Primed to vocalize: Wild-derived male house mice increase vocalization rate and diversity after a previous encounter with a female

Abstract

Males in a wide variety of taxa, including insects, birds and mammals, produce vocalizations to attract females. Male house mice emit ultrasonic vocalizations (USVs), especially during courtship and mating, which are surprising complex. It is often suggested that male mice vocalize at higher rates after interacting with a female, but the evidence is mixed depending upon the strain of mice. We conducted a study with wild-derived house mice (Mus musculus musculus) to test whether male courtship vocalizations (i.e., vocalizations emitted in a sexual context) are influenced by a prior direct interaction with a female, and if so, determine how long the effect lasts. We allowed sexually naïve males to directly interact with a female for five minutes (sexual priming), and then we recorded males'vocalizations either 1, 10, 20, or 30 days later when presented with an unfamiliar female (separated by a perforated partition) and female scent. We automatically detected USVs and processed recordings using the Automatic Mouse Ultrasound Detector (A-MUD version 3.2), and we describe our improved version of this tool and tests of its performance. We measured vocalization rate and spectro-temporal features and we manually classified USVs into 15 types to investigate priming effects on vocal repertoire diversity and composition. After sexual priming, males emitted nearly three times as many USVs, they had a larger repertoire diversity, and their vocalizations had different spectro-temporal features (USV length, slope and variability in USV frequency) compared to unprimed controls. Unprimed control males had the most distinctive repertoire composition compared to the primed groups. Most of the effects were found when comparing unprimed to all primed males (treatment models), irrespective of the time since priming. Timepoint models showed that USV length increased 1 day after priming, that repertoire diversity increased 1 and 20 days after priming, and that the variability of USV frequencies was lower 20 and 30 days after priming. Our results show that wild-derived male mice increased the number and diversity of courtship vocalizations if they previously interacted with a female. Thus, the USVs of house mice are not only context-dependent, they depend upon previous social experience and perhaps the contexts of these experiences. The effect of sexual priming on male courtship vocalizations is likely mediated by neuro-endocrine-mechanisms, which may function to advertise males' sexual arousal and facilitate social recognition.

Fig 1. Classification of USVs: USV types, their abbreviations, a spectrogram’s example and definitions following the classification of [, –56].

Fig 2. Histogram depicting variation in ultrasonic vocalization rate (number of USVs per 10 min) among individuals.

Approximately half of the males emitted less than 50 USVs during the 10 min trials, though some mice were very vocal (n = 50).

Fig 3. Number of USVs emitted with or without sexual priming.

Boxplots of the number of USVs emitted by unprimed (control) and primed males. Boxes around the median (horizontal line) show the interquartile range (quartile 1 to 3) and whiskers extend to 1.5 times this range, or to the most extreme point, whichever is closer to the median. Extreme points are shown as circles. Different letters denote significant differences (p<0.05).

Fig 4. Number of all vocalizations (sonic and ultrasonic) emitted with or without sexual priming.

Boxplots with medians of the number of the overall vocalizations emitted by unprimed (control) and primed males. Different letters denote significant differences (p<0.05).

Fig 5. USV length with or without priming.

Boxplots with medians of USV lengths emitted by unprimed (0) and primed males (≥1). Different letters denote significant differences (p<0.05).

Fig 6. Spectrogram examples of an unprimed (0d) and a sexually primed (1d) male.

The two spectrograms show a 10 s continuous sequence of the males that emitted most USVs in both groups, (A) the unprimed group and (B) in the group recorded 1d after priming. All lines of the spectrograms are continuous and each line shows 2 s (50 ms interval) of the 10 s sequence. Y-axes represent frequencies between 0–150 kHz with intervals of 25 kHz. Letters indicate examples of vocalization types, following the definitions and abbreviations in Fig 1. LFV = low-frequency vocalization.

Fig 7. USV slope with or without priming.

Boxplots with medians of USV slopes emitted by unprimed (control) and primed males. Different letters denote significant differences (p<0.05).

Fig 8. Variability in USV frequency between primed and unprimed males.

Boxplots of absolute deviations of the USV frequency from the overall median. Different letters denote significant differences (p<0.05).

Fig 9. Repertoire diversity of USV with or without priming.

Boxplots with medians of repertoire diversity of unprimed (0) and primed males (≥1). Different letters denote significant differences (p<0.05).

Fig 10. Non-metric multi-dimensional clustering of USV type according to priming groups (nMDS: Stress = 0.13).

Mice are clustered according to the amount of each vocalization type emitted, and spectrograms of vocalization types are depicted on the legend on the right (see also Fig 1). Mice are color-coded by groups: unprimed males in orange triangles, 1d primed males in blue circles, 10d primed males in dark green circles, 20d primed males in green circles and 30d primed males in light green circles.

Fig 11. Proportions of the different types of vocalizations emitted by mice in the treatment and control groups.

Pie charts show the mean proportions (%) of the occurrence of vocalization types emitted by each group, and the legend shows the 15 vocalization types (see also Fig 1).