Evaluating the potential of cetacean blow for non-invasive dietary DNA analysis

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Kita, Koji Kanda, Atsushi Yoshinaka, Hiroshi Ohizumi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7191423/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study proposes the utility of blow sampling for DNA analysis as a non-invasive approach to examine the feeding ecology of cetaceans. Conventional methods such as stomach content analysis and stable isotope profiling, although informative, are inherently invasive and have notable limitations. In contrast, blow analysis offers a non-invasive alternative and, when integrated with unmanned aerial vehicles, facilitates the assessment of endocrine function and health status in free-ranging whales. In this study, fish- and bacteria-derived DNA was detected in the blows of captive orcas and dolphins, indicating that breath may contain trace amounts of dietary DNA. Additionally, detection accuracy was enhanced through DNA purification and repeated PCR amplification. These findings suggest that blow sampling represents a promising tool for dietary analysis in wild cetaceans. Biological sciences/Biological techniques Biological sciences/Ecology Earth and environmental sciences/Ecology Earth and environmental sciences/Ocean sciences Biological sciences/Zoology Cetacean blow food-derived DNA metagenomic analysis non-invasive method Figures Figure 1 Introduction Understanding the feeding ecology of marine mammals is essential for elucidating their habitat use and life history strategies 1 , 2 . In small cetaceans, dietary information can be obtained using five primary methods: (1) stomach content analysis 3 , 4 , (2) stable isotope analysis 4 – 7 , (3) fatty acid analysis 7 , (4) fecal mitochondrial DNA (mtDNA) analysis 8 , and (5) direct observation 5 , 9 . Among these, stomach content analysis is the most widely used, yet it has several limitations. Prey identification based on undigested hard parts, such as otoliths, bones, and cephalopod beaks, is prone to bias due to the rapid and differential digestion rates of various prey items 10 . Furthermore, given the scarcity of deceased individuals from top predators such as whales, the dietary data derived from methods (1) to (3) may not be representative of the population as a whole. Non-invasive techniques, including fecal mtDNA analysis and direct observation, have gained attention as alternatives. However, fecal sampling (method 4) requires immediate collection post-defecation, limiting sampling opportunities, while direct observation (method 5) is only feasible in warm waters where researchers can swim alongside the animals. Cetaceans, like other mammals, exhale forcefully when surfacing, producing a visible "blow" or "spout" composed of exhaled condensate. Over the past decade, this blow has been utilized as a non-invasive biological sampling source 11 . The first application of blow sampling in cetacean research was by Hogg (2005) 12 , who used it to examine reproductive hormones 12 , 13 . Since then, blow analysis has proven useful for assessing the health and disease status of wild whales 14 . More recently, unmanned aerial systems (UAS) have enabled the collection of exhaled breath from free-ranging whales, allowing endocrine assessments, DNA profiling, microbial metabarcoding, and metagenomic analyses 15 . In contrast, studies on small odontocetes remain limited. Although DNA has been successfully recovered from the blow of captive bottlenose dolphins ( Tursiops truncatus ) 16 , only a single instance of successful detection in a wild individual, specifically a short-beaked common dolphin ( Delphinus delphis ), has been reported to date 17 . Our research team conducts field surveys in regions such as the Nemuro Strait off Rausu (Shiretoko) and the Muroran coast to investigate the ecology of wild cetaceans, including killer whales ( Orcinus orca ) and Pacific white-sided dolphins ( Lagenorhynchus obliquidens ) that inhabit the waters around Hokkaido 18 – 20 . In ecological studies, non-invasive techniques are indispensable for population genetic analyses. As part of our ongoing efforts to establish such methods using the exhalations of captive cetaceans, we detected DNA likely originating from their dietary intake. Here, we report our findings on this novel application of breath sampling for dietary analysis. Results Metagenomic analysis using unpurified DNA products Meta-barcoding analysis of exhaled DNA without purification yielded 24,627 reads from a captive killer whale ( O. orca 1; Oror-1) and 37,965 reads from a captive Pacific white-sided dolphin ( L. obliquidens 1; Laob-1) (Table 1 ). Of these, host-derived sequences accounted for 24,116 reads (Oror-1; 97.9%) and 36,962 reads (Laob-1; 97.1%), while cetacean-specific sequences totaled 445 reads (Oror-1) and 947 reads (Laob-1). In Oror-1, two bacterial taxa, Alcaligenes sp. (50 reads) and Pseudomonas sp. (16 reads), were identified. In Laob-1, sequences corresponding to two fish species ( Trachurus japonicus , 35 reads; Scomber japonicus , 29 reads) and four bacterial taxa ( Bacteroides sp., 22 reads; Morganella sp., 38 reads; Proteus sp., 2 reads; Vibrio sp., 13 reads) were detected (Table 2 ). Table 1 Summary of DNA purification steps, the number of first-round PCR replicates, and the sequencing reads obtained from blow samples of captive killer whales and Pacific white-sided dolphins. "y" indicates "yes"; "n" indicates "no." Species ID Cleanup DNA 1stPCR Num. Read Orcinus orca Oror-1 n 1 24,627 Oror-2 y 8 34,794 Lagenorhynchus obliquidens Laob-1 n 1 37,965 Laob-2 y 8 40,727 Table 2 Taxonomic assignment of host, dietary, bacterial, and tick DNA detected in blow samples via DNA metabarcoding, along with the number of sequencing reads obtained per sample. Classification Species Oror-1 Oror-2 Laob-1 Laob-2 Total host Orcinus orca 24,116 33,914 - - 58,030 Lagenorhynchus obliquidens - - 36,879 39,962 76,841 cetacean 445 763 947 723 2,878 dietary Ammodytes sp. - - - 6 6 Pleurogrammus azonus - 102 - 6 108 Trachurus japonicus - - 35 26 61 Scomber japonicus - - 29 - 29 bacteria Alcaligenes sp. 50 - - - 50 Bacteroides sp. - - 22 - 22 Morganella sp. - - 38 - 38 Proteus sp. - - 2 - 2 Pseudomonas sp. 16 - - - 16 Vibrio sp. - - 13 - 13 tick Tyrophagus sp. - - - 4 4 Contamination Human - 15 - - 15 Total 24,627 34,794 37,965 40,727 138,113 Metagenomic analysis following DNA purification and first-round PCR with eight replicates Meta-barcoding analysis of exhaled DNA following PCR inhibitor removal and eight replicates of first-round PCR yielded 34,794 reads from Oror-2 and 40,727 reads from Laob-2 (Table 1 ). Of these, host-derived sequences represented 33,914 reads (Oror-2; 97.5%) and 39,962 reads (Laob-2; 98.1%), while cetacean-specific sequences accounted for 763 reads (Oror-2) and 723 reads (Laob-2). In Oror-2, Pleurogrammus azonus (102 reads) and human-derived sequences (15 reads) were identified. In Laob-2, sequences from three fish taxa, Ammodytes sp. (6 reads), P. azonus (6 reads), and T. japonicus (26 reads), and one mite species ( Tyrophagus sp., 4 reads) were detected (Table 2 ). Discussion On the day of blow sample collection, the killer whale at the Port of Nagoya Public Aquarium was fed 39 kg/day of P. azonus (52.7%), 13 kg/day of Clupea pallasii (17.6%), and 22 kg/day of S. japonicus (29.7%). At Noboribetsu Marine Park Nixe, the Pacific white-sided dolphin received 1.3 kg/day of Ammodytes sp. (21.7%), 2.3 kg/day of P. azonus (38.3%), 1.2 kg/day of T. japonicus (20.0%), and 1.2 kg/day of S. japonicus (20.0%). In the killer whale, only the Oror-2 sample, which underwent cleanup purification and was subjected to eight replicates of the first PCR, yielded detectable levels of P. azonus . In the Pacific white-sided dolphin, two species were detected in Laob-1 (unpurified), whereas three species were identified in Laob-2 (purified sample) (Table 2 ). Cetaceans exhibit a cranial morphology characterized by the telescoping of skull bones, resulting in a dorsally positioned larynx that maintains near-complete anatomical separation from the pharynx 21 , 22 . However, recent findings have revealed the presence of an “oral plug” in the fin whale ( Balaenoptera physalus ), which passively separates the larynx from the pharynx 23 . Additionally, small food particles have occasionally been observed in exhaled droplets from captive individuals (Kanda, pers. comm.). While not observed in the individuals used in this study, some captive killer whales have been noted to release air through the oral cavity (Fig. 1 ). These observations suggest that odontocetes, unlike mysticetes, may lack a complete septum separating the airway and esophagus at the pharyngeal level. In the present study, although blow sampling from the killer whale was performed in a pool, the individual expelled residual seawater from the blowhole prior to sample collection. For the Pacific white-sided dolphin, sampling was conducted while the individual was in a stationary position at the poolside, under trainer instruction. Given these controlled conditions, the likelihood of seawater contamination by environmental DNA (e.g., fecal matter) was considered minimal. Collectively, these findings support the hypothesis that cetacean blow may contain trace amounts of prey-derived DNA. In samples without DNA purification, bacterial sequences were detected; however, after purification, sequences derived from ticks and humans were identified, with no bacterial DNA present. In the killer whale, prey-derived sequences were observed only following DNA purification, whereas in the Pacific white-sided dolphin, purification increased the number of prey species detected (Table 2 ). PCR inhibition is commonly attributed to interference with DNA polymerase activity by environmental contaminants, such as humic substances (e.g., humic acid, fulvic acid, and tannic acid). These inhibitors are prevalent in aquatic environments and must be efficiently removed during nucleic acid extraction to ensure reliable environmental DNA (eDNA) analysis 24 – 29 . Additionally, strong PCR bias can occur during the first amplification step, particularly when using gene-specific primers such as those targeting the mtDNA cytochrome c oxidase I ( COI ) gene. Therefore, it is recommended to conduct at least four, and preferably eight, PCR replicates to enhance detection accuracy 30 . Accordingly, for metagenomic analysis of blow-derived DNA, it is essential to eliminate potential inhibitors and perform a minimum of eight first-round PCR replicates. Despite these precautions, only one prey species (33.3%) was detected in the killer whale sample (Oror-2), whereas all four species were detected in the Pacific white-sided dolphin samples. Notably, the number of sequencing reads for Ammodytes sp. and P. azonus was limited to six each, possibly too low to be considered robust detections (Table 2 ). In fecal DNA-based dietary analyses, mitochondrial markers such as COI , 12S rRNA, and 16S rRNA are commonly employed 31 , often alongside blocking primers designed to suppress host DNA amplification 8 , 32 , 33 . Although blocking primers were not utilized in this study, prey-derived sequences were detected at proportions of 0.17% in Laob-1 (unpurified dolphin sample), and 0.29% and 0.09% in Oror-2 and Laob-2 (purified killer whale and dolphin samples, respectively, with eight PCR replicates). These values may exceed those commonly obtained from fecal DNA, suggesting that the application of blocking primers could further enhance prey DNA detection in future analyses. Importantly, the relative proportions of detected prey species did not reflect the actual feed composition on the day of sampling. In the killer whale, only Atka mackerel ( P. azonus ) was detected in Oror-2 (read distribution: P. azonus = 102; C. pallasii = 0; S. japonicus = 0). For the Pacific white-sided dolphin, Laob-1 revealed the presence of T. japonicus and S. japonicus (reads: 35 and 29, respectively), while Laob-2 identified Ammodytes sp., P. azonus , and T. japonicus (reads: 6, 6, and 26, respectively), with S. japonicus undetected. As highlighted by Thomas et al. (2014) 34 , tissue-specific differences in lipid, protein, ash, and moisture content necessitate the application of Tissue Correction Factors (TCFs) and Digestion Correction Factors (DCFs) when interpreting dietary DNA results. Consequently, the number of sequencing reads in this study is unlikely to directly correlate with the biomass of prey consumed. This study is the first to report the detection of prey-derived DNA sequences in cetacean blow. Future research should address potential sources of environmental contamination, such as by feeding prey species not typically used in aquaria, and assess the utility of host- and human-specific blocking primers for improving detection sensitivity. Taken together, these findings demonstrate that blow sampling offers a promising, non-invasive tool for dietary analysis in wild cetaceans. Methods All experimental procedures were approved by the Tokai University Animal Experiment Committee (Approval No. 221049). Blow samples were collected from one killer whale ( O. orca ) at the Port of Nagoya Public Aquarium and one Pacific white-sided dolphin ( L. obliquidens ) at Noboribetsu Marine Park Nixe. The animals had been pre-trained by their respective handlers to exhale on cue. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Blow samples were collected into 50 mL tubes, as described previously 16 , and preserved in 1 mL of ATL buffer (QIAGEN Inc.). Genomic DNA was extracted using the QIAamp DNA Investigator Kit (QIAGEN Inc.) according to the manufacturer’s protocol (QIAamp DNA Investigator Handbook, January 2020). The first round of PCR targeted the COI region and was performed by Bioengineering Lab Co., Ltd. (Kanagawa, Japan) following established protocols 35 , 36 . A second PCR was conducted to append multiplex identifier (MID) tags. To evaluate the impact of PCR inhibitors on detection sensitivity, genomic DNA was divided into two groups: one subjected to inhibitor removal using the DNeasy PowerClean Pro Cleanup Kit (QIAGEN Inc.) and one untreated. For the inhibitor-removal group, the first PCR was performed in eight replicates (Table 1 ). Library quality was assessed using the Synergy H1 (Agilent Technologies Inc.) in conjunction with the QuantiFluor dsDNA System (Promega Inc.). Paired-end sequencing (2 × 300 bp) was carried out using the MiSeq Reagent Kit v3 on the MiSeq platform (Illumina Inc., San Diego, CA, USA) at Bioengineering Lab Co., Ltd. Only reads with exact matches to primer sequences at the read start were retained using the fastx_barcode_splitter tool from FASTX-Toolkit ver. 0.0.14 ( http://hannonlab.cshl.edu/fastx_toolkit/ ). Primer sequences and the 3′-terminal 50 bases were trimmed using fastx_trimmer. Low-quality reads (Phred score < 20) were removed using Sickle ver. 1.33 37 , and reads shorter than 40 bp, along with their paired reads, were discarded. Paired-end reads were merged using FLASH ver. 1.2.11 38 with a minimum required overlap of 10 bp. Chimeric and low-quality sequences were filtered using the DADA2 plugin in QIIME2 ver. 2023.7 ( https://forum.qiime2.org/t/qiime-2-2023-7-is-now-available/27432 ). Representative sequences and an amplicon sequence variant (ASV) table were subsequently generated. Taxonomic assignment of representative sequences was performed using BLASTN ver. 2.13.0 ( https://software.cqls.oregonstate.edu/updates/blast-2.13.0/ ) against the NCBI nt database (Supplementary Table 1). Declarations Competing interests The authors declare no competing interests. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS KAKENHI) Grant Number 21H02217 and the Research and Study Project of the Tokai University Research Organization Grant number PJ2022-5. Author Contribution All authors contributed to the conception and design of the study. Sample collection was performed by Kita Y.F., Kanda K., Yoshinaka A., and Ohizumi H. Data acquisition and analysis were carried out by Kita Y.F. Fig. 1 was prepared by Kanda K. The first draft of the manuscript was prepared by Kita Y.F., and all authors provided feedback on earlier versions. All authors read and approved the final manuscript. Acknowledgement We thank the Port of Nagoya Public Aquarium, Noboribetsu Marine Park Nixe, and the members of the Kita and Ohizumi Laboratories at Tokai University for their assistance in collecting blow samples. We are also grateful to Bioengineering Lab. Co., Ltd., for support with DNA analyses. This study was partially supported by JSPS KAKENHI (grant number 21H02217) and the Research and Study Project of the Tokai University Research Organization (grant number PJ2022-5). Part of the analysis was conducted at the Hokkaido Regional Research Center, Tokai University (THRRC). Data Availability The list of species detected based on blow analysis, including ASV tables and taxonomic assignments, is available in Supplementary Table 1. References Amir, O. A., Berggren, P., Ndaro, S. G. M. & Jiddawi, N. S. Feeding ecology of the Indo-Pacific bottlenose dolphin (Tursiops aduncus) incidentally caught in the gillnet fisheries off Zanzibar, Tanzania. Estuar. Coast. 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Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Kita","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3PsWoCMRzH8Z8I5xK89Q6k10f4S8DJ4qvkCFyXCk5O1QrCuRy43mMUhM6RQLrIvYI3dWqho4NDrylCp5xjofkOCfnDhySAz/cX6xWACIAQ6AJkZ8JN2OGHxPnVJHpoloaQ+SbXRGpqPuv54oYf15pOMyThCm+1m1SyFNUrH5lApgVhWCrck5PsC440N+mLYVwxQucZyCIn0cySp13O+P5MmLQTY8mjoIBx2dyStpL40JcQlRqWJpN8QJEsdctf+scP3TnNl0m41jp+P4/vtpsiq13kVtlNX87Nk7oscwkkK7stf896xkl8Pp/v3/UF1FRIrgqryqEAAAAASUVORK5CYII=","orcid":"","institution":"Tokai University","correspondingAuthor":true,"prefix":"","firstName":"Yuki","middleName":"F.","lastName":"Kita","suffix":""},{"id":500099592,"identity":"ebe9ec1f-b067-4078-aabf-623878eb4408","order_by":1,"name":"Koji Kanda","email":"","orcid":"","institution":"Port of Nagoya Public Aquarium, Nagoya Port Foundation","correspondingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Kanda","suffix":""},{"id":500099593,"identity":"5dfc1358-cf7f-4a43-a55d-29a38640eb9e","order_by":2,"name":"Atsushi Yoshinaka","email":"","orcid":"","institution":"Noboribetsu Marine Park Nixe","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Yoshinaka","suffix":""},{"id":500099594,"identity":"626b3c5b-ee04-4c26-9ace-7b5f91499ee5","order_by":3,"name":"Hiroshi Ohizumi","email":"","orcid":"","institution":"Tokai University","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Ohizumi","suffix":""}],"badges":[],"createdAt":"2025-07-23 02:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7191423/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7191423/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89484423,"identity":"ea9b1c5e-6dac-4ebd-a289-9da4ac2a41fb","added_by":"auto","created_at":"2025-08-20 12:34:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104336,"visible":true,"origin":"","legend":"\u003cp\u003eCaptive killer whales releasing air from their mouths. Photograph taken by Setoguchi at the Port of Nagoya Public Aquarium on January 27, 2019.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7191423/v1/25a6b2173231b36e9ab412c7.jpeg"},{"id":101492995,"identity":"90415ee6-0359-4da4-9402-ff61a4a3ba45","added_by":"auto","created_at":"2026-01-30 11:25:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":702281,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7191423/v1/29f6958a-8089-4de4-ac22-e3cc647eb098.pdf"},{"id":89484420,"identity":"417a140a-50a7-4b3b-9152-6bcab34c0125","added_by":"auto","created_at":"2025-08-20 12:34:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23586,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7191423/v1/24c41020bab4c3a56153c500.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating the potential of cetacean blow for non-invasive dietary DNA analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUnderstanding the feeding ecology of marine mammals is essential for elucidating their habitat use and life history strategies\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In small cetaceans, dietary information can be obtained using five primary methods: (1) stomach content analysis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, (2) stable isotope analysis\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, (3) fatty acid analysis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, (4) fecal mitochondrial DNA (mtDNA) analysis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and (5) direct observation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAmong these, stomach content analysis is the most widely used, yet it has several limitations. Prey identification based on undigested hard parts, such as otoliths, bones, and cephalopod beaks, is prone to bias due to the rapid and differential digestion rates of various prey items\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Furthermore, given the scarcity of deceased individuals from top predators such as whales, the dietary data derived from methods (1) to (3) may not be representative of the population as a whole. Non-invasive techniques, including fecal mtDNA analysis and direct observation, have gained attention as alternatives. However, fecal sampling (method 4) requires immediate collection post-defecation, limiting sampling opportunities, while direct observation (method 5) is only feasible in warm waters where researchers can swim alongside the animals.\u003c/p\u003e\u003cp\u003eCetaceans, like other mammals, exhale forcefully when surfacing, producing a visible \"blow\" or \"spout\" composed of exhaled condensate. Over the past decade, this blow has been utilized as a non-invasive biological sampling source\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The first application of blow sampling in cetacean research was by Hogg (2005)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, who used it to examine reproductive hormones\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Since then, blow analysis has proven useful for assessing the health and disease status of wild whales\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. More recently, unmanned aerial systems (UAS) have enabled the collection of exhaled breath from free-ranging whales, allowing endocrine assessments, DNA profiling, microbial metabarcoding, and metagenomic analyses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In contrast, studies on small odontocetes remain limited. Although DNA has been successfully recovered from the blow of captive bottlenose dolphins (\u003cem\u003eTursiops truncatus\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, only a single instance of successful detection in a wild individual, specifically a short-beaked common dolphin (\u003cem\u003eDelphinus delphis\u003c/em\u003e), has been reported to date\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur research team conducts field surveys in regions such as the Nemuro Strait off Rausu (Shiretoko) and the Muroran coast to investigate the ecology of wild cetaceans, including killer whales (\u003cem\u003eOrcinus orca\u003c/em\u003e) and Pacific white-sided dolphins (\u003cem\u003eLagenorhynchus obliquidens\u003c/em\u003e) that inhabit the waters around Hokkaido\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In ecological studies, non-invasive techniques are indispensable for population genetic analyses. As part of our ongoing efforts to establish such methods using the exhalations of captive cetaceans, we detected DNA likely originating from their dietary intake. Here, we report our findings on this novel application of breath sampling for dietary analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMetagenomic analysis using unpurified DNA products\u003c/h2\u003e\u003cp\u003eMeta-barcoding analysis of exhaled DNA without purification yielded 24,627 reads from a captive killer whale (\u003cem\u003eO. orca\u003c/em\u003e 1; Oror-1) and 37,965 reads from a captive Pacific white-sided dolphin (\u003cem\u003eL. obliquidens\u003c/em\u003e 1; Laob-1) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of these, host-derived sequences accounted for 24,116 reads (Oror-1; 97.9%) and 36,962 reads (Laob-1; 97.1%), while cetacean-specific sequences totaled 445 reads (Oror-1) and 947 reads (Laob-1). In Oror-1, two bacterial taxa, \u003cem\u003eAlcaligenes\u003c/em\u003e sp. (50 reads) and \u003cem\u003ePseudomonas\u003c/em\u003e sp. (16 reads), were identified. In Laob-1, sequences corresponding to two fish species (\u003cem\u003eTrachurus japonicus\u003c/em\u003e, 35 reads; \u003cem\u003eScomber japonicus\u003c/em\u003e, 29 reads) and four bacterial taxa (\u003cem\u003eBacteroides\u003c/em\u003e sp., 22 reads; \u003cem\u003eMorganella\u003c/em\u003e sp., 38 reads; \u003cem\u003eProteus\u003c/em\u003e sp., 2 reads; \u003cem\u003eVibrio\u003c/em\u003e sp., 13 reads) were detected (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of DNA purification steps, the number of first-round PCR replicates, and the sequencing reads obtained from blow samples of captive killer whales and Pacific white-sided dolphins. \"y\" indicates \"yes\"; \"n\" indicates \"no.\"\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCleanup DNA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1stPCR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNum. Read\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eOrcinus orca\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOror-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e24,627\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOror-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e34,794\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLagenorhynchus obliquidens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLaob-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e37,965\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLaob-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e40,727\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTaxonomic assignment of host, dietary, bacterial, and tick DNA detected in blow samples via DNA metabarcoding, along with the number of sequencing reads obtained per sample.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClassification\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOror-1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOror-2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLaob-1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLaob-2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehost\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eOrcinus orca\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24,116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33,914\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e58,030\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLagenorhynchus obliquidens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36,879\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e39,962\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e76,841\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecetacean\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e445\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e763\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e947\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e723\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2,878\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edietary\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAmmodytes\u003c/em\u003e sp.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePleurogrammus azonus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e108\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTrachurus japonicus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eScomber japonicus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebacteria\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAlcaligenes\u003c/em\u003e sp.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eBacteroides\u003c/em\u003e sp.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMorganella\u003c/em\u003e sp.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eProteus\u003c/em\u003e sp.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eVibrio\u003c/em\u003e sp.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etick\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTyrophagus\u003c/em\u003e sp.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eContamination\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHuman\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24,627\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34,794\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e37,965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40,727\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e138,113\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMetagenomic analysis following DNA purification and first-round PCR with eight replicates\u003c/h3\u003e\n\u003cp\u003eMeta-barcoding analysis of exhaled DNA following PCR inhibitor removal and eight replicates of first-round PCR yielded 34,794 reads from Oror-2 and 40,727 reads from Laob-2 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of these, host-derived sequences represented 33,914 reads (Oror-2; 97.5%) and 39,962 reads (Laob-2; 98.1%), while cetacean-specific sequences accounted for 763 reads (Oror-2) and 723 reads (Laob-2). In Oror-2, \u003cem\u003ePleurogrammus azonus\u003c/em\u003e (102 reads) and human-derived sequences (15 reads) were identified. In Laob-2, sequences from three fish taxa, \u003cem\u003eAmmodytes\u003c/em\u003e sp. (6 reads), \u003cem\u003eP. azonus\u003c/em\u003e (6 reads), and \u003cem\u003eT. japonicus\u003c/em\u003e (26 reads), and one mite species (\u003cem\u003eTyrophagus\u003c/em\u003e sp., 4 reads) were detected (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOn the day of blow sample collection, the killer whale at the Port of Nagoya Public Aquarium was fed 39 kg/day of \u003cem\u003eP. azonus\u003c/em\u003e (52.7%), 13 kg/day of \u003cem\u003eClupea pallasii\u003c/em\u003e (17.6%), and 22 kg/day of \u003cem\u003eS. japonicus\u003c/em\u003e (29.7%). At Noboribetsu Marine Park Nixe, the Pacific white-sided dolphin received 1.3 kg/day of \u003cem\u003eAmmodytes\u003c/em\u003e sp. (21.7%), 2.3 kg/day of \u003cem\u003eP. azonus\u003c/em\u003e (38.3%), 1.2 kg/day of \u003cem\u003eT. japonicus\u003c/em\u003e (20.0%), and 1.2 kg/day of \u003cem\u003eS. japonicus\u003c/em\u003e (20.0%). In the killer whale, only the Oror-2 sample, which underwent cleanup purification and was subjected to eight replicates of the first PCR, yielded detectable levels of \u003cem\u003eP. azonus\u003c/em\u003e. In the Pacific white-sided dolphin, two species were detected in Laob-1 (unpurified), whereas three species were identified in Laob-2 (purified sample) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Cetaceans exhibit a cranial morphology characterized by the telescoping of skull bones, resulting in a dorsally positioned larynx that maintains near-complete anatomical separation from the pharynx\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, recent findings have revealed the presence of an \u0026ldquo;oral plug\u0026rdquo; in the fin whale (\u003cem\u003eBalaenoptera physalus\u003c/em\u003e), which passively separates the larynx from the pharynx\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Additionally, small food particles have occasionally been observed in exhaled droplets from captive individuals (Kanda, pers. comm.). While not observed in the individuals used in this study, some captive killer whales have been noted to release air through the oral cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These observations suggest that odontocetes, unlike mysticetes, may lack a complete septum separating the airway and esophagus at the pharyngeal level. In the present study, although blow sampling from the killer whale was performed in a pool, the individual expelled residual seawater from the blowhole prior to sample collection. For the Pacific white-sided dolphin, sampling was conducted while the individual was in a stationary position at the poolside, under trainer instruction. Given these controlled conditions, the likelihood of seawater contamination by environmental DNA (e.g., fecal matter) was considered minimal. Collectively, these findings support the hypothesis that cetacean blow may contain trace amounts of prey-derived DNA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn samples without DNA purification, bacterial sequences were detected; however, after purification, sequences derived from ticks and humans were identified, with no bacterial DNA present. In the killer whale, prey-derived sequences were observed only following DNA purification, whereas in the Pacific white-sided dolphin, purification increased the number of prey species detected (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PCR inhibition is commonly attributed to interference with DNA polymerase activity by environmental contaminants, such as humic substances (e.g., humic acid, fulvic acid, and tannic acid). These inhibitors are prevalent in aquatic environments and must be efficiently removed during nucleic acid extraction to ensure reliable environmental DNA (eDNA) analysis\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, strong PCR bias can occur during the first amplification step, particularly when using gene-specific primers such as those targeting the mtDNA cytochrome c oxidase I (\u003cem\u003eCOI\u003c/em\u003e) gene. Therefore, it is recommended to conduct at least four, and preferably eight, PCR replicates to enhance detection accuracy\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Accordingly, for metagenomic analysis of blow-derived DNA, it is essential to eliminate potential inhibitors and perform a minimum of eight first-round PCR replicates.\u003c/p\u003e\u003cp\u003eDespite these precautions, only one prey species (33.3%) was detected in the killer whale sample (Oror-2), whereas all four species were detected in the Pacific white-sided dolphin samples. Notably, the number of sequencing reads for \u003cem\u003eAmmodytes\u003c/em\u003e sp. and \u003cem\u003eP. azonus\u003c/em\u003e was limited to six each, possibly too low to be considered robust detections (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In fecal DNA-based dietary analyses, mitochondrial markers such as \u003cem\u003eCOI\u003c/em\u003e, 12S rRNA, and 16S rRNA are commonly employed\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, often alongside blocking primers designed to suppress host DNA amplification\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Although blocking primers were not utilized in this study, prey-derived sequences were detected at proportions of 0.17% in Laob-1 (unpurified dolphin sample), and 0.29% and 0.09% in Oror-2 and Laob-2 (purified killer whale and dolphin samples, respectively, with eight PCR replicates). These values may exceed those commonly obtained from fecal DNA, suggesting that the application of blocking primers could further enhance prey DNA detection in future analyses.\u003c/p\u003e\u003cp\u003eImportantly, the relative proportions of detected prey species did not reflect the actual feed composition on the day of sampling. In the killer whale, only Atka mackerel (\u003cem\u003eP. azonus\u003c/em\u003e) was detected in Oror-2 (read distribution: \u003cem\u003eP. azonus\u003c/em\u003e\u0026thinsp;=\u0026thinsp;102; \u003cem\u003eC. pallasii\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0; \u003cem\u003eS. japonicus\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0). For the Pacific white-sided dolphin, Laob-1 revealed the presence of \u003cem\u003eT. japonicus\u003c/em\u003e and \u003cem\u003eS. japonicus\u003c/em\u003e (reads: 35 and 29, respectively), while Laob-2 identified \u003cem\u003eAmmodytes\u003c/em\u003e sp., \u003cem\u003eP. azonus\u003c/em\u003e, and \u003cem\u003eT. japonicus\u003c/em\u003e (reads: 6, 6, and 26, respectively), with \u003cem\u003eS. japonicus\u003c/em\u003e undetected. As highlighted by Thomas et al. (2014)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, tissue-specific differences in lipid, protein, ash, and moisture content necessitate the application of Tissue Correction Factors (TCFs) and Digestion Correction Factors (DCFs) when interpreting dietary DNA results. Consequently, the number of sequencing reads in this study is unlikely to directly correlate with the biomass of prey consumed.\u003c/p\u003e\u003cp\u003eThis study is the first to report the detection of prey-derived DNA sequences in cetacean blow. Future research should address potential sources of environmental contamination, such as by feeding prey species not typically used in aquaria, and assess the utility of host- and human-specific blocking primers for improving detection sensitivity. Taken together, these findings demonstrate that blow sampling offers a promising, non-invasive tool for dietary analysis in wild cetaceans.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e All experimental procedures were approved by the Tokai University Animal Experiment Committee (Approval No. 221049). Blow samples were collected from one killer whale (\u003cem\u003eO. orca\u003c/em\u003e) at the Port of Nagoya Public Aquarium and one Pacific white-sided dolphin (\u003cem\u003eL. obliquidens\u003c/em\u003e) at Noboribetsu Marine Park Nixe. The animals had been pre-trained by their respective handlers to exhale on cue. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Blow samples were collected into 50 mL tubes, as described previously\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and preserved in 1 mL of ATL buffer (QIAGEN Inc.). Genomic DNA was extracted using the QIAamp DNA Investigator Kit (QIAGEN Inc.) according to the manufacturer\u0026rsquo;s protocol (QIAamp DNA Investigator Handbook, January 2020).\u003c/p\u003e\u003cp\u003eThe first round of PCR targeted the \u003cem\u003eCOI\u003c/em\u003e region and was performed by Bioengineering Lab Co., Ltd. (Kanagawa, Japan) following established protocols\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. A second PCR was conducted to append multiplex identifier (MID) tags. To evaluate the impact of PCR inhibitors on detection sensitivity, genomic DNA was divided into two groups: one subjected to inhibitor removal using the DNeasy PowerClean Pro Cleanup Kit (QIAGEN Inc.) and one untreated. For the inhibitor-removal group, the first PCR was performed in eight replicates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLibrary quality was assessed using the Synergy H1 (Agilent Technologies Inc.) in conjunction with the QuantiFluor dsDNA System (Promega Inc.). Paired-end sequencing (2 \u0026times; 300 bp) was carried out using the MiSeq Reagent Kit v3 on the MiSeq platform (Illumina Inc., San Diego, CA, USA) at Bioengineering Lab Co., Ltd. Only reads with exact matches to primer sequences at the read start were retained using the fastx_barcode_splitter tool from FASTX-Toolkit ver. 0.0.14 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hannonlab.cshl.edu/fastx_toolkit/\u003c/span\u003e\u003cspan address=\"http://hannonlab.cshl.edu/fastx_toolkit/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Primer sequences and the 3\u0026prime;-terminal 50 bases were trimmed using fastx_trimmer. Low-quality reads (Phred score\u0026thinsp;\u0026lt;\u0026thinsp;20) were removed using Sickle ver. 1.33\u003csup\u003e37\u003c/sup\u003e, and reads shorter than 40 bp, along with their paired reads, were discarded. Paired-end reads were merged using FLASH ver. 1.2.11\u003csup\u003e38\u003c/sup\u003e with a minimum required overlap of 10 bp. Chimeric and low-quality sequences were filtered using the DADA2 plugin in QIIME2 ver. 2023.7 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://forum.qiime2.org/t/qiime-2-2023-7-is-now-available/27432\u003c/span\u003e\u003cspan address=\"https://forum.qiime2.org/t/qiime-2-2023-7-is-now-available/27432\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Representative sequences and an amplicon sequence variant (ASV) table were subsequently generated. Taxonomic assignment of representative sequences was performed using BLASTN ver. 2.13.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://software.cqls.oregonstate.edu/updates/blast-2.13.0/\u003c/span\u003e\u003cspan address=\"https://software.cqls.oregonstate.edu/updates/blast-2.13.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) against the NCBI nt database (Supplementary Table\u0026nbsp;1).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Japan Society for the Promotion of Science (JSPS KAKENHI) Grant Number 21H02217 and the Research and Study Project of the Tokai University Research Organization Grant number PJ2022-5.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the conception and design of the study. Sample collection was performed by Kita Y.F., Kanda K., Yoshinaka A., and Ohizumi H. Data acquisition and analysis were carried out by Kita Y.F. Fig. 1 was prepared by Kanda K. The first draft of the manuscript was prepared by Kita Y.F., and all authors provided feedback on earlier versions. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the Port of Nagoya Public Aquarium, Noboribetsu Marine Park Nixe, and the members of the Kita and Ohizumi Laboratories at Tokai University for their assistance in collecting blow samples. We are also grateful to Bioengineering Lab. Co., Ltd., for support with DNA analyses. This study was partially supported by JSPS KAKENHI (grant number 21H02217) and the Research and Study Project of the Tokai University Research Organization (grant number PJ2022-5). Part of the analysis was conducted at the Hokkaido Regional Research Center, Tokai University (THRRC).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe list of species detected based on blow analysis, including ASV tables and taxonomic assignments, is available in Supplementary Table 1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmir, O. A., Berggren, P., Ndaro, S. G. M. \u0026amp; Jiddawi, N. S. Feeding ecology of the Indo-Pacific bottlenose dolphin (Tursiops aduncus) incidentally caught in the gillnet fisheries off Zanzibar, Tanzania. \u003cem\u003eEstuar. Coast. 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FLASH: fast length adjustment of short reads to improve genome assemblies. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e, 2957\u0026ndash;2963. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btr507\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btr507\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cetacean blow, food-derived DNA, metagenomic analysis, non-invasive method","lastPublishedDoi":"10.21203/rs.3.rs-7191423/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7191423/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study proposes the utility of blow sampling for DNA analysis as a non-invasive approach to examine the feeding ecology of cetaceans. Conventional methods such as stomach content analysis and stable isotope profiling, although informative, are inherently invasive and have notable limitations. In contrast, blow analysis offers a non-invasive alternative and, when integrated with unmanned aerial vehicles, facilitates the assessment of endocrine function and health status in free-ranging whales. In this study, fish- and bacteria-derived DNA was detected in the blows of captive orcas and dolphins, indicating that breath may contain trace amounts of dietary DNA. Additionally, detection accuracy was enhanced through DNA purification and repeated PCR amplification. These findings suggest that blow sampling represents a promising tool for dietary analysis in wild cetaceans.\u003c/p\u003e","manuscriptTitle":"Evaluating the potential of cetacean blow for non-invasive dietary DNA analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 12:33:57","doi":"10.21203/rs.3.rs-7191423/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e6e17ef-5004-4956-b66a-fc298b4f8570","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53126122,"name":"Biological sciences/Biological techniques"},{"id":53126123,"name":"Biological sciences/Ecology"},{"id":53126124,"name":"Earth and environmental sciences/Ecology"},{"id":53126125,"name":"Earth and environmental sciences/Ocean sciences"},{"id":53126126,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-01-30T11:24:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-20 12:33:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7191423","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7191423","identity":"rs-7191423","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}