Cuba. 2 Facultad de Biología, Unive

Cuba.
2 Facultad de Biología, Universidad de La Habana, UH. Calle 25 #455 e/ J y H, Vedado, Plaza, La Habana, Cuba.




ABSTRACT

The present work estimated the genetic variability and relatedness index of the fifth stock of Pacific white shrimp, Litopenaeus vannamei, imported into Cuba for farming purposes from the US Shrimp Improvement System (SIS). Genetic variability was estimated by genotyping 33 samples for four microsatellite loci: M1, Pvan 1815, Pvan 0040 and Pvan 1758. This stock had average expected and observed heterozygosities of 0.37 and 0.27 respectively; the lowest of all stocks previously introduced in Cuba. The above, together with the low amount of allelic variants detected for each microsatellite, was suggestive of low genetic variability. In addition, pairwise relatedness coefficients clustered around unity, indicating a high degree of consanguinity. Taken as a whole, the data suggests that this breeding stock should be crossed first with other individuals from a different source or with higher genetic variability.

Keywords: genetic variability, Litopenaeus vannamei, microsatellites, shrimp.


RESUMEN

Se estimó la variabilidad genética y el índice de parentesco entre lotes de camarón blanco del Pacífico, Litopenaeus vannamei, introducidos por quinta ocasión en Cuba, procedentes del Centro de mejora del camarón, de Estados Unidos (Shrimp Improving System: SIS), para su cultivo. La variabilidad genética se estimó mediante el genotipo de 33 muestras con cuatro loci microsatélites: M1, Pvan 1815, Pvan 0040 y Pvan 1758. El quinto lote de Litopenaeus vannamei tuvo los valores promedios de heterocigosidad esperada y observada, más bajos de todos los introducidos en Cuba: 0.37 y 0.27, respectivamente. Esto, unido a la poca cantidad de variantes alélicas para cada región microsatélite, indica una escasa variabilidad genética. Los valores del coeficiente de parentesco alrededor de la unidad expresan una alta consanguinidad. Todo ello sugiere el cruce de los individuos de esta introducción con otros de diferente origen o más variables genéticamente.

Palabras clave: variabilidad genética, Litopenaeus vannamei, microsatélites, camarón.










INTRODUCTION

Shrimp, as many other fishing resources, has received the impact of overfishing and environmental deterioration. Natural populations have shrunk, turning shrimp farming into a useful alternative for the obtention of this important protein source.

During the year 2003, two stocks of Pacific white shrimp (Litopenaeus vannamei) from the US Shrimp Improvement System (SIS), denominated here as scotcks 1 and 2, were introduced for the first time in Cuba [1], followed by the gradual implementation of techniques for the handling, health, nutrition and assessment of the genetic variability of this species in hatcheries and farms previously involved with Litopenaeus schmitti, an autochthonous shrimp. Two additional stocks were introduced during successive years (denominated here as stocks 3 and 4), all characterized using microsatellite markers [2, 3].

A fifth stock of this species, obtained from the same source, was introduced in October 2008. Its genetic composition and allele polymorphism are yet to be exa- mined, however. Although their consanguinity was low, the genetic variability of stocks 1 and 2 was already smaller than in natural populations [2]; stocks 3 and 4, likewise, exhibited a decreased genetic variability [3].

Using allozymes or microsatellites and a number of different shrimp species, it has been demonstrated that loss of genetic variability correlates with significant decreases in productivity (as for instance in Marsupenaeus japonicus [4], Litopenaeus stylirostris [5], P. monodon [6] and L. vannamei [7, 8]). The use of microsatellite markers, however, is better suited for the follow-up and assessment of genetic variability in farmed populations, due to its higher resolution and sensitivity [2, 9].

The objectives of the present work, therefore, are to determine genetic variability and relatedness indexes in a sample from the fifth stock of L. vannamei introduced in Cuba, employing four microsatellite loci and comparing them with all of the previously introduced stocks.

MATERIALS AND METHODS

Sampling

Pleopod samples were taken from the fourth pair, between the exopodito and the endopodito, of 40 randomly chosen individuals evenly split between males and females. The sampling procedure was performed on previously acclimatized shrimp from the hatcheries of the Shrimp Genetic Center in Mariel, Cuba.

Genotyping of microsatellite loci

DNA extraction procedures, microsatellite loci selected for analysis (M1, obtained by Wolfus et al. [8], Pvan 1758, Pvan 1815 and Pvan 0040, isolated by Cruz et al.[10]), amplification programs and electrophoresis conditions for genotyping runs have all been described in Borrell et al. [2].

DNA was extracted with 5% Chelex resin, and the amplified fragments were separated under a running voltage of 2000 mV in 6% bis-acrylamide/acrylamide gels which were later stained with 0.1% silver nitrate-0.05% formaldehyde, fixed with 10% acetic acid and developed with 3% sodium carbonate/0.05% formaldehyde and 20 µg/mL sodium thiosulfate. Samples previously genotyped by these authors [2] were used as controls for each microsatellite locus, with sizes ranging from 206 to 240 for M1, 140 to 146 for Pvan 0040, 110 to 136 for Pvan 1815 and 174 to 188 for Pvan 1758. PGEM® was also included as a conventional size marker.

Statistical processing

Determining the number of alleles per locus, the frequency of each allele and the values for observed (Ho) and expected (He, according to Nei [11]) heterozygosity for each locus, as well as whether the populations were in Hardy-Weinberg equilibrium, was performed with the GeneAlEx (version 6.1) software application [12]. Any locus with at least two alleles where the frequency of the most common allele did not exceed 95% was considered polymorphic [13].

Deviations from equilibrium were corroborated by calculating FIS [14], following the formula FIS = 1 - (Ho/He), with the FSTAT software application (version 2.9.3) [15]. Although it depends on population size, it is unaffected by the presence of multiple alleles per locus, the number of individuals per population or the number of populations.

The genetic relatedness coefficient (r) [16] for a single pair of individuals was also calculated with GeneAlEx ver. 6.1 [12]. This coefficient is calculated for codominant markers, using the following formula:






where: x stands for the individuals; k for the loci; l for allelic positions (two for diploids and one for haploids), Px is the frequency of individual “x” for locus k and allelic position l, Py is the frequency of allele “y” in the group or individual compared to x; and P* is the total frequency of the allele in the population. The genetic relatedness coefficient must be r ≤ 0 for non-related individuals; r = 0.25 for half brothers and r ≥ 0.5 for brothers [16].



RESULTS AND DISCUSSION

Genetic variability of the fifth stock of L. vannamei compared to previous stocks

The present work analyzed the genetic variability of the fifth stock of L. vannamei introduced in Cuba for shrimp farming, using four microsatellite loci: M1, isolated from L. vannamei [8], and Pvan 0040, Pvan 1758 and Pvan 1815, isolated from the same species [10].

The table contains the main calculated parameters: number of alleles (Na), observed and expected heterozygosity (Ho and He) and deviations from Hardy-Weinberg equilibrium (FIS), together with the values estimated during previous studies. According to the results of the analysis of linkage disequilibrium with FSTAT (version 2.9.3), these loci are not genetically linked in L. vannamei [2, 9, 17].

Expected and observed heterozygosity

Average observed heterozygosity for the fifth stock of L. vannamei introduced in Cuba yielded a value of 0.271; the lowest figure compared to previous introduced stocks (Figure 1). In addition, this value is below all previous intervals reported by other researchers employing microsatellites to study peneaid shrimps.

A now classic review [9] on the use of microsatellite loci for natural populations from four different species described observed heterozygosities that ranged from 0.425 to 0.964, yielding a mean of 0.666 which fell below the expected average (0.927). The same author observed heterozygosities ranging from 0.45 to 1.00 for three species of farmed peneaids, yielding a mean of 0.594, just below the mean for expected heterozygosity (0.674).

However, studies on P. stylirostris cultured for 22 and 24 generations [5] have produced much smaller values: Ho = 0.32 to 0.48; He = 0.46 to 0.61. Still, the values from the fifth stock introduced in Cuba (Ho = 0.271; He = 0.367) are below these figures (Figure 1), even though heterozygosity for all previous introductions of L. vannamei is within the above intervals. However, we agree that heterozygosity is an imperfect measure for variability, as it can yield high values with just two alleles, and, therefore, their quality also matters [17-19].

Allele frequencies and deviations from Hardy-Weinberg equilibrium

One single locus, Pvan 0040, is responsible to a large extent for the decrease in heterozygosity of the fifth stock, as it appears to be monomorphic in this case (as it did for the fourth Litopenaeus vannamei introduction). The other remaining three loci are still polymorphic, with five alleles each for M1 and Pvan 1758 and four alleles for Pvan 1815.

Allele frequencies for the loci of the fifth stock are shown in figure 2. Molecular weights for the observed alleles coincide within the intervals published by other authors, including those who first isolated them from a genomic library [10] and others who have later used them to characterize populations of this species [2, 3, 8, 17-19].

In the present work th

Kết quả (Việt) 1:

Cuba. 2 Facultad de Biología, Universidad de La Habana, UH. Calle 25 #455 e / J y H, Vedado, Plaza, La Habana, Cuba. TÓM TẮT Công việc hiện nay ước tính di truyền biến đổi và relatedness chỉ số của các cổ phiếu thứ năm của Thái Bình Dương trắng tôm, Litopenaeus vannamei, nhập khẩu vào Cuba cho nông nghiệp mục đích từ chúng tôi tôm cải thiện hệ thống (SIS). Biến đổi di truyền được ước tính của gen 33 mẫu cho bốn microsatellite loci: M1, Pvan năm 1815, Pvan 0040 và Pvan 1758. Cổ phiếu này có trung bình dự kiến và quan sát heterozygosities 0,37 và 0,27 tương ứng; thấp nhất của tất cả các cổ phiếu đã được giới thiệu ở Cuba. Ở trên, cùng với số lượng thấp của các biến thể allelic phát hiện cho mỗi microsatellite, được gợi của thấp biến đổi di truyền. Ngoài ra, Hệ số cử relatedness tập trung xung quanh thành phố thống nhất, chỉ ra một mức độ cao của consanguinity. Thực hiện như một toàn thể, các dữ liệu cho thấy rằng cổ phiếu chăn nuôi này nên được vượt qua lần đầu tiên với các cá nhân từ một nguồn khác nhau hoặc với sự biến đổi di truyền cao. Từ khoá: biến đổi di truyền, Litopenaeus vannamei, microsatellites, tôm. RESUMEN Se estimó la variabilidad genética y el índice de parentesco entre lotes de camarón blanco del Pacífico, Litopenaeus vannamei, introducidos por quinta ocasión en Cuba, procedentes del Centro de mejora del camarón, de Estados Unidos (Shrimp Improving System: SIS), para su cultivo. La variabilidad genética se estimó mediante el genotipo de 33 muestras con cuatro loci microsatélites: M1, Pvan 1815, Pvan 0040 y Pvan 1758. El quinto lote de Litopenaeus vannamei tuvo los valores promedios de heterocigosidad esperada y observada, más bajos de todos los introducidos en Cuba: 0.37 y 0.27, respectivamente. Esto, unido a la poca cantidad de variantes alélicas para cada región microsatélite, indica una escasa variabilidad genética. Los valores del coeficiente de parentesco alrededor de la unidad expresan una alta consanguinidad. Todo ello sugiere el cruce de los individuos de esta introducción con otros de diferente origen o más variables genéticamente. Palabras clave: variabilidad genética, Litopenaeus vannamei, microsatélites, camarón. INTRODUCTION Shrimp, as many other fishing resources, has received the impact of overfishing and environmental deterioration. Natural populations have shrunk, turning shrimp farming into a useful alternative for the obtention of this important protein source. During the year 2003, two stocks of Pacific white shrimp (Litopenaeus vannamei) from the US Shrimp Improvement System (SIS), denominated here as scotcks 1 and 2, were introduced for the first time in Cuba [1], followed by the gradual implementation of techniques for the handling, health, nutrition and assessment of the genetic variability of this species in hatcheries and farms previously involved with Litopenaeus schmitti, an autochthonous shrimp. Two additional stocks were introduced during successive years (denominated here as stocks 3 and 4), all characterized using microsatellite markers [2, 3]. A fifth stock of this species, obtained from the same source, was introduced in October 2008. Its genetic composition and allele polymorphism are yet to be exa- mined, however. Although their consanguinity was low, the genetic variability of stocks 1 and 2 was already smaller than in natural populations [2]; stocks 3 and 4, likewise, exhibited a decreased genetic variability [3]. Using allozymes or microsatellites and a number of different shrimp species, it has been demonstrated that loss of genetic variability correlates with significant decreases in productivity (as for instance in Marsupenaeus japonicus [4], Litopenaeus stylirostris [5], P. monodon [6] and L. vannamei [7, 8]). The use of microsatellite markers, however, is better suited for the follow-up and assessment of genetic variability in farmed populations, due to its higher resolution and sensitivity [2, 9]. The objectives of the present work, therefore, are to determine genetic variability and relatedness indexes in a sample from the fifth stock of L. vannamei introduced in Cuba, employing four microsatellite loci and comparing them with all of the previously introduced stocks. MATERIALS AND METHODS Sampling Pleopod samples were taken from the fourth pair, between the exopodito and the endopodito, of 40 randomly chosen individuals evenly split between males and females. The sampling procedure was performed on previously acclimatized shrimp from the hatcheries of the Shrimp Genetic Center in Mariel, Cuba. Genotyping of microsatellite loci DNA extraction procedures, microsatellite loci selected for analysis (M1, obtained by Wolfus et al. [8], Pvan 1758, Pvan 1815 and Pvan 0040, isolated by Cruz et al.[10]), amplification programs and electrophoresis conditions for genotyping runs have all been described in Borrell et al. [2]. DNA was extracted with 5% Chelex resin, and the amplified fragments were separated under a running voltage of 2000 mV in 6% bis-acrylamide/acrylamide gels which were later stained with 0.1% silver nitrate-0.05% formaldehyde, fixed with 10% acetic acid and developed with 3% sodium carbonate/0.05% formaldehyde and 20 µg/mL sodium thiosulfate. Samples previously genotyped by these authors [2] were used as controls for each microsatellite locus, with sizes ranging from 206 to 240 for M1, 140 to 146 for Pvan 0040, 110 to 136 for Pvan 1815 and 174 to 188 for Pvan 1758. PGEM® was also included as a conventional size marker.
Statistical processing

Determining the number of alleles per locus, the frequency of each allele and the values for observed (Ho) and expected (He, according to Nei [11]) heterozygosity for each locus, as well as whether the populations were in Hardy-Weinberg equilibrium, was performed with the GeneAlEx (version 6.1) software application [12]. Any locus with at least two alleles where the frequency of the most common allele did not exceed 95% was considered polymorphic [13].

Deviations from equilibrium were corroborated by calculating FIS [14], following the formula FIS = 1 - (Ho/He), with the FSTAT software application (version 2.9.3) [15]. Although it depends on population size, it is unaffected by the presence of multiple alleles per locus, the number of individuals per population or the number of populations.

The genetic relatedness coefficient (r) [16] for a single pair of individuals was also calculated with GeneAlEx ver. 6.1 [12]. This coefficient is calculated for codominant markers, using the following formula:






where: x stands for the individuals; k for the loci; l for allelic positions (two for diploids and one for haploids), Px is the frequency of individual “x” for locus k and allelic position l, Py is the frequency of allele “y” in the group or individual compared to x; and P* is the total frequency of the allele in the population. The genetic relatedness coefficient must be r ≤ 0 for non-related individuals; r = 0.25 for half brothers and r ≥ 0.5 for brothers [16].



RESULTS AND DISCUSSION

Genetic variability of the fifth stock of L. vannamei compared to previous stocks

The present work analyzed the genetic variability of the fifth stock of L. vannamei introduced in Cuba for shrimp farming, using four microsatellite loci: M1, isolated from L. vannamei [8], and Pvan 0040, Pvan 1758 and Pvan 1815, isolated from the same species [10].

The table contains the main calculated parameters: number of alleles (Na), observed and expected heterozygosity (Ho and He) and deviations from Hardy-Weinberg equilibrium (FIS), together with the values estimated during previous studies. According to the results of the analysis of linkage disequilibrium with FSTAT (version 2.9.3), these loci are not genetically linked in L. vannamei [2, 9, 17].

Expected and observed heterozygosity

Average observed heterozygosity for the fifth stock of L. vannamei introduced in Cuba yielded a value of 0.271; the lowest figure compared to previous introduced stocks (Figure 1). In addition, this value is below all previous intervals reported by other researchers employing microsatellites to study peneaid shrimps.

A now classic review [9] on the use of microsatellite loci for natural populations from four different species described observed heterozygosities that ranged from 0.425 to 0.964, yielding a mean of 0.666 which fell below the expected average (0.927). The same author observed heterozygosities ranging from 0.45 to 1.00 for three species of farmed peneaids, yielding a mean of 0.594, just below the mean for expected heterozygosity (0.674).

However, studies on P. stylirostris cultured for 22 and 24 generations [5] have produced much smaller values: Ho = 0.32 to 0.48; He = 0.46 to 0.61. Still, the values from the fifth stock introduced in Cuba (Ho = 0.271; He = 0.367) are below these figures (Figure 1), even though heterozygosity for all previous introductions of L. vannamei is within the above intervals. However, we agree that heterozygosity is an imperfect measure for variability, as it can yield high values with just two alleles, and, therefore, their quality also matters [17-19].

Allele frequencies and deviations from Hardy-Weinberg equilibrium

One single locus, Pvan 0040, is responsible to a large extent for the decrease in heterozygosity of the fifth stock, as it appears to be monomorphic in this case (as it did for the fourth Litopenaeus vannamei introduction). The other remaining three loci are still polymorphic, with five alleles each for M1 and Pvan 1758 and four alleles for Pvan 1815.

Allele frequencies for the loci of the fifth stock are shown in figure 2. Molecular weights for the observed alleles coincide within the intervals published by other authors, including those who first isolated them from a genomic library [10] and others who have later used them to characterize populations of this species [2, 3, 8, 17-19].

In the present work th