The genetic basis of mammalian behavior has been studied in a limited set of species (Flint et al., 2005
; Kendler and Greenspan, 2006
). The model choice was determined in large part by such criteria as availability of information on the genome, genetic tools, well defined genetically inherited behavioral phenotypes, and the opportunity for experimental breeding. Previously, among mammals, mainly rodent species could satisfy all these requirements. Information on the genomes of species representing different mammalian groups (http://www.broad.mit.edu/mammals/
) has become available in the last few years. This progress in genome biology now creates an opportunity for genetic investigations in species that are well-established as models for behavioral studies but, until recently, lacked the major genetic tools (Scott and Fuller, 1965
; Trut, 1999
; Williamson et al., 2003
; Suomi, 2006
In a long-term experiment at the Institute of Cytology and Genetics of the Russian Academy of Sciences (ICG), specific strains of silver fox with markedly different behavioral phenotypes have been selected (Trut, 1980a
; Trut 1999
and Trut et al., 2004
). Foxes bred for docility demonstrate a friendly response to humans similar to that of domestic dogs. In contrast, foxes from a strain selected for aggressive behavior are aggressive toward humans and difficult to handle. Inter-specific aggression “in defense of the subject's own bodily integrity” is classified as defensive aggression (Blanchard and Blanchard, 2005
). These tame and aggressive fox strains have been bred separately for over 40 generations under strong selection for their respective phenotypes, but in a manner designed to deliberately minimize inbreeding. Inbreeding coefficients during selection remained in the range 0.02−0.07 (Trut, 1999
; Trut et al., 2004
), and this low level of inbreeding has been confirmed in recent analysis with microsatellite markers (Kukekova et al., 2004
). The genetic nature of these fox behavioral phenotypes is well established (Trut, 1980a
). Because these genetically determined behavioral differences segregate in very large pedigrees of a single species, they offer an opportunity to map and identify the genes responsible. The evolutionary closeness of fox and dog (Wayne et al., 1997
) enables the exploitation of canine molecular genetic tools, particularly the 7.6× dog genome sequence (Lindblad-Toh et al., 2005
), to facilitate construction of a fox meiotic linkage map and to undertake genetic mapping in foxes (Kukekova et al., 2004
Measurement of behavior presents several challenges, particularly in attempting to map the underlying genetic loci. Because behavior is clearly phenotypically complex, but also has indisputable heritable aspects, it likely reflects a complex interaction of multiple genetic loci and environmental factors. To identify genes responsible for behavioral variation, it is first highly desirable to identify, among the wide spectrum of behavioral expression, those specific, independent, and presumably simpler aspects that can be measured objectively and quantitatively and that can be demonstrated to be inherited.
In the course of selection of foxes for behavior, two scoring systems for assignment of fox behavioral phenotypes have been used: one for the tame and another for the aggressive population (reviewed in Trut, 1980a
; Kukekova et al., 2005
). Although the original farm-fox population showed a continuous variation in behavior from “relatively less aggressive and fearful” to “extremely aggressive”, very quickly the phenotypes in the selected tame and aggressive populations no longer overlapped. Foxes from the tame population were scored by ranking them based on a repertoire of tame behaviors which were either shown or not during interaction with an experimenter under the standardized conditions. Scores of tame foxes reflect the intensity of the fox's friendly response toward the experimenter: the tamest foxes are assigned scores of 3.5−4.0; the least tame score 0.5−1.0. Behavioral assessment in the tame population was further refined by evaluating a comprehensive set of measures for scoring behaviors contributing to “tameness” by principal-components analysis (Vasilieva and Trut, 1990
). In contrast, the major criterion for measuring behavior in the aggressive population was the critical distance between the experimenter and the caged animal when the animal first demonstrates an aggressive reaction and intensity of the fox aggressive response (Trut, 1980a
; Kukekova et al., 2005
). Animals demonstrating the most aggressive response to humans are scored −4; those showing the least aggressive response score −0.5. The systems for measuring behavior in the tame and aggressive strains yield objective and reproducible behavioral assessment of individuals in both populations and were used to select animals exhibiting the most tame and the most aggressive behaviors for breeding the next generation. The continued improvement in scores with selection over multiple generations (Trut 1999
) is the best evidence for the reliability and utility of these scoring systems for measuring behavior in the tame and aggressive strains.
To study the genetic basis of fox behavioral phenotypes, three-generation experimental pedigrees have been established by breeding tame to aggressive founders to produce an F1 generation, and then backcrossing to the tame strain (Acland et al., 2004
; Kukekova et al., 2005
). Assignment of behavioral phenotypes in F1 clearly demonstrates that the traditional scoring systems established for selection of foxes for behavior has limited resolution for measuring behavior as a continuous variable in the cross-bred pedigrees. Broadly, F1 foxes exhibit a wide range of behaviors; substantial percent of foxes had low values on both the “tame” and “aggressive” scales (Trut, 1980a
; Kukekova et al., 2005
). Furthermore, behavioral patterns characteristic of the founder populations become fragmented or reshuffled in the cross-breed offspring. Thus, before attempting to map or identify genes underlying behavioral variations segregating in these fox strains, we needed a high-resolution, objective, quantitative system that defined behavior of foxes from both the tame and aggressive strains, and that enabled assignment of behavioral phenotypes in both founder and experimental populations.
In the current study we developed and tested a new system for assignment of fox behavioral phenotypes. To capture those fox behavioral components which had been selected for in the development of the founder populations, this new system is rooted in the traditional behavioral tests developed at ICG (Trut et al., 2004
; Vasilieva and Trut, 1990
). The behavior of the foxes was evaluated as in the traditional methods, and videotaped. A comprehensive primary set of binary (present, absent or yes, no) objective observations was then developed for scoring the physical manifestations of fox behavior during the test from video records. Statistical analyses, including principal-components analysis (PCA), were used to dissect out the independent, resegregating traits underlying the phenotypic variation expressed in these multiply correlated observations. To validate this new system for measuring behavior we evaluated the concordance between the ICG behavioral assignment and this new system. Moreover, a useful system for measuring behavior in experimental cross-bred pedigrees has to distinguish between the behavior of foxes from the tame and aggressive strains as well as cover a range of values for the F1 and backcross-to-tame generations that are intermediate to the parental strains. Consistency of this new system was tested between independent data sets obtained by different observers and the reproducibility was tested among repeated tests.
The system described herein provides an essential tool for quantitative analysis of these fox behavioral phenotypes. Together with the newly available tools for genomic research, this now makes it feasible to map the loci and genes implicated in tame and aggressive fox behavior. Determination of the genetic basis for specific behavioral phenotypes in these foxes promises to yield broader insights into the genetics of complex behavior and its underlying molecular mechanisms.