DIETARY ANALYSIS: FISH
Barnett A., Redd K. S., Frusher S. D., Stevens J. D., Semmens J. M. (2010): Non-lethal method to obtain stomach samples from a large marine predator and the use of DNA analysis to improve dietary information. Journal of Experimental Marine Biology and Ecology 393: 188-192.
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Dietary information of apex predators is crucial to understanding community dynamics and ecosystem processes. However, as dietary studies traditionally involve lethal sampling, obtaining this essential information can have repercussions on predator populations and the structure and functioning of marine ecosystems. With stronger emphasis being placed on conservation of species that are vulnerable to overexploitation, the need for non-destructive methods of sampling is imperative, as is the requirement to maximize the information obtained from each sample. Stomach flushing (gastric lavage) and DNA analysis of stomach contents methods were tested on the broadnose sevengill shark Notorynchus cepedianus Peron 1807. Acoustic tracking and recaptures of sharks implied high survivorship post-fishing and stomach flushing. From 85 prey items collected, 36 (43%) could be identified to species level using morphological analysis. After DNA analysis, a further 35 items were identified to species level, doubling the information obtained from these stomachs. The number of N. cepedianus that were confirmed to have eaten gummy sharks Mustelus antarcticus Gunther 1870 also doubled after DNA analysis. Without DNA analysis (of stomach contents) the importance of M. antarcticus in the diets of N. cepedianus would have been substantially underestimated. In addition, the non-lethal approach provides an opportunity to obtain meaningful information from non-harvested, endangered or rare species or sampling of species within protected areas.
Hammerschlag N., Sulikowski J. (2011): Killing for conservation: the need for alternatives to lethal sampling of apex predatory sharks. Endangered Species Research 14: 135-140.
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Top oceanic predators, especially large predatory sharks (TOPS), appear to be experiencing varying degrees of population declines. Life history data (e.g. diet, reproductive status, age and growth, mortality) are critical for developing effective conservation strategies for TOPS. Presently, lethal sampling remains the most effective and accurate means of gathering these data. To meet such challenges, many scientists have utilized specimens obtained from recreational and commercial fisheries, but have needed to supplement those data with fishery-independent sampling. However, there is growing public and scientific debate as to whether lethal sampling of TOPS is justified for obtaining conservation data. Here we describe the development and use of non-lethal alternatives for collecting data on (1) trophodynamics; (2) maturity state and fecundity; and (3) growth and mortality rates necessary to enact conservation measures for threatened or even data-deficient TOPS.
Beckmann M. C., Gilliam J. F., Langerhans R. B. (2015): X-ray imaging as a time-saving, non-invasive technique for diet analysis. Fisheries Research 161: 1-7.
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Dietary patterns of animals have a long-recognized importance in ecology and evolution, with numerous and diverse applications. While many methods of diet assessment exist, the most common method of direct diet examination for most small vertebrates is stomach-content analysis, using labor-intensive surgical removal of the gut following death. Methods that can reduce the time required to collect diet information without necessarily sacrificing specimens could prove invaluable for a range of applications. We evaluated digital X-ray imaging as a non-invasive method for examination of stomach contents of small fishes. Based on both a feeding experiment and examination of field-collected preserved specimens, we found that digital radiography consistently revealed the presence of moderate- to high-density prey items in the stomach, such as small arthropods. Moreover, X-ray imaging allowed for rapid identification of some particular prey items such as detritus, dipteran larvae, ostracods, hard-shelled molluscs, and small fish. However, this method failed to detect some low-density prey items present in some stomachs, and could not be used for precise taxonomic identifications in most cases. Overall, we found that digital X-ray images can be quickly acquired from anesthetized or preserved animals, permit rapid identification of certain prey items, and facilitate digital data archives. Future studies that employ this method should first “ground-truth” the radiological signatures of prey items observed within a given study using stomach-content analysis, which then permits rapid data collection strictly using X-ray images. This method can provide information useful for determining the inclusion of certain prey items in diets, even quantifying particular taxonomic groups of prey (% occurrence, % by number). Thus our results indicate that for certain study goals, X-ray radiography may provide a time reducing, non-invasive technique for diet analysis of small vertebrates.
Corse E., Valladares S., Planas M., Chamorro A., Pintado J. (2015): Analysis of the diet of the long‐snouted seahorse Hippocampus guttulatus by 18S rDNA amplification of prey in faeces. Aquaculture Nutrition 21: 528-540.
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Breeding in captivity for research or exhibition (e.g. in aquaria) can replace the capture of wild specimens of endangered species and allow controlled reinforcement of wild populations. With this aim, diet analysis and establishing the adequate prey are determinant factors for breeding success. However, non‐invasive approaches such as faecal DNA analysis are advisable for analysing the diet of these species. Therefore, the aim of the present study was to demonstrate the usefulness of faecal DNA analysis by specific PCR amplification of prey DNA for assessing the diet of the seahorse Hippocampus guttulatus. In a comparison of the suitability of different genes (COI, 18SrRNA and 28SrRNA), 18SrRNA was found to be the most suitable for designing specific primers for the prey types fed to seahorses (Artemia, Palaemonetes and Mysidae). The technique was assessed in feeding experiments in which prey ingestion was recorded daily, and faeces were collected for DNA extraction and presence/absence PCR analysis. Amplification of the prey DNA in faeces was consistent with the sequence of prey supplied (prey eaten the day before was always detected). Differences in the time between feeding and detection in faeces suggested prey‐specific gut passage times, which were shorter for Palaemonetes than for Mysidae. This fact highlights the importance of feeding trials to avoid overestimating the consumption of prey with long gut retention when PCR‐based methods are used. This molecular technique is thus applicable for studying the feeding behaviour of captive seahorses and could be adapted for use in other marine species.
Elston C., von Brandis R. G., Cowley P. D. (2015): Gastric lavage as a non-lethal method for stingray (Myliobatiformes) diet sampling. African Journal of Marine Science 37: 415-419.
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Understanding the trophic interactions of ecologically important mesopredators such as Myliobatiformes (stingrays) is vital, but, given their susceptibility to overexploitation, the collection of stomach contents for dietary analysis necessitates the use of non-lethal methods. We provide a detailed method of using gastric lavage to collect stomach contents from the ‘Vulnerable’ porcupine ray Urogymnus asperrimus at the St Joseph Atoll of the Amirantes Group, Seychelles. Rays were captured by hand, inverted and restrained while a plastic hose, connected to a bilge pump, was inserted into the stomach to flush out the contents. A high percentage (94.5%) of gastric lavage attempts resulted in the collection of stomach contents. There was no visual evidence of stress or short-term, post-release mortality and, given the success and relative ease of this technique, we conclude that the described method of gastric lavage is an effective, non-lethal means of obtaining stomach contents from stingrays.
Correa S. B., Anderson J. T. (2016): A non‐destructive sampling protocol for field studies of seed dispersal by fishes. Journal of Fish Biology 88: 1989-2003.
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This paper presents a standardized protocol for the non‐lethal capture of fishes, sampling of stomach contents and quantification of seed dispersal efficiency by frugivorous fishes. Neotropical pacu Piaractus mesopotamicus individuals were collected with fruit‐baited hooks. The diets of 110 fish were sampled using a lavage method, which retrieved >90% of stomach contents of both juveniles and adults and allowed individuals to recover within 5 min of treatment. The proportional volume of six food categories was similar for stomachs and whole digestive tracts retrieved by dissection. Fruit pulp was proportionally lower in the stomach. The abundance and species richness of intact seeds increased with fish size independent of whether only stomachs or whole digestive tracts were analysed. The analysis of stomach contents accounted for 62·5% of the total species richness of seeds dispersed by P. mesopotamicus and 96% of common seeds (seed species retrieved from more than one fish). Germination trials revealed that seed viability was similar for seeds collected from the stomach via lavage and seeds that passed through the entire digestive tract. Therefore, stomach contents provide an unbiased representation of the dietary patterns and seed dispersal of frugivorous fishes.
Braga R. R., Ribeiro V. M., Bornatowski H., Abilhoa V., Vitule J. R. S. (2017): Gastric lavage for dietary studies of small fishes: efficiency, survival and applicability. Acta Ichthyologica et Piscatoria 47: 97–100.
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Studies on feeding ecology provide information of great importance to the understanding of aquatic ecosystems. However, these studies usually require the sacrifice of animals. In view of this problem, the presently reported study aims to evaluate the effectiveness and applicability of the gastric lavage method for small catfishes. There was no mortality recorded for the 80 specimens of Rhamdia quelen (Quoy et Gaimard, 1824) and after recovery from anaesthesia all fish presented a normal swimming pattern. With the gastric lavage method we were able to retrieve 71.4% of the stomach contents of 34 specimens of Pimelodella pappenheimi Ahl, 1925. These results serve as a stimulus for studies prioritizing non-lethal techniques. These methods would ensure greater knowledge about ecology and conservation of smaller fishes, especially to populations of small and rare species that are the most endangered in freshwater systems.
Corse E., Meglecz E., Archambaud G., Ardisson M., Martin J. F., Tougard C., Chappaz R., Dubut V. (2017): A from‐benchtop‐to‐desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies. Molecular Ecology Resources 17: e146-e159.
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The main objective of this work was to develop and validate a robust and reliable “from-benchtop-to-desktop” metabarcoding workflow to investigate the diet of invertebrate-eaters. We applied our workflow to faecal DNA samples of an invertebrate-eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of nonarbitrary thresholds established from control samples and from molecular replicates to address the elimination of cross-contamination, PCR/sequencing errors and mistagging artefacts. This resulted in a conservative and informative metabarcoding data set. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semiquantitative statistic in our diet study, the minimum number of individuals, which is based on the number of distinct variants in each sample. The metabarcoding approach described in this article may guide future diet studies that aim to produce robust data sets associated with a fine and accurate identification of prey items.
Guillerault N., Bouletreau S., Iribar A., Valentini A., Santoul F. (2017): Application of DNA metabarcoding on faeces to identify European catfish Silurus glanis diet. Journal of Fish Biology 90: 2214-2219.
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In this study, the results of conventional stomach-content analysis are compared with the recent DNA metabarcoding approach on faeces to identify fish species consumed by non-native European catfish Silurus glanis in the Garonne River (south-western France), with a special emphasis on anadromous prey. Fourteen prey species were identified in the stomach contents or faeces, including four anadromous fish species. Despite higher intestine than stomach emptiness, more species were identified through faecal analysis (11 of 14) than through stomach-content analysis (five of 14) suggesting that DNA metabarcoding on faeces is an efficient, non-intrusive technique to study the diet of predatory fishes.
Hoenig B. D., Trevelline B. K., Nuttle T., Porter B. A. (2021): Dietary DNA metabarcoding reveals seasonal trophic changes among three syntopic freshwater trout species. Freshwater Biology 66: 509-523.
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Providing taxonomically precise dietary characterisations for freshwater fish species is critical for gaining a deeper understanding of the trophic dynamics present in freshwater ecosystems. However, our current understanding of freshwater trophic ecology has relied almost entirely upon direct observation of foraging attempts or morphological identification of partially digested prey. Due to the limitations of morphological dietary characterisations of soft-bodied arthropod prey, these techniques offer dietary descriptions that can lack satisfactory taxonomic resolution and may bias our interpretations of freshwater food webs. Recent advancements in DNA-based prey identification have allowed for species-level prey characterisations for many terrestrial insectivores, although these techniques have seldom been applied to understand the diets of freshwater fish. This study used DNA metabarcoding with high-throughput, next-generation sequencing to provide species-level descriptions of prey composition for three naturally reproducing, syntopic freshwater trout species. Our study supports previous findings that suggested that brook trout (Salvelinus fontinalis), brown trout (Salmo trutta), and rainbow trout (Oncorhynchus mykiss) are generalist predators that display a high degree of seasonal dietary flexibility. Prey composition varied significantly across sampling periods, with detection frequency of terrestrial prey greater in the spring/summer period compared to the autumn period. Pollution-sensitive aquatic macroinvertebrates were detected frequently across both sampling periods, highlighting the importance of high-quality streams that support such arthropod prey. DNA metabarcoding also detected a high richness of soft-bodied, Lepidoptera prey species, a taxonomic group that has been largely underrepresented in previous trout dietary studies that used traditional morphological techniques. This study demonstrates the applicability of dietary DNA metabarcoding for the detection and species-level identification of arthropods found in freshwater fish lavage samples and highlights the importance of taxonomically precise techniques when attempting to better understand trophic interactions within freshwater communities.
Johnson N. S., Lewandoski S. A., Merkes C. (2021): Assessment of sea lamprey (Petromyzon marinus) diet using DNA metabarcoding of feces. Ecological Indicators 125: 107605.
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Sea lamprey (Petromyzon marinus) are invasive in the Laurentian Great Lakes, parasitize large-bodied fishes, and therefore are the focus of an international control program. However, damage caused by sea lamprey to modern day fish stocks remains uncertain because diet analysis of juvenile sea lamprey has been challenging; they feed on blood and are difficult to randomly sample in the lakes. Here, both challenges were addressed by showing that DNA metabarcoding of fecal material can be used to identify the diet of actively feeding juvenile sea lamprey, and can also be used to determine what non-feeding adult sea lamprey captured in streams fed on while parasitizing fish. Fecal samples from juvenile sea lamprey that were feeding on lake trout in northern Lake Huron overwhelmingly contained lake trout (Salvelinus namaycush) DNA (90%), while smaller percentages contained lake whitefish (Coregonus clupeaformis; 5%) and longnose sucker (Catostomus catostomus; 5%) DNA. Fecal samples from adult sea lamprey captured from a tributary to northern Lake Huron overwhelmingly contained longnose and white sucker DNA (Catostomus spp.; 80%), while a smaller percentage contained lake trout DNA (10%). Diet composition of adult sea lamprey sampled in the tributary (Black Mallard Creek) was more diverse than juvenile diet composition. DNA metabarcoding suggests that Catostomus spp. may be an important host fish in northern Lake Huron for sea lamprey prior to spawning. Future research could investigate how diet varies across years and lakes and the prevalence and sources of DNA contamination. Application of DNA metabarcoding for diet assessment may be practical for identifying populations of invasive sea lamprey that feed on highly valued fishes and help guide restoration of lampreys worldwide.
Lazic T., Pierri C., Corriero G., Balech B., Cardone F., Deflorio M., Fosso B., Gissi C., Marzano M., Nonnis Marzano F., Pesole G., Santamaria M., Gristina M. (2021): Evaluating the efficiency of DNA metabarcoding to analyze the diet of Hippocampus guttulatus (Teleostea: Syngnathidae). Life 11: 998.
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Seahorses are considered a flagship species for conservation efforts and due to their conservation status, improving knowledge on their dietary composition while applying a non-invasive approach, could be useful. Using Hippocampus guttulatus as a case study, the present study represents pioneering research into investigating the diet of seahorses by NGS-based DNA metabarcoding of fecal samples. The study developed and tested the protocol for fecal DNA metabarcoding during the feeding trials where captive seahorses were fed on a diet of known composition; the process was subsequently applied on fecal samples collected from wild individuals. The analysis of samples collected during the feeding trials indicated the reliability of the applied molecular approach by allowing the characterization of the effectively ingested prey. In the field study, among detected prey species, results revealed that the majority of the seahorse samples contained taxa such as Amphipoda, Decapoda, Isopoda, and Calanoida, while less common prey taxa were Gastropoda and Polyplacophora. As only a small amount of starting fecal material is needed and the sampling procedure is neither invasive nor lethal. The present study indicates DNA metabarcoding as useful for investigating seahorse diet and could help define management and conservation actions.
van Zinnicq Bergmann M. P., Postaire B. D., Gastrich K., Heithaus M. R., Hoopes L. A., Lyons K., Papastamatiou Y. P., Schneider E. V. C., Strickland B. A., Talwar B. S., Chapman D. D., Bakker J. (2021): Elucidating shark diets with DNA metabarcoding from cloacal swabs. Molecular Ecology Resources 21: 1056-1067.
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Animal dietary information provides the foundation for understanding trophic relationships, which is essential for ecosystem management. Yet, in marine systems, high-resolution diet reconstruction tools are currently under-developed. This is particularly pertinent for large marine vertebrates, for which direct foraging behaviour is difficult or impossible to observe and, due to their conservation status, the collection of stomach contents at adequate sample sizes is frequently impossible. Consequently, the diets of many groups, such as sharks, have largely remained unresolved. To address this knowledge gap, we applied metabarcoding to prey DNA in faecal residues (fDNA) collected on cotton swabs from the inside of a shark’s cloaca. We used a previously published primer set targeting a small section of the 12S rRNA mitochondrial gene to amplify teleost prey species DNA. We tested the utility of this method in a controlled feeding experiment with captive juvenile lemon sharks (Negaprion brevirostris) and on free-ranging juvenile bull sharks (Carcharhinus leucas). In the captive trial, we successfully isolated and correctly identified teleost prey DNA without incurring environmental DNA contamination from the surrounding seawater. In the field, we were able to reconstruct high-resolution teleost dietary information from juvenile C. leucas fDNA that was generally consistent with expectations based on published diet studies of this species. While further investigation is needed to validate the method for larger sharks and other species, it is expected to be broadly applicable to aquatic vertebrates and provides an opportunity to advance our understanding of trophic interactions in marine and freshwater systems.
Villsen K., Corse E., Meglécz E., Archambaud‐Suard G., Vignes H., Ereskovsky A. V., Chappaz R., Dubut V. (2022): DNA metabarcoding suggests adaptive seasonal variation of individual trophic traits in a critically endangered fish. Molecular Ecology 31: 5889-5908.
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Dietary studies are critical for understanding foraging strategies and have important applications in conservation and habitat management. We applied a robust metabarcoding protocol to characterize the diet of the critically endangered freshwater fish Zingel asper (the Rhone streber). We conducted modelling and simulation analyses to identify and characterize some of the drivers of individual trophic trait variation in this species. We found that population density and ontogeny had minor effects on the trophic niche of Z. asper. Instead, our results suggest that the majority of trophic niche variation was driven by seasonal variation in ecological opportunity. The total trophic niche width of Z. asper seasonally expanded to include a broader range of prey. Furthermore, null model simulations revealed that the increase of between-individual variation in autumn indicates that Z. asper become more opportunistic relative to summer and spring, rather than being associated with a seasonal specialization of individuals. Overall, our results suggest an adaptive variation of individual trophic traits in Z. asper: the species mainly consumes a few ephemeropteran taxa (Baetis fuscatus and Ecdyonurus) but seems to be capable of adapting its foraging strategy to maintain its body condition. This study illustrates how metabarcoding data obtained from faeces can be validated and combined with individual-based modelling and simulation approaches to explore inter- and intrapopulational individual trophic traits variation and to test hypotheses in the conventional analytic framework of trophic ecology.
Campbell L. J., Castillo N. A., Shenker J., Owens L. A., Santos R. O., Adams A. J., Rehage J. S., Denton K. E., Goldberg T. L. (2023): Bone appétit: DNA metabarcoding as a non-lethal alternative to morphological dietary assessment in Atlantic bonefish (Albula vulpes). Environmental Biology of Fishes 106: 337-348.
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Traditional approaches to dietary assessment in fish necessitate the collection of stomach contents through either gastric lavage or lethal sampling. The Atlantic bonefish (Albula vulpes) is an economically important sportfish in the western central Atlantic region for which a minimally invasive, non-lethal alternative to morphological dietary assessment would be extremely useful. Here, we compare dietary DNA metabarcoding from cloacal swabs of 16 A. vulpes to dietary composition data obtained using traditional morphological classification techniques and metabarcoding of homogenized stomach contents. Further, we compare the performance of two commonly used barcoding genes (18S rRNA and COI) at inferring A. vulpes diet composition. We found that detection of taxa and the resolution of taxonomic annotation varied between markers, suggesting a multi-marker approach is likely to provide the most complete results. Importantly however, the number of dietary OTUs identified and the taxonomic composition of the core diet were not significantly different between molecular markers. Dietary compositions identified using metabarcoding approaches differed in both diversity and composition from matched dietary data obtained from morphologically analyzed stomach contents; however, the same core prey classes were identified using both methods, suggesting that metabarcoding does indeed offer a viable alternative to morphological dietary assessment. Importantly, dietary compositions identified by metabarcoding of cloacal swabs did not differ significantly from those identified by metabarcoding of stomach contents. Metabarcoding of minimally invasive cloacal swabs should be considered for dietary studies of bonefish and other fish species for which invasive or lethal sampling is problematic.