environmental-dna-sampling-from-whale-watching-vessels-for-cetacean

Environmental DNA Sampling from Whale-Watching Vessels for Cetacean Monitoring

Environmental DNA (eDNA) offers a non-invasive approach for monitoring cetaceans, yet its widespread application from operational platforms requires ...

University of Innsbruck

WasserCluster Lunz - Biologische Station GmbH

Molecular methods allow noninvasive assessment of vertebrate predator-prey systems at high taxonomic resolution by examining dietary samples such as faeces and pellets. To facilitate the interpretation of field-derived data, feeding trials, investigating the impacts of biological, methodological and environmental factors on prey DNA detection, have been conducted. The effect of meal size, however, has not yet been explicitly considered for vertebrate consumers. Moreover, different noninvasively obtained sample types remain to be compared in such experiments. Here, we present a feeding trial on abundant piscivorous birds, Great Cormorants (Phalacrocorax carbo), to assess meal size effects on postfeeding prey DNA detection success. Faeces and pellets were sampled twice a day after the feed of large (350-540 g), medium (190-345 g) and small (15-170 g) fish meals contributing either a large (>79%) or small (<38%) share to the daily consumption. Samples were examined for prey DNA and fish hard parts. Molecular analysis of faeces revealed that both large meal size and share had a significantly positive effect on prey DNA detection rate postfeeding. Furthermore, large meals were detectable for a significantly longer time span with a detection limit at ~76 hr and a 50% detection probability at ~32 hr postfeeding. In pellets, molecular methods reliably identified the meal consumed the previous day, which was not possible via morphological analysis or when examining individual faeces. The less reliable prey DNA detection of small meals or meal shares in faeces signifies the importance of large numbers of dietary samples to obtain reliable trophic data.

The influence of meal size on prey DNA detectability in piscivorous birds.

Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory.

Rapid plant identification using species- and group-specific primers targeting chloroplast DNA.

When using the eddy covariance (EC) method for measuring the ecosystem-atmosphere exchange of sensible and latent heat, it is not uncommon to find that these two energy fluxes fall short of available energy by 20-30 %. As the causes for the energy imbalance are still under discussion, it is currently not clear how the energy balance should be closed. The objective of the present paper is to use independent measurements of evapotranspiration (ET) for empirically devising on how to best close the energy balance. To this end ET of a temperate mountain grassland was quantified during two measurement campaigns using both an open- and a closed-path EC system, lysimeters and an approach scaling up leaf-level stomatal conductance to canopy level transpiration. Our study showed that both EC systems underestimated ET measured independently by lysimeters and the up-scaling approach. Best correspondence to independently measured ET was achieved by assigning the entire energy imbalance to ET and by adjusting ET according to the average energy balance ratio during the first and second measurement campaign, respectively. Due to a large spatial variability in ET during the first measurement campaign and given large differences in spatial scale between the EC and the independent methods, we are more confident with the comparison of approaches during the second measurement campaign and thus recommend forcing energy balance closure by adjusting for the average energy balance ratio.

Multiple constraints on grassland evapotranspiration: implications for closing the energy balance.

Diet analysis is an important aspect when investigating the ecology of fish-eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time-consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two-step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species-specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field-collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost-effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity.

Molecular prey identification in Central European piscivores.

Avian carcasses can provide important information on the trophic ecology of birds. Usually, the number of carcasses available for examination is limited and therefore it is important to gain as much dietary information per specimen as possible. In piscivorous birds and raptors, the stomach has been the primary source of dietary information, whereas the gut (intestine) has so far been neglected as it usually contains only a few morphologically identifiable hard parts of prey. Molecular approaches have the potential to retrieve dietary information from the gut, although this has not yet been verified. As well as identifying the prey, it is important to estimate any secondary predation to avoid food web errors in dietary analyses. The assignment of accidentally consumed prey is notoriously difficult regardless of the prey identification approach used. In the present study, morphological and molecular analyses were, for the first time, combined to maximize the dietary information retrievable from the complete digestive tract of Great Cormorants . Moreover, a novel approach based on predator-prey size ratios was applied to these piscivorous birds to minimize the number of samples that might contain secondarily predated prey. The stomach contents of the examined birds were found to provide the most dietary information when morphological and molecular analyses were used in combination. However, compared with the morphological approach, the molecular analysis increased the number of fish species detected by 39%. The molecular approach also permitted the identification of fish DNA in the Cormorant guts. Predator-prey size ratios derived from morphological analysis of fish hard parts can reduce the incidence of potential confounding influence of secondarily predated prey by 80%. Our findings demonstrate that a combination of morphological and molecular approaches maximizes the trophic information retrievable from bird carcasses.

Maximizing dietary information retrievable from carcasses of Great Cormorants  using a combined morphological and molecular analytical approach.

In trophic studies on piscivorous birds, it is vital to know which kind of dietary sample provides the information of interest and how the prey can be identified reliably and efficiently. Often, noninvasively obtained dietary samples such as regurgitated pellets, feces, and regurgitated fish samples are the preferred source of information. Fish prey has usually been identified via morphological analysis of undigested hard parts, but molecular approaches are being increasingly used for this purpose. What remains unknown, however, is which dietary sample type is best suited for molecular diet analysis and how the molecular results compare to those obtained by morphological analysis. Pellets, feces, and regurgitated fish samples of Great Cormorants () were examined for prey using both morphological hard part analysis and molecular prey identification. The sample types and methods were compared regarding number of species detected (overall and per sample) as well as the prey species composition and its variability among individual samples. Via molecular analysis, significantly higher numbers of prey species were detected in pellets, feces, and fish samples. Of the three sample types, pellets contained the most comprehensive trophic information and could be obtained with the lowest sampling effort. Contrastingly, dietary information obtained from feces was least informative and most variable. For all sample types, the molecular approach outperformed morphological hard part identification regarding the detectable prey spectrum and prey species composition. We recommend the use of pellets in combination with molecular prey identification to study the diet of piscivorous birds.

Diet analysis in piscivorous birds: What can the addition of molecular tools offer?

Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next-generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high-quality DNA extraction procedures for obtaining the minute quantities of short-fragmented food DNA. Automatic high-throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole-body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high-throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor-intensive, low-throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost-efficient and innovative methodology at low contamination risk also in trophic ecology.

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples.

Piscivorous birds frequently display sex-specific differences in their hunting and feeding behavior, which lead to diverging impacts on prey populations. Cormorants (Phalacrocoracidae), for example, were previously studied to examine dietary differences between the sexes and males were found to consume larger fish in coastal areas during autumn and winter. However, information on prey partitioning during breeding and generally on sex-specific foraging in inland waters is missing. Here, we assess sex-specific prey choice of Great Cormorants () during two subsequent breeding seasons in the Central European Alpine foreland, an area characterized by numerous stagnant and flowing waters in close proximity to each other. We developed a unique, noninvasive approach and applied it to regurgitated pellets: molecular cormorant sexing combined with molecular fish identification and fish-length regression analysis performed on prey hard parts. Altogether, 364 pellets delivered information on both, bird sex, and consumed prey. The sexes differed significantly in their overall prey composition, even though , , and  spp. represented the main food source for both. Albeit prey composition did not indicate the use of different water bodies by the sexes, male diet was characterized by higher prey diversity within a pellet and the consumption of larger fish. The current findings show that female and male cormorants to some extent target the available prey spectrum at different levels. Finally, the comprehensive and noninvasive approach has great potential for application in studies of other piscivorous bird species.

Sex-specific prey partitioning in breeding piscivorous birds examined via a novel, noninvasive approach.

Potamodromous fish are considered important indicators of habitat connectivity in freshwater ecosystems, but they are globally threatened by anthropogenic impacts. Hence, non-invasive techniques are necessary for monitoring during spawning migrations. The use of environmental DNA (eDNA) potentially facilitates these efforts, albeit quantitative examinations of spawning migrations remain so far mostly uncharted. Here, we investigated spawning migrations of Danube bleak, Alburnus mento, and Vimba bream, Vimba vimba, and found a strong correlation between daily visual fish counts and downstream eDNA signals obtained from filtered water samples analysed with digital PCR and end-point PCR coupled with capillary electrophoresis. By accounting for daily discharge fluctuations, it was possible to predict eDNA signal strength from the number of migrating fish: first, the whole spawning reach was taken into account. Second, the model was validated using eDNA signals and fish counts obtained from the upper half of the examined river stretch. Consequently, fish counts and their day-to-day changes could be described via an eDNA-based time series model for the whole migration period. Our findings highlight the capability of eDNA beyond delivering simple presence/absence data towards efficient and informative monitoring of highly dynamic aquatic processes such as spawning migrations of potamodromous fish species.

Monitoring spawning migrations of potamodromous fish species via eDNA.

Fish are both consumers and prey, and as such part of a dynamic trophic network. Measuring how they are trophically linked, both directly and indirectly, to other species is vital to comprehend the mechanisms driving alterations in fish communities in space and time. Moreover, this knowledge also helps to understand how fish communities respond to environmental change and delivers important information for implementing management of fish stocks. DNA-based methods have significantly widened our ability to assess trophic interactions in both marine and freshwater systems and they possess a range of advantages over other approaches in diet analysis. In this review we provide an overview of different DNA-based methods that have been used to assess trophic interactions of fish as consumers and prey. We consider the practicalities and limitations, and emphasize critical aspects when analysing molecular derived trophic data. We exemplify how molecular techniques have been employed to unravel food web interactions involving fish as consumers and prey. In addition to the exciting opportunities DNA-based approaches offer, we identify current challenges and future prospects for assessing fish food webs where DNA-based approaches will play an important role.

Fish as predators and prey: DNA-based assessment of their role in food webs.

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.

Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.

Piscivorous birds in aquatic ecosystems exert predation pressure on fish populations. But the site-specific impact on fish populations, including stocked and commercially used fish species, remains disputed. One of the key questions for the management of piscivorous birds and fish is determining the origin of prey and thus which fish populations are targeted by the birds. We addressed this question by provenancing otoliths (earstones) of fish obtained from regurgitated pellets of piscivorous birds by otolith microchemistry analysis. We retrieved otoliths from regurgitated pellets of great cormorants () collected every 2 weeks for 2 years from breeding and roosting colonies at Chiemsee in Bavaria, Germany, and classified them according to family or species. We collected water samples from Chiemsee and potential surrounding foraging grounds. We measured the strontium (Sr) Sr/Sr isotope ratio and Sr mass fraction of water and otoliths using (laser ablation) inductively coupled plasma-mass spectrometry. We assigned otoliths from regurgitated pellets to habitat clusters of origin by comparing the Sr isotopic and elemental composition of otoliths and waterbodies. In 36% of cormorant pellets collected at Chiemsee, prey was assigned to waterbodies distinct from Chiemsee. Furthermore, cormorants used different foraging sites during 1 day. Microchemical provenancing of prey remains can contribute to identifying foraging sites of piscivorous birds and to what extend the birds switched among foraging sites.

Microchemical provenancing of prey remains in cormorant pellets reveals the use of diverse foraging grounds.

The AquaEcOmics meeting brought together 280 scientists applying omics tools to aquatic research in March 2025 (Evian-les-Bains, France). We synthesised here the main outcomes from the 167 presentations which were given. A similar number of presentations were about micro- and macroorganisms. While studies on macroorganisms mostly focused on stakeholder-driven research and methodology development, studies on microorganisms tended to focus on fundamental scientific questions. These questions mostly contributed to community ecology studies using metabarcoding; functional ecology studies using metagenomics, metatranscriptomics and metabolomics; and to a lesser degree population genomics studies using metabarcoding and RADseq, ddRADseq. Stakeholder-driven research presentations could be clustered in two groups: those using omics to replace existing standardised monitoring methods-also termed 'retrofitting'-to meet regulatory frameworks (e.g., fish biomonitoring), and those investigating anthropogenic pressures by harnessing the full power of omics, for instance by sequencing the entire microbial diversity or by detecting resistance genes to antibiotics and metals in rivers. The carbon footprint and applicability of omics were also questioned, sparking animated debates among attendees. Finally, some presentations focused on methodological developments and encompassed sampling strategies and devices, issues related to reference libraries (completeness, curation), comparison of short reads versus long reads, and in situ detection and quantification of organisms (dPCR, models). The use of omics to study aquatic ecosystems is a fast-evolving field. Meeting attendees agreed to maintain the generated momentum to promote omics as tools to improve the monitoring and protection of our environment.

Omics to Study and Manage Aquatic Environments: A Snapshot From the AquaEcOmics Meeting (Evian-les-Bains, 2025).

Marine biodiversity faces challenges due to climate change, pollution, and human activities. Conservation efforts are often constrained by limited resources and public disengagement. This study evaluates the potential benefits of incorporating environmental DNA (eDNA) sampling and citizen science into whale-watching tours to raise environmental awareness and promote marine conservation. A Likert-scale questionnaire evaluating attitudes, knowledge, and willingness to contribute to conservation through financial support, behavioral changes, and volunteering was administered to 224 participants across the Pelagos Sanctuary (Italy), the Azores (Portugal), and Skjálfandi Bay (Iceland). Environmental awareness, interest in environmental DNA (eDNA), and higher education levels were identified as significant predictors of willingness to pay (WTP). Conversely, higher ticket prices negatively impacted financial commitment. The majority of respondents reported an increased awareness of marine biodiversity and eDNA, along with a strong sense of personal responsibility toward marine protection. The study outlined various participant profiles, indicating how age, education, and ecotourism experience influence conservation attitudes. Specifically, younger individuals who are highly educated and have prior whale-watching experience were more inclined to provide financial support and engage in citizen science activities. These findings demonstrate that integrating citizen science-based activities and environmental DNA (eDNA) sampling, into whale-watching tours is an effective way to foster meaningful pro-environmental engagement. This model presents a scalable and cost-effective approach to enhancing public participation in marine conservation and biodiversity monitoring, with potential applications extending to broader marine governance and ecotourism settings.

Article	Year
The influence of meal size on prey DNA detectability in piscivorous birds. Molecular ecology resources\| PubMed ID: 28776942	2017
Rapid plant identification using species- and group-specific primers targeting chloroplast DNA. PloS one\| PubMed ID: 22253728	2012
Multiple constraints on grassland evapotranspiration: implications for closing the energy balance. Vadose zone journal : VZJ\| PubMed ID: 24339743	2010
Molecular prey identification in Central European piscivores. Molecular ecology resources\| PubMed ID: 26053612	2016
Maximizing dietary information retrievable from carcasses of Great Cormorants using a combined morphological and molecular analytical approach. The Ibis\| PubMed ID: 26877544	2016
Diet analysis in piscivorous birds: What can the addition of molecular tools offer? Ecology and evolution\| PubMed ID: 28331605	2017
Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples. Ecology and evolution\| PubMed ID: 28861241	2017
Sex-specific prey partitioning in breeding piscivorous birds examined via a novel, noninvasive approach. Ecology and evolution\| PubMed ID: 30271560	2018
Monitoring spawning migrations of potamodromous fish species via eDNA. Scientific reports\| PubMed ID: 31659232	2019
Fish as predators and prey: DNA-based assessment of their role in food webs. Journal of fish biology\| PubMed ID: 32441321	2021
Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions. PloS one\| PubMed ID: 34297715	2021
Microchemical provenancing of prey remains in cormorant pellets reveals the use of diverse foraging grounds. The Journal of wildlife management\| PubMed ID: 36246203	2022
Omics to Study and Manage Aquatic Environments: A Snapshot From the AquaEcOmics Meeting (Evian-les-Bains, 2025). Molecular ecology\| PubMed ID: 40698877	2025
Willingness to engage in marine conservation through eDNA-informed citizen science on whale-watching platforms. Scientific reports\| PubMed ID: 41290929	2025

Bettina Thalinger

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