Mass selection and inbreeding effects on a cultivated strain of Penaeus (Litopenaeus) vannamei in Venezuela

Aquaculture 247 (2005) 159 – 167 www.elsevier.com/locate/aqua-online

Mass selection and inbreeding effects on a cultivated strain of Penaeus (Litopenaeus) vannamei in Venezuela Marcos De Donatoa,T, Ramon Manriqueb, Rau´l Ramirezb, Luis Mayerb, Chris Howellb a

Lab. Gene´tica Molecular, Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas, Universidad de Oriente, Cumana, estado Sucre, Venezuela b Aquamarina de la Costa Shrimp Farm, Caracas, Venezuela Received 15 November 2003; received in revised form 1 February 2005; accepted 1 February 2005

Abstract Artificial selection can significantly improve animal performance in culture, but one of the major concerns in genetic programs is inbreeding, which can affect fitness-related traits, and may have a significant negative impact on production. We present the analysis of production records for 11 generations (1990–2001) of a Venezuelan strain of Penaeus vannamei under mass selection and maintained in a closed reproductive cycle. Symptoms of IHHNV disease were reported during the first years of selection. The reproductive stock of the farm was established from three different populations mixed in successive generations (Mexico first generation, Panama second generation, and Colombia third generation). Reproductive stocks were collected from 1-ha ponds selecting the biggest animals showing no signs of disease nor deformities. Production related parameters exhibited significant improvements through time: yearly averages for survival changed from 59% to 76%, growth rate from 0.76 to 0.87 g/week, feed conversion ratio (FCR) from 1.86 to 1.51:1, production from 1.20 to 2.10 ton/ha, and percentage of deformities from 29% to 1%. The coefficient of variation on final weight changed from 19.7% to 11.6%, during the period analyzed. The high prevalence of deformities during the first years was due to IHHNV disease and probably an effect of accumulated inbreeding of the Mexican population due to a small population size. There were statistically significant relationships between the percentage of deformities and the other variables analyzed, except for growth rate. The effects of an inbreeding depression during the first generations could have decrease until disappearing, probably due to the elimination of deleterious alleles from the genetic pool, as can be inferred from the high degree of relationship between the percentage of deformities and F t . However, no signs of deterioration on the fitness-related traits have been seen in the last generations. The absence of symptoms for IHHNV disease in the last generations, could suggests that this strain may be tolerant, or even resistant to this pathogen; however, experiments should be carried out to prove this hypothesis. The results in this and other studies on the Venezuelan stocks of P. vannamei, have proven that this is an important resource for shrimp culture in the Americas. D 2005 Elsevier B.V. All rights reserved. Keywords: Mass selection; Penaeus vannamei; IHHNV; Inbreeding

T Corresponding author. Tel.: +58 293 4302140; fax: +58 293 4521297. E-mail address: [email protected] (M. De Donato). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.02.005

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1. Introduction The advancements in the reproductive biology of many economically important aquatic species in the last two decades have made possible maintaining the complete life cycle under farm conditions and have also allowed the possibility for domestication and artificial selection (Bentsen and Olesen, 2002). However, only in the last decade substantial genetic improvement and increases in production efficiency have been achieved in some farmed fish species such as Salmonids and tilapia (Hulata, 2001). This improvement has been achieved through the implementation of quantitative genetic principles facilitated by improved animal tagging systems. Since a faster growing animal will possess an improved feed conversion ratio and overall health, growth rate has been the first trait targeted for improvement by selective breeding. In Penaeid shrimp, early genetics work was related to domestication and culturing in closed cycles, and recent efforts are focused on artificial selection for growth and growth-related traits as well as resistance to viral infection (Benzie et al., 1997; Pe´rez-Rostro et al., 1999; Bierne et al., 2000; Hetzel et al., 2000; Argue et al., 2002; Crocos et al., 2002; Goyard et al., 2002a,b; Preston et al., 2002; Sua´rez et al., 2002). The power of artificial selection to significantly improve animal performance in culture could be counterbalanced by a rapid growth in the inbreeding coefficient, which can affect fitness-related traits such as survival, reproduction, growth, presence of deformities, and a decrease of genetic variability, resulting from careless genetic management of the populations. The negative effects of these traits could also have a significant negative impact on production in the form of inbreeding depression. Besides the fact that many aquaculture operations are already working with partially inbred animals, inbreeding depression has not been extensively investigated in fish and hardly investigated at all in other aquaculture organisms (Bentsen and Olesen, 2002). The Venezuelan strain of Penaeus (Litopenaeus) vannamei has been incorporated into the reproductive stocks of genetic improvement programs of other Latin American countries such as Brazil, Colombia, and Mexico (CENIACUA, 1999; Herna´ndez, 2002; ABCC, 2003; Sua´rez et al., 2002), due to

its good performance and the absence of wide spread diseases. Thus, in order to determine the effect of mass selection and inbreeding during the process of domestication, we analyzed 11 generations of production records from a shrimp farm in Venezuela.

2. Materials and methods 2.1. Founder reproductive stock The data used for this study were collected from the production records of the ponds over a period of 11 generations, from September of 1990 through November of 2001. Pond area started from 150 ha in 1991, increased to 350 in 1994 and finally reached 438 ha in 1997. Aquamarina de la Costa shrimp farm (Venezuela) used a culture system based on closed production cycle of P. vannamei. The reproductive stock of the farm was established with three different populations and different contributions: shrimps originated in the Pacific coast of south Mexico, which have been cultivated for 6 generations in other farms with a small effective population size (actual data is not available), and were the first animals to arrived (64% of the stock). In the second generation, wild animals from the Pacific coast of Panama (20% of the stock) were incorporated into the broodstock, and finally, 3rd generation cultivated shrimps from the Pacific coast of Colombia (16% of the stock) were incorporated into the third generation. The founder population started with 44 animals (22 of each sex) and increased to around 100 individuals. 2.2. Rearing conditions The hatchery was set 120 km from the pond area. Nauplii were raised in concrete, epoxy-coated tanks, of 10–12 ton at a density of 120–150 nauplii/l, and fed with microalgae, commercial liquid larval diets and Artemia nauplii. The larvae were raised to the postlarval stage with a minimum size of 5–6 mm (usually to PL8–PL11 after 7 to 11 days). The post larvae were then shipped to the pond area in either 10-l bags filled with sea water and oxygen or in 2000-l tanks with aeration provided by a portable blower. There, they were placed inside 55,000-l concrete, tanks at a density of 35 PLs/l, and fed with commercial diets,

M. De Donato et al. / Aquaculture 247 (2005) 159–167 100

Several sets of the matings were used for the next selection cycle and the rest of the animals were used for production. For the calculation of the effective population size (Ne t ), which is the number of parents contributing to the offspring of the next generation, we used the equation of the harmonic mean from Falconer and Mackay (1996) as follows:

80 Survival (%)

70 60 50 40 30 20 10 1992

1994

1996

1998

2000

2002

Fig. 1. Yearly average survival (x) during growout in the ponds for the studied period, estimated from tons produced and average shrimp weight.

with 35–50% protein content according to their age, for 2 to 4 weeks depending on the demand. The tanks had continuous air supply and the level of oxygen and the temperature were monitored twice a day. After that, a sample of the juveniles was weighed (initial weight), and they were stocked at a density of around 17 individuals/m2 in ponds of 8.8 ha on average. The juveniles were fed with a commercial pond pellet diet having 35% protein content. Production ponds were harvested after 121.9F12.2 days (range 91–144 days), calculating final weight and survival (per harvest). Survival was estimated from the total weight produced per pond and the average shrimp weight. Yearly averages were calculated by averaging the data from the ponds harvested that year. Mass selection was carried out on shrimps grown in 1-ha ponds stocked at a density of around 1.5 individuals/m2. Usually after 90 days, the pond was harvested and 1/2 of the number of animals were stocked into a newly prepared 1-ha pond, eliminating the animals that were too small, or showed deformities or signs of disease. This operation was repeated three more times until the animals reached about 10 months of age, harvesting 1500 animals in the last selection process to be used as a broodstock for the next generation. The animals were transported back to the hatchery, where they were maintained in concrete, circular tanks with aeration at a density of 6 individuals/m2 until they reached sexual maturity. The largest and healthiest mature males and females were put together to mate and the eggs were collected with a mesh and transferred to fiberglass, 50-l tanks for hatching. Nauplii were grown as described before.

t Net ¼ P t 1 N i i¼1 where t=generations. The inbreeding coefficient ( F t ) by generation was calculated following Falconer and Mackay (1996) as: F t ¼ 1  ð1  DF Þt where DF=1/2Ne A zero inbreeding coefficient was assumed in the base population.

3. Results The analysis of the production records have shown that the production related parameters exhibited differences when compared over time, most of them exhibiting significant improvements. The all-time averages for these parameters were as follows: survival of 72.6%, growth rate at harvest of 0.88 g/ week, feed conversion ratio (FCR) at harvest of 1.68:1, production of 1.86 ton/ha, coefficient of variation on weight of 16.3%, and percentage of individuals with deformities of 14.2. Yearly averages 1.20 1.00 Growth Rate (g/week)

90

0 1990

161

0.80 0.60 0.40 0.20 0.00 1990

1992

1994

1996

1998

2000

2002

Fig. 2. Yearly average growth rate (x; grams per week) during growout in the ponds for the studied period.

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M. De Donato et al. / Aquaculture 247 (2005) 159–167 0.60

2 1.9

0.50 COR (Survival x GR/FCR)

FCR (g food/g shrimp)

1.8 1.7 1.6 1.5 1.4 1.3 1.2

0.40 0.30 0.20 0.10

1.1 1 1990

0.00 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 1992

1994

1996

1998

2000

2002

Fig. 3. Yearly average value of feed conversion ratio (x; FCR) calculated from the total shrimp produced divided by the total food added to the pond during growout in the ponds for the studied period.

Fig. 5. Yearly average value of culture optimization rate (x; COR) calculated multiplying survival by the ratio of growth divided by feed conversion ratio. This rate integrates the three most important factors that affect production and it can be used to compare among years of production within a farm.

for survival during growout in the ponds changed from 59% in 1991 to 76% in 2001 (Fig. 1), which represents 28.8% increase. Growth rate in the ponds improved from 0.76 g/week in 1991 to a record high of 0.97 g/week in 1993, oscillating after that, to end at 0.87 g/week in 2001 (Fig. 2), which represents an increase of 14.5%. FCR oscillated the first 5 years and later decreased from 1.83 in 1995 to 1.51 in 2001 (Fig. 3), which represents 18.8% improvement. FCR was probably affected by management policies, in order to decrease it after 1996, which could have also affected growth rate so that there was a slight decrease of 0.08 g/week in growth rate from that year to 2001. Production, measured as tons of shrimp harvested per hectare of cultivated pond, increased significantly from 1.20 in 1991 to 2.10 in 2001 (74.9% increase), although there was a production decrease for 1997

and 1998 related to predation, disease, and unfavorable environmental conditions (Fig. 4). In order to determine if there was improvement in the traits that had the greatest effect on production, such as survival, growth rate, and FCR, a culture optimization rate (COR) was devised, calculated by multiplying survival with the ratio of growth rate and FCR. This rate can be used for comparison among years in order to determine if the system is improving, but it should be used only for in-farm comparisons, because of its relative nature. In this case, COR improved significantly through the years from 0.241 to 0.438 (Fig. 5), which represents an increase of 81.6%. A regression analysis between production and COR showed a highly significant relationship ( F=42.70, P=0.0001, R 2=82.59).

2.5 35

2.25 Frequency of Deformities (%)

Production (ton/ha)

2 1.75 1.5 1.25 1 0.75 0.5 0.25 0 1990

1992

1994

1996

1998

2000

2002

Fig. 4. Yearly average production (x; ton/ha) adjusted for stocking density (which varied slightly) during growout in the ponds for the studied period.

30 25 20 15 10 5 0 1990

1992

1994

1996

1998

2000

2002

Fig. 6. Yearly average percentage of individuals with deformities observed at harvest.

M. De Donato et al. / Aquaculture 247 (2005) 159–167

10.45%

163

1.80%

Deformed Tail 19.05%

2.60% 2.01% 0.63%

Deformed Head Deformed Antennae

8.98%

Twisted Rostrum

Short Rostrum Bifid Rostrum Twisted Digestive Tract 14.86%

Bulky Segments Displaced Segment

39.62%

Fig. 7. Types of deformities observed at harvest. Percentages were calculated as averages of each type from the years 1994–1998. There were no significant changes among the years for each type.

The percentage of deformities decreased drastically from around 30% in 1991–1994 to about 1% after 1999 (Fig. 6), which represents 96.6% decrease. The main types of deformities observed in shrimps at harvest were: deformed head (19.05%), stunted antennae (14.86%), twisted rostrum (39.62%), short rostrum (8.98%), and displaced abdominal segments (10.45%; Fig. 7). Runt deformity syndrome (RDS), which is caused by the Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV), was reported in the population early in 1992, and presumably brought with the wild stocks from Panama or Colombia. Several of the types of deformities observed in the shrimps were concordant 25

20

with the symptoms reported for this disease (Lightner, 1996). Currently, no animals with deformities are seen in the population and only less than 1% of dwarfism is seen. The coefficient of variation (CoV) on final weight, calculated as the ratio of the standard deviation on harvest weight to its average value, expressed in percentage, progressively decreased through time from 19.7%, at the beginning, and stabilizing at around 11.4% after 2000 (Fig. 8), which represents a decrease of 42.2%. A regression analysis, carried out to determine the relationship between the percentage of deformities in the population at the time of harvest and the other variables analyzed in this study, showed statistically significant relationships with all variables except for growth rate (Table 1). One of the strongest relationships was with the coefficient of variation (R 2=84.09), showing a pattern of a logarithmic curve (Fig. 9).

CoV

15

Table 1 Regression analysis between the percentage of deformities in the population and other variables

10

5

0 1990

1992

1994

1996

1998

2000

2002

Fig. 8. Yearly average value of the coefficient of variation (CoV) during growout in the ponds for the studied period, which was calculated as the ratio of the harvest weight standard deviation and its average, shown as a percentage, which measures the dispersion of the individual data.

Variable

F value

P

Correlation coefficient

R2

Survival Growth rate FCR Production COR CoV Ne t Ft

6.19 0.10 14.44 13.39 8.25 47.57 10.48 74.40

0.0346 0.7589 0.0042 0.0052 0.0184 0.0001 0.0102 N0.0001

0.6382 0.1049 0.7849 0.7733 0.6917 0.9170 0.7335 0.9445

40.73 1.10 61.60 59.80 47.84 84.09 53.80 89.21

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M. De Donato et al. / Aquaculture 247 (2005) 159–167

90

1.00

Survival

Growth Rate

85 0.95 80 0.90

75

0.85

g/wk

%

70 65 60

0.80 0.75

55 0.70

50

0.65

45

0.60

40 0

5

2.45

10

15

20

25

30

35

0

Production

10

Survival x GR/FCR

2.05 1.85 1.65

20

25

30

35

COR

0.4 0.3 0.2 0.1

1.45 1.25

0 0

5

10

2

15

20

25

30

35

0

5

10

22

FCR

15

20

25

30

35

20

25

30

35

20

25

30

35

CoV

1.9 20

1.8 1.7

18

1.6 1.5

%

g food/g shrimp

15

0.5

2.25

ton/ha

5

0.6

16

1.4 14

1.3 1.2

12

1.1 1

10 0

5

10

0.07

15

20

25

30

35

0

5

10

100

F

15

Ne

90

0.06 80 0.05

70 60

0.04

50 0.03

40

0.02

30 20

0.01

10 0

0 0

5

10

15

20

25

30

35

0

5

10

15

Fig. 9. Regression analysis between the percentage of deformities (X-axis) in the population and other variables analyzed in this study.

M. De Donato et al. / Aquaculture 247 (2005) 159–167 0.12

100 90

0.10

80

Ne

0.06

60 50 40 30 20

F

0.08

70

0.04 Ne F

0.02 0.00

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

Fig. 10. Values of the harmonic mean of the effective population size through the generations (Ne t ; *) and the coefficient of inbreeding ( F t ; x) for the studied period, calculated as stated in the Materials and methods.

The effective population size (Ne) started with 44 individuals, founders of the population, and increased to 100 after 1995. The harmonic mean (Ne t ) increased from its lowest value to 90.4 in 2001 (Fig. 10), showing a stabilization pattern around that value (asymptotic pattern). The coefficient of inbreeding ( F t ) per generation showed the lowest values of 0.011 in 1991 and increased to 0.059 in 2001 (Fig. 10), displaying a steady rate of increase. Additionally, a significant relationship was detected between the percentage of deformities and Ne t (Table 1).

4. Discussion Mass selection has been, by far, the most commonly used type of artificial selection in aquaculture due to its simplicity, but its results have not always been successful (Doyle, 2002). However, in shrimp culture, there are cases of successful improvement by mass selection; in the present study, we demonstrated the success of mass selection for improving the production related parameters analyzed, especially significant for the percentage of individuals with deformities and production. The deformities seen at first generations were caused by the high prevalence of IHHNV alone or by the effects of both virus and inbreeding. After 1994, the percentage of deformities decreased rapidly, and the absence of symptoms for IHHNV disease in the population for the last generations suggests that this strain may be tolerant, or even resistant to this pathogen. However, specific experiments should be

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carried out to prove this hypothesis, such as measuring for RDS after exposure to IHHNV. An IHHNVresistant strain of Penaeus stylirostris has been developed in Venezuela, promoted as SuperShrimpk, which had a significant impact on shrimp farm production in Mexico and other countries (Persyn, 1999). Another IHHNV-resistant strain of P. stylirostris had been reported before (Be´dier et al., unpublished results). After 1994, the effects of the depression could have decreased until disappearing, as can be inferred from the high correlation between the percentage of deformities and F t . This can be explained by a purging effect, which usually occurs with high levels of inbreeding (Keller and Waller, 2002). Current level of inbreeding is low ( F=5.6%), however, it may be an underestimation due to accumulated inbreeding in the founder Mexican population. Additionally, elimination of deleterious alleles from the genetic pool of the population may be enhanced by the process of mass selection. The genetic variability of this stock may have been significantly reduced very early in the domestication process, and it is very likely that inbreeding contributed to some extent to the increase of the percentage of deformities and to the negative effects shown on growth rate and FCR between 1993 and 1995. However, no signs of deterioration on fitnessrelated traits have been observed in the last generations after that period. In 1996, there were high peaks for survival, growth rate, and production probably due to optimal environmental conditions. In this population, the phenotypic variation was reduced as the process of mass selection and possibly optimization of the culture system took place. The reduction in the coefficient of variation in this population may be also related to inbreeding and genetic drift acting together. Sbordoni et al. (1987) were the first to try monitoring the genetic changes that could occur after several generations of closed-cycle reproduction in a shrimp species. They found a progressive reduction in the level of genetic variability, using allozyme markers, from the F1 to the F5 generation of hatchery-produced stocks of Penaeus japonicus, resulted from the inadvertent selection for early spawning and bigger weight. This reduction of genetic variability was highly correlated to the reduction of

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the mean hatching rate (about 80%), which is attributable to inbreeding depression. More recently published results show that the magnitude of inbreeding depression may vary considerably depending on the species and the trait, and that one experiment may show inbreeding depression for some traits but not for others (Bentsen and Olesen, 2002). A reduction in genetic variation was also reported for the F7 generation of domesticated populations of P. vannamei from Ecuador, compared to wild stocks, using microsatellites markers to characterize the populations (Wolfus et al., 1997). These researchers suggest that further depletion of genetic variation could occur if adequate management of this population is not performed. A significant positive correlation was detected between microsatellite heterozygosity and growth rate in Tahitian cultivated stocks of P. stylirostris, which had been genetically isolated from wild founders for 17 generations. These results suggest that heterozygosity at neutral marker loci is sufficiently well correlated with individual inbreeding coefficients to reveal a significant residual inbreeding load for growth rate in these stocks (Bierne et al., 2000). Crocos et al. (2002) evaluated the effect of inbreeding on growth, survival and reproductive performance in domesticated lines of P. japonicus, and found inbreeding depression associated with final weight, survival, number of eggs per spawning and of nauplii per female per month in F4 and F5 generations of inbred lines compared to outbred ones. In the present study, a 14.5% improvement in growth rate was realized after 11 generations of mass selection. Other studies have shown an increase in growth rate of 21% at the fifth generation of mass selection, as reported by Goyard et al. (2002a) for P. stylirostris. An update in this research showed 35% increase in growth rate at the sixth generation, and presented improved FCR for the selected strain (Goyard et al., 2002b). Here we devised a coefficient that integrates the values of growth rate, survival, and FCR, which are the major determinants of production in the ponds. COR has the advantage of monitoring the improvement of the system because the patterns of production can be more easily obscured by artifacts from management procedures, social problems and environmental effects affecting the system but not the

animals. In this sense, after 2000, a family selection program was established in order to maximize the improvement on growth for this strain. The analysis of the data of three generations of selection showed that there was an average response to selection of over 15% per generation (De Donato et al., unpublished results). The additional fact that the strain is being cultivated in a region that, to this point, has been free of diseases such as Taura Syndrome Virus (TSV), White Spot Syndrome Virus (WSSV), and yellow head makes this strain a great resource for shrimp culture in the Americas.

Acknowledgements We want to acknowledge the great support to the shrimp culture industry in Venezuela given by Rodolfo Luzardo and Federico Rivero. They are guided by a vision of the future in shrimp culture and the potential of genetic improvement techniques on current shrimp culture systems. We also want to acknowledge Harvey Persyn, Amber Persyn, and Reginald Loy Markham, who contributed significantly in the development and optimization of the culture system at various points.

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