Different syllable organizations in 129 and B6 strains of mice
Male mice USVs were induced by introducing a B6 female at estrus stage. Male mice exhibited sniffing, chasing, and mounting behaviors, as previously reported [13
]. USVs emitted during sniffing behaviors were compared to avoid potentially confounding effects with different behaviors [14
USV patterns were characterized by visual inspection of sonograms and it was found that129 and B6 males had mutually exclusive phenotypes. Based on previously published criteria for syllable classification [10
] it was determined that 129 mice frequently repeated chevron-type syllables, whereas B6 mice used short-type syllables in their repeats (Figure ). The chevron-rich repeats are referred to as "chevron-wave" USVs, while the short-rich repeats are referred to as "staccato" USVs. These results suggest that B6 and 129 mice have different syllable preferences during the generation of courtship USVs.
Figure 1 Syllabic composition of the USVs of 129, B6, and F1 mice. (a) up: staccato (short repeats) (red) and chevron-wave (chevron repeats) (blue) of 129 (left), B6 (center), and F1 (right). The box represents short or chevron adjacently repeated regions. down (more ...)
Quantification of syllable composition indicated that chevron-type syllables were infrequently used by B6 mice, but they were dominant in 129 mice. Short-type syllables were significantly more commonly used by B6 mice compared with 129 mice (Figure ; F(14,476) = 30, p < 0.001 for strain × syllable type; for chevron or short, p < 0.001 for 129 vs. B6, Student-Newman-Keuls (SNK test) multiple comparison test), although they had the capacity to generate both syllable types (Figure ). Both strains showed no significant differences in the emission of other syllable types (Figure and Table ; p > 0.4 for Sh, Up, Dn, Fl, Cx, OS, or MS, for 129 vs. B6, SNK test).
Parameters for syllables produced by 129, B6, and F1.
Furthermore, there were no significant differences in the number of sniffs or syllables emitted, which reflects the motivational effect on USV emission (Figure ; sniffing: p = 0.52, t-test; number of syllables: p = 0.18, Student t-test). These results suggest that the chevron-wave and staccato phenotypes of B6 and 129 mice strains were associated with specific preferences in these strains for selecting chevrons or short syllables, despite their similar responsiveness in females.
F1 males produce a B6-like USV
The genetic relationship between the staccato and chevron-wave phenotypes was addressed by generating F1 mice using both 129 ♀ × B6 ♂, and B6 ♀ × 129 ♂, matings. Interestingly, the proportion of short and chevron USV syllables produced by F1 males was similar to that produced by B6 males and differ from 129 (F(14,476) = 30.4, p < 0.001; short: p < 0.001 for 129 vs. B6 or F1 and p = 0.33 for B6 vs. F1; chevron: p < 0.001 for 129 vs. B6 and p = 0.86 for B6 vs. F1). The B6-like USVs of F1 mice were also unaffected by the paternal genotype (p = 0.47, B6 ♀ × 129 ♂, vs. 129 ♀ × B6 ♂,). A high proportion of frequently repeated short-type syllables was produced by the F1 mice (Figure ), as found in B6 mice (Figure ). F1 mice exhibited a similar amount of sniffing behavior (Figure ; F(2,60) = 2.5, p = 0.08) and emitted a lower number of syllables during sniffing behavior when compared with their parental strains (Figure ; ANOVA on ranks, p = 0.29; p < 0.05 for F1 vs. B6 or 129). These results suggest that staccato is the dominant phenotype, while chevron-wave is recessive.
USV composition was not dependent on the female strain
The different USV patterns of B6 and 129 males could be attributable to differences in their responsiveness to B6 females, i.e., B6 males might be exposed to females with the same genetic background, whereas 129 males might encounter females of different strains. To address this issue, we compared their responsiveness to 129 females. However, the relative syllable composition of each strain was not significantly affected by 129 females (Figure ; 129 male: F(1,210) = 0, p = 1, two way repeated measures ANOVA; p = 0.972 for 129 vs. B6 female, SNK test; B6 male: F(1,224) = 0, p = 1; p = 0.4 for 129 vs. B6, SNK test). These results suggest it was unlikely that differences in the USV phenotypes of B6 and 129 males (Figure ) were due to motivational preferences associated with female stains.
Figure 2 Syllable composition of B6 and 129 males was not affected by the female strain. (a) The ratio of each syllable type for 129 and B6 male mice stimulated by a 129 female. The syllable composition pattern was consistent with Fig. 1 (stimulated by B6 female). (more ...)
Repetition of a specific syllable in B6, 129 and F1mice
To compare the degree of repetition of either short and chevron-type syllable among genotypes, we first measure the onset probability of those syllables along USV sequences (Figure and ). Strain B6 or F1 produced more short repeats than 129 (Figure left; F(2,710) = 165.1, p < 0.001; p < 0.001 for 129 vs. B6 or F1, p = 0.5 B6 vs. F1, SNK test; 1 ×-4 × short repeats, p < 0.001 for 129 vs. B6 or F1, SNK test). Chevron repeats were more frequent in 129 compared with B6 or F1 (Figure right; F(2,710) = 50.0, p < 0.001; p <0.001 for 129 vs. B6 or F1, p = 0.14 for B6 vs. F1; 1 ×-4 × short repeats, p < 0.001 for 129 vs. B6 and F1, SNK method].
Figure 3 Sequential syllable structure for USVs of B6, 129, and F1 mice. (a) Syllable repetition in B6, 129, and F1 mice. The number of events of differing length was normalized with the total number of syllables. B6 and 129 mice emitted different amounts of short (more ...)
The biased preference for syllables was also found to be expressed in repeated syllables. For example, all B6 or F1 mice had the capacity to generate more than 4 × short repeats, whereas only 17% of the 129 mice produced 4 × short repeats (Figure left; p < 0.05 for 129 vs. B6 or F1, chi-squared test). In contrast, all 129 males had the capacity to produce four or more chevron repeats (≥ 4 × chevron repeats), while few of the B6 (4%) or F1(4%) mice produced 4 × chevron repeats (Figure right; p < 0.01 for 129 vs. B6 or F1, chi-squared test). These results suggest that the different USV patterns found in 129 and B6 mice (Figure and ) were associated with different syllable preferences of mice, both in terms of random selection and syllable repetition.
Short-rich and chevron-rich phenotypes were segregated in F2 generations
USVs of F2 mice obtained from the mating of F1 mice were analyzed to determine whether the mutually exclusive staccato and chevron-wave USV phenotypes were different alleles at the same genetic locus (Figure ). B6 and F1 USVs were exclusively composed of short-type syllables, whereas the chevron-type was preferred by 129 mice (Figure , white, black and red dots); however, F2 mice produced a more diverse composition of short-type and chevron-type syllables (Figure , red dots). Many F2 mice emitted 129-like USVs (Figure and , 129-type), although a larger proportion of F2 mice produced B6-like USVs (B6-type). Furthermore, new types of mice were found that produced mixed-type USVs, while others lacked both staccato and chevron-wave repeats (novel-type) in their USVs. This suggests that at least two separate genetic loci determine the preference for syllable usage, i.e., one for the chevron-wave and another for the staccato repeat phenotypes.
Figure 4 F2 USVs were diversified into B6-like, 129-like, and mixed phenotypes. (a) USV patterns of F2 mice. Representative sonograms of the four types of F2 mice are shown. Chevron repeat (red) and short repeats (blue) are highlighted (box). (b) Proportions of (more ...)
Syllable repetition phenotypes were diversified also. Only c. 25% of the F2 mice exhibited the repetition of chevron syllables more than four times (Figure ; 12 of 41 total animals; χ2 = 0.2, p = 0.65, chi-squared test, 1/4 vs. 12/41), so the allele for chevron repetition preference appears to be recessive. Approximately 75% of the F2 mice produced USVs containing short-rich syllables repeated more than four times (Figure ; 32 of 41; χ2 = 0.07, p = 0.79 for chi-squared test 3/4 vs. 32/41), which indicates the dominance of this phenotype as also observed in the F1 generation.