Pseudogenes and DNA-based diet analyses: a cautionary tale from a relatively well sampled predator-prey system.

Mitochondrial ribosomal DNA is commonly used in DNA-based dietary analyses. In such studies, these sequences are generally assumed to be the only version present in DNA of the organism of interest. However, nuclear pseudogenes that display variable similarity to the mitochondrial versions are common in many taxa. The presence of nuclear pseudogenes that co-amplify with their mitochondrial paralogues can lead to several possible confounding interpretations when applied to estimating animal diet. Here, we investigate the occurrence of nuclear pseudogenes in fecal samples taken from bottlenose dolphins (Tursiops truncatus) that were assayed for prey DNA with a universal primer technique. We found pseudogenes in 13 of 15 samples and 1-5 pseudogene haplotypes per sample representing 5-100% of all amplicons produced. The proportion of amplicons that were pseudogenes and the diversity of prey DNA recovered per sample were highly variable and appear to be related to PCR cycling characteristics. This is a well-sampled system where we can reliably identify the putative pseudogenes and separate them from their mitochondrial paralogues using a number of recommended means. In many other cases, it would be virtually impossible to determine whether a putative prey sequence is actually a pseudogene derived from either the predator or prey DNA. The implications of this for DNA-based dietary studies, in general, are discussed.

A molecular approach to identify prey of the southern rock lobster.

We demonstrate the use of molecular techniques to detect specific prey consumed by the southern rock lobster (Jasus edwardsii). A quick and non-lethal method was used to collect rock lobster faecal material and a molecular protocol was employed to isolate prey DNA from faecal samples. The isolated DNA was amplified using the polymerase chain reaction (PCR) with PCR primers designed to target specific prey items. Feeding experiments determined that DNA from black-lipped abalone (Haliotis rubra) and sea urchins (Centrostephanus rodgersii and Heliocidaris erythrogramma) can be detected in rock lobster faecal samples within seven hours and remains present for up to 60 h after ingestion.

DNA as a dietary biomarker in Antarctic krill, Euphausia superba.

The diet of Antarctic krill (Euphausia superba) has been studied using a variety of techniques, but current methods still suffer from problems that are difficult to solve. This study examined an alternative approach utilizing DNA as a prey biomarker. Methods were developed for the preservation, extraction, and identification of prey DNA from krill collected in the field. Group-specific polymerase chain reaction (PCR) was used to amplify diatom prey (Phylum: Bacillariophyta) and the results from DNA clone libraries were compared with microscopic diet analysis. DNA analysis was superior to microscopy for prey detection. However, differences in prey relative abundance estimates between the two techniques suggested some bias in the DNA-based estimates. Quantification showed that large amounts of prey DNA had been successfully preserved and extracted. Overall the results suggest that the application of DNA-based diet analysis to krill warrants further investigation, particularly for prey that are difficult to study using other methods.

Molecular scatology as a tool to study diet: analysis of prey DNA in scats from captive Steller sea lions.

The DNA of prey present in animal scats may provide a valuable source of information for dietary studies. We conducted a captive feeding trial to test whether prey DNA could be reliably detected in scat samples from Steller sea lions (Eumetopias jubatus). Two sea lions were fed a diet of fish (five species) and squid (one species), and DNA was extracted from the soft component of collected scats. Most of the DNA obtained came from the predator, but prey DNA could be amplified using prey-specific primers. The four prey species fed in consistent daily proportions throughout the trial were detected in more than 90% of the scat DNA extractions. Squid and sockeye salmon, which were fed as a relatively small percentage of the daily diet, were detected as reliably as the more abundant diet items. Prey detection was erratic in scats collected when the daily diet was fed in two meals that differed in prey composition, suggesting that prey DNA is passed in meal specific pulses. Prey items that were removed from the diet following one day of feeding were only detected in scats collected within 48 h of ingestion. Proportions of fish DNA present in eight scat samples (evaluated through the screening of clone libraries) were roughly proportional to the mass of prey items consumed, raising the possibility that DNA quantification methods could provide semi-quantitative diet composition data. This study should be of broad interest to researchers studying diet since it highlights an approach that can accurately identify prey species and is not dependent on prey hard parts surviving digestion.

Genetic screening for prey in the gut contents from a giant squid (Architeuthis sp.).

Giant squids (Architeuthis sp.) remain mysterious; they have evaded observation and are rarely taken from their deep sea habitat. Information on the diet of Architeuthis is scarce due to the limited number of specimens with morphologically recognizable remains in their digestive tracts. We explored the use of polymerase chain reaction (PCR)-based methods for detection of DNA in the prey remains and amorphous slurry from an Architeuthis gut sample. The DNA region amplified varied in size, allowing separation of fish and squid components. Sequence comparisons identified fish prey as Macruronus novaezelandiae. Isolation of Architeuthis DNA from an ingested tentacle and the presence of chitin fragments indicate cannibalism occurs in giant squid. Denaturing gradient gel electrophoresis was used to screen for less common DNA types, revealing a high frequency of PCR-generated false alleles, but no additional prey species.

Group-specific polymerase chain reaction for DNA-based analysis of species diversity and identity in dietary samples.

Unique DNA sequences are present in all species and can be used as biomarkers for the detection of cells from that species. These DNA sequences can most easily be detected using the polymerase chain reaction (PCR), which allows very small quantities of target DNA sequence to be amplified even when the target is mixed with large amounts of nontarget DNA. PCR amplification of DNA markers that are present in a wide range of species has proven very useful for studies of species diversity in environmental samples. The taxonomic range of species to be identified from environmental samples may often need to be restricted to simplify downstream analyses and to ensure that less abundant sequences are amplified. Group-specific PCR primer sets are one means of specifying the range of taxa that produce an amplicon in a PCR. We have developed a range of group-specific PCR primers for studying the prey diversity found in predator stomach contents and scats. These primers, their design and their application to studying prey diversity and identity in predator diet are described.

A DNA-based method for identification of krill species and its application to analysing the diet of marine vertebrate predators.

Accurate identification of species that are consumed by vertebrate predators is necessary for understanding marine food webs. Morphological methods for identifying prey components after consumption often fail to make accurate identifications of invertebrates because prey morphology becomes damaged during capture, ingestion and digestion. Another disadvantage of morphological methods for prey identification is that they often involve sampling procedures that are disruptive for the predator, such as stomach flushing or lethal collection. We have developed a DNA-based method for identifying species of krill (Crustacea: Malacostraca), an enormously abundant group of invertebrates that are directly consumed by many groups of marine vertebrates. The DNA-based approach allows identification of krill species present in samples of vertebrate stomach contents, vomit, and, more importantly, faeces. Utilizing samples of faeces from vertebrate predators minimizes the impact of dietary studies on the subject animals. We demonstrate our method first on samples of Adelie penguin (Pygoscelis adeliae) stomach contents, where DNA-based species identification can be confirmed by prey morphology. We then apply the method to faeces of Adelie penguins and to faeces of the endangered pygmy blue whale (Balaenoptera musculus brevicauda). In each of these cases, krill species consumed by the predators could be identified from their DNA present in faeces or stomach contents.

28S rDNA evolution in the Eumalacostraca and the phylogenetic position of krill.

The Malacostraca are an ancient and morphologically diverse class of Crustacea. The phylogenetic position of one order within this class, the Euphausiacea ("krill," subclass Eumalacostraca) was investigated using 28S rDNA sequences from representatives of several malacostracan orders. Phylogenies for these sequences were estimated by maximum-likelihood and maximum-parsimony analysis. The results of these analyses produced a new scheme for evolution within the Eumalacostraca. The new phylogenies suggested that Euphausiacea are most closely related to the Mysida and not the Decapoda, as is generally thought. Furthermore, the Mysida were found not to be closely related to the Lophogastrida, which are often considered their sister taxon. These hypotheses were tested against the hypotheses of monophyly for the Eucarida, Mysidacea, and Peracarida and found to be significantly better on the basis of the 28S rDNA data.