Field data collection

Data from North Atlantic humpback whales were collected off the coast of Massachusetts, in and around the Stellwagen Bank National Marine Sanctuary in the southern Gulf of Maine in July/August 2022 and July 2023. As part of a long-term behavioral study, humpback whales were tagged with suction-cup acoustic recording tags (DTAGs [19]; CATS tags [20]) that remained attached for up to ~33 hours (minimum ~5 minutes). Additionally, data from North Atlantic right whales were collected in southern New England in May 2023 and in Cape Cod Bay in March 2024 using the same tag platforms and methodology. Tags on North Atlantic right whales remained attached for up to ~30 hours (minimum ~30 minutes). Tag deployments were approved by the Institutional Animal Care and Use Committee of Syracuse University (IACUC approval numbers 20-002 and 23-002).

Tags were adrift in the ocean for various time periods before retrieval (~5 minutes – ~ 18 hours). Upon retrieval of the tag, suction-cups were wiped with sterile foam-tipped swabs (Puritan, cat. no. 25-1506 1PF) regardless of visible skin. During later stages of the protocol development, two swabs per tag were collected (one from the front two suction cups and one from the back two suction cups) to provide additional data for validation. The swabs were then placed into sample vials, labelled with the deployment details, and immediately moved to a -20 freezer located onboard the vessel. Upon returning to land, samples were stored in a -80°C freezer.

DNA extraction methodologies

Genomic DNA was extracted from collected tissue samples using the MyTaq Extract-PCR kit (Meridian Bioscience, cat. no. BIO-21126) or the DNeasy Blood and Tissue Kit (Qiagen, cat. no. 69504) following the manufacturer protocols. Briefly, the swab tip of the collection swab was detached from the applicator shaft using a clean razor blade. The swab tip was returned to the original storage vial and DNA extraction was performed. DNA extraction using the MyTaq Extract-PCR kit followed the manufacturer’s standard protocol. DNA extraction using the DNeasy Blood and Tissue Kit was performed following the manufacturer’s standard protocol, with the following exceptions: 1) during lysis, samples were incubated with the provided lysis buffer and proteinase K solution for 3 hours in a hot water bath at 37°C; 2) tissue lysate was incubated on the DNA-binding spin column membrane for 5 minutes to allow DNA to bind before centrifuging; and 3) purified DNA was eluted from the DNA-binding spin column in 100 μL of Elution Buffer two times, to yield a dilute solution of purified DNA at a final volume of 200 µL. Samples were then dried under vacuum for 10 hours at ambient temperature using a Vacufuge Plus vacuum centrifuge (Eppendorf, Hamburg, Germany). The resulting DNA sample was re-solubilized in 20 µL of ultrapure water.

The DNA yield (ng/µL) and sample purity (A260/A280 value) were measured using a Nanodrop Spectrophotometer 2000/2000c (Thermo Fisher Scientific, Wilmington, NC). Samples with an A260/A280 value of 1.8–2.0 were considered pure and continued to PCR analysis. The DNA samples extracted using the MyTaq Extract-PCR kit did not undergo pre-amplification screening for yield and purity. Samples of extracted DNA were stored at -20°C until downstream analysis.

Primers

This study used primer sequences specifically targeting the homologous ZFX/XFY (zinc-finger protein genes located on both X and Y chromosomes) regions and specific oligonucleotide primer sequences targeting the Y-linked sex-determining region Y (SRY) that were previously designed and validated for sex determination of cetaceans and dugongs [21]. Lyophilized oligonucleotide primers targeting the SRY gene (forward: CCC ATG AAC GCA TTC ATT GTG TGG; reverse: ATT TTA GCC TTC CGA CGA GGT CGA TA) and ZFX/ZFY (forward: ATA ATC ACA TGG AGA GCC ACA AGC T; reverse: GCA CTT CTT TGG TAT CTG AGA AAG T) were synthesized by Integrated DNA Technologies (Coralville, IA). The oligonucleotides were re-solubilized in ultrapure water to a final concentration of 100 uM and stored at -20°C.

PCR

PCR reactions were carried out in 8-tube strips of 0.2-mL PCR-grade reaction tubes in a final reaction volume of 25 µL containing 1X OneTaq Standard Reaction Buffer (New England Biolabs, cat. no. B9022SVIAL), 0.4 µM primers (ZFX/ZFY forward and reverse and SRY forward and reverse primers), 200 µM dNTPs (Thermo Fisher Scientific, cat. no. 18427013), 2–5 µL extracted DNA, and 2.5 U OneTaq DNA polymerase (New England Biolabs, cat. no. M0480S) in deionized water. The PCR amplification was performed using a MyCycler thermal cycler (Bio-Rad, Hercules, CA). Samples underwent 32 total cycles of PCR amplification at the following conditions: 1 cycle of initial denaturation at 95°C for 180 seconds, 30 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 20 seconds, and 1 cycle for a final extension at 72°C for 300 seconds. Reaction products were resolved by electrophoresis in a 2% agarose gel stained with 1X SYBR Safe DNA gel stain (Invitrogen, cat. no. S33102). An amplicon was expected at 210–224 bp for the presence of the SRY gene. An amplicon at 442–445 was expected for the presence of the ZFX/ZFY gene.

Presence of amplicons for both genes indicated analyzed DNA was isolated from a male individual, whereas absence of the amplicon for the Y-linked SRY gene indicated analyzed DNA was isolated from a female individual, as females do not carry the chromosome (although absence can also indicate amplification failure – see below). Because this test is based upon the absence of the SRY gene, inclusion of primers targeting ZFX/ZFY gene regions also served as an internal reference control for amplification and viable DNA presence for analysis [21,22].

Assessing protocol performance

Validation assessment method.

The probability of an accurate test result was calculated by comparing test results to the known sex of individuals. This was accomplished for all known-sex samples that met extraction criteria, and produced visible amplification of the ZFX/ZFY control gene in gels, using an application of Bayes’ Theorem similar to assessing the diagnostic accuracy of disease testing [25]. A true positive test represents the PCR detection of a Y chromosome for a known male individual, and a true negative test when the Y chromosome is absent for a known female.

Test performance was calculated such that:

(1)

Where P(TS) is the probability of a true sex determination result (for males, the odds of a true positive and for females the odds of a true negative), P_o(S) is the probability of the sex being assessed occurring in the dataset (i.e., proportion of individuals of a sex that that met protocol criteria divided by the total number of individuals assessed), P(~TS) represents the odds of a false result (false negative for males and false positive for females), and P_o(~S) represents the probability of the opposite sex occurring in the dataset.

As a Bayesian methodology, the probabilities of a test result correctly determining the sex of an individual are intended to be updated with new information [25]. To refine our results, data collected from deployments on known-sex individuals in future field seasons will be used to update the probabilities in the associated protocol for this publication. This will allow robust information on the reliability of this method with increasing sample sizes and future protocol improvements.

Validation

Of the 21 samples that yielded results, all but one matched the known sex of the individual whose sex was validated by genetic analysis of a tissue biopsy, as described above (Table 1). In that case, a female was misidentified as a male according to the PCR results. The misidentified sample was one of two that originated from the same tag deployment, and the other sample matched the known sex, highlighting the importance of taking multiple samples from the same tag deployment to assess the potential for error.

The probability of a true positive in the dataset (e.g., the individual was a known male and the SRY gene was present) was 1.0, while the probability of a false negative (e.g., the individual was a known male and the SRY gene was absent) was 0. The probability of obtaining a true negative in the validation dataset (e.g., the individual was a known female and the SRY gene was absent) was 0.92, whereas the probability of obtaining a false positive (e.g., the individual was a known female and the SRY gene was detected) was 0.08. Based on Bayes’ formula (Equation 1), the probability that a sample will be sexed as male when the known sex is male was 0.91 given the true sex ratio of 0.4 males to 0.5 females in this dataset. In other words, the chance of an accurate test result in the present study for males was 91%. Female protocol was found to be 100% accurate in the current dataset.

There are some opportunities in this protocol to introduce contamination which may have produced the single misidentified sample, including during the handling of the tag upon retrieval. Additionally, after release from a whale, the tag suction cups may have been exposed to whale DNA in the environment (eDNA). The risk of other animal tissue contacting and thus contaminating the suction cups is harder to mitigate, particularly in large groups or during social interactions. Care should be taken to only use samples taken from the area in direct contact with the animal (i.e., the inside of the suction cups) to reduce this risk.

Despite these potential issues, the contamination of samples when working with sloughed skin is thought to be minimal [9]. It is possible that the incorrect sex determination was instead the result of an error in the lab process, such as mishandling or amplification/read errors. Further refinements to this protocol could improve the efficiency of DNA extraction and the accuracy of results, including the use of more specific primers. Future studies on the efficacy of the provided protocol should include an analysis of additional environmental impacts on sample viability considering salinity, sun exposure, and time may all have an impact on DNA retention. Lastly, more detailed validation of tag-based results against other data (e.g., mtDNA) would be beneficial.