The ability to successfully sequence trace quantities of environmental DNA (eDNA) has provided unprecedented opportunities to use genetic analyses to elucidate animal ecology, behavior, and population structure without affecting the behavior, fitness, or welfare of the animal sampled. Hair associated with an animal track in the snow, the shed exoskeleton of an insect, or a swab of animal scat are all examples of non-invasive methods to collect eDNA. Despite the seemingly uncomplicated definition of "non-invasive" as proposed by Taberlet et al. , Lefort et al.  highlight that its appropriate application to sampling methods in practice is not so straightforward. For example, collecting scat left behind on the forest floor by a mammal could be invasive if feces is used by that species to mark territorial boundaries. Other collection strategies such as baited DNA traps to collect hair, capturing and handling an individual to swab or stimulate emission of a body fluid, or removal of a presumed non essential body part like a feather, fish scale, or even a leg from an insect are often described as "non-invasive" sampling methods. However, such methods cannot be considered truly non-invasive. At a minimum, attracting or capturing and handling an animal to obtain a DNA sample interrupts its normal behavioral routine, but additionally can cause both acute and long-lasting physiological and behavioral stress responses and other effects. Even invertebrates exhibit long-term hypersensitization after an injury, which manifests as heightened vigilance and enhanced escape responses [3-5].
Through an extensive analysis of 380 papers published from 2013-2018, Lefort et al.  document the widespread misapplication of the term "non-invasive" to methods used to sample DNA. An astonishing 58% of these papers employed the term incorrectly. A big part of the problem is that "non-invasive" is usually used by authors in the medical or veterinary sense of not breaking the skin or entering the body , rather than in the broader, ecological sense of Taberlet et al. . The authors argue that correct use of the term matters, because it may lead naive readers – one can imagine students, policy makers, and the general public – to incorrectly assume a particular method is safe to use in a situation where disturbing the animal could affect experimental results or raise animal welfare concerns. Such assumptions can affect experimental design, as well as interpretations of one's own or others' data.
The importance of the Lefort et al.  paper lies in part on the authors' call for the research community to be much more careful when applying the term "non-invasive" to methods of DNA sampling. This call cannot be shrugged off as a minor problem in a few papers – as their literature review demonstrates, "non-invasive" is being applied incorrectly more often than not. The authors recognize that not all DNA sampling must be non-invasive to be useful or ethical. Examples include taking samples for DNA extraction from museum specimens, or opportunistically from carcasses of animals hunted either legally or seized by authorities from poachers. In many cases, there may be no viable non-invasive method to obtain DNA, but a researcher strives to collect samples using methods that, although they may involve taking a sample directly from the animal's body, do not disrupt, or only slightly disrupt behavior, fitness, or welfare of the animal. Thus, the other important contribution by Lefort et al.  is to propose the terms "non-disruptive" and "minimally-disruptive" to describe such sampling methods, which are not strictly non-invasive. While gray areas undoubtedly remain, as acknowledged by the authors, answering the call for correct use of "non-invasive" and applying the proposed new terms for certain types of invasive sampling with a focus on level of disruption, will go a long way in limiting misconceptions and misinterpretations caused by the current confusion in terminology.
 Taberlet P., Waits L. P. and Luikart G. 1999. Noninvasive genetic sampling: look before you leap. Trends Ecol. Evol. 14: 323-327. doi: 10.1016/S0169-5347(99)01637-7
 Lefort M.-C., Cruickshank R. H., Descovich K., Adams N. J., Barun A., Emami-Khoyi A., Ridden J., Smith V. R., Sprague R., Waterhouse B. R. and Boyer S. 2019. Blood, sweat and tears: a review of non-invasive DNA sampling. bioRxiv, 385120, ver. 4 peer-reviewed and recommended by PCI Ecology. doi: 10.1101/385120
 Khuong T. M., Wang Q.-P., Manion J., Oyston L. J., Lau M.-T., Towler H., Lin Y. Q. and Neely G. G. 2019. Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila. Science Advances 5: eaaw4099. doi: 10.1126/sciadv.aaw4099
 Crook, R. J., Hanlon, R. T. and Walters, E. T. 2013. Squid have nociceptors that display widespread long-term sensitization and spontaneous activity after bodily injury. Journal of Neuroscience, 33(24), 10021-10026. doi: 10.1523/JNEUROSCI.0646-13.2013
 Walters E. T. 2018. Nociceptive biology of molluscs and arthropods: evolutionary clues about functions and mechanisms potentially related to pain. Frontiers in Physiololgy 9: doi: 10.3389/fphys.2018.01049
 Garshelis, D. L. 2006. On the allure of noninvasive genetic sampling-putting a face to the name. Ursus 17: 109-123. doi: 10.2192/1537-6176(2006)17[109:OTAONG]2.0.CO;2
DOI or URL of the preprint: https://www.biorxiv.org/content/10.1101/385120v2
Version of the preprint: 2
Dear Dr. Lefort,
Thank you for revising your pre-print, "Blood, sweat and tears...", and resubmitting to PCI Ecology for review. I returned it to the original reviewer #2, who is satisfied with most of the revisions. However, he/she still has concerns about the issue of citing papers as being out of compliance with the definition of non-invasive DNA sampling when animals were killed for methods-development purposes, and even when there was no claim of non-invasive sampling in the paper itself.
In addition, I have the following comments for you to consider when revising:
Regarding Supplementary Table 1: Sorting by first author followed by a quick visual scan, I notice there are at least 7 duplicate entries in this table: entries 48/49; 50/51; 70/71; 80/81; 83/84; 119/120; 187/188. This will change the total number of papers sampled from 342 to 335, and the percentages, proportions, etc. will have to be recalculated. Please check closely and make sure there are no additional duplicates. In addition, for the duplicate pair 80/81, the entries in the "Sampling_Method" column differ, so please revisit the paper and decide which is correct.
Also for Supp. Table 1, please include a column with the publication information for the paper (i.e., journal, volume, page numbers). And several places in the table, "Mixte" should be replaced by "Mixed."
Line 103, consider "information to draw conclusions about the specific"
Lines 124-25, consider "section of each paper"
Line 145, "Marking" should be "Mark"
Line 186, consider "When the terminology for DNA sampling is misapplied as being non-invasive when it is not,"
Line 191, consider "for judging the validity"
Line 237, should be "encounters"
Line 239, should be "effects"
Line 261, should be "on animal"
Line 286, should be "adults"
For the "Sin 7" title (line 319), please consider changing to "Equating a non-invasive procedure with non-invasive sampling". This is a more descriptive and intuitive construction than the current title.
Line 362, "reproduction" should be "reproductive"; also, should be "as proxies for"
Line 434, should be "effects"
Line 458, should be "experimentation" (i.e., not plural)
Line 467 and 475, consider "that must be" (instead of "need to" or "needs to")
Line 495, should be "animals being more attracted"
L503, consider "being indistinguishable from that of captured"
L519, should be "to obtain"
Lines 863 and 878 indicate the literature review was through May 2018, but line 865 indicates it was conducted in July 2018. Which is correct? In the method section (line 100) it says July 2018.
Line 872, consider "width of the rectangles is proportional to the number of"
Line 880, consider "width of the bars is proportional to the number of"
After making these revisions, I will examine the paper one more time and consider for formal recommendation by PCI Ecology if the revisions are satisfactory. Please let me know of any questions. I look forward to seeing your revised version.
Best Wishes, --Tom Sappington
DOI or URL of the preprint: https://doi.org/10.1101/385120
Dear Dr. Lefort,
Thank you for requesting a peer-review of your pre-print, "Blood, sweat and tears...", by PCI Ecology. I apologize for the long delay in providing the reviews, we had some difficulty finding reviewers followed by slow response from one of them.
You will see that the comments of reviewer #1 are favorable. The comments of reviewer #2 are more thorough and critical. They are essentially favorable, but suggest a major revision and restructuring of the paper before being acceptable. Although the requested revisions will require some fundamental changes in how your ideas for a different terminology in the field of DNA sampling from living animals are presented and explained, I think it would be worth the effort, and encourage you to consider doing so. If you can make such revisions, I will be happy to review the paper afresh and consider for formal recommendation by PCI Ecology.
Please let me know of any questions. I look forward to seeing your revised version. If you choose not to revise, let me know if you would, please.