DIETARY ANALYSIS: MOLLUSCS
BIVALVES
Tran B. T., Kim K. Y., Heo J. S., Park S. J., Park H. K., Choi Y. H. (2022): Determination of the Pacific oyster Magallana gigas (Crassostrea gigas) diet composition in two aquaculture farms by fecal DNA metabarcoding. Aquaculture 552: 738042.
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Abstract
Fecal DNA metabarcoding is a noninvasive, accurate, rapid, and cost-effective tool to investigate diet composition. However, most fecal DNA metabarcoding studies have been conducted in mammals and birds; few have focused on marine animals, including bivalves. We performed a pilot study of the diet composition and its variation over four seasons in the Pacific oyster Magallana gigas, using eight fecal samples from two aquaculture farms, Tongyeong and Gadeokdo, in South Korea. The diversity and stability of M. gigas dietary composition were higher in Tongyeong than in Gadeokdo; they were highest in fall and lowest in winter. Taxonomic analysis identified 14 phyla, 23 classes, 43 orders, 53 families, 56 genera, and 82 species. Among them, Dinoflagellates (Dinoflagellata) and diatoms (Bacillariophyta) were predominant, together with green algae (Chlorophyta), protists (Bigyra and Cercozoa), parasites (Apicomplexa), and flatworms (Platyhelminthes) in both Tongyeong and Gadeokdo at the phylum level, based on the frequency of occurrence (FOO). High proportions of Arthropoda and Mollusca were found, indicating no selective feeding by M. gigas. At the species level, many harmful dinoflagellates were detected in both Tongyeong and Gadeokdo. In addition, there were strong similarities in the M. gigas diet composition between Tongyeong and Gadeokdo according to seasons. An additional step to cleave or block oyster DNA and avoid cross-contamination are recommended. Our findings indicated the ability of fecal DNA metabarcoding to provide qualitative and semi-quantitative information regarding the M. gigas diet, which will assist in the formulation of aquaculture management guidelines.
GASTROPODS
Boyer S., Yeates G. W., Wratten S. D., Holyoake A., Cruickshank R. H. (2011): Molecular and morphological analyses of faeces to investigate the diet of earthworm predators: Example of a carnivorous land snail endemic to New Zealand. Pedobiologia 54: S153-S158.
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Ecosystem services provided by earthworms include increasing soil fertility, recycling organic material and provision of food for numerous predatory species. Estimating the role of earthworms in food webs is essential for a thorough evaluation of the ecosystem services they provide. Although their soft bodies are rapidly digested, earthworm remains can be detected via their chaetae in the faeces of predators. This study proposes a method for the identification of predated earthworm species using only predator faeces. We conducted morphological and molecular analyses of earthworm remains in the faeces of a highly endangered carnivorous land snail (Powelliphanta augusta) endemic to a restricted area of New Zealand. Microscopic examination of faecal strings from P. augusta showed the presence of earthworm chaetae in 47 of the 49 strings examined, suggesting an important role for earthworms in snail diets. Although earthworm tissue was not detectable in snail faeces, earthworm DNA was still present in sufficient quantity for molecular identification. Results showed that this land snail feeds on at least five species of New Zealand endemic earthworms. The method developed here is particularly appropriate for investigating the diet of potential invertebrate and vertebrate predators for which feeding behaviour is difficult to observe and gut content analysis is not possible. Such studies can help to develop a more complete understanding of the ecosystem services provided by earthworm communities. In this case it also helps the conservation of a rare and endangered predatory snail species.
Boyer S., Wratten S. D., Holyoake A., Abdelkrim J., Cruickshank R. H. (2013): Using next-generation sequencing to analyse the diet of a highly endangered land snail (Powelliphanta augusta) feeding on endemic earthworms. Plos One 8: e75962.
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Predation is often difficult to observe or quantify for species that are rare, very small, aquatic or nocturnal. The assessment of such species’ diet can be conducted using molecular methods that target prey DNA remaining in predators’ guts and faeces. These techniques do not require high taxonomic expertise, are applicable to soft-bodied prey and allow for identification at the species level. However, for generalist predators, the presence of mixed prey DNA in guts and faeces can be a major impediment as it requires development of specific primers for each potential prey species for standard (Sanger) sequencing. Therefore, next generation sequencing methods have recently been applied to such situations. In this study, we used 454-pyrosequencing to analyse the diet of Powelliphanta augusta, a carnivorous landsnail endemic to New Zealand and critically endangered after most of its natural habitat has been lost to opencast mining. This species was suspected to feed mainly on earthworms. Although earthworm tissue was not detectable in snail faeces, earthworm DNA was still present in sufficient quantity to conduct molecular analyses. Based on faecal samples collected from 46 landsnails, our analysis provided a complete map of the earthworm-based diet of P. augusta. Predated species appear to be earthworms that live in the leaf litter or earthworms that come to the soil surface at night to feed on the leaf litter. This indicates that P. augusta may not be selective and probably predates any earthworm encountered in the leaf litter. These findings are crucial for selecting future translocation areas for this highly endangered species. The molecular diet analysis protocol used here is particularly appropriate to study the diet of generalist predators that feed on liquid or soft-bodied prey. Because it is non-harmful and non-disturbing for the studied animals, it is also applicable to any species of conservation interest.
Waterhouse B. R., Boyer S., Wratten S. D. (2014): Pyrosequencing of prey DNA in faeces of carnivorous land snails to facilitate ecological restoration and relocation programmes. Oecologia 175: 737-746.
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Identifying and understanding predator diets is of high importance in biological conservation. This is particularly true for the introduction, establishment and maintenance of predator populations in newly created or modified ecological communities, such as translocation sites or restored habitats. Conservation status of predators may not permit captive feeding trials or intrusive gut-content methods, so non-intrusive diet assessment is required, such as faecal analysis. However, prey such as earthworms leave no morphological clues suitable for accurately discriminating between species consumed through visual faecal analysis. This study uses non-intrusive molecular methods on earthworm DNA extracted from the faeces of the carnivorous land snail Powelliphanta patrickensis to identify its earthworm diet and any seasonal trends. Data from 454-pyrosequencing revealed earthworm DNA in all samples (n = 60). Sequences were compared to a DNA library created from published and unpublished studies of New Zealand’s endemic earthworms and online databases. Unidentified earthworm sequences were clustered into molecular operational taxonomic units (MOTUs). Twenty-six MOTUs were identified, 17 of which matched the library, whereas nine did not. Similarity indices indicate that there were seasonal differences (P < 0.05) in the earthworm communities represented in the summer and the winter diets. This study highlights the importance of utilising the vast body of data generated by pyrosequencing to investigate potential temporal diet shifts in protected species. The method described here is widely applicable to a wide range of predatory species of conservation interest and can further inform habitat restoration and relocation programmes to optimize the long-term survival of the target species.
O’Rorke R., Cobian G. M., Holland B. S., Price M. R., Costello V., Amend A. S. (2015): Dining local: the microbial diet of a snail that grazes microbial communities is geographically structured. Environmental Microbiology 17: 1753-1764.
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Achatinella mustelina is a critically endangered tree snail that subsists entirely by grazing microbes from leaf surfaces of native trees. Little is known about the fundamental aspects of these microbe assemblages: not taxonomic composition, how this varies with host plant or location, nor whether snails selectively consume microbes. To address these questions, we collected 102 snail faecal samples as a proxy for diet, and 102 matched‐leaf samples from four locations. We used Illumina amplicon sequencing to determine bacterial and fungal community composition. Microbial community structure was significantly distinct between snail faeces and leaf samples, but the same microbes occurred in both. We conclude that snails are not ‘picky’ eaters at the microbial level, but graze the surface of whatever plant they are on. In a second experiment, the gut was dissected from non‐endangered native tree snails in the same family as Achatinella to confirm that faecal samples reflect gut contents. Over 60% of fungal reads were shared between faeces, gut and leaf samples. Overall, location, sample type (faeces or leaf) and host plant identity all significantly explained the community composition and variation among samples. Understanding the microbial ecology of microbes grazed by tree snails enables effective management when conservation requires captive breeding or field relocation.
O’Rorke R., Holland B. S., Cobian G. M., Gaughen K., Amend A. S. (2016): Dietary preferences of Hawaiian tree snails to inform culture for conservation. Biological Conservation 198: 177-182.
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One strategy to safeguard endangered species against extinction is raising subpopulations in ex situ facilities. Feeding animals ex situ is difficult when their diet is cryptic. We present a combined molecular and behavioral approach to assess the diet of Achatinella, a critically endangered genus of tree snail, to determine how diet of captive snails differs from wild snails. Cultured snails are currently fed biofilms growing on leaf surfaces, as well as a Cladosporium fungus isolated from this same habitat. Amplicon sequencing of DNA extracted from feces of wild and cultured snails confirms that this Cladosporium is abundant in the wild (~ 1.5% of sequences), but it dominates the ex situ snails’ diet (~ 38%) and the diet of captive snails is still significantly less diverse than wild snails. To test the hypothesis that snails have diet preferences, we conducted feeding trials. These used a surrogate snail species, Auriculella diaphana, which is a confamilial Oahu endemic, though non-federally listed. Contrary to our expectations we found that snails do have feeding preferences. Furthermore, our feeding preference trials show that over all other feeding options snails most preferred the “no-microbe” control, which consisted only of potato dextrose agar (PDA). PDA is rich in simple carbohydrates, in contrast to the oligotrophic environment of wild tree-snails. These results suggest further research should focus on calorie budgets of snails, devising new approaches to supplementing their ex situ diet and determining whether a wild diet is an optimum diet.
O’Rorke R., Tooman L., Gaughen K., Holland B. S., Amend A. S. (2017): Not just browsing: an animal that grazes phyllosphere microbes facilitates community heterogeneity. The ISME Journal 11: 1788-1798.
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Although grazers have long been recognized as top–down architects of plant communities, animal roles in determining microbial community composition have seldom been examined, particularly in aboveground systems. To determine the extent to which an animal can shape microbial communities, we conducted a controlled mesocosm study in situ to see if introducing mycophageous tree snails changed phyllosphere fungal community composition relative to matched control mesocosms. Fungal community composition and change was determined by Illumina sequencing of DNA collected from leaf surfaces before snails were introduced, daily for 3 days and weekly for 6 weeks thereafter. Scanning electron microscopy was used to confirm that grazing had occurred, and we recorded 3.5 times more cover of fungal hyphae in control mesocosms compared with those containing snails. Snails do not appear to vector novel microbes and despite grazing, a significant proportion of the initial leaf phyllosphere persisted in the mesocosms. Within-mesocosm diversities of fungi were similar regardless of whether or not snails were added. The greatest differences between the snail-treated and control mesocosms was that grazed mesocosms showed greater infiltration of microbes that were not sampled when the experiment commenced and that the variance in fungal community composition (beta diversity) was greater between leaves in snail-treated mesocosms indicating increased community heterogeneity and ecosystem fragmentation.
Price M. R., O’Rorke R., Amend A. S., Hadfield M. G. (2017): Diet selection at three spatial scales: Implications for conservation of an endangered Hawaiian tree snail. Biotropica 49: 130-136.
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Several recent studies suggest local adaptation in multiple taxa across Hawaii’s steep environmental gradients. Restoration efforts in devastated tropical island ecosystems may be deficient if we lack an understanding of the interactions and dependencies in communities that occur along these gradients. Endangered Hawaiian tree snails are part of a snail–epiphyte–plant system where they graze fungi and other microbes on the leaf surface, a process difficult to observe using conventional techniques. Tree snails have undergone catastrophic decline due to introduced predators, removal by shell collectors, and human‐influenced habitat degradation. Prior to this study, little was known about the relationship among tree‐snails, their host plants, and the epiphytic microbes on which they feed. In this study, we identified scale‐dependent selection of substrates in Achatinella sowerbyana and Achatinella lila across the species’ ranges. We assessed: (1) within‐plant diet selection using high‐throughput DNA sequencing (micro‐scale); (2) among‐plant selection of tree host species (small‐scale); (3) and the influence of climate on this system (macro‐scale). Selection of substrates occurred at two scales: fungal communities in fecal samples differed in composition from those available on leaf surfaces; and at all sites, snail occurrence on Metrosideros polymorpha, a foundational forest plant, was significantly higher than expected based on availability. Habitat restoration efforts should focus on out‐planting of M. polymorpha, the preferred snail host tree, in degraded habitat. Fungal differences across sites suggest relocation efforts to predator‐free enclosures may be hindered by microbial shifts associated with geographic distance or differing environments.