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Growth and developmental instability
The Veterinary Journal The Veterinary Journal 166 (2003) 19–27 www.elsevier.com/locate/tvjl
Review
Growth and developmental instability Anders Pape Møller *, John Manning Laboratoire de Parasitologie Evolutive, CNRS UMR 7103, Universit e Pierre et Marie Curie, B^ at. A, 7 eme etage, 7 quai St. Bernard, Case 237, F-75252 Paris Cedex 05, France Population Biology Research Group, School of Biological Sciences, Nicholson Building, University of Liverpool, Liverpool L69 3BX, UK Accepted 23 September 2002
Abstract Developmental stability reflects the ability of an individual to develop a regular phenotype under given environmental and genetic conditions. Measures of developmental instability include the degree of fluctuating asymmetry and the frequency of phenodeviants. Endocrine, neural and circulatory mechanisms that control similar development of morphological characters on the two sides of the body are also involved in controlling overall development. Cross-validation studies have shown that measures of developmental instability are positively correlated with other measures of welfare such as tonic immobility in poultry. Asymmetric animals grow less rapidly than symmetric individuals. Eleven studies have investigated the relationship between growth rate and developmental instability, and the observed effect size (Pearson correlation coefficient adjusted for sample size) is )0.15. Studies of chickens have shown that asymmetry increases as a response to selection for increased growth rate. As conditions for rearing deteriorate by higher density, fluctuating asymmetry increases and growth rate decreases, both within and among farms. Fluctuating asymmetry can be considered a measure of animal welfare since larger values reflect worse environmental conditions as experienced by the individual animal itself. Since growth rate and fluctuating asymmetry are negatively correlated, we can infer that improvement of rearing conditions leading to reduced asymmetry will both benefit the producer (in terms of increased growth), but also the animals in terms of better conditions for rearing. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Chickens; Environmental conditions; Fluctuating asymmetry; Phenodeviants; Welfare
1. Introduction Modern industrialised agriculture has dramatically increased productivity over the last century. For animal production this has been achieved through animal breeding for increased growth rates, improved feed quality, veterinary intervention and the use of substances such as growth hormones. This change has come at the expense of animal welfare, which only during the last couple of decades has entered to some extent as an important additional factor modifying agricultural practices. The consequences of increased productivity for the animals have thus only recently become a central issue of concern.
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Corresponding author. Tel.: +33-1-44-27-25-94; fax: +33-1-44-2735-16. E-mail address: [email protected] (A. Pape Møller).
Assessment of the quality of living conditions as seen from the perspective of the animal is thus a crucial point. One such measure is developmental instability (Møller et al., 1999; Møller and Swaddle, 1997), which reflects the inability of an individual to produce a regular phenotype under given environmental conditions (Møller and Swaddle, 1997; Parsons, 1990; Zakharov, 1989). All organisms have regular external phenotypes, and these have been modified by natural selection for mechanical efficiency. Perturbing factors during development may disrupt developmental processes and cause deviations from a regular phenotype, while regulatory processes such as feedback may restore the growth trajectory towards the regular phenotype. Developmental stability is a concept of internal developmental mechanisms that control development, and this cannot readily be estimated. However, deviations from regular development such as small, randomly directed asymmetries (so-called fluctuating asymmetry) and minor physical
1090-0233/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1090-0233(02)00262-9
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anomalies such as a missing character on one side of the body (phenodeviants) can readily be assessed. Fluctuating asymmetry is characterised by randomly directed deviations from perfect symmetry, with most individuals only having little or no asymmetry. Frequency distributions of signed left-minus-right character values are near normal (Fig. 1). It seems that leptokurtic distributions predominate in empirical examples (Fig. 1; i.e., there are many asymmetries near the mean of zero and in the tails of the distribution, and fewer than expected observations at intermediate levels of asymmetry), probably because composite distributions reflecting individual differences in developmental control are not normal, but leptokurtic (Gangestad and Thornhill, 1999). Hence, the degree of leptokurtosis provides a direct estimate of the amount of individual differences in developmental control in a population, with higher leptokurtosis reflecting a larger degree of individual differences. Since fluctuating asymmetries are small (on average less than 1% in animals (Møller and Swaddle, 1997)) and normally distributed, measurement error must be assessed to ascertain that signal differs from noise. Such assessment of measurement error can be done using different statistical techniques (Palmer, 1994; Van Dongen et al., 1999). Fluctuating asymmetry and phenodeviants are increased under deviant environmental conditions such as heat or cold, poor food quality or quantity, deviant light
Fig. 1. (A) A normal frequency distribution of signed left-minus-right character values and (B) a leptokurtic frequency distribution for comparison.
conditions, elevated electromagnetic and radioactive radiation, parasitism, and even perceived risk of predation (review in Møller and Swaddle, 1997). A number of genetic factors are also associated with increased developmental instability, including inbreeding, mutations, sometimes heterozygosity and quantitative genetic variation (review in Møller and Swaddle, 1997). Thus, the regularity of the phenotype will integrate the environmental conditions experienced by an individual in relation to its genetic constitution. Asymmetry in any single trait only provides a very imprecise estimate of developmental instability because a large number of factors including completely random ones may affect the development of such a trait (Gangestad and Thornhill, 1999). Asymmetries of different traits have a small, but highly consistent positive correlation of around 0.075, which reflects the underlying common control of the development of the entire phenotype (Gangestad and Thornhill, 1999; Polak et al., 2002). The repeatability of fluctuating asymmetry (Van Dongen, 1998; Whitlock, 1998) is the correlation between the asymmetry of a trait across hypothetical individuals allowed to develop on two different occasions. It reflects the proportion of variance in fluctuating asymmetry of a single trait caused by individual differences rather than the stochastic nature of developmental errors of single traits. Obviously individuals develop only once and hence this correlation must be estimated rather than measured directly. As the percentage of variance in fluctuating asymmetry due to individual differences increases, the leptokurtosis of the distribution of signed fluctuating asymmetry becomes larger (Gangestad and Thornhill, 1999) and, correspondingly, the coefficient of variation of unsigned fluctuating asymmetry increases (Whitlock, 1998). Based on leptokurtosis of a number of species, ranging from plants over insects to birds, mammals and humans for which large sample sizes were available, Gangestad and Thornhill (1999) estimated that this amount of variation accounted for only 7% of the variance in fluctuating asymmetry of a single trait, although underlying individual differences in developmental instability are substantial (with the coefficient of variation of these individual differences being ca. 22–24 across a wide range of species). Therefore, the repeatability of fluctuating asymmetry of a single trait is only about 0.07, and this may be further reduced by measurement error. The repeatability of fluctuating asymmetry of a single trait puts an upper limit on the correlation between fluctuating asymmetry of two traits. This correlation between fluctuating asymmetry of two traits should only be about 0.07, even if their developmental instability is fully shared. Therefore, composite indices of asymmetry even for as few as 3–5 traits may greatly improve the estimate of asymmetry reflecting developmental instability because the precision of this estimate
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increases rapidly with the number of traits measured (Leung et al., 2000). Why is developmental instability important? The importance derives primarily from the ability of scientists to assess the quality of living conditions as perceived by the individual organism. There are few such measures available, and in studies of animal welfare various groups have advocated behavioural, endocrinological or other indicators of adverse conditions. Fluctuating asymmetry and other measures of developmental instability differ from the other indicators in two important ways. Firstly, the same measure can be used in all kinds of organisms including viruses, plants and animals, whereas the other measures are restricted to one taxon. Secondly, measures of developmental instability already have a pre-defined, in-built optimum, namely symmetry, while the optimum for the other measures must be determined from first principles, preferably using optimality models. Therefore, a population of animals that has a high level of asymmetry can be considered to have experienced sub-optimal conditions during development. This assumption could be tested experimentally since individuals reared under conditions associated with high levels of asymmetry are predicted to work harder to escape their current situation in a condition experiment, as compared to control individuals. Measures of developmental instability are also important because they tie into important components of fitness that are relevant for the maintenance of viable, free-living populations and for productivity of farmed animals. Møller (1999) reviewed the literature on the relationship between developmental instability and growth, survival and fecundity, respectively, and found mean Pearson correlations adjusted for sample size of )0.15, )0.25 and )0.35. Similarly, Møller and Cuervo (2002) in a meta-analysis of the relationship between developmental instability and sexual selection (mating success or sexual attractiveness) reported correlations of )0.17. Since most of these studies we based on asymmetry of a single character, relationships between developmental instability and fitness is likely to be much stronger. This has important implications for animal welfare because animals reared in conditions that give rise to high levels of asymmetry will also tend to grow less well, to survive less well, to have reduced fecundity and to have problems with mating. How is fluctuating asymmetry estimated? There is a large literature on indices of asymmetry and how to estimate them (reviews in Palmer, 1994; Møller and Swaddle, 1997). Traits typically measured in chickens and other poultry include length and width of tibia, tarso-metatarsus, wing length and length of the longest primary feather on the two sides of the body. Traits measured in cattle include length and width of ears, width of the calcaneous and metatarsal bones, width of
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the hock joint, and the medial corner of the eye to the nostril. The number of repeat measurements is typically two, where first the right side is measured, then the left side, then the right side again, and finally the left side. Signed asymmetry is simply right-minus-left character size, while unsigned asymmetry is this difference disregarding the sign of the difference. A number of composite indices of asymmetry for more than a single character also exist (Leung et al., 2000). A useful composite index is based on the sum of the standardised signed asymmetry, which is simply standardised to a standard normal deviate with a mean of zero and a variance of one. The aim of the present review was to assess the relationship between developmental instability and growth. We do that by firstly describing the microscopic mechanisms of ontogeny that link developmental instability to macroscopic fluctuating asymmetry. Secondly, we assess the general relationship between developmental instability and growth. Thirdly, we describe in detail a number of studies investigating the relationship between growth and developmental instability in chickens. Finally, we list a number of future directions for further study.
2. Mechanisms linking microscopic patterns of growth to developmental instability Bilaterally symmetric multi-cellular species derive from a single, fertilised cell that subsequently divides a very large number of times. The extraordinary feat that, for example, the right and left hands of a human are almost perfect mirror images, suggests that there are developmental mechanisms that keep the two copies of an organ or a body part within very restricted boundaries. These developmental mechanisms at the microscopic level that give rise to the quasi-symmetric, macroscopic phenotype have been the basis for a number of models of development (Aparicio, 1998; Graham et al., 1993; Hallgrımsson, 1993, 1999; Klingenberg, 2002; Klingenberg and Nijhout, 1999; Møller and Pagel, 1998). Several models assume that there are homeostatic mechanisms at work providing such a feedback between sides. In fact, communication between body parts must act either through the neural, endocrine or circulatory systems. Any irregularity in the constituent cells of these systems will affect the ease of communication, and even the directionality of communication (Møller and Pagel, 1998). Thus, it is not surprising that some mechanism of quality control at the cell level has to be invoked. Numerous cells emerge that are never incorporated into an organ because they are discarded through apoptosis at an early stage. Available information suggests that apoptosis depends on the quality of a cell as determined by its signalling properties (review in Møller and Pagel,
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1998). Cells that signal at very low levels, thus showing signs of poor quality, or cells signalling at a very high level, thus showing signs reminiscent of cancer cells, are generally selected against (see, for example, Edelman, 1987; Godin et al., 1991; Klekowski, 1988; Michaelson, 1989, 1991; Sachs, 1988). Cells that through their intermediate level of signalling reveal their quality are incorporated into tissues and organs. Such a selective mechanism results in the production of functional tissues and organs with proper communication between the sides of the body and hence in a quasi-symmetric phenotype (Møller and Pagel, 1998). If the proposed developmental mechanisms giving rise to a symmetric phenotype are at work, then we should expect phenotypes at both the microscopic and the macroscopic levels to show similarities in level of morphological regularity. Indeed, asymmetric morphological characters consist of asymmetric and irregular growth increments (Møller, 1996b). Similarly, tibial dyschondroplasia, which is a pathological leg disorder in chickens (Edwards and Veltman, 1983), is more severe in chickens with high levels of fluctuating asymmetry (Møller et al., 1999). Interestingly, the arrangement of cells in the tarsometatarsus becomes increasingly more irregular as the severity of tibial dyschondroplasia increases (A.P. Møller unpublished data). Thus, microscopic and macroscopic patterns of morphological regularity are clearly positively associated.
3. Patterns of growth and developmental instability There is accumulating evidence that asymmetric animals grow less rapidly than symmetric individuals. The magnitude of a relationship between two variables across a sample of studies can be assessed from metaanalysis (e.g., Rosenthal, 1991). Møller (1999) reported a mean effect size adjusted for sample size of the relationship between asymmetry and growth rate of PearsonÕs r ¼ 0:146 (95% C.I. )0.096, )0.196), P < 0:001, for 11 studies. There was statistically significant heterogeneity in effect size among studies included in this meta-analysis ðv2 ¼ 27:46; P < 0:01Þ. The robustness of this finding was assessed from the fail-safe number, which provides an estimate of the number of unknown cases needed to eliminate the overall significance of the weighted mean effect size at the 5% significance level when the mean effect size of the unknown cases is zero. Alternatively, the fail-safe number can be considered to represent the number of unpublished studies in file drawers that is needed to nullify the result. This fail-safe number was 168, which exceeds the minimum number of 65 studies required for a firm conclusion (according to the generally accepted criterion by Rosenthal (1991, p. 106). Using mean values for species as the unit of analysis (if studies of the same species are not considered
statistically independent), the effect size was )0.156 (95% C.I. )0.106, )0.206), P < 0:001 for 10 species. Again, there was significant heterogeneity among studies ðv2 ¼ 25:03; P < 0:01Þ. The fail-safe number equalled 150, which exceeded the minimum requirement of 55 studies. Thus, at the study level the relationship between growth and asymmetry accounted for 2.1% of the variance, and at the species level for 2.4% of the variance. General conclusions based on meta-analysis may be seriously biased if the available studies represent a biased sample of all studies conducted (Møller and Jennions, 2001). There are a number of direct and indirect tests available for studies of publication bias. A study assessing bias in meta-analyses in biology revealed little evidence of bias for the meta-analysis of developmental instability and growth rate (Jennions and Møller, 2002). What is the relationship between developmental stability and growth rate? If we assume that underlying individual differences in developmental stability are of the order of magnitude of 0.07 (Gangestad and Thornhill, 1999, 2002) this value is remarkably similar for organisms ranging from plants over insects to birds and mammals, including domesticated animals and humans), and if the correlation between growth and developmental instability is )0.15 (and hence the amount of variance explained is 0.0225 (0.15 squared), then the relationship between developmental stability and growth is 0.0225/0.07 ¼ 0.321. Thus, underlying developmental stability across phenotypic characters accounts for almost a third of all variance in growth among individuals, which must by all measures be considered a significant amount.
4. Selection, population density and growth in chickens Domestic animals have been selected for rapid growth. Directional selection may disrupt developmental stability because of selection against genetic modifiers that control developmental processes (Møller and Pomiankowski, 1993; Møller and Swaddle, 1997). More recently, Klingenberg and Nijhout (1999) used a simple developmental model to show that heritable variation in fluctuating asymmetry can result without a separation of trait genes and modifiers. Extensive studies of the Australian sheep blow fly Lucilia cuprina model system is a good example of this phenomenon. In diazinone-resistant strains of this fly the modifier gene is a homologue of the Notch gene which encodes components of signalling pathways associated with the number and the asymmetry of sternopleural bristles (Davies et al., 1996; Long et al., 1998). Thus, resistant flies have more asymmetry, but also a different number of bristles, as compared to controls. Although this observation of a trait subject to strong stabilising selection is consistent with the hypothesis proposed by Klingenberg and
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Nijhout (1999), there is currently a dearth of empirical information on this subject. The general observation that there is no significant correlation between effect size (strength of the relationship, measured in terms of Pearson product-moment correlation) of response of size and asymmetry to stress across 20 different studies, implies that size and asymmetry of the same characters index different phenomena (N. Cadee et al., unpublished data). This is particularly surprising given that larger species generally have disproportionately high degrees of asymmetry (Hallgrımsson, 1998). Clearly, more studies are needed to address why that is the case. The developmental models discussed in the previous paragraph imply that improvements in growth due to selection may initially come at a cost in terms of irregular development. Møller et al. (1995) contrasted levels of fluctuating asymmetry in three strains of chickens, a slow growth strain and two fast growing, selected strains, and compared these to asymmetries in wild jungle fowl, which are the ancestors of domestic chickens. While asymmetry only accounted for on average 0.5% of the length of six morphological characters in jungle fowl, it accounted for 3.0% in the slow growing strain and 3.9% and 4.1% in the two fast growing strains. These values can be compared to mean relative asymmetries of less than 1% in animals (Møller and Swaddle, 1997). Yang et al. (1998) reported that asymmetry in a selection line for low body mass was 4.9%, while asymmetry decreased to 3.1% after four generations of relaxed selection in a sub-line. Similarly, Nestor et al. (2000) reported for lines of turkeys that had been selected for increased body mass, shank width and egg laying that they had significantly higher levels of relative asymmetry than did control lines. Furthermore, these differences among lines were better explained by differences in body mass than by differences in homozygosity among lines. Trends in the same direction were reported by Yalcßin et al. (2001) for three lines of chickens differing in growth rates. These findings are consistent with the hypotheses proposed by Møller and Pomiankowski (1993) and Klingenberg and Nijhout (1999). However, we must consider the findings carefully for any bias. Yang et al. (1998) reported an increase in mortality in their selected lines, and since viability selection generally acts against asymmetric individuals (Møller, 1999), the actual differences between lines are likely to be greater than reported. Furthermore, developmental selection at early life stages also usually acts against asymmetric individuals (Møller, 1997), potentially causing further bias. Thus, the reported findings are likely to be conservative. The relationship between growth and asymmetry of individual chickens was )0.071 in one study (Møller et al., 1995) and )0.027 in another study (Sanotra, 1999). These values are bound to underestimate the relationship between developmental stability and
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growth, and the reported relationship between mean asymmetry among chickens and mean growth across 24 Danish chicken farms of )0.358 (Sanotra, 1999; Fig. 2) is likely to provide a better estimate. A key point of concern for animal welfare has been the density at which animals are reared. Møller et al. (1995) conducted an experiment testing the relationship between fluctuating asymmetry and density. Chickens were reared at 20, 24 and 28 per square metre, where the highest density is that commonly used on chicken farms. Mean relative asymmetry for six morphological characters increased from 3.4% at the lowest density over 4.0% at the intermediate density to 4.5% at highest density. This increase by 32% across treatments is very large compared to what has been recorded in other studies of asymmetry (Møller and Swaddle, 1997), even though the experiment was conducted in a benign environment (a poultry research facility with maximum animal care). For commercial farms Sanotra (1999) found for 23 chicken farms a positive correlation of 0.287 between density and mean asymmetry of chickens. For comparison mean relative asymmetry on these farms ranged from 1.7 to 5.7%. There was no response in terms of size of the same characters, supporting the developmental model by Møller and Pomiankowski (1993) rather than that proposed by Klingenberg and Nijhout (1999). Most commercially raised poultry are reared under constant light regimes although this deviates from the ancestral conditions. In an experiment conducted on commercial chicken farms, Møller et al. (1999) analysed the effect of light conditions on mean relative asymmetry in three morphological characters. Chickens were either
Fig. 2. The relationship between mean body mass (g; an indirect measure of growth rate since individuals were not fully grown) and mean fluctuating asymmetry in three leg characters (length, diameter and thickness of tarsometatarsus, each adjusted to a mean of zero and a variance of one) of chickens. Values are farm means. The relationship is significantly negative (Kendall rank order correlation: tau ¼ 0:282; N ¼ 27 farms, P ¼ 0:039). Data from Sanotra (1999).
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reared under a 16:8 light–dark cycle, a changing light regime (starting at constant light being gradually reduced to 16 h during days 4–7, and from days 25 to 29 light again being increased gradually to constant light), or constant light. Mean relative fluctuating asymmetry increased from 3.6% in a 16:8 light–dark cycle, 3.3% in the changing light regime and 4.7% in constant light. Chickens reared under the extreme condition were 36% more asymmetric than chickens in the two benign environments. Again, there was no response to treatment in terms of size of the same characters, supporting the developmental model by Møller and Pomiankowski (1993) rather than that proposed by Klingenberg and Nijhout (1999). Developmental instability is not only reflected in morphology since any aspect of the phenotype will show signs of irregular development. Since the neural, endocrine and circulatory systems are involved in both the regular development of morphology (Møller and Pagel, 1998), and in the production of behaviour, we can consider variation in behaviour to be a by-product of irregularities in the underlying anatomy. Studies of behaviour suggest that abnormal behaviour or irregular sequences of behaviour are associated with fluctuating asymmetry and phenodeviants (Furlow et al., 1997; Møller, 1998; Møller and Swaddle, 1997; Thornhill and Møller, 1997). Gait quality in chickens could be directly affected by asymmetry in leg morphology, and Møller et al. (1999) found a significant reduction in gait quality with increasing asymmetry. Tonic immobility is a standard measure of fearfulness in chickens, with increasing tonic immobility reflecting greater levels of fearfulness (Jones and Faure, 1981). Tonic immobility was positively correlated with relative asymmetry in three breeds of chickens studied by Møller et al. (1995) and in the study by Møller et al. (1999). However, Campo et al. (2000) only reported a significant positive relationship between tonic immobility and asymmetry in one of five breeds of chickens. Asymmetries may have a direct link to profitability of chicken farming if asymmetric individuals weigh less, and are more likely to die or become discarded from consumption for product quality reasons. We have already treated the relationship between body mass and asymmetry above. Sanotra (1999) found a weak negative relationship between mortality during the first week and mean asymmetry in three different characters across 24 chicken farms ðr ¼ 0:230Þ and an even weaker relationship for total mortality ðr ¼ 0:134Þ. The relationship between the percentage of chickens discarded at slaughter for quality reasons and mean asymmetry was stronger and accounted for 13.2% of the variance ðr ¼ 0:348Þ. Similarly, a preliminary study of milk production in dairy cows showed that more symmetric individuals had considerably higher milk yield than the average cow (J.T. Manning et al. unpublished data).
Thus, there should even be economic incentives to decrease asymmetry among chickens on farms.
5. Measures of developmental instability and other measures of animal welfare How do fluctuating asymmetry and other measures of developmental instability perform as an indicator of welfare compared to other measures? Traditionally, animal welfare has been assessed using physiological measures such as corticosterone levels or heat shock protein levels, or behavioural measures such as tonic immobility in poultry. Several studies have shown positive covariance between these different measures. For example, Satterlee et al. (2000) showed that lines of quail selected for increased stress resistance had both low corticosterone levels and reduced levels of fluctuating asymmetry, as predicted. In a similar vein, Roberts and Feder (1999) showed for Drosophila that individuals from lines with increased genes for heat shock protein induction also developed a larger degree of fluctuating asymmetry. Møller et al. (1995, 1999) showed for chickens that fluctuating asymmetry indeed was correlated with tonic immobility as predicted. Finally, M. Bakken (personal communication) injected chicken eggs with corticosterone and found significantly increased asymmetry in chickens that hatched from experimental as compared to control eggs. Body size is generally of great importance in animal production, because larger animals may yield greater returns on capital investment. Hence, it might be of general interest to investigate how efficiently size and fluctuating asymmetry of the same characters reflect stress. In a review of 20 studies of abiotic stress where both size and asymmetry were assessed, mean effect size (adjusted for sample size) was of a similar magnitude for the two types of characters (N. Cadee et al., unpublished data). Since fluctuating asymmetry only vaguely reflects developmental instability, we can infer that developmental instability is much more sensitive to stress than is size of the same characters. Surprisingly, there was no significant correlation between the effect sizes of the response of two kinds of characters to stress, which implies that size and asymmetry of the same characters index different phenomena (N. Cadee et al., unpublished data). Clearly, more studies that attempt to cross-validate different indicators of stress are needed. We note that this is not only a problem for studies of developmental instability, since cross-validation studies are equally rare for corticosterone, heat shock proteins and behaviour such as tonic immobility. Although other measures of animal welfare may perform as well as measures of developmental instability by indexing rearing conditions, they suffer from at least one problem. We do not know a priori what is the optimal level
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of the measure. Hence, this optimum has to be determined before conclusions can be drawn. What are the benefits and costs of using measures of developmental instability rather than alternative methods to assess stress? We have shown above that fluctuating asymmetry is just as efficient in indexing environmental stress as size of the same morphological characters. The relative efficiency of other methods remain to be quantified when more studies become available. The use of fluctuating asymmetry has the advantage that it can be assessed relatively easily in a non-invasive way by technicians, while assessment of stress by means of corticosterone or heat shock proteins involve relatively complicated laboratory analyses. All these other methods suffer from the additional disadvantage that there is no a priori optimum given, contrary to what is the case for fluctuating asymmetry. Assessment of specific kinds of stress experienced by animals may only be possible with the use of specific methods. Fluctuating asymmetry is generally considered a non-specific response (just as heat shock proteins or corticosterone). However, recent findings based on extensive analyses of very large data sets from numerous organisms suggest that morphological traits involved in locomotion, and that secondary sexual characters important in sexual selection, differ in susceptibility to stress from other characters (Polak et al., 2002). Therefore, fluctuating asymmetry may both provide specific and general information about stress susceptibility. Clearly, no single method is suitable to assess stress in all situations and conditions. However, we suggest that fluctuating asymmetry and other measures of developmental instability have at least as great potential as several traditional methods, and that fluctuating asymmetry is currently not used in proportion to its potential in studies of welfare.
6. Directions for future study The study of developmental instability in the context of veterinary science, animal breeding and animal welfare has barely begun. Thus, there are many opportunities for further study. Many of these are directly related to novel farming practices, and we list a few such examples here. What is the relationship between growth and developmental instability when the use of growth hormones is applied? What is the relationship when genetically engineered strains are considered? This latter question is particularly relevant because insertion of novel alleles into a genome may disrupt co-adapted gene complexes with severe consequences for developmental instability as shown in cases of evolution of pesticide resistance (e.g., Davies et al., 1996; review in Møller and Swaddle, 1997). How can developmental instability be
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reduced when selection for increased growth is the target? One possibility is to select for increased stress resistance as already done for quail and other domestic animals. Indeed, Satterlee et al. (2000) have shown that lines of quail selected for increased stress resistance, as reflected by their low corticosterone levels, have reduced levels of fluctuating asymmetry, as predicted. What are the behavioural consequences of selection for rapid growth? We have briefly described some examples above. Studies of cannibalism in chickens have demonstrated significant differences in asymmetry between cannibalistic and cannibalised chickens (Yngvesson and Keeling, 2000), suggesting a direct link with developmental instability. Particularly relevant to veterinarians is the link between developmental instability and susceptibility to parasites and disease. Does selection for rapid growth cause an increase in the frequency of disease conditions, and how is this relationship associated with fluctuating asymmetry? Thornhill and Møller (1997) provide an extensive review of the literature on developmental instability and parasitism and disease for humans, while Møller (1996a,b) reviews the literature suggesting general relationships between parasitism and increased levels of asymmetry. Numerous other studies have appeared in the medical and evolutionary literature since these reviews appeared.
7. Conclusions Measures of developmental instability can be considered to reflect the suitability of conditions for rearing as assessed from the point of view of the individual animal. This assertion follows from the evidence briefly presented in this review. Hence, estimates of fluctuating asymmetry and the frequency of phenodeviants can be considered to represent a measure of the level of animal welfare, as we have illustrated with some examples above. Estimates of fluctuating asymmetry of individual traits are poor indicators of underlying developmental stability, but composite measures based on a relatively small number of traits can radically improve the predictive power of such tests. Relationships between productivity, stress and developmental instability suggest that the interests of domestic animals, farmers and scientists to some extent are congruent, and that improvements in developmental instability through artificial selection and superior rearing conditions will favour all parties involved. References Aparicio, J.M., 1998. Patterns of fluctuating asymmetry in developing primary feathers: a test of the compensational growth hypothesis. Proceedings of the Royal Society of London B 265, 2353–2357.
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Møller, A.P., 1996a. Parasitism and developmental stability of hosts: a review. Oikos 77, 189–196. Møller, A.P., 1996b. Development of fluctuating asymmetry in tail feathers of the barn swallow Hirundo rustica. Journal of Evolutionary Biology 9, 677–694. Møller, A.P., 1997. Developmental selection against developmentally unstable offspring and sexual selection. Journal of Theoretical Biology 185, 415–422. Møller, A.P., 1998. Developmental instability as a general measure of stress. Advances in the Study of Behavior 27, 181–213. Møller, A.P., 1999. Asymmetry as a predictor of growth, fecundity and survival. Ecology Letters 2, 149–156. Møller, A.P., Cuervo, J.J., 2002. Asymmetry, size and sexual selection: meta-analysis, publication bias and factors affecting variation in relationships. In: Polak, M. (Ed.), Developmental Instability. Oxford University Press, New York. Møller, A.P., Jennions, M.D., 2001. Testing and adjusting for publication bias. Trends in Ecology and Evolution 16, 580–586. Møller, A.P., Pagel, M., 1998. Developmental stability and signalling among cells. Journal of Theoretical Biology 193, 497–506. Møller, A.P., Pomiankowski, A., 1993. Punctuated equilibria or gradual evolution: the importance of fluctuating asymmetry. Journal of Theoretical Biology 161, 359–367. Møller, A.P., Sanotra, G.S., Vestergaard, K.S., 1995. Developmental stability in relation to population density and breed of chickens Gallus gallus. Poultry Science 74, 1761–1771. Møller, A.P., Sanotra, G.S., Vestergaard, K.S., 1999. Developmental stability and light regime in chickens Gallus gallus. Applied Animal Behaviour Science 62, 57–71. Møller, A.P., Swaddle, J.P., 1997. Asymmetry, Developmental Stability and Evolution. Oxford University Press, Oxford. Nestor, K.E., Anderson, J.W., Patterson, R.A., 2000. Effects of selection for increased body weight, egg production, and shank width on developmental stability in turkeys. Poultry Science 79, 937–945. Palmer, A.R., 1994. Fluctuating asymmetry analyses: a primer. In: Markow, T.A. (Ed.), Developmental Instability: Its Origins and Evolutionary Implications. Kluwer Academic Publishers, Dordrecht, pp. 355–363. Parsons, P.A., 1990. Fluctuating asymmetry: an epigenetic measure of stress. Biological Reviews 65, 131–145. Polak, M., Møller, A.P., Kroeger, D.E., Gangestad, S.W., Manning, J.T., Thornhill, R., 2002. Statistical covariance in fluctuating asymmetry among traits? A meta-analysis. In: Polak, M. (Ed.), Developmental Instability. Oxford University Press, New York. Roberts, S.P., Feder, M.E., 1999. Natural hypothermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster. Oecologia 121, 323–329. Rosenthal, R., 1991. Meta-analytic Procedures for Social Research. Sage, New York. Sachs, T., 1988. Epigenetic selection: an alternative mechanism of pattern formation. Journal of Theoretical Biology 134, 547– 559. Sanotra, G.S., 1999. Recording actual strength of legs in farmed chickens (In Danish). Frederiksberg C, Denmark, Dyrenes Beskyttelse (www.dyrenes-beskyttelse.dk). Satterlee, D.G., Cadd, G.G., Jones, R.B., 2000. Developmental instability in Japanese quail genetically selected for contrasting adrenocortical responsiveness. Poultry Science 79, 1710–1714. Thornhill, R., Møller, A.P., 1997. Developmental stability, disease and medicine. Biological Reviews 72, 497–548. Van Dongen, S., 1998. How repeatable is the estimation of developmental stability by fluctuating asymmetry? Proceedings of the Royal Society of London B 265, 1423–1427. Van Dongen, S., Molenberghs, G., Matthysen, E., 1999. The statistical analysis of fluctuating asymmetry: REML estimation
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Yang, A., Dunnington, E.A., Siegel, P.B., 1998. Developmental stability in different genetic stocks of White Rock chickens. Journal of Heredity 89, 260–264. Yngvesson, J., Keeling, L.J., 2000. Body size and fluctuating asymmetry in relation to cannibalistic behaviour in laying hens. Animal Behaviour 61, 609–615. Zakharov, V.M., 1989. Future prospects for population phenogenetics. Soviet Scientific Reviews. F. Physiology and General Biology 4, 1–79.
Review
Growth and developmental instability Anders Pape Møller *, John Manning Laboratoire de Parasitologie Evolutive, CNRS UMR 7103, Universit e Pierre et Marie Curie, B^ at. A, 7 eme etage, 7 quai St. Bernard, Case 237, F-75252 Paris Cedex 05, France Population Biology Research Group, School of Biological Sciences, Nicholson Building, University of Liverpool, Liverpool L69 3BX, UK Accepted 23 September 2002
Abstract Developmental stability reflects the ability of an individual to develop a regular phenotype under given environmental and genetic conditions. Measures of developmental instability include the degree of fluctuating asymmetry and the frequency of phenodeviants. Endocrine, neural and circulatory mechanisms that control similar development of morphological characters on the two sides of the body are also involved in controlling overall development. Cross-validation studies have shown that measures of developmental instability are positively correlated with other measures of welfare such as tonic immobility in poultry. Asymmetric animals grow less rapidly than symmetric individuals. Eleven studies have investigated the relationship between growth rate and developmental instability, and the observed effect size (Pearson correlation coefficient adjusted for sample size) is )0.15. Studies of chickens have shown that asymmetry increases as a response to selection for increased growth rate. As conditions for rearing deteriorate by higher density, fluctuating asymmetry increases and growth rate decreases, both within and among farms. Fluctuating asymmetry can be considered a measure of animal welfare since larger values reflect worse environmental conditions as experienced by the individual animal itself. Since growth rate and fluctuating asymmetry are negatively correlated, we can infer that improvement of rearing conditions leading to reduced asymmetry will both benefit the producer (in terms of increased growth), but also the animals in terms of better conditions for rearing. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Chickens; Environmental conditions; Fluctuating asymmetry; Phenodeviants; Welfare
1. Introduction Modern industrialised agriculture has dramatically increased productivity over the last century. For animal production this has been achieved through animal breeding for increased growth rates, improved feed quality, veterinary intervention and the use of substances such as growth hormones. This change has come at the expense of animal welfare, which only during the last couple of decades has entered to some extent as an important additional factor modifying agricultural practices. The consequences of increased productivity for the animals have thus only recently become a central issue of concern.
*
Corresponding author. Tel.: +33-1-44-27-25-94; fax: +33-1-44-2735-16. E-mail address: [email protected] (A. Pape Møller).
Assessment of the quality of living conditions as seen from the perspective of the animal is thus a crucial point. One such measure is developmental instability (Møller et al., 1999; Møller and Swaddle, 1997), which reflects the inability of an individual to produce a regular phenotype under given environmental conditions (Møller and Swaddle, 1997; Parsons, 1990; Zakharov, 1989). All organisms have regular external phenotypes, and these have been modified by natural selection for mechanical efficiency. Perturbing factors during development may disrupt developmental processes and cause deviations from a regular phenotype, while regulatory processes such as feedback may restore the growth trajectory towards the regular phenotype. Developmental stability is a concept of internal developmental mechanisms that control development, and this cannot readily be estimated. However, deviations from regular development such as small, randomly directed asymmetries (so-called fluctuating asymmetry) and minor physical
1090-0233/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1090-0233(02)00262-9
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anomalies such as a missing character on one side of the body (phenodeviants) can readily be assessed. Fluctuating asymmetry is characterised by randomly directed deviations from perfect symmetry, with most individuals only having little or no asymmetry. Frequency distributions of signed left-minus-right character values are near normal (Fig. 1). It seems that leptokurtic distributions predominate in empirical examples (Fig. 1; i.e., there are many asymmetries near the mean of zero and in the tails of the distribution, and fewer than expected observations at intermediate levels of asymmetry), probably because composite distributions reflecting individual differences in developmental control are not normal, but leptokurtic (Gangestad and Thornhill, 1999). Hence, the degree of leptokurtosis provides a direct estimate of the amount of individual differences in developmental control in a population, with higher leptokurtosis reflecting a larger degree of individual differences. Since fluctuating asymmetries are small (on average less than 1% in animals (Møller and Swaddle, 1997)) and normally distributed, measurement error must be assessed to ascertain that signal differs from noise. Such assessment of measurement error can be done using different statistical techniques (Palmer, 1994; Van Dongen et al., 1999). Fluctuating asymmetry and phenodeviants are increased under deviant environmental conditions such as heat or cold, poor food quality or quantity, deviant light
Fig. 1. (A) A normal frequency distribution of signed left-minus-right character values and (B) a leptokurtic frequency distribution for comparison.
conditions, elevated electromagnetic and radioactive radiation, parasitism, and even perceived risk of predation (review in Møller and Swaddle, 1997). A number of genetic factors are also associated with increased developmental instability, including inbreeding, mutations, sometimes heterozygosity and quantitative genetic variation (review in Møller and Swaddle, 1997). Thus, the regularity of the phenotype will integrate the environmental conditions experienced by an individual in relation to its genetic constitution. Asymmetry in any single trait only provides a very imprecise estimate of developmental instability because a large number of factors including completely random ones may affect the development of such a trait (Gangestad and Thornhill, 1999). Asymmetries of different traits have a small, but highly consistent positive correlation of around 0.075, which reflects the underlying common control of the development of the entire phenotype (Gangestad and Thornhill, 1999; Polak et al., 2002). The repeatability of fluctuating asymmetry (Van Dongen, 1998; Whitlock, 1998) is the correlation between the asymmetry of a trait across hypothetical individuals allowed to develop on two different occasions. It reflects the proportion of variance in fluctuating asymmetry of a single trait caused by individual differences rather than the stochastic nature of developmental errors of single traits. Obviously individuals develop only once and hence this correlation must be estimated rather than measured directly. As the percentage of variance in fluctuating asymmetry due to individual differences increases, the leptokurtosis of the distribution of signed fluctuating asymmetry becomes larger (Gangestad and Thornhill, 1999) and, correspondingly, the coefficient of variation of unsigned fluctuating asymmetry increases (Whitlock, 1998). Based on leptokurtosis of a number of species, ranging from plants over insects to birds, mammals and humans for which large sample sizes were available, Gangestad and Thornhill (1999) estimated that this amount of variation accounted for only 7% of the variance in fluctuating asymmetry of a single trait, although underlying individual differences in developmental instability are substantial (with the coefficient of variation of these individual differences being ca. 22–24 across a wide range of species). Therefore, the repeatability of fluctuating asymmetry of a single trait is only about 0.07, and this may be further reduced by measurement error. The repeatability of fluctuating asymmetry of a single trait puts an upper limit on the correlation between fluctuating asymmetry of two traits. This correlation between fluctuating asymmetry of two traits should only be about 0.07, even if their developmental instability is fully shared. Therefore, composite indices of asymmetry even for as few as 3–5 traits may greatly improve the estimate of asymmetry reflecting developmental instability because the precision of this estimate
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increases rapidly with the number of traits measured (Leung et al., 2000). Why is developmental instability important? The importance derives primarily from the ability of scientists to assess the quality of living conditions as perceived by the individual organism. There are few such measures available, and in studies of animal welfare various groups have advocated behavioural, endocrinological or other indicators of adverse conditions. Fluctuating asymmetry and other measures of developmental instability differ from the other indicators in two important ways. Firstly, the same measure can be used in all kinds of organisms including viruses, plants and animals, whereas the other measures are restricted to one taxon. Secondly, measures of developmental instability already have a pre-defined, in-built optimum, namely symmetry, while the optimum for the other measures must be determined from first principles, preferably using optimality models. Therefore, a population of animals that has a high level of asymmetry can be considered to have experienced sub-optimal conditions during development. This assumption could be tested experimentally since individuals reared under conditions associated with high levels of asymmetry are predicted to work harder to escape their current situation in a condition experiment, as compared to control individuals. Measures of developmental instability are also important because they tie into important components of fitness that are relevant for the maintenance of viable, free-living populations and for productivity of farmed animals. Møller (1999) reviewed the literature on the relationship between developmental instability and growth, survival and fecundity, respectively, and found mean Pearson correlations adjusted for sample size of )0.15, )0.25 and )0.35. Similarly, Møller and Cuervo (2002) in a meta-analysis of the relationship between developmental instability and sexual selection (mating success or sexual attractiveness) reported correlations of )0.17. Since most of these studies we based on asymmetry of a single character, relationships between developmental instability and fitness is likely to be much stronger. This has important implications for animal welfare because animals reared in conditions that give rise to high levels of asymmetry will also tend to grow less well, to survive less well, to have reduced fecundity and to have problems with mating. How is fluctuating asymmetry estimated? There is a large literature on indices of asymmetry and how to estimate them (reviews in Palmer, 1994; Møller and Swaddle, 1997). Traits typically measured in chickens and other poultry include length and width of tibia, tarso-metatarsus, wing length and length of the longest primary feather on the two sides of the body. Traits measured in cattle include length and width of ears, width of the calcaneous and metatarsal bones, width of
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the hock joint, and the medial corner of the eye to the nostril. The number of repeat measurements is typically two, where first the right side is measured, then the left side, then the right side again, and finally the left side. Signed asymmetry is simply right-minus-left character size, while unsigned asymmetry is this difference disregarding the sign of the difference. A number of composite indices of asymmetry for more than a single character also exist (Leung et al., 2000). A useful composite index is based on the sum of the standardised signed asymmetry, which is simply standardised to a standard normal deviate with a mean of zero and a variance of one. The aim of the present review was to assess the relationship between developmental instability and growth. We do that by firstly describing the microscopic mechanisms of ontogeny that link developmental instability to macroscopic fluctuating asymmetry. Secondly, we assess the general relationship between developmental instability and growth. Thirdly, we describe in detail a number of studies investigating the relationship between growth and developmental instability in chickens. Finally, we list a number of future directions for further study.
2. Mechanisms linking microscopic patterns of growth to developmental instability Bilaterally symmetric multi-cellular species derive from a single, fertilised cell that subsequently divides a very large number of times. The extraordinary feat that, for example, the right and left hands of a human are almost perfect mirror images, suggests that there are developmental mechanisms that keep the two copies of an organ or a body part within very restricted boundaries. These developmental mechanisms at the microscopic level that give rise to the quasi-symmetric, macroscopic phenotype have been the basis for a number of models of development (Aparicio, 1998; Graham et al., 1993; Hallgrımsson, 1993, 1999; Klingenberg, 2002; Klingenberg and Nijhout, 1999; Møller and Pagel, 1998). Several models assume that there are homeostatic mechanisms at work providing such a feedback between sides. In fact, communication between body parts must act either through the neural, endocrine or circulatory systems. Any irregularity in the constituent cells of these systems will affect the ease of communication, and even the directionality of communication (Møller and Pagel, 1998). Thus, it is not surprising that some mechanism of quality control at the cell level has to be invoked. Numerous cells emerge that are never incorporated into an organ because they are discarded through apoptosis at an early stage. Available information suggests that apoptosis depends on the quality of a cell as determined by its signalling properties (review in Møller and Pagel,
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1998). Cells that signal at very low levels, thus showing signs of poor quality, or cells signalling at a very high level, thus showing signs reminiscent of cancer cells, are generally selected against (see, for example, Edelman, 1987; Godin et al., 1991; Klekowski, 1988; Michaelson, 1989, 1991; Sachs, 1988). Cells that through their intermediate level of signalling reveal their quality are incorporated into tissues and organs. Such a selective mechanism results in the production of functional tissues and organs with proper communication between the sides of the body and hence in a quasi-symmetric phenotype (Møller and Pagel, 1998). If the proposed developmental mechanisms giving rise to a symmetric phenotype are at work, then we should expect phenotypes at both the microscopic and the macroscopic levels to show similarities in level of morphological regularity. Indeed, asymmetric morphological characters consist of asymmetric and irregular growth increments (Møller, 1996b). Similarly, tibial dyschondroplasia, which is a pathological leg disorder in chickens (Edwards and Veltman, 1983), is more severe in chickens with high levels of fluctuating asymmetry (Møller et al., 1999). Interestingly, the arrangement of cells in the tarsometatarsus becomes increasingly more irregular as the severity of tibial dyschondroplasia increases (A.P. Møller unpublished data). Thus, microscopic and macroscopic patterns of morphological regularity are clearly positively associated.
3. Patterns of growth and developmental instability There is accumulating evidence that asymmetric animals grow less rapidly than symmetric individuals. The magnitude of a relationship between two variables across a sample of studies can be assessed from metaanalysis (e.g., Rosenthal, 1991). Møller (1999) reported a mean effect size adjusted for sample size of the relationship between asymmetry and growth rate of PearsonÕs r ¼ 0:146 (95% C.I. )0.096, )0.196), P < 0:001, for 11 studies. There was statistically significant heterogeneity in effect size among studies included in this meta-analysis ðv2 ¼ 27:46; P < 0:01Þ. The robustness of this finding was assessed from the fail-safe number, which provides an estimate of the number of unknown cases needed to eliminate the overall significance of the weighted mean effect size at the 5% significance level when the mean effect size of the unknown cases is zero. Alternatively, the fail-safe number can be considered to represent the number of unpublished studies in file drawers that is needed to nullify the result. This fail-safe number was 168, which exceeds the minimum number of 65 studies required for a firm conclusion (according to the generally accepted criterion by Rosenthal (1991, p. 106). Using mean values for species as the unit of analysis (if studies of the same species are not considered
statistically independent), the effect size was )0.156 (95% C.I. )0.106, )0.206), P < 0:001 for 10 species. Again, there was significant heterogeneity among studies ðv2 ¼ 25:03; P < 0:01Þ. The fail-safe number equalled 150, which exceeded the minimum requirement of 55 studies. Thus, at the study level the relationship between growth and asymmetry accounted for 2.1% of the variance, and at the species level for 2.4% of the variance. General conclusions based on meta-analysis may be seriously biased if the available studies represent a biased sample of all studies conducted (Møller and Jennions, 2001). There are a number of direct and indirect tests available for studies of publication bias. A study assessing bias in meta-analyses in biology revealed little evidence of bias for the meta-analysis of developmental instability and growth rate (Jennions and Møller, 2002). What is the relationship between developmental stability and growth rate? If we assume that underlying individual differences in developmental stability are of the order of magnitude of 0.07 (Gangestad and Thornhill, 1999, 2002) this value is remarkably similar for organisms ranging from plants over insects to birds and mammals, including domesticated animals and humans), and if the correlation between growth and developmental instability is )0.15 (and hence the amount of variance explained is 0.0225 (0.15 squared), then the relationship between developmental stability and growth is 0.0225/0.07 ¼ 0.321. Thus, underlying developmental stability across phenotypic characters accounts for almost a third of all variance in growth among individuals, which must by all measures be considered a significant amount.
4. Selection, population density and growth in chickens Domestic animals have been selected for rapid growth. Directional selection may disrupt developmental stability because of selection against genetic modifiers that control developmental processes (Møller and Pomiankowski, 1993; Møller and Swaddle, 1997). More recently, Klingenberg and Nijhout (1999) used a simple developmental model to show that heritable variation in fluctuating asymmetry can result without a separation of trait genes and modifiers. Extensive studies of the Australian sheep blow fly Lucilia cuprina model system is a good example of this phenomenon. In diazinone-resistant strains of this fly the modifier gene is a homologue of the Notch gene which encodes components of signalling pathways associated with the number and the asymmetry of sternopleural bristles (Davies et al., 1996; Long et al., 1998). Thus, resistant flies have more asymmetry, but also a different number of bristles, as compared to controls. Although this observation of a trait subject to strong stabilising selection is consistent with the hypothesis proposed by Klingenberg and
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Nijhout (1999), there is currently a dearth of empirical information on this subject. The general observation that there is no significant correlation between effect size (strength of the relationship, measured in terms of Pearson product-moment correlation) of response of size and asymmetry to stress across 20 different studies, implies that size and asymmetry of the same characters index different phenomena (N. Cadee et al., unpublished data). This is particularly surprising given that larger species generally have disproportionately high degrees of asymmetry (Hallgrımsson, 1998). Clearly, more studies are needed to address why that is the case. The developmental models discussed in the previous paragraph imply that improvements in growth due to selection may initially come at a cost in terms of irregular development. Møller et al. (1995) contrasted levels of fluctuating asymmetry in three strains of chickens, a slow growth strain and two fast growing, selected strains, and compared these to asymmetries in wild jungle fowl, which are the ancestors of domestic chickens. While asymmetry only accounted for on average 0.5% of the length of six morphological characters in jungle fowl, it accounted for 3.0% in the slow growing strain and 3.9% and 4.1% in the two fast growing strains. These values can be compared to mean relative asymmetries of less than 1% in animals (Møller and Swaddle, 1997). Yang et al. (1998) reported that asymmetry in a selection line for low body mass was 4.9%, while asymmetry decreased to 3.1% after four generations of relaxed selection in a sub-line. Similarly, Nestor et al. (2000) reported for lines of turkeys that had been selected for increased body mass, shank width and egg laying that they had significantly higher levels of relative asymmetry than did control lines. Furthermore, these differences among lines were better explained by differences in body mass than by differences in homozygosity among lines. Trends in the same direction were reported by Yalcßin et al. (2001) for three lines of chickens differing in growth rates. These findings are consistent with the hypotheses proposed by Møller and Pomiankowski (1993) and Klingenberg and Nijhout (1999). However, we must consider the findings carefully for any bias. Yang et al. (1998) reported an increase in mortality in their selected lines, and since viability selection generally acts against asymmetric individuals (Møller, 1999), the actual differences between lines are likely to be greater than reported. Furthermore, developmental selection at early life stages also usually acts against asymmetric individuals (Møller, 1997), potentially causing further bias. Thus, the reported findings are likely to be conservative. The relationship between growth and asymmetry of individual chickens was )0.071 in one study (Møller et al., 1995) and )0.027 in another study (Sanotra, 1999). These values are bound to underestimate the relationship between developmental stability and
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growth, and the reported relationship between mean asymmetry among chickens and mean growth across 24 Danish chicken farms of )0.358 (Sanotra, 1999; Fig. 2) is likely to provide a better estimate. A key point of concern for animal welfare has been the density at which animals are reared. Møller et al. (1995) conducted an experiment testing the relationship between fluctuating asymmetry and density. Chickens were reared at 20, 24 and 28 per square metre, where the highest density is that commonly used on chicken farms. Mean relative asymmetry for six morphological characters increased from 3.4% at the lowest density over 4.0% at the intermediate density to 4.5% at highest density. This increase by 32% across treatments is very large compared to what has been recorded in other studies of asymmetry (Møller and Swaddle, 1997), even though the experiment was conducted in a benign environment (a poultry research facility with maximum animal care). For commercial farms Sanotra (1999) found for 23 chicken farms a positive correlation of 0.287 between density and mean asymmetry of chickens. For comparison mean relative asymmetry on these farms ranged from 1.7 to 5.7%. There was no response in terms of size of the same characters, supporting the developmental model by Møller and Pomiankowski (1993) rather than that proposed by Klingenberg and Nijhout (1999). Most commercially raised poultry are reared under constant light regimes although this deviates from the ancestral conditions. In an experiment conducted on commercial chicken farms, Møller et al. (1999) analysed the effect of light conditions on mean relative asymmetry in three morphological characters. Chickens were either
Fig. 2. The relationship between mean body mass (g; an indirect measure of growth rate since individuals were not fully grown) and mean fluctuating asymmetry in three leg characters (length, diameter and thickness of tarsometatarsus, each adjusted to a mean of zero and a variance of one) of chickens. Values are farm means. The relationship is significantly negative (Kendall rank order correlation: tau ¼ 0:282; N ¼ 27 farms, P ¼ 0:039). Data from Sanotra (1999).
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reared under a 16:8 light–dark cycle, a changing light regime (starting at constant light being gradually reduced to 16 h during days 4–7, and from days 25 to 29 light again being increased gradually to constant light), or constant light. Mean relative fluctuating asymmetry increased from 3.6% in a 16:8 light–dark cycle, 3.3% in the changing light regime and 4.7% in constant light. Chickens reared under the extreme condition were 36% more asymmetric than chickens in the two benign environments. Again, there was no response to treatment in terms of size of the same characters, supporting the developmental model by Møller and Pomiankowski (1993) rather than that proposed by Klingenberg and Nijhout (1999). Developmental instability is not only reflected in morphology since any aspect of the phenotype will show signs of irregular development. Since the neural, endocrine and circulatory systems are involved in both the regular development of morphology (Møller and Pagel, 1998), and in the production of behaviour, we can consider variation in behaviour to be a by-product of irregularities in the underlying anatomy. Studies of behaviour suggest that abnormal behaviour or irregular sequences of behaviour are associated with fluctuating asymmetry and phenodeviants (Furlow et al., 1997; Møller, 1998; Møller and Swaddle, 1997; Thornhill and Møller, 1997). Gait quality in chickens could be directly affected by asymmetry in leg morphology, and Møller et al. (1999) found a significant reduction in gait quality with increasing asymmetry. Tonic immobility is a standard measure of fearfulness in chickens, with increasing tonic immobility reflecting greater levels of fearfulness (Jones and Faure, 1981). Tonic immobility was positively correlated with relative asymmetry in three breeds of chickens studied by Møller et al. (1995) and in the study by Møller et al. (1999). However, Campo et al. (2000) only reported a significant positive relationship between tonic immobility and asymmetry in one of five breeds of chickens. Asymmetries may have a direct link to profitability of chicken farming if asymmetric individuals weigh less, and are more likely to die or become discarded from consumption for product quality reasons. We have already treated the relationship between body mass and asymmetry above. Sanotra (1999) found a weak negative relationship between mortality during the first week and mean asymmetry in three different characters across 24 chicken farms ðr ¼ 0:230Þ and an even weaker relationship for total mortality ðr ¼ 0:134Þ. The relationship between the percentage of chickens discarded at slaughter for quality reasons and mean asymmetry was stronger and accounted for 13.2% of the variance ðr ¼ 0:348Þ. Similarly, a preliminary study of milk production in dairy cows showed that more symmetric individuals had considerably higher milk yield than the average cow (J.T. Manning et al. unpublished data).
Thus, there should even be economic incentives to decrease asymmetry among chickens on farms.
5. Measures of developmental instability and other measures of animal welfare How do fluctuating asymmetry and other measures of developmental instability perform as an indicator of welfare compared to other measures? Traditionally, animal welfare has been assessed using physiological measures such as corticosterone levels or heat shock protein levels, or behavioural measures such as tonic immobility in poultry. Several studies have shown positive covariance between these different measures. For example, Satterlee et al. (2000) showed that lines of quail selected for increased stress resistance had both low corticosterone levels and reduced levels of fluctuating asymmetry, as predicted. In a similar vein, Roberts and Feder (1999) showed for Drosophila that individuals from lines with increased genes for heat shock protein induction also developed a larger degree of fluctuating asymmetry. Møller et al. (1995, 1999) showed for chickens that fluctuating asymmetry indeed was correlated with tonic immobility as predicted. Finally, M. Bakken (personal communication) injected chicken eggs with corticosterone and found significantly increased asymmetry in chickens that hatched from experimental as compared to control eggs. Body size is generally of great importance in animal production, because larger animals may yield greater returns on capital investment. Hence, it might be of general interest to investigate how efficiently size and fluctuating asymmetry of the same characters reflect stress. In a review of 20 studies of abiotic stress where both size and asymmetry were assessed, mean effect size (adjusted for sample size) was of a similar magnitude for the two types of characters (N. Cadee et al., unpublished data). Since fluctuating asymmetry only vaguely reflects developmental instability, we can infer that developmental instability is much more sensitive to stress than is size of the same characters. Surprisingly, there was no significant correlation between the effect sizes of the response of two kinds of characters to stress, which implies that size and asymmetry of the same characters index different phenomena (N. Cadee et al., unpublished data). Clearly, more studies that attempt to cross-validate different indicators of stress are needed. We note that this is not only a problem for studies of developmental instability, since cross-validation studies are equally rare for corticosterone, heat shock proteins and behaviour such as tonic immobility. Although other measures of animal welfare may perform as well as measures of developmental instability by indexing rearing conditions, they suffer from at least one problem. We do not know a priori what is the optimal level
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of the measure. Hence, this optimum has to be determined before conclusions can be drawn. What are the benefits and costs of using measures of developmental instability rather than alternative methods to assess stress? We have shown above that fluctuating asymmetry is just as efficient in indexing environmental stress as size of the same morphological characters. The relative efficiency of other methods remain to be quantified when more studies become available. The use of fluctuating asymmetry has the advantage that it can be assessed relatively easily in a non-invasive way by technicians, while assessment of stress by means of corticosterone or heat shock proteins involve relatively complicated laboratory analyses. All these other methods suffer from the additional disadvantage that there is no a priori optimum given, contrary to what is the case for fluctuating asymmetry. Assessment of specific kinds of stress experienced by animals may only be possible with the use of specific methods. Fluctuating asymmetry is generally considered a non-specific response (just as heat shock proteins or corticosterone). However, recent findings based on extensive analyses of very large data sets from numerous organisms suggest that morphological traits involved in locomotion, and that secondary sexual characters important in sexual selection, differ in susceptibility to stress from other characters (Polak et al., 2002). Therefore, fluctuating asymmetry may both provide specific and general information about stress susceptibility. Clearly, no single method is suitable to assess stress in all situations and conditions. However, we suggest that fluctuating asymmetry and other measures of developmental instability have at least as great potential as several traditional methods, and that fluctuating asymmetry is currently not used in proportion to its potential in studies of welfare.
6. Directions for future study The study of developmental instability in the context of veterinary science, animal breeding and animal welfare has barely begun. Thus, there are many opportunities for further study. Many of these are directly related to novel farming practices, and we list a few such examples here. What is the relationship between growth and developmental instability when the use of growth hormones is applied? What is the relationship when genetically engineered strains are considered? This latter question is particularly relevant because insertion of novel alleles into a genome may disrupt co-adapted gene complexes with severe consequences for developmental instability as shown in cases of evolution of pesticide resistance (e.g., Davies et al., 1996; review in Møller and Swaddle, 1997). How can developmental instability be
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reduced when selection for increased growth is the target? One possibility is to select for increased stress resistance as already done for quail and other domestic animals. Indeed, Satterlee et al. (2000) have shown that lines of quail selected for increased stress resistance, as reflected by their low corticosterone levels, have reduced levels of fluctuating asymmetry, as predicted. What are the behavioural consequences of selection for rapid growth? We have briefly described some examples above. Studies of cannibalism in chickens have demonstrated significant differences in asymmetry between cannibalistic and cannibalised chickens (Yngvesson and Keeling, 2000), suggesting a direct link with developmental instability. Particularly relevant to veterinarians is the link between developmental instability and susceptibility to parasites and disease. Does selection for rapid growth cause an increase in the frequency of disease conditions, and how is this relationship associated with fluctuating asymmetry? Thornhill and Møller (1997) provide an extensive review of the literature on developmental instability and parasitism and disease for humans, while Møller (1996a,b) reviews the literature suggesting general relationships between parasitism and increased levels of asymmetry. Numerous other studies have appeared in the medical and evolutionary literature since these reviews appeared.
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