Assistance in Determining Net Fitness Component Values

Relative Mating Success (m_j) and (f_i)
As with juvenile viability, mating success of each genotype can be determined either directly or indirectly. Direct observation is the simplest, but may not be possible in all species. With direct observation, staged mating trials are conducted that allow for both mate competition and mate choice. The mating frequency of each genotype is recorded.^4,5 Because the effect of the transgene on mating success can vary with its relative frequency, such trials should also be performed using different ratios of genotypes.

With indirect observation, transgenic and wild type males and females in various ratios are placed together in a setting and allowed to mate. Muir and Howard call these trials `mating sets.' Several different mating sets, and replications of mating sets, are conducted. After mating, the identity of the male parent is inferred from the genotypes of the progeny. In the simplest case, a single wild-type female would be introduced into a tank containing a transgenic and wild-type male. Any transgenic offspring would immediately identify the genotype of the male parent. Assume for this example an equal mating success of transgenic and wild-type males and females. (Values must be positive for males)

Male Fertility (r_j)
Fertility is more difficult to determine than fecundity and is inferred from the number of eggs fertilized by alternative genotypes. We examined the ability of the male genotype to fertilize eggs with a simple, completely randomized design experiment in which 10 transgenic males and 10 wild-type females were randomly single-pair mated with wild-type females in separate 40-liter tanks for eight days. The first three egg masses produced by each female in that eight-day period were collected and incubated in a hatching tank. Twenty-four hours later, the eggs were examined under a dissecting microscope and classified as fertile or infertile based on presence or absence of embryo development. Assume for the medaka example that both wild-type and transgenic males have fertility rates of 95%. A value of 1.0 indicates 100% success; .9, 90%; and so on. (Values must be greater than or equal to 0 and less than or equal to 1)

Female Fecundity (c_j)
Fecundity is straightforward. For medaka, estimating fecundity is a simple matter of counting the number of eggs produced from each genotype. The genotypes should be the same age and several fish should be measured. For this example, assume wild-type fish produce an average of 8.8 eggs per spawn and transgenic produce 11.4. (Values must be greater than or equal to 2)

Number Of Males or Females In Initial Population
Wild type (ww): Values must be greater than or equal to 1000;
Tw and TT values must be greater than or equal to 0.

Juvenile Viability (v_j)
Juvenile viability is simply defined as survival from the embryo to the age of sexual maturity (or approaching sexual maturity). There are several ways to estimate this component. The simplest experiment would be to establish two pure breeding lines (TT and ww) and, starting with a known number of fertile eggs, count the number that survive to sexual maturity. The experiment should be conducted under environmental conditions that closely approximate the natural environment into which they might escape. This experiment should be replicated several times (2 to 10). The average percent survival of each genotype (v'_j) is converted to per day viabilities (v_j) occurring between consecutive census time periods (a_t+1 and a_t) by assuming a log-linear reduction in daily viability between time periods. Viability can then be described by the following equation:

For example, if 10 replicates of 1000 fry results in an average of 175 transgenic and 250 wild-type individuals surviving to 56 days of age, the per day survival rate is calculated thus:

An alternative method that addresses the issue of background genotype, but does not require the offspring to be genotyped, is given by Muir and Howard.^1,2 This method is based on the theory that the only difference between survival of an intercross and a backcross is the segregation ratio of 3:1 vs. 1:1. If the viabilities of each genotype are the same, then the expected survival for each cross would be the same. If viability of the transgenic genotype is less than that of wild type, then total survival of the intercross will be less than that of a backcross. Preferably the wild-type line is representative of the native fish in the area into which the fish might escape. In this way, the background genotype of both transgenic and wild-type fish are taken into account.

The procedure is to cross the homozygous transgenic line with a wild-type strain to produce the F₁ generation. The cross F₁ is then intercrossed to produce the F₂generation and the F₁ is also backcrossed to the wild type stock to produce the BC₁ generation. The number of fish that survive from hatching to sexual maturity (or approaching sexual maturity) is recorded. The relative viabilities are then found by the method of maximum likelihood as shown by Muir and Howard.¹

Adult Viability (u_j)
Ideally, adult survival of each genotype would be measured in as natural an environment as possible until most fish die. Because this may take a very long time, an alternative method is to assume a log-linear reduction in daily viability and observe only enough time periods to establish a trend.

For example, assume 1000 fish of each genotype are observed for 100 days past sexual maturity. The proportion surviving at the termination of the experiment of each genotype (u'_j) is 90% for wild-type and transgenic individuals. The daily reduction in survival (u_j) is determined using the following equation:

In our example, a_t+1- a_t = 100 days. Thus

Age at Sexual Maturity (s_j)
Age at sexual maturity can be straightforward or difficult, depending on the species. For medaka, age at sexual maturity was recorded as the age at which females first produced eggs. We assumed the males to be mature at the same age. For other species, it may be necessary to sacrifice the animals at various ages and observe gonadal development. Assume for this example that ages at sexual maturity are 56 and 49 days after hatching for wild type and transgenic fish, respectively. Values must be greater than or equal to 1.