.

TABLE 3.
The karyotype analysis in six mitotic metaphases
of triploid of M. mercenaria.

 Relative
 No. of length Arm Ratio
Chromosome (X [+ or -] SD) (X [+ or -] SD)

 1 7.13 [+ or -] 0.28 1.23 [+ or -] 0.03
 2 6.64 [+ or -] 0.17 1.55 [+ or -] 0.03
 3 6.07 [+ or -] 0.21 1.38 [+ or -] 0.03
 4 5.93 [+ or -] 0.10 1.53 [+ or -] 0.55
 5 5.71 [+ or -] 0.12 1.50 [+ or -] 0.03
 6 5.65 [+ or -] 0.15 1.29 [+ or -] 0.03
 7 5.57 [+ or -] 0.10 1.50 [+ or -] 0.04
 8 5.41 [+ or -] 0.13 1.22 [+ or -] 0.01
 9 5.33 [+ or -] 0.21 1.78 [+ or -] 0.09
 10 5.25 [+ or -] 0.14 1.33 [+ or -] 0.02
 11 5.16 [+ or -] 0.06 1.74 [+ or -] 0.04
 12 5.10 [+ or -] 0.08 1.37 [+ or -] 0.04
 13 4.79 [+ or -] 0.13 1.12 [+ or -] 0.01
 14 4.69 [+ or -] 0.05 1.25 [+ or -] 0.09
 15 4.71 [+ or -] 0.10 1.35 [+ or -] 0.02
 16 4.63 [+ or -] 0.10 1.75 [+ or -] 0.04
 17 4.48 [+ or -] 0.17 1.81 [+ or -] 0.09
 18 4.10 [+ or -] 0.19 1.43 [+ or -] 0.03
 19 3.38 [+ or -] 0.39 1.21 [+ or -] 0.02

 No. of Centromere Type of
Chromosome Index Chromosome

 1 44.84 [+ or -] 0.60 m
 2 39.22 [+ or -] 0.46 m
 3 42.02 [+ or -] 0.53 m
 4 39.53 [+ or -] 0.78 m
 5 40.00 [+ or -] 0.48 m
 6 43.67 [+ or -] 0.58 m
 7 40.00 [+ or -] 0.64 m
 8 45.05 [+ or -] 0.21 m
 9 35.97 [+ or -] 1.29 sm
 10 42.92 [+ or -] 0.37 m
 11 36.50 [+ or -] 0.54 sm
 12 42.19 [+ or -] 0.71 m
 13 39.37 [+ or -] 0.62 m
 14 47.17 [+ or -] 0.22 m
 15 42.55 [+ or -] 0.36 m
 16 36.36 [+ or -] 0.54 sm
 17 35.59 [+ or -] 1.14 sm
 18 41.32 [+ or -] 0.51 m
 19 45.25 [+ or -] 0.41 m

m, metacentric; sm, submetacentric.
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Author:Lin, Zhi-hua; Lu, Zhen-Ming; Chai, Xue-Liang; Fang, Jun; Zhang, Jiong-Ming
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:9CHIN
Date:Apr 1, 2008
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