Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication,diet and diabetes

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch‐rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed‐dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus.     

Genome Sequencing Highlights the Dynamic Early History of Dogs

To identify genetic changes underlying dog domestication and reconstruct their early evolutionary history, we generated high-quality genome sequences from three gray wolves, one from each of the three putative centers of dog domestication, two basal dog lineages (Basenji and Dingo) and a golden jackal as an outgroup. Analysis of these sequences supports a demographic model in which dogs and wolves diverged through a dynamic process involving population bottlenecks in both lineages and post-divergence gene flow. In dogs, the domestication bottleneck involved at least a 16-fold reduction in population size, a much more severe bottleneck than estimated previously. A sharp bottleneck in wolves occurred soon after their divergence from dogs, implying that the pool of diversity from which dogs arose was substantially larger than represented by modern wolf populations. We narrow the plausible range for the date of initial dog domestication to an interval spanning 11–16 thousand years ago, predating the rise of agriculture. In light of this finding, we expand upon previous work regarding the increase in copy number of the amylase gene (AMY2B) in dogs, which is believed to have aided digestion of starch in agricultural refuse. We find standing variation for amylase copy number variation in wolves and little or no copy number increase in the Dingo and Husky lineages. In conjunction with the estimated timing of dog origins, these results provide additional support to archaeological finds, suggesting the earliest dogs arose alongside hunter-gathers rather than agriculturists. Regarding the geographic origin of dogs, we find that, surprisingly, none of the extant wolf lineages from putative domestication centers is more closely related to dogs, and, instead, the sampled wolves form a sister monophyletic clade. This result, in combination with dog-wolf admixture during the process of domestication, suggests that a re-evaluation of past hypotheses regarding dog origins is necessary.     

Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds

A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon‐Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon‐Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs.     

Linked genetic variants on chromosome 10 control ear morphology and body mass among dog breeds

Background

The domestic dog is a rich resource for mapping the genetic components of phenotypic variation due to its unique population history involving strong artificial selection. Genome-wide association studies have revealed a number of chromosomal regions where genetic variation associates with morphological characters that typify dog breeds. A region on chromosome 10 is among those with the highest levels of genetic differentiation between dog breeds and is associated with body mass and ear morphology, a common motif of animal domestication. We characterised variation in this region to uncover haplotype structure and identify candidate functional variants.

Results

We first identified SNPs that strongly associate with body mass and ear type by comparing sequence variation in a 3 Mb region between 19 breeds with a variety of phenotypes. We next genotyped a subset of 123 candidate SNPs in 288 samples from 46 breeds to identify the variants most highly associated with phenotype and infer haplotype structure. A cluster of SNPs that associate strongly with the drop ear phenotype is located within a narrow interval downstream of the gene MSRB3, which is involved in human hearing. These SNPs are in strong genetic linkage with another set of variants that correlate with body mass within the gene HMGA2, which affects human height. In addition we find evidence that this region has been under selection during dog domestication, and identify a cluster of SNPs within MSRB3 that are highly differentiated between dogs and wolves.

Conclusions

We characterise genetically linked variants that potentially influence ear type and body mass in dog breeds, both key traits that have been modified by selective breeding that may also be important for domestication. The finding that variants on long haplotypes have effects on more than one trait suggests that genetic linkage can be an important determinant of the phenotypic response to selection in domestic animals.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1702-2) contains supplementary material, which is available to authorized users.     

Analysis of structural diversity in wolf-like canids reveals post-domestication variants

Background

Although a variety of genetic changes have been implicated in causing phenotypic differences among dogs, the role of copy number variants (CNVs) and their impact on phenotypic variation is still poorly understood. Further, very limited knowledge exists on structural variation in the gray wolf, the ancestor of the dog, or other closely related wild canids. Documenting CNVs variation in wild canids is essential to identify ancestral states and variation that may have appeared after domestication.

Results

In this work, we genotyped 1,611 dog CNVs in 23 wolf-like canids (4 purebred dogs, one dingo, 15 gray wolves, one red wolf, one coyote and one golden jackal) to identify CNVs that may have arisen after domestication. We have found an increase in GC-rich regions close to the breakpoints and around 1 kb away from them suggesting that some common motifs might be associated with the formation of CNVs. Among the CNV regions that showed the largest differentiation between dogs and wild canids we found 12 genes, nine of which are related to two known functions associated with dog domestication; growth (PDE4D, CRTC3 and NEB) and neurological function (PDE4D, EML5, ZNF500, SLC6A11, ELAVL2, RGS7 and CTSB).

Conclusions

Our results provide insight into the evolution of structural variation in canines, where recombination is not regulated by PRDM9 due to the inactivation of this gene. We also identified genes within the most differentiated CNV regions between dogs and wolves, which could reflect selection during the domestication process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-465) contains supplementary material, which is available to authorized users.     

Individual differences in AMY1 gene copy number, salivary α-amylase levels, and the perception of oral starch

Background

The digestion of dietary starch in humans is initiated by salivary α-amylase, an endo-enzyme that hydrolyzes starch into maltose, maltotriose and larger oligosaccharides. Salivary amylase accounts for 40 to 50% of protein in human saliva and rapidly alters the physical properties of starch. Importantly, the quantity and enzymatic activity of salivary amylase show significant individual variation. However, linking variation in salivary amylase levels with the oral perception of starch has proven difficult. Furthermore, the relationship between copy number variations (CNVs) in the AMY1 gene, which influence salivary amylase levels, and starch viscosity perception has not been explored.

Principal Findings

Here we demonstrate that saliva containing high levels of amylase has sufficient activity to rapidly hydrolyze a viscous starch solution in vitro. Furthermore, we show with time-intensity ratings, which track the digestion of starch during oral manipulation, that individuals with high amylase levels report faster and more significant decreases in perceived starch viscosity than people with low salivary amylase levels. Finally, we demonstrate that AMY1 CNVs predict an individual''s amount and activity of salivary amylase and thereby, ultimately determine their perceived rate of oral starch viscosity thinning.

Conclusions

By linking genetic variation and its consequent salivary enzymatic differences to the perceptual sequellae of these variations, we show that AMY1 copy number relates to salivary amylase concentration and enzymatic activity level, which, in turn, account for individual variation in the oral perception of starch viscosity. The profound individual differences in salivary amylase levels and salivary activity may contribute significantly to individual differences in dietary starch intake and, consequently, to overall nutritional status.     

The <Emphasis Type="Italic">IGF1</Emphasis>small dog haplotype is derived from Middle Eastern grey wolves

Background

A selective sweep containing the insulin-like growth factor 1 (IGF1) gene is associated with size variation in domestic dogs. Intron 2 of IGF1 contains a SINE element and single nucleotide polymorphism (SNP) found in all small dog breeds that is almost entirely absent from large breeds. In this study, we surveyed a large sample of grey wolf populations to better understand the ancestral pattern of variation at IGF1 with a particular focus on the distribution of the small dog haplotype and its relationship to the origin of the dog.

Results

We present DNA sequence data that confirms the absence of the derived small SNP allele in the intron 2 region of IGF1 in a large sample of grey wolves and further establishes the absence of a small dog associated SINE element in all wild canids and most large dog breeds. Grey wolf haplotypes from the Middle East have higher nucleotide diversity suggesting an origin there. Additionally, PCA and phylogenetic analyses suggests a closer kinship of the small domestic dog IGF1 haplotype with those from Middle Eastern grey wolves.

Conclusions

The absence of both the SINE element and SNP allele in grey wolves suggests that the mutation for small body size post-dates the domestication of dogs. However, because all small dogs possess these diagnostic mutations, the mutations likely arose early in the history of domestic dogs. Our results show that the small dog haplotype is closely related to those in Middle Eastern wolves and is consistent with an ancient origin of the small dog haplotype there. Thus, in concordance with past archeological studies, our molecular analysis is consistent with the early evolution of small size in dogs from the Middle East.See associated opinion by Driscoll and Macdonald: http://jbiol.com/content/9/2/10

     

The legacy of domestication: accumulation of deleterious mutations in the dog genome

Dogs exhibit more phenotypic variation than any other mammal and are affected by a wide variety of genetic diseases. However, the origin and genetic basis of this variation is still poorly understood. We examined the effect of domestication on the dog genome by comparison with its wild ancestor, the gray wolf. We compared variation in dog and wolf genes using whole-genome single nucleotide polymorphism (SNP) data. The d(N)/d(S) ratio (omega) was around 50% greater for SNPs found in dogs than in wolves, indicating that a higher proportion of nonsynonymous alleles segregate in dogs compared with nonfunctional genetic variation. We suggest that the majority of these alleles are slightly deleterious and that two main factors may have contributed to their increase. The first is a relaxation of selective constraint due to a population bottleneck and altered breeding patterns accompanying domestication. The second is a reduction of effective population size at loci linked to those under positive selection due to Hill-Robertson interference. An increase in slightly deleterious genetic variation could contribute to the prevalence of disease in modern dog breeds.     

Evolutionary genomics of dog domestication

We review the underlying principles and tools used in genomic studies of domestic dogs aimed at understanding the genetic changes that have occurred during domestication. We show that there are two principle modes of evolution within dogs. One primary mode that accounts for much of the remarkable diversity of dog breeds is the fixation of discrete mutations of large effect in individual lineages that are then crossed to various breed groupings. This transfer of mutations across the dog evolutionary tree leads to the appearance of high phenotypic diversity that in actuality reflects a small number of major genes. A second mechanism causing diversification involves the selective breeding of dogs within distinct phenotypic or functional groups, which enhances specific group attributes such as heading or tracking. Such progressive selection leads to a distinct genetic structure in evolutionary trees such that functional and phenotypic groups cluster genetically. We trace the origin of the nuclear genome in dogs based on haplotype-sharing analyses between dogs and gray wolves and show that contrary to previous mtDNA analyses, the nuclear genome of dogs derives primarily from Middle Eastern or European wolves, a result more consistent with the archeological record. Sequencing analysis of the IGF1 gene, which has been the target of size selection in small breeds, further supports this conclusion. Finally, we discuss how a black coat color mutation that evolved in dogs has transformed North American gray wolf populations, providing a first example of a mutation that appeared under domestication and selectively swept through a wild relative.     

Allelic Combinations of Promoter and Exon 2 in <Emphasis Type="Italic">DQB1</Emphasis> in Dogs and Wolves

Polymorphism of PBRs of the major histocompatibility complex (MHC) genes is well recognized, but the polymorphism also extends to proximal promoter regions. Examining DQB1 variability in dogs and wolves, we identified 7 promoter variants and 13 exon 2 alleles among 89 dogs, including a previously unknown DQB1 exon 2 allele, and 8 promoter variants and 9 exon 2 alleles among 85 wolves. As expected from previous studies and from a close chromosomal location, strong linkage disequilibrium was demonstrated in both wolves and dogs by having significantly fewer promoter/exon 2 combinations than expected from simulations of randomized data sets. Interestingly, we noticed weaker haplotypic associations in dogs than in wolves. Dogs had twice as many promoter/exon 2 combinations as wolves and an almost 2-fold difference in the number of exon 2 alleles per promoter variant. This difference was not caused by an admixture of breeds in our group of dogs because the high ratio of observed to expected number of haplotypes persisted within a single dog breed, the German Shepherd. Ewens-Watterson tests indicated that both the promoter and exon 2 are under the balancing selection, and both regions appear to be more recently derived in the dog than in the wolf. Hence, although reasons for the differences are unknown, they may relate to altered selection pressure on patterns of expression. Deviations from normal MHC expression patterns have been associated with autoimmune diseases, which occur frequently in several dog breeds. Further knowledge about these deviations may help us understand the source of such diseases.     

Mitochondrial DNA from prehistoric canids highlights relationships between dogs and South-East European wolves

The question of the origins of the dog has been much debated. The dog is descended from the wolf that at the end of the last glaciation (the archaeologically hypothesized period of dog domestication) was one of the most widespread among Holarctic mammals. Scenarios provided by genetic studies range from multiple dog-founding events to a single origin in East Asia. The earliest fossil dogs, dated approximately 17-12,000 radiocarbon ((14)C) years ago (YA), were found in Europe and in the Middle East. Ancient DNA (a-DNA) evidence could contribute to the identification of dog-founder wolf populations. To gain insight into the relationships between ancient European wolves and dogs we analyzed a 262-bp mitochondrial DNA control region fragment retrieved from five prehistoric Italian canids ranging in age from approximately 15,000 to approximately 3,000 (14)C YA. These canids were compared to a worldwide sample of 547 purebred dogs and 341 wolves. The ancient sequences were highly diverse and joined the three major clades of extant dog sequences. Phylogenetic investigations highlighted relationships between the ancient sequences and geographically widespread extant dog matrilines and between the ancient sequences and extant wolf matrilines of mainly East European origin. The results provide a-DNA support for the involvement of European wolves in the origins of the three major dog clades. Genetic data also suggest multiple independent domestication events. East European wolves may still reflect the genetic variation of ancient dog-founder populations.     

Metagenomic dissection of the canine gut microbiota: insights into taxonomic,metabolic and nutritional features

Domestication of dogs from wolves is the oldest known example of ongoing animal selection, responsible for generating more than 300 dog breeds worldwide. In order to investigate the taxonomic and functional evolution of the canine gut microbiota, a multi-omics approach was applied to six wild wolves and 169 dog faecal samples, the latter encompassing 51 breeds, which fully covers currently known canine genetic biodiversity. Specifically, 16S rRNA gene and bifidobacterial Internally Transcribed Spacer (ITS) profiling were employed to reconstruct and then compare the canine core gut microbiota to those of wolves and humans, revealing that artificial selection and subsequent cohabitation of dogs with their owners influenced the microbial population of canine gut through loss and acquisition of specific bacterial taxa. Moreover, comparative analysis of the intestinal bacterial population of dogs fed on Bones and Raw Food (BARF) or commercial food (CF) diet, coupled with shotgun metagenomics, highlighted that both bacterial composition and metabolic repertoire of the canine gut microbiota have evolved to adapt to high-protein or high-carbohydrates intake. Altogether, these data indicate that artificial selection and domestication not only affected the canine genome, but also shaped extensively the bacterial population harboured by the canine gut.     

Demographically-Based Evaluation of Genomic Regions under Selection in Domestic Dogs

Controlling for background demographic effects is important for accurately identifying loci that have recently undergone positive selection. To date, the effects of demography have not yet been explicitly considered when identifying loci under selection during dog domestication. To investigate positive selection on the dog lineage early in the domestication, we examined patterns of polymorphism in six canid genomes that were previously used to infer a demographic model of dog domestication. Using an inferred demographic model, we computed false discovery rates (FDR) and identified 349 outlier regions consistent with positive selection at a low FDR. The signals in the top 100 regions were frequently centered on candidate genes related to brain function and behavior, including LHFPL3, CADM2, GRIK3, SH3GL2, MBP, PDE7B, NTAN1, and GLRA1. These regions contained significant enrichments in behavioral ontology categories. The 3^rd top hit, CCRN4L, plays a major role in lipid metabolism, that is supported by additional metabolism related candidates revealed in our scan, including SCP2D1 and PDXC1. Comparing our method to an empirical outlier approach that does not directly account for demography, we found only modest overlaps between the two methods, with 60% of empirical outliers having no overlap with our demography-based outlier detection approach. Demography-aware approaches have lower-rates of false discovery. Our top candidates for selection, in addition to expanding the set of neurobehavioral candidate genes, include genes related to lipid metabolism, suggesting a dietary target of selection that was important during the period when proto-dogs hunted and fed alongside hunter-gatherers.     

MHC class II genes in European wolves: a comparison with dogs

The genome of the grey wolf, one of the most widely distributed land mammal species, has been subjected to both stochastic factors, including biogeographical subdivision and population fragmentation, and strong selection during the domestication of the dog. To explore the effects of drift and selection on the partitioning of MHC variation in the diversification of species, we present nine DQA, 10 DQB, and 17 DRB1 sequences of the second exon for European wolves and compare them with sequences of North American wolves and dogs. The relatively large number of class II alleles present in both European and North American wolves attests to their large historical population sizes, yet there are few alleles shared between these regions at DQB and DRB1. Similarly, the dog has an extensive array of class II MHC alleles, a consequence of a genetically diverse origin, but allelic overlap with wolves only at DQA. Although we might expect a progression from shared alleles to shared allelic lineages during differentiation, the partitioning of diversity between wolves and dogs at DQB and DRB1 differs from that at DQA. Furthermore, an extensive region of nucleotide sequence shared between DRB1 and DQB alleles and a shared motif suggests intergenic recombination may have contributed to MHC diversity in the Canidae.     

Studies of modern Italian dog populations reveal multiple patterns for domestic breed evolution

Through thousands of years of breeding and strong human selection, the dog (Canis lupus familiaris) exists today within hundreds of closed populations throughout the world, each with defined phenotypes. A singular geographic region with broad diversity in dog breeds presents an interesting opportunity to observe potential mechanisms of breed formation. Italy claims 14 internationally recognized dog breeds, with numerous additional local varieties. To determine the relationship among Italian dog populations, we integrated genetic data from 263 dogs representing 23 closed dog populations from Italy, seven Apennine gray wolves, and an established dataset of 161 globally recognized dog breeds, applying multiple genetic methods to characterize the modes by which breeds are formed within a single geographic region. Our consideration of each of five genetic analyses reveals a series of development events that mirror historical modes of breed formation, but with variations unique to the codevelopment of early dog and human populations. Using 142,840 genome‐wide SNPs and a dataset of 1,609 canines, representing 182 breeds and 16 wild canids, we identified breed development routes for the Italian breeds that included divergence from common populations for a specific purpose, admixture of regional stock with that from other regions, and isolated selection of local stock with specific attributes.     

Rethinking the starch digestion hypothesis for AMY1 copy number variation in humans

Alpha‐amylase exists across taxonomic kingdoms with a deep evolutionary history of gene duplications that resulted in several α‐amylase paralogs. Copy number variation (CNV) in the salivary α‐amylase gene (AMY1) exists in many taxa, but among primates, humans appear to have higher average AMY1 copies than nonhuman primates. Additionally, AMY1 CNV in humans has been associated with starch content of diets, and one known function of α‐amylase is its involvement in starch digestion. Thus high AMY1 CNV is considered to result from selection favoring more efficient starch digestion in the Homo lineage. Here, we present several lines of evidence that challenge the hypothesis that increased AMY1 CNV is an adaptation to starch consumption. We observe that α‐ amylase plays a very limited role in starch digestion, with additional steps required for starch digestion and glucose metabolism. Specifically, we note that α‐amylase hydrolysis only produces a minute amount of free glucose with further enzymatic digestion and glucose absorption being rate‐limiting steps for glucose availability. Indeed α‐amylase is nonessential for starch digestion since sucrase‐isomaltase and maltase‐glucoamylase can hydrolyze whole starch granules while releasing glucose. While higher AMY1 CN and CNV among human populations may result from natural selection, existing evidence does not support starch digestion as the major selective force. We report that in humans α‐amylase is expressed in several other tissues where it may have potential roles of evolutionary significance.     

MHC variability supports dog domestication from a large number of wolves: high diversity in Asia

The process of dog domestication is still somewhat unresolved. Earlier studies indicate that domestic dogs from all over the world have a common origin in Asia. So far, major histocompatibility complex (MHC) diversity has not been studied in detail in Asian dogs, although high levels of genetic diversity are expected at the domestication locality. We sequenced the second exon of the canine MHC gene DLA–DRB1 from 128 Asian dogs and compared our data with a previously published large data set of MHC alleles, mostly from European dogs. Our results show that Asian dogs have a higher MHC diversity than European dogs. We also estimated that there is only a small probability that new alleles have arisen by mutation since domestication. Based on the assumption that all of the currently known 102 DLA–DRB1 alleles come from the founding wolf population, we simulated the number of founding wolf individuals. Our simulations indicate an effective population size of at least 500 founding wolves, suggesting that the founding wolf population was large or that backcrossing has taken place.     

Dispelling dog dogma: an investigation of heterochrony in dogs using 3D geometric morphometric analysis of skull shape

Heterochrony is an evolutionary mechanism that generates diversity via perturbations of the rate or timing of development that requires very little genetic innovation. As such, heterochrony is thought to be a common evolutionary mechanism in the generation of diversity. Previous research has suggested that dogs evolved via heterochrony and are paedomorphic wolves. This study uses three-dimensional landmark-based coordinate data to investigate heterochronic patterns within the skull morphology of the domestic dog. A total of 677 adult dogs representing 106 different breeds were measured and compared with an ontogenetic series of 401 wolves. Geometric morphometric analysis reveals that the cranial shape of none of the modern breeds of dogs resembles the cranial shapes of adult or juvenile wolves. In addition, investigations of regional heterochrony in the face and neurocranium also reject the hypothesis of heterochrony. Throughout wolf cranial development the position of the face and the neurocranium remain in the same plane. Dogs, however, have a de novo cranial flexion in which the palate is tilted dorsally in brachycephalic and mesaticephalic breeds or tilted ventrally in dolichocephalic and down-face breeds. Dogs have evolved very rapidly into an incredibly morphologically diverse species with very little genetic variation. However, the genetic alterations to dog cranial development that have produced this vast range of phylogenetically novel skull shapes do not coincide with the expectations of the heterochronic model. Dogs are not paedomorphic wolves.     

Measurements of Salivary Alpha Amylase and Salivary Cortisol in Hominoid Primates Reveal Within-Species Consistency and Between-Species Differences

Salivary alpha amylase (sAA) is the most abundant enzyme in saliva. Studies in humans found variation in enzymatic activity of sAA across populations that could be linked to the copy number of loci for salivary amylase (AMY1), which was seen as an adaptive response to the intake of dietary starch. In addition to diet dependent variation, differences in sAA activity have been related to social stress. In a previous study, we found evidence for stress-induced variation in sAA activity in the bonobos, a hominoid primate that is closely related to humans. In this study, we explored patterns of variation in sAA activity in bonobos and three other hominoid primates, chimpanzee, gorilla, and orangutan to (a) examine if within-species differences in sAA activity found in bonobos are characteristic for hominoids and (b) assess the extent of variation in sAA activity between different species. The results revealed species-differences in sAA activity with gorillas and orangutans having higher basal sAA activity when compared to Pan. To assess the impact of stress, sAA values were related to cortisol levels measured in the same saliva samples. Gorillas and orangutans had low salivary cortisol concentrations and the highest cortisol concentration was found in samples from male bonobos, the group that also showed the highest sAA activity. Considering published information, the differences in sAA activity correspond with differences in AMY1 copy numbers and match with general features of natural diet. Studies on sAA activity have the potential to complement molecular studies and may contribute to research on feeding ecology and nutrition.