Assays of Kin Recognition

A number of different assay designs have been used to study kin recognition and it is important to understand why they are used and what they show. This is by no means an exhaustive list, and each assay described below has a multitude of variations, but the following section covers the most common assay designs and what they are designed to examine.

1.1.8.1 Cross-fostering

To remove the effects of prior association a number of studies of kin recognition have used a cross-fostering design (reviewed in Todrank & Heth, 2001; Mateo & Holmes, 2004). Cross-fostering involves moving infants from their biological parents to foster parents. Infants could be transferred to unrelated members of their own species (e.g. Holmes & Sherman, 1982) or to a different species (e.g. Kendrick et al., 1998). Either entire litters or individuals can be cross fostered in experimental studies. Often litters are reciprocally cross-fostered so that young are swapped between two sets of parents. The inability of cross-fostered individuals to discriminate kin from non-kin suggests a prior association mechanism, whilst the ability of cross-fostered individuals to recognise own biological kin signals the use of self-referential phenotype matching. The behaviour of cross-fostered individuals towards foster-siblings can also reveal information about the importance of familiarity and the interaction between individual and kin recognition. One flaw with the cross-fostering design is that it is difficult to prevent any learning prior to fostering. Whilst most studies transfer kin within one day of birth, learning could potentially occur during the hours immediately following birth or even pre-natal, subsequently influencing experimental results and interpretation (Hepper, 1991).

1.1.8.2 Scent assays

Olfactory kin recognition has been widely documented and therefore there have been a number of tests designed to examine olfactory discrimination. Habituation-dishabituation tests are often used to demonstrate that an animal can distinguish a difference between two odours. In the habituation phase individuals are presented (often over multiple trials) with the same habituation odour (or samples from the same habituation stimulus animal). The subject’s response to the odour decreases over multiple trials as it quickly recognises an

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odour with which it is already familiar. In the dishabituation phase, a new odour is presented to the individual instead of the habituation odour. If the response to the new odour is significantly higher than the last habituation odour response then the habituation odour and the test odour are perceived by the subject as being different from each other. This method has been used, for example, to demonstrate the ability of laboratory mice to discriminate between urine samples from different strains (Isles et al., 2002), and those that differ at only one MHC locus (Penn & Potts, 1998b).

Habituation-discrimination tests are a variation of the habituation-dishabituation design. The habituation phase proceeds as normal, but instead of a single dishabituation odour, in the discrimination phase two odours are presented – the same habituation odour (or another sample from the same habituation stimulus animal) and a novel odour. The subject’s duration of investigation towards both odours is then measured. If the subject investigates the novel odour significantly more than the habituation odour then the subject perceives that there is a difference between the two odours. Using this design Newman & Halpin (1988) showed that prairie voles (M. ochrogaster) can discriminate between the urine of male and female conspecifics.

Odour-genes covariance tests are another variant on the habituation-dishabituation tests (reviewed in Todrank & Heth, 2003). Whilst habituation-dishabituation/discrimination tests are used to demonstrate that two odours are perceived as being different from each other, odour-genes covariance tests assess odour similarities (Heth & Todrank, 2000). During the habituation phase a subject is presented with a habituation odour. The habituation odour is removed and the subject is presented with two novel discrimination odours (this can be simultaneously or consecutively). As both discrimination odours are unfamiliar to the subject, the subject should spend an equal amount of time sniffing each odour. However, if one of the discrimination odours is related to, or otherwise shares a common feature with the habituation odour, then the subject may spend less time investigating the related discrimination odour than the second discrimination odour. The habituation and related discrimination odour can then be said to appear to the subject as more similar to each other than the habituation odour and the unrelated discrimination odour. In a study of golden hamsters (M. auratus) by Todrank et al. (1998), hamsters perceived more similarity between two sibling odours than between unrelated odours. Similarly, female blind mole rats (Spalax galili) treat the odours of more closely related

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individuals as more similar compared to the odours of more genetically distant individuals (Tzur et al., 2009).

In the three scent assays described so far the stimulus odours used could be entirely unrelated to the subject, for example laboratory mice have been used to detect odour similarities between related chacma baboons (Papio ursinus) (Celerier et al., 2010). A fourth design is often used to assess individual response to the odours of own relatives compared to the unrelated odours – a scent discrimination and/or attraction assay. There is no habituation phase and instead individuals are presented with two odours simultaneously (normally one related to the subject and one unrelated) and their response to each odour is recorded. The assay measures the ability of an individual to discriminate spontaneously between two odours, and whether individuals demonstrate any attraction towards either odour. Individuals are often predicted to spend longer investigating unrelated odours, as related odours may contain elements with which the individual is already familiar. For example, neonate smooth snakes (C. austriaca) investigate unrelated odour more than related odour (as measured by the number of tongue flicks directed towards each odour; Pernetta et al., 2009), and female golden hamsters (M. auratus) spend longer with their nose in close proximity to unrelated than related male odours (Mateo & Johnston, 2000). Where kin are expected to aggregate, individuals are predicted to show an attraction towards related over unrelated odours. In an odour choice flume assay juvenile zebrafish (D. rerio) were found to spend longer on the related side than the unrelated side (Gerlach & Lysiak, 2006). Whilst the first three scent assay designs are therefore useful for understanding how animals perceive similarities and differences between odours, the scent discrimination and attraction assay is useful for understanding how animals spontaneously respond to odours.

1.1.8.3 Proximity/preference assays

Kin aggregation or proximity to kin is often used as an indication of kin recognition. For example, sibling star ascidian tunicate (B. schlosseri) planktonic larvae settle in closer proximity to each other than to non-sibling larvae (Grosberg & Quinn, 1986). By giving a subject access to two stimulus animals (or groups), experimenters can measure affiliation and attraction. For example, when given the choice both cascade frog (R. cascadae) tadpoles and juvenile cichlids (N. pulcher) spend more time in proximity to more closely related conspecifics (Blaustein & O'Hara, 1982; Le Vin et al., 2010). Inbreeding avoidance

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studies tend to look for a reverse pattern – a tendency to spend more time in proximity to unrelated individuals of the opposite sex. For example, male bank voles (M. glareolus) spend more time in close proximity to unrelated females than to full sisters (Lemaitre et al., 2012).

1.1.8.4 Interaction assays

Social responses often change with relatedness. Researchers often use dyadic interactions to demonstrate kin discriminative behaviours. Two individuals are often placed in a neutral test arena and their behaviours towards each other measured. Kin selection theory predicts that relatives may be less aggressive towards each other than unrelated individuals; for example reduced aggression during related interactions has been shown in parasitoid wasp (G. Legneri) (Lizé et al., 2012) and fire salamander (S. infraimmaculata) larvae (Markman et al., 2009). Male white-footed deer mice (Peromyscus leucopus) chase unrelated males more than related males (Grau, 1982). Conversely, amicable interactions are often predicted to increase with relatedness, as shown in European polecats (M. putorius) and marmots (M. monax) (Lode, 2008)

1.1.8.5 Inbreeding avoidance assays

A number of inbreeding avoidance studies use assays of the style described above (e.g. Bateson, 1982; Partridge, 1983; Lemaitre et al., 2012). Whilst these studies are informative, measures such as odour attraction or time spent in proximity do not necessarily predict reproductive choice. Therefore a number of studies allow animals to mate and measure pre-copulatory behaviour (e.g. time to mating; Whitehorn et al., 2009), reproductive success (e.g. number of litters; Boyd & Blaustein, 1985), or post-copulatory behaviours (e.g. time taken to re-mate; Välimäki et al., 2011). ‘No-choice’ studies are often used to examine inbreeding avoidance where individuals are paired and reproductive success is measured. Reproductive success between pairs of individuals with varying relatedness is often then compared (e.g. Barnard & Fitzsimons, 1989). Free-breeding experiments are also used to assess inbreeding avoidance. This is where several animals are allowed to interact and breed freely (often in a semi-natural enclosure). Any resulting offspring are then genotyped to calculate the parentage and therefore reveal the individuals that successfully mated (e.g. Lucia & Keane, 2012). Whilst such studies directly measure inbreeding avoidance, one problem is that they are often expensive in terms of time taken to run and animal costs and can produce excess animals that may have to be culled.

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Additionally they also provide limited information on the mechanisms that lead to successful reproduction, including which sex chooses mating partners.