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In the MUP marker group, females were presented with urine from a stimulus female that matched at MUP (matching) and urine from an equally related stimulus female that was different at MUP (dissimilar). There was no difference between the match-to-self and match- to-maternal template groups for female investigatory behaviour, as measured by the amount of time spent sniffing urine (F(1,20) = 0.45, p = 0.51; Figure 4.2a) and the total amount of time females spent directly under urine marks (F(1,20) = 0.39, p = 0.54; Figure 4.2b). Subject females did not spend longer sniffing urine from dissimilar stimulus females than urine from matching stimulus females (F(1,20) = 0.07, p = 0.80; Figure 4.2a, nor did they spend longer directly under urine from dissimilar stimulus females compared to urine from matching stimulus females (F(1,20) = 0.19, p = 0.67; Figure 4.2b).

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Subject female attraction to the stimulus females was measured by the amount of time females spent on each side of the test cage. Overall females showed no significant attraction to either the matching or dissimilar stimulus urine side (F(1,20) = 1.52, p = 0.23; Figure 4.3), however there was a significant interaction between time spent on each side and whether matching was to self or maternal templates (F(1,20) = 7.66, p = 0.01). Further investigation revealed that in the match-to-self template group females did not spend longer on the matching stimulus urine side than the dissimilar stimulus urine side (t(13) = 1.38, p = 0.19, paired t-test; Figure 4.3). However in the match-to-maternal template group there was a non- significant trend for females to spend longer in the dissimilar stimulus urine side than in the matching stimulus urine side (t(7) = -2.21, p = 0.06, paired t-test; Figure 4.3).

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b)

Figure 4.2: Investigation of urine from matching and dissimilar stimulus females in the MHC/ESP and MUP marker groups.

Investigation of urine from matching (grey bars) and dissimilar (open bars) stimulus females in the MHC/ESP (Self n = 8, Maternal n = 12) and MUP (self n = 14, Maternal n = 8) marker groups. Female discrimination ability measured by a) time spent sniffing urine (seconds, mean + standard error) and b) the total time spent directly under urine (seconds, mean + standard error).

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Figure 4.3: Attraction towards urine from matching and dissimilar stimulus females in the MHC/ ESP and MUP marker groups.

Female attraction towards urine from matching (grey bars) and dissimilar (open bars) stimulus females in the MHC/ESP (Self n = 8, Maternal n = 12) and MUP (Self n = 14, Maternal n = 8) marker groups. Attraction measured by the total amount of time spent on each side of the test cage (seconds, mean + standard error).

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4.4.2 Nest Partner Choice

4.4.2.1 Match to MHC/ESP

Females were given a choice of nest partner over 36 hours based on matching of MHC/ESP. There was no difference in the proportion of time females spent in the matching stimulus female cage between the match-to-self and match-to-maternal template groups (z = -0.92, p = 0.38, two-tailed; Figure 4.4). Overall females spent more time in the matching stimulus cage than in the dissimilar stimulus cage (z = -2.03, p = 0.02, one-tailed; Figure 4.4). There was a non-significant trend for more subject females (68%) to choose the matching stimulus cage than the dissimilar stimulus cage (p = 0.07; Figure 4.4).

4.4.2.2 Match to MUP

Over 36 hours females were given a choice of nest partner between a MUP matching stimulus female and a MUP dissimilar stimulus female. There was no difference in the proportion of time females spent in the matching stimulus female cage between the match-to- self and match-to-maternal template groups (z = -1.57, p = 0.13, two-tailed; Figure 4.4). Subject females spent longer in the matching stimulus female cage than in the dissimilar stimulus female cage (z = -1.87, p = 0.03, one-tailed; Figure 4.4). More females (73%) chose to nest in the matching stimulus female cage than in the dissimilar stimulus female cage (p = 0.03; Figure 4.4).

Inspection of the data suggested that there might be a difference in female response between the match-to-self and match-to-maternal template groups. To investigate this further the groups were analysed separately. Subject females in the match-to-self template group spent more time in the matching stimulus female cage (z = -1.96, p = 0.03, one-tailed; Figure 4.4). However, females in the match-to-maternal template group did not spend longer in the matching stimulus female cage compared to the dissimilar stimulus female cage (z = -0.87, p = 0.21, one-tailed; Figure 4.4).

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Figure 4.4: Nest partner choice in the MHC/ESP and MUP marker groups.

Time (minutes) spent in the matching (grey boxes), centre (hashed boxes) and dissimilar (open boxes) stimulus female cages in the MHC/ESP (Self n = 12, maternal n = 10) and MUP (Self n = 9, maternal n = 13) marker groups.

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4.4.3 Odour-Genes Covariance

4.4.3.1 Match to MHC/ESP

Females from the unrelated line were tested using an odour-genes covariance assay to assess whether neutral females perceive a similarity between urine from females that match for MHC/ESP genotype compared to females that have a dissimilar MHC/ESP genotype. Investigation was assessed by the amount of time females spent sniffing each discrimination urine mark and the total amount of time spent directly under each discrimination urine mark. There was no difference in the proportion of time females spent sniffing the dissimilar discrimination urine between the match-to-self and match-to-maternal template groups (z = - 1.17, p = 0.28, two-tailed; Figure 4.5a). Subject females did not spend longer sniffing the dissimilar discrimination urine than the matching discrimination urine (z = -0.73, p = 0.25, one-tailed; Figure 4.5a).

There was a significant difference in the proportion of time females spent directly under the dissimilar urine between the match-to-self and match-to-maternal template groups (z = -2.20, p = 0.03, two-tailed; Figure 4.5b). The total time spent directly under each urine mark was therefore analysed separately for the match-to-self and match-to-maternal template groups. Females in the match-to-self template group did not spend longer under dissimilar discrimination urine than under matching discrimination urine (z = -0.84, p = 0.23, one- tailed; Figure 4.5b). However in the match-to-maternal template group there was a non- significant trend for females to spend longer under the dissimilar stimulus urine than under the matching stimulus urine (z = -1.75, p = 0.06, one-tailed; Figure 4.5b).

4.4.3.2 Match to MUP

In the MUP marker group females did not spend longer sniffing the dissimilar discrimination urine than the matching discrimination urine (F(1,11) = 0.86, p = 0.37; Figure 4.5a). There was no difference in the proportion of time females spent sniffing the dissimilar discrimination urine between the match-to-self and match-to-maternal groups (F(1,11) = 0.002, p = 0.97; Figure 4.5a). However, the amount of sniffing behaviour differed between the match-to-self and match-to-maternal template groups and females spent longer sniffing both urine marks in the match-to-maternal template group than in the match-to-self group (F(1,11) = 4.91, p = 0.05; Figure 4.5a). Similarly, there was a non-significant trend for females to spend longer under both urine marks in the match to maternal cue group than in the match to self cue group (F(1,11) = 3.78, p = 0.08; Figure 4.5b). Subject females did not spent longer directly under

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urine from the dissimilar discrimination female compared to urine from the matching discrimination female (F(1,11) = 0.70, p = 0.42; Figure 4.5b), and there was no difference in the proportion of time females spent sniffing the dissimilar discrimination urine between the match-to-self and match-to-maternal groups (F(1,11) = 0.04, p = 0.84; Figure 4.5b).

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b)

Figure 4.5: Investigation of odour-genes covariance discrimination urine in the MHC/ESP and MUP marker groups.

Total time (seconds, mean + standard error) subject females spent a) sniffing urine or b) under urine from the matching (grey bars) and dissimilar (open bars) stimulus females in the MHC/ESP (Self n = 8, Maternal n = 5) and MUP (Self n = 6, Maternal n = 7) marker groups.

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Table 4.9: Summary of molecular marker results.

Marker Assay Behaviour Measured Template Origin

(Maternal vs. Self)

Marker Similarity (Matching vs. Dissimilar)

MHC/ESP Scent Discrimination and Attraction Sniff Urine (discrimination) p = 0.18a p = 0.92a

Under Urine (discrimination) p = 0.51a p = 0.77a Cage Side (attraction) p = 0.25a p = 0.76a

Nest Partner Choice Time in stimulus cage p = 0.38b p = 0.02c (M>D)

Odour-Genes Covariance Sniff Urine p = 0.28b p = 0.25c

Under Urine p = 0.03b Mat: p = 0.06c (d>m) Self: p = 0.23c

MUP Scent Discrimination and Attraction Sniff Urine (discrimination) p = 0.51a p = 0.80a

Under Urine (discrimination) p = 0.54a p = 0.67a

Cage Side (attraction) p = 0.01a Mat: p = 0.06d (d>m) Self: p = 0.19d

Nest Partner Choice Time in stimulus cage p = 0.13b p = 0.03c (M>D)

Odour-Genes Covariance Sniff Urine p = 0.97a p = 0.37a

Under Urine p = 0.08a p = 0.42a

Difference in subject female response between the template origin groups (Mat – Match-To-Maternal, Self – Match-To-Self) towards stimulus females of differing marker similarity (M/m – Matching, D/d – Dissimilar). Statistical tests performed: a – repeated measures GLM, b – Mann-Whitney U test, c – Wilcoxon Signed Ranks test, d – paired t-test.

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4.5 Discussion

Female house mice recognise and preferentially associate with unfamiliar full sisters over unfamiliar unrelated females, and show an attraction toward the urine of full sisters (Chapter 2). This suggests that genetic markers of relatedness are present in female house mouse urine. The MHC and MUP multigene families influence mouse odour and both have been proposed as potential molecular markers of kin recognition. Recently it was found that differences in a third polymorphic multigene family, ESP, can be detected by mice. In this study MHC, ESP and MUP were investigated by directly testing female kin recognition ability of females that matched or were dissimilar at each marker. The results (Table 4.9) suggest that all three families may be involved in kin recognition, although due to the mirroring of MHC and ESP haplotypes it is currently impossible to disentangle their separate effects.