PREDATOR AVOIDANCE, SPAWNING AND FORAGING ABILITY OF TRANSGENIC CHANNEL CATFISH WITH RAINBOW TROUT GROWTH HORMONE GENE

Rex A. Dunham^a, T.T. Chen^b, Dennis A. Powers^c, Amy Nichols^a, Brad Argue and C. Chitmanat

^aDepartment of Fisheries and Allied Aquacultures, Alabama Agricultural Experiment Station, Auburn University, AL 36849; ^bUniversity of Connecticut; and ^cHopkins Marine Station, Stanford University

SUMMARY

Channel catfish, Ictalurus punctatus, containing rainbow trout growth hormone cDNA were compared to negative controls for spawning ability, foraging ability and predator avoidance. Spawning rates were similar between transgenic and control catfish when stocked communally for competitive spawning. Transgenic males had slightly higher spawning rates than non-transgenic males. This may have been a result of their being larger with better secondary sexual characteristics at the time of spawning. In aquaria, transgenic individuals grew 33% faster than controls when fed artificial catfish diets. No differences in growth were observed in ponds with only natural food items and no supplemental feeding. Additionally, survival of transgenic fry was lower than controls in this environment. Angling vulnerablility was the same for transgenic and control channel catfish. No overall differences were observed for avoidance of sunfish and bass predators. However, transgenic families had better predator avoidance in four trials, controls in four trials and no difference in two trials. This may indicate that family differences in predator avoidance are more important for avoiding predators than possession of the transgene. If transgenes are transferred into families with superior genes for predator avoidance, the frequency of the transgene could increase after escapement into the natural environment. The relative predator avoidance of wild channel catfish should be compared to domestic transgenic catfish.

INTRODUCTION

Transgenic channel catfish possessing foreign growth hormone genes have been produced (Dunham et al., 1987; Hayat et al., 1991; Dunham et al., 1992). The transgenic catfish containing salmonid growth hormone gene produce salmonid growth hormone and transmit the recombinant gene to their progeny (Dunham et al., 1992). Transgenic catfish grow 25% faster than controls.

For the aquaculture industry to benefit from this research and future research, the transgenic fish need to be released and utilized by the private sector. Before this can be done the potential risk of transgenic catfish to natural populations and the natural environment must be determined. Our long term goals include the study of a variety of traits of transgenic fish, especially fitness traits such as spawning ability, foraging ability and predator avoidance which are critically related to survival and competitiveness in the natural environment.

Our laboratory has transferred salmonid growht hormone genes, RSVLTR-rtGH₁ cDNA, RSVLTR-rtGH₂ cDNA and RSVLTR-csGH gene, to channel catfish. One-nine copies of the foreign DNA were inserted in either head-to-tail tandem array at single insertion sites or single copies at multiple insertion sites. All P₁ transgenic catfish evaluated produced salmonid growth hormone regardless of the construct. Five P₁ x P₁ matings have been accomplished. The spawning rate and fertility of these P₁transgenics in artificial spawning conditions was comparable to that of normal channel catfish. In two of three years, 100% spawning and 100% hatch were obtained. Percent transgenic progeny observed in the five matings were 20, 52, 7, 47, and 0% which was lower than the 75% inheritance expected assuming the P₁ brood stock had at least one copy of the foreign gene integrated and were not mosaics in the germ-line. At least seven of ten P₁ were mosaics and a minimum of 2 of 10 P₁ did not possess the salmonid growth hormone genein their germ-line.

P₁ transgenics grew at the same rate as their non-transgenic full-siblings which is not surprising since P₁ were mosaics. F₁ transgenic progeny in two families possessing RSVLTR-csGH grew 26% faster to 40-50 g than their non-transgenic full-siblings when evaluated communally. One F₁ progeny group produced by a RSVLTR-rtGH₁ cDNA x RSVLTR-csGH mating and one F₁ progeny group (parents either RSVLTR-rtGH₁ cDNA or RSVLTR-csGH) grew at the same rate as normal full-siblings when grown communally to 25 g. In families where F₁ progeny grew faster than controls, the range in body weight and coefficient of variation for the transgenic full-siblings was less than that for controls. In families where F₁progeny grew at the same rate as controls, range in body weight and coefficient of varioation were similar for transgenic and normal individuals. The percent deformities observed in P₁ transgenics, 13.65, was higher that in microinjected P₁ non-transgenics, 5.1%. Percent deformities in transgenic and control F₁channel catfish was not different, 0.5 and 2.8%, respectively.

The RSV-LTR (Rous sarcoma virus-long terminal repeat) promoter, a constitutive promoter used in these experiments, appears to be effective in fish. Channel catfish and common carp express salmonid growth hormone genes driven by RSV-LTR. Northern pike, Esox lucius, and goldfish, Carassius auratus, also express recombinant genes driven by the RSV-LTR promoter (Schneider et al., 1989; Yoon et al., 1990). The mouse metallothionean promoter does not appear to consistently drive expression in fish (McEvoy et al., 1988; Rokkones et al., 1989). The SV-40 promoter usually does not allow expression (Guyomard et al., 1988; Stuart et al., 1988) with the lone exception being the successful expression of catalase in transgenic tilapia (Indiq and Moav, 1988).

Results we have obtained for F₁ transgenic common carp (Zhang et al., 1990; Chen et al., 1993) and channel catfish (Hayat et al., 1991; Dunham et al., 1992) containing salmonid trout growth hormone gene were similar. The presence or absence of increased growth can vary among families and may be related to family effects, genetic background, epistasis or dosage effeccts of the foreign growth hormone gene expression. Apparently, a combination of both family selection as well as gene transfer is needed to optimize growth increase from the insertion of salminid growth hormone genes.

Factors Affecting Establishment of New Genotypes in Established Natural Populations. Several factors might affect the establishment of domestic populations by accidental or intentional release and their opportunity or ability to interact with and influence wild populations. These include size of fish, number of fish stocked, number of repeat stockings or releases, timing of stocking or release, selective value of the new genotype and other environmental variables. These variables have not been completely evaluated, but a growing base of data illustrates their importance and function (Dunham et al., 1992b; Isley et al., 1987; Kulzer et al., 1985; Norgren et al., 1986; Plosila, 1977; Pycha and King, 1967; Smitherman et al., 1989a; 1989b; Maceina et al., 1988; Philipp et al., 1985). These studies indicate that it is difficult to establish a new genotype, even wild rather than domesticated, in an established natural population (Norgren et al., 1986; Smitherman et al., 1989a; 1989b; Wirgin, 1990; Wirgin et al., 1990; Fraser, 1972).

One example illustrating this point is that of the massive stockings of Florida largemouth bass, Micropterus salmoides floridanus, into established northern or native largemouth bass, M. salmoides salmoides, populations (Dunham et al., 1992b; Kulzer et al., 1985; Norgren et al., 1986). Genes from Florida largemouth bass were established in these populations at varying levels, and in some cases not at all. Key factors in the establishment of Florida alleles were total numbers of fish stocked, number of years since initial stocking, number of repeat stockings, elevation, age of the lake and water clarity.

Size of fingerling or sub-adult fish stocked has an effect that is not well defined. Kulzer et al. (1985) did nto find a correlation between size of fingerling or sub-adult largemouth bass stocked and the success oif the introduction in largemouth bass. However, the results were confounded by lack of replication and a multitude of additional variables. Studies on the success of stocking large and small sub-adult trout had contradictory results (Plosila, 1977; Pycha and King, 1967; MacLean et al., 1981; Anderson, 1962; Buettner, 1962). The large sub-adults had greater survival when introduced in one study and lower survival in another.

The main conclusion of these studies is that it is difficult to genetically impact established natural populations of fish.

Reproductively Isolated Sympatric Populations. Another possible interaction between domestic and wild populations of fish is the establishment of sympatric, but reproductively isolated populations. Although strains of fish usually so not have reproductive isolating mechanisms preventing them from interbreeding, occasionally behavioral mating blocks prevetn or decrease the rate of inter-strain matings. We have found that Marion channel catfish females preferentially mated with their own strain rather than Kansas males (Smitherman et al., 1984), and Ghana strain of Oreochromis niloticus was more likely to mate with its own strain than other strains (Smitherman et al., 1988). The existence of reproductively isolated, sympatric populations of trout (Lerder et al., 1984; Brown et al., 1981; Ryman and Stahl, 1981), especially brown trout, Salmo trutta, is well-documented. Some strains of domestic and wild rainbow trout are sympatric, but reproductively or near to reproductive isolation. This occurs because of behavioral differences including temporal or spatial differences in spawning (Smitherman et al., 1988).

Reproduction. In addition to other factors, size often plays an important role in reaching sexual maturity and reproductive success. Often the fastest growing individuals within a population will reach sexual maturity the earliest (Gall, 1986; Dunham, 1990), although early sexual maturity does not necessarily correspond to an increased growth rate alone (Wirgin et al., 1991; Gall, 1986; Dunham, 1990; Childers, 1967; Dunham and Smitherman, 1984; 1987). Hulata et al. (1985) reported a negative association between growth rate and onset of sexual maturity in Cyprinus carpio, and this same relationship between growth rate and sexual maturity has been documented in channel catfish (Dunham, 1990; Dunham and Smitherman, 1987).

Egg production also is related to size in channel catfish, and it is likely that transgenic channel catfish may produce more eggs than a normal channel catfish of the same size. However, we have observed that the maximum relative size difference between the transgenic and non-transgenic carp occurs by the time they reach 200 g. When these fish are three years old and have reached a size of 2 kg, the relative size difference is the same or slightly less, and preliminary evidence in catfish indicates that relative size differences of transgenic and control fish occur early, adn then remain unchanged. This growth rate relationship would help negate any size-related reproductive advantage of the transgenic catfish.

To gain a reproductive advantage in the natural environment, a specific genotype would need to be vastly superior to that of other genotypes. The ability to produce large numbers of offspring is not necessarily advantageous because of the tremendous mortality of young fish in the natural environment. In a typical Alabama reservoir, only 0.04% of the channel catfish larvae produced survive to be adults. Among the factors causing natural mortalities and affecting the survival of catfish during their early life history stages are predation and availability of food. Unless a large reroductive advantage existed, the ability to forage and avoid predators are likely of more importance than reproduction for fitness in the natural environment.

Predator Avoidance. Predator avoidance probably has both behavioral and growth components. Aggressiveness or lack of warinness can lead to mortatlity due predation by insects, fish, animals, angling or netting. Additionally, increased growth can be disadvantageous making a fish susceptible to selective harvest (Hulata et al., 1985; Ricker, 1975) by either animal predators or man. Conversely, rapid growth at certain life stages is probably important to reach a size that allows avoidance of small predators.

Foraging Ability. The ability to forage and find feed is, of course, critical to survival in natural settings. The ability of a genotype well adapted or selected for feeding in the culture environment is not necessarily well adapted for feeding in the natural environment since the genetic value or rank of a particular genotype may change as the environment changes. The best genotype for one environment may not be the best for another.

Genotype-environment interactions are prevalent in aquaculture experiments when different genotypes are compared utilizing either natural food organisms or artificial supplemental feeds (Dunham, 1992; Khater, 1985; Wohlfarth et al., 1975; Wohlfarth et al., 1983). High performance triploid oysters have no advantage compared to normal oysters when food is limiting.

Catfish is the major aquaculture species in the United States accounting for over 50% of all U.S. aquaculture. Production in 1992 is estimated to be 500 million lbs. This crop is worth 1.5 billion dollars including value added. The benefit of utilization of high performance transgenic fish, if it can be demonstrated they do not pose risk to the natural environment, would be worth billions of dollars as the catfish and aquaculture industries continue growth.

The primary goal of the research is to determine the genetic risk of transgenic channel catfish, Ictalurus punctatus, which possess the rainbow trout growth hormone gene to wild populations of channel catfish. If transgenic channel catfish containing the rainbow trout growth hormone gene were to be accidentally released into the natural environment from either a research or commercial aquaculture facility, several possible scenarios exist. These transgenic fish may be eliminated by predation or starvation before they reproduce. They may not be able to reproduce or spawn effectively. They may reproduce but catfish possessing the rainbow trout growth hormone gene (or other growth hormone genes) would be eliminated by random genetic drift because of their low population numbers. These transgenic fish could spawn and the foreign gene be established at low frequency based upon initial gene frequencies resulting from the surviving and reproducing escaped fish. The transgenic fish could segregate, spawn with themselves and form a reproductively isolated, sympatric population. Lastly, the transgenic catfish could increase in numbers or porportion compared to non-transgenic catfish.

The last example has the greatest potential for genetic impact, be that positive or negative, on natural populations. The last scenario, increase in transgenic fish numbers in the wild, will only occur if the transgenic fish possess traits that allow them greater predator avoidance (therefore, increased ability to survive to sexual maturity), greater ability to forage on natural food, or a greater ability to spawn and produce young. These traits are the key to fitness, the ability to transmit ones genes to the next generation.

The elucidation of these traits should allow the prediction with a high level of accuracy whether or not transgenic catfish possessing foriegn growth hormone genes, specifically salmonid growth hormone gene, have the potential to genetically impact natural populations of channel catfish.

MATERIALS AND METHODS

Experimental Units. Environmental risk data for transgenic fish has not previously been attempted. The proposed experiments were conducted in 0.04 ha earthen ponds that have been designed specifically for confinement of and approved by USDA for transgenic fish research. It is important to conduct such risk data in confined ponds to mimic as closely as possible the natural environment. This makes ponds a more appropriate test environment than aquaria or tanks since ponds can more closely represent the ecological complexity and diversity of the natural environment. This consideration is especially important because of the potential of genotype-environment interactions. The relative performance of genotypes can change with change in environment. Genotype-environment interactions are common in fish experiments (Ricker, 1981; Dunham, 1992).

Experimental ponds were fertilized, habitat, prey and predators introduced to simulate as closely as possible natural conditions.

Foraging Ablility. Ponds were prepared and fertilized to provide a natural food base and plankton levels similar to that of the natural environment. Transgenic fry and controls were stocked in each pond at swim-up, the stage of first feeding. Samples of the surviving fish from each pond were individually weighed and fin clipped for DNA analysis. Mean body weight and survival of transgenic and control catfish were determined.

Predation Experiments. Ponds were stocked with transgenic and control fry. Ponds were fertilized, a natural forage base established, and natural cover and habitat provided. Aquatic insects, green sunfish, Lepomis cajanellus, and largemouth bass, Micropterus salmoides, were established in each pond at natural densities prior to stocking of the fry. These are the primary predators that fry must escape to survive the early life stages.

The catfish in the ponds were sampled, fin clips (for DNA analysis), and weights were obtained.

Vulnerability to Human Predation. Transgenic and control catfish were stocked into two 0.04 ha ponds. Each fish was individually marked with a heat brand, weighed and sexed. Angling experiments were conducted.

DNA Analysis for Rainbow Trout cDNA. Genomic DNA was extracted from pectoral fin clips of presumptive transgenic fish following the methods of Maniatis et al. (1982) with some modifications. Pectoral fin tissue was lysed in 4 ml of 10mM Tris buffer (pH 8.0) containing 0.1 M EDTA (pH 7.0), 0.5% SDS and 200 µg/ml proteinase K, and digested at 50C for 15 hr. Samples were extracted with phenol, phenol/chloroform and chloroform/isoamly alcohol. Then DNase free RNase A was added at a final concentration of 100 µg/ml and samples will be incubated for 3 hrs. at 37C. DNA was extracted again with phenol/

chloroform and chloroform, and dialysed against several changes of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). After ethanol precipitation, the DNA pellet was dissolved in TE buffer and concentration determined.

To detect the presence of rainbow trout cDNA in presumptive transgenic fish, the polymerase chain reaction (PCR) amplification/Southern blot hybridization method was used. In this method, two synthetic oligonucleotide fragments (20 mers) derived from the sequences flanking the RSVLTR and rtGHcDNA were used as amplification primers and the genomic DNA of the presumptive transgenic fish as a template for PCR amplification. The amplification conditions are: denaturation at 90C for 2 min, annealing at 50C for 2 min and synthesis at 72C for 2 min, and amplifying for 30 cycles. Following amplification, the products were resolved by electrophoresis on 1.0% agarose gels, transferred to nylon membranes and hybridized to radio-labelled rtGHcDNA.

Genomic DNA (18 g) of some individuals was denatured in 0.5 M NaOH at 37C for 10 min and spotted onto a nylon membrane using a commercial dot blot apparatus (BRL, Bethesda, MD). The membrane was soaked in 1.5 M NaCl, 0.5 M NaOH for 2 min, neutralized in 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2), 1 mM EDTA for 5 min. After air drying, the membrane was irradiated on a short wavelength UV transilluminator for 5 min.

The Nde I-Hind III and Kpn I-Hind III restriction fragments isolated from the pRSVrtGHcDNA construct was labelled with [ -³²P] dCTP either by nick translation (Maniatis et al., 1982) or by random priming (Dunham and Smitherman, 1987), then purified through a sephadex G-50 spin dialysis column and used as probes. Prehybridization was carried out in 20 mM Tris-HCl, pH 7.5, 0.1% SDS, 5x SSC, 10x Denhardt and 100 µg/ml of denatured calf thymus DNA at 42C with constant shaking for 3 hr. Hybridization was carried out in the same buffer containing denatured probe at 42C with constant shaking overnight. Membranes were washed twice in a solution containing 2x SSC and 0.1% SDS for 15 min at room temperature, and twice in a solution of 0.2x SSC, 0.1% SDS for 15 min at 65C.

For Southern blot analyses, DNA (18 g) was digested with Hind III or BamH I under conditions indicated by the supplier (Boehringer Manheim) and electrophoresed on a 0.8% agarose gel. DNA samples in the gel were transferred to nylon membranes by diffusion, and hybridized to ³²P labelled DNA probes prepared as above.

Data Analysis. Growth rate of transgenic and control fish was compared with student's t test within each experiment. Genotype-environment interactions were evaluated with analysis of variance. Spawning percentage, percent successful hatches, and survival in all experiments for transgenic and control fish were compared with chi-square analysis.

RESULTS

Channel catfish, Ictalurus punctatus, containing rainbow trout growth hormone cDNA were compared to negative controls for spawning ability, foraging ability and predator avoidance. Spawning rates were similar between transgenic and control catfish when stocked communally for competitive spawning. Transgenic males had slightly higher spawning rates than non-transgenic males. This may have been a result of their being larger with better secondary sexual characteristics at the time of spawning. In aquaria, transgenic individuals grew 33% faster than controls when fed artificial catfish diets. No differences in growth were observed in ponds with only natural food items and no supplemental feeding. Additionally, survival of transgenic fry was lower than controls in this environment. Angling vulnerability was the same for transgenic and control channel catfish. No overall differences were observed for avoidance of sunfish and bass predators. However, transgenic families had better predator avoidance in four trials, controls in four trials, and no difference in two trials. This may indicate that family differences in predator avoidance are more important than possession of the transgene for avoiding predators. If transgenes are transferred into families with superior genes for predator avoidance, the frequency of the transgene could increase after escapement into the natural environment. The relative predator avoidance of wild channel catfish should be compared to domestic transgenic catfish.

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