Given that the N. vitripennis methylome has recently been sequenced (Wang et al. 2013; Beeler et al. 2014), future research will determine which methylated DNA sequences are involved in sex allocation.

As we have recently shown that there is (so far) no detectable differential gene expres- sion associated with facultative sex allocation in Nasonia (N. Cook, U. Trivedi, B. Pannebakker, M. Blaxter, M. Ritchie, E. Tauber, T. Sneddon, and D. Shuker, unpublished man- uscript), it would appear that disrupting DNA methylation is not having its effect by changing patterns of gene expres- sion during oviposition, but rather the effect must be up- stream, perhaps changing the sex allocation machinery prior to the female actually having to allocate sex.

We are still some way off of uncovering what this machinery is.

More generally, our data show how elucidating the mechanism of familiar behaviors can enrich our understanding of them, in this case, showing that we may need to consider genomic conflict in our models of sex allocation under LMC.

Fur- thermore, our data confirm that new theoretical and em- pirical insights are still there to be discovered, even in well- studied systems such as sex allocation in parasitoid wasps.


The use of DNA demethylating agents has been popular in epigenetic studies.

Recently, Cook and colleagues, in a 2015 American Naturalist article, claimed an effect of 5-aza-20-deoxycytidine (5-aza-dC) on the sex ratio of a parasitoid wasp without verifying its effect on DNA methylation.

We repeated the 5-aza-dC feeding treatment to test its effectiveness.

We used bisulfite amplicon sequencing of 10 genes that either were heavily methylated, previously showed a response to 5-aza-dC, or were suggested to regulate fatty acid synthesis epigenetically, and we demonstrate that wasps fed 5-aza-dC did not show reduced DNA methylation at these loci.

Therefore, the conclusion that demethylation shifts sex ratios upward needs reconsideration.


Epigenetics is a rapidly expanding field of research.

In an increasing number of developmental, reproductive, and behavioral processes, epigenetic variation has been shown to affect the phenotype and its plasticity [1][2][3][4].

Epigenetic DNA modifications affect the accessibility of genomic regions for regulatory proteins or protein complexes through mechanisms such as altered DNA methylation patterns or altered histone modification.

A large number of studies have reported variation between populations in the overall degree of DNA methylation patterns, which were linked to phenotypic differences [5].

Also, the use of demethylating agents such as 5-aza-20-deoxycytidine (5-aza-dC) has been popular in showing the impact of DNA methylation on the phenotype.

The 5-aza-dC is incorporated into the DNA, where it inhibits DNA methylation [6], as has been shown in plants [1][7], invertebrates [8][9][10], and vertebrates [11][12].


Recently, Cook et al. ( 2015) applied 5-aza-dC to test for an effect of DNA demethylation on the sex ratio response of the parasitoid wasp Nasonia vitripennis, which is known to adjust the sex ratio of its clutch based on the number of founding females in a host brood [13][14].

In this experiment, wasps that were fed 5-aza-dC in a 20% sucrose solution showed a slight but consistent shift in sex ratio in the direction predicted by theory.

However, no validation of the actual demethylating effect of 5-aza-dC was carried out.

In fact, demethylation efficacy should be verified for each organism and each concentration at which 5-aza-dC is applied because 5-aza-dC is known to have a wide range of antimetabolic activities that could confound its demethylating effect [6].

Therefore, the lack of such evidence in the study by Cook and colleagues precludes the observed change in sex ratio in N. vitripennis to be attributed to a decrease in DNA methylation.


Several other studies followed Cook et al. ( 2015) and administered 5-aza-dC in an identical manner to the same or other parasitoid species, with no [15] or very limited [16] quantification of its effect on DNA methylation.

In the latter study, only four CpG sites were tested, each one in a different gene.

Although subtle changes were seen in methylation status for three out of the four loci, loci were not tested separately for significant changes, and the number of CpG sites tested was too low to obtain a representative estimate of the effect of 5-aza-dC on the epigenome [16].

Here we want to emphasize the importance of validating the demethylating effect of 5-aza-dC when testing hypotheses about the relationship between DNA methylation and phenotypic expression.

In addition to sex allocation [13] and induction of diapause [16], several other phenotypes have been implied in parasitoid wasps to be regulated epigenetically, including caste determination [17] and fatty acid composition [15].

For a more reliable interpretation of all experiments with orally administered 5-aza-dC to parasitoids, it will be necessary to have a better idea of the extent and pervasiveness of demethylating effect of this method.


Therefore, we applied oral 5-aza-dC treatment on N. vitripennis in the same dose as previous studies [13][16] and tested its effect on DNA methylation of 155 CpG sites distributed over 10 different genes.

We included (i) genes that were heavily methylated based on several published genome methylation scans in this species [18][19][16], (ii) genes that were previously shown by quantitative polymerase chain reaction (PCR) to change their CpG methylation status in response to 5-aza-dC [16], and (iii) genes that are implied to be regulated epigenetically based on the lack of sequence variation despite different expression levels and phenotypes [20][21][22].

For the latter category, we chose genes that are involved in the fatty acid synthesis pathway, as N. vitripennis lacks the ability to convert dietary sugars into fatty acids [23][20], similar to most other parasitoid wasps [24][25][26].

No apparent gene loss or pseudogenization has been observed in pathways relevant to lipogenesis [27], and DNA methylation has been suggested as a regulator of fatty acid synthesis in Drosophila melanogaster [21].

Therefore, we hypothesize that N. vitripennis may regain lipid production after demethylation.


Methylation status of the genes was assessed using high-throughput bisulfite amplicon sequencing (BSAS) on an Illumina platform [28].

With this method it is possible to accurately estimate the percentage of methylated CpGs per site at very high coverage (on average 12,000 times per sample in our experiment).

We tested the hypothesis that 5-aza-dC treatment following established methods in N. vitripennis [13] results in significant DNA demethylation.

In addition, we measured lipid reserves of wasps in control and 5-aza-dC treatments over a 9-day period.

These phenotypic measurements served a twofold purpose: first, if 5-aza-dC treatments result in significant changes in DNA methylation, we can test whether there is a concomitant change in lipid levels.

Second, if no effect of 5-aza-dC treatments on DNA methylation is found, the phenotypic measurements allow us to ascertain the general effectiveness of our 5-aza-dC treatment, as the antimetabolic activity of the demethylation agent is expected to cause a decrease in physical condition of the wasps, for which lipid reserves are a general and sensitive measure [24][29].


The fully homozygous wild-type strain AsymCX of Nasonia vitripennis of which the genome sequence is available [27] was chosen for our experiment.

These wasps were reared on Calliphora vomitoria pupae at 25°C, 16L:8D photoperiod, and 75% relative humidity.

To test the effect of 5-aza-dC on N. vitripennis, newly emerged females (<4 h old) were randomly assigned to either of two treatments: 5-aza-dC and control.

Each treatment consisted of four biological replicates, with each replicate consisting of 10 pooled females.

Wasps were kept in a plastic tube (height×diameter = 75.0 mm×23.5 mm) with a sponge stopper.

Wasps in the control treatment were fed a 20% sucrose solution, while the wasps in the 5-aza-dC treatment were fed a 20% sucrose solution to which 10 mM of 5-aza-dC was added, following the methods applied by previous studies [13][16].

In both treatments, wasps were kept without food for several hours before giving them access to the sucrose solution to ensure a high motivation to feed.

Wasps were observed to start feeding immediately when food was offered.

This is important because 5-aza-dC has low solution stability and a half-life of about 25 min in human cytoplasma due to high activity of cytidine deaminase [6], an enzyme for which the gene is also present in N. vitripennis (LOC107980937).

After 2 days, wasps were put in 70% ethanol and stored at -20°C for further analysis.

No individuals were removed or excluded from the experiment because all survived our treatments.


As a positive control of 5-aza-dC effectiveness, another set of wasps was given the same feeding treatments as above in parallel.

After feeding for 2 days on the same solutions as above, these wasps were given clean sucrose solution for another week to test for an extended effect of 5-aza-dC incorporation.

Randomly chosen samples of wasps from both treatments were frozen on days 0 (emergence), 2, and 9.

We measured their per capita lipid levels following standard methods [30][24][26].

All data on lipid levels have been deposited in the Dryad Digital Repository: https://dx.doi.org/10.5061/dryad.d305qm1 [31].


All four biological replicates (each consisting of 10 wasps) were washed with 70% ethanol, vacuum dried, and crushed in 100 µL of phosphate-buffered saline.

For DNA isolation, we followed a modified protocol of Promega.

In short, we added 100 µL of Nuclei Lysis Solution (Promega) and 2 µL of Proteinase K (20 mg/mL; Roche) and incubated the lysate for 60 min at 60°C.

After incubation, we added 170 µL of Lysis Buffer (Promega), vortexed shortly, and centrifuged the mixture for 10 min at 14,000 rpm.

We used spin columns and DNA wash buffer (Promega) to isolate the DNA from the supernatant.

For each sample, an aliquot of DNA was converted with bisulfite following the protocol of the EZ DNA Methylation-Gold kit (Zymo).

We selected 10 different gene fragments for measuring the DNA methylation level.

Genes aa and rnapol were chosen based on their consistently heavy methylation pattern in both Wang et al. ( 2013) and Beeler et al. ( 2014).

Genes perq and wdr36 contain highly methylated sites in the study by Pegoraro et al. ( 2016) and had reduced DNA methylation in their dmnt3-knockdown experiments.

Genes acc, fabd, fasn1, fasn2, fasn3, and mcd are part of the fatty acid metabolic pathway, which has been suggested to be epigenetically regulated [20][21].


For every fragment, we developed primers on the bisulfite-converted sequence.

Furthermore, we added nonconverted PCR products of aa and rnapol to counterbalance the low CG content in the sequencing library due to bisulfite conversion of C → T. Primers and PCR conditions are listed in table 2.

For PCR, we used the GoTaq Flexi DNA Polymerase protocol (Promega) according to the manufacturer’s specifications.

PCR products were loaded on a 1.5% agarose gel, and bands of the correct size were cut out.

PCR fragments were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).

DNA concentrations of all 96 (two treatments, 12 amplicons, four replicates per amplicon) purified PCR fragments were measured with Qubit DNA-HS technology (ThermoFisher Scientific).

Amplicons were diluted to a concentration of 0.2 ng/mL. We prepared dual index libraries following the Nextera XT DNA Library prep kit and the Nextera Index kit (Illumina).

Ninety out of 96 samples passed our quality check on an Agilent 2100 Bioanalyzer system, and these were normalized and pooled to a final pool of 3 nM. The pooled amplicon library was sequenced at Macrogen using Illumina MiSeq 150 bp paired-end sequencing technology.

No reads mapping to fasn2 were recovered, indicating that the library preparation failed for this amplicon.

The raw short-read sequence data are available in the National Center for Biotechnology Information Short Read Archive under BioProject number PRJNA52613.


Differences in lipid levels of wasps were tested using ANCOVA with feeding treatment and time as independent variables and fat-free dry mass as the covariate.

ANCOVAs were used for every day separately as post hoc tests (with a p < 0.05/3) to verify on which day the lipid levels diverged between treatments.

Raw sequence data were checked for quality with FastQC version 0.11.3 [32].

Illumina primers were trimmed with Trimmomatic version 0.35 [33] and demultiplexed with a Python script.

Reads were mapped to target gene sequences using Bowtie 2 version 2.2.6 [34], and single-nucleotide polymorphisms (SNPs) were called using the mpileup function in SAMtools version 0.1.19 [35] and summarized using BCFtools.

Because we used an entirely homozygous strain (AsymCX), SNP calling of reads against the reference genome gives only sites with varying methylation level plus a small number of sequencing errors.

Efficiency of the bisulfite conversion was measured on the proportion of C → T SNPs in non-CpG sites.

SNPs in non-CpG sites were removed from this data set.

Only CpG sites with >100 times the coverage were retained.

We calculated the proportion of methylated cytosines per site and plotted the results in R version 3.2.5 [36].

Using this pipeline, we obtained a data set of per-site methylation proportions for all amplicons and treatments.

Differences in methylation level per site were tested separately for each amplicon using a two-way ANOVA with treatment and site as factors, including interactions.

It is important to emphasize that site was included as a fixed effect in the model with each site assigned a unique code, so we did not test for a nonzero slope.


The lipid content of wasps is plotted in figure 1.

Despite feeding on sucrose, lipid levels decreased continuously over time in both treatments (ANCOVA, F1, 219 = 102.3, P < 0.001), which conforms to expectations for fatty acid auxotrophs.

Feeding treatment had a significant effect on lipid levels (ANCOVA, F1, 219 = 6.657, P = 0.011), with a larger decrease in lipid content in the 5-aza-dC feeding treatment.

On day 0, there was no difference between lipid levels between treatments (ANCOVA, F1, 74 = 0.907, P = 0.344), while on day 2 (ANCOVA, F1, 69 = 6.775, P = 0.011) and day 9 (ANCOVA, F1, 72 = 14.91, P < 0.001) lipid levels were significantly lower in the 5-aza-dC feeding treatment than in the control treatment.


After quality filtering and trimming, the DNA sequence data set consisted of 1,513,412 paired-end reads in total.

Of these, 90.8% mapped to the reference gene sequences.

We obtained 2,101 SNPs in total, of which 1,357 had a coverage of >100 times per sample.

Bisulfite conversion was highly effective at a rate of 98.86% ± 0.39% (mean ± SE).

No C → T SNPs were observed in the non-bisulﬁte-converted amplicons.

Figure 2 shows a histogram of the coverage per site for all amplicons combined.

Of the 155 CpG sites of our target amplicons, 148 were SNPs in our data set.

Some CpG sites had a lower proportion of cytosine than predicted by the bisulfite conversion efficiency (i.e., these sites are probably never methylated in situ).

The proportion of methylated cytosines is plotted per CpG site per amplicon in figure 3.


For a substantial proportion of the 155 individual CpG sites, methylation level increased or decreased in the 5-aza-dC relative to the control, but we tested changes in methylation status at the whole amplicon level (table 3).

In all amplicons we found significant differences in methylation level per site over the length of the amplicon (all P < 0.05).

There were no significant differences in methylation levels between 5-aza-dC and control treatments (all P > 0.1) and no significant interactions between treatment and site on the amplicon (all P > 0.05).


We expected 5-aza-dC-fed wasps to have a lower proportion of methylated CpG sites compared to the controls.

However, no significant differences between 5-aza-dC-fed and control treatments were found in any of the genes we examined at the 10-mM doses provided, the dose commonly used in other studies in this model system [13][16].

Although for a substantial proportion of the 155 individual CpG sites we noted a decrease or increase in methylation status, our results demonstrate that 5-aza-dC does not consistently reduce DNA methylation in the nine amplicons that we tested.

This result is corroborated by a recent genome-wide methylation study in Nasonia vitripennis, also showing a lack of uniform demethylating effect of 5-aza-dC treatment across sites [37].

We observed the same result for each of the three categories of genes we tested: there was no reduction in DNA methylation in highly methylated genes, nor in genes previously shown to have varying methylation levels, nor in genes involved in lipogenesis, a metabolic process that has been suggested to be epigenetically regulated.


We found a negative effect of 5-aza-dC treatment on lipid levels of N. vitripennis, most likely due to the detrimental effects of antimetabolic activity of 5-aza-dC [6].

Therefore, previous studies that attributed phenotypic changes in 5-aza-dC-fed wasps to a decrease in DNA methylation should be reconsidered in the light of the current results.

The high proportion (98.86%) of C → T SNPs in non-CpG sites proved that the bisulfite conversion efficiency in our study was high, also compared to other methods [38].

In CpG sites, the average methylation level was 0.09%–80.73% per site.

These two observations imply that the lack of a demethylating effect of 5-aza-dC was not due to inefficient bisulfite conversion or improper detection of DNA methylation.


Two CpG sites were tested in both our BSAS study and the MethylQuant-based study by Pegoraro et al. ( 2016).

The MethylQuant-tested sites correspond in our study to sites 113 and 220 for perq and wdr36, respectively.

Site 113 for wdr36 is the only site in this amplicon with a detectable methylation level, with considerable variation between samples.

Site 220 for perq seems to have a higher methylation level in 5-aza-dC-treated wasps.

However, most other sites in this amplicon show no difference, similar to the rest of the data set.

These differences could be life stage specific, a general pattern also found by Wang et al. ( 2013).


In plants and fungi, genome-wide DNA methylation levels have repeatedly been shown to decrease after 5-aza-dC treatment, often with associated changes in phenotypes such as growth, flowering phenology, or secondary metabolite production [39][1][7][40].

The evidence for a demethylating effect of 5-aza-dC in arthropods is much less strong.

This is partly because the major insect genetic model Drosophila lacks a DNA methylation system [41].

In other arthropod model systems, DNA methylation is present.

In Daphnia, for instance, 5-aza-dC had a clear demethylating effect on four out of the five tested genes, which differed among CpG sites [10].

However, few insect studies have shown a consistent effect of 5-aza-dC. In bumble bees, 5-aza-dC has an effect on overall DNA methylation, but its effect is erratic, as hyper- and hypomethylation were both observed after 5-aza-dC treatment [9].

A similar variable effect of 5-aza-dC treatment on methylation status of individual CpG sites was also found in a genome-wide study of methylation in N. vitripennis [37].

Our study adds to this growing body of literature by showing a lack of consistent demethylating effect of 5-aza-dC in N. vitripennis.


There may be several causes for this lack of a demethylating effect of 5-aza-dC in N. vitripennis and in insects in general.

The 5-aza-dC is usually administered via the food, and one explanation may be that the animals did not feed enough.

In our study, however, we observed all wasps to feed within minutes after the food was offered, ensuring that 5-aza-dC was ingested by all wasps.

Also, the phenotypic effect of 5-aza-dC treatment on lipid reserves indicates that the 5-aza-dC solution was effective.

An alternative explanation for the lack of demethylating effect of 5-aza-dC could be that there is a paucity of cell division in adult insects [42].

Incorporation of 5-aza-dC into the DNA happens only during cell division and is necessary to inactivate the DNA methyltransferase 1 enzyme [6].

In adult insects, only a few tissues contain regenerative cells, such as cells involved in neurogenesis in the brain of the house cricket Acheta domesticus [43], intestinal stem cells in the midgut of Drosophila melanogaster [44], and germ cells continuously dividing in ovaria of N. vitripennis [45].

Compared to organisms with continuous growth, the amount of cell division in adult insects is low, yet an effect of 5-aza-dC on DNA methylation would have been possible.


We showed a small but significant negative effect of 5-aza-dC on lipid levels in N. vitripennis.

Lipid levels decreased over time in wasps in both treatments, but the rate of decrease was higher in 5-aza-dC-treated wasps.

However, this is probably not biologically significant but due mainly to detrimental effects of 5-aza-dC on wasp health.

The five genes involved in the fatty acid synthesis contain many CpG sites, but methylation levels varied substantially among genes.

In FASN1, FASN3, and ACC, methylation levels at CpG sites were on average less than 1%, whereas MCD and FabD showed high methylation levels at CpG sites on various locations in the amplicon.

However, 5-aza-dC did not influence the DNA methylation levels in any of these genes.

Our results suggest that phenotypic differences observed in adult wasps after administering 5-aza-dC arise through a mechanism independent of DNA methylation—for instance, an effect on other metabolic processes.

Even though we did not quantify sex ratio in our study, our results warrant further investigation of the previously reported relationship between DNA methylation and sex allocation in N. vitripennis [13].

Due to the inconsistent effect of 5-aza-dC for DNA demethylation, it would not be advisable for future studies on the link between CpG methylation and phenotypes in N. vitripennis.

Pupal knockdown of DNA methyltransferases could be a good alternative, although this will also remove methylation of sites that could be essential for successful metamorphosis and thus lead to premature mortality.

A second alternative is the use of the nonspecific DMNT1 inhibitor RG108 [46], but as far as we know this method remains untested in N. vitripennis.

In all scenarios, the study should always include evidence showing consistent changes in DNA methylation.


We previously demonstrated that treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) alters the offspring sex ratios produced by females of the parasitoid wasp Nasonia vitripennis [1].

Females allocate offspring sex ratio in line with Local Mate Competition theory, producing more or less female-biased sex ratios as the number of other females laying eggs on a patch varies, thereby reducing competition amongst their sons for mates.

Interestingly, treatment with 5-aza-dC did not ablate the facultative sex allocation response.

Instead, sex ratios became less female-biased, a shift in the direction of the optimum sex ratio for paternally-inherited alleles according to genomic conflict theory.

This was the first (albeit indirect) experimental evidence for genomic conflict over sex allocation.

Ellers et al. ( 2019) have since assayed the effects of 5-aza-dC on DNA methylation in ten Nasonia genes, finding no evidence of demethylation in these 10 genes, from which they conclude that 5-aza-dC has no demethylating capability in Nasonia vitripennis.

Quantifying the efficacy of 5-aza-dC in terms of demethylation is indeed crucial to in-depth interpretation of studies using 5-aza-dC to link phenotypes to epigenetic regulation.

Here, we outline the mode of action of 5-aza-dC and demonstrate that determining the efficacy of 5-aza-dC in insect systems requires a whole-genome approach.


DNA methylation, sex allocation and 5-aza-2′-deoxycytidine In a previous study, we demonstrated that treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) alters the sex allocation response of Nasonia vitripennis females [1].

As with all Hymenoptera, N. vitripennis is a haplodiploid species, with males arising from unfertilized, haploid eggs and females developing from fertilized, diploid eggs.

Females are in putative control of sex allocation, either laying unfertilized eggs that will become sons or releasing sperm to fertilize eggs that will become daughters.

Female Nasonia allocate sex broadly in line with Local Mate Competition (LMC) theory [2][3][4]; females laying eggs alone will produce very female-biased sex ratios to minimize competition amongst their sons for mates.

As the number of laying females (foundresses) on a patch increases though, the level of LMC consequently declines and less female-biased sex ratios are produced.

Recent theory, however, has demonstrated that maternally- and paternally-inherited alleles should have differing patterns of optimal sex allocation under LMC, with maternally-inherited alleles selected to produce more female-biased sex ratios [5].

Importantly, parent-of-origin information must be associated with each allele, for instance via epigenetic marks such as DNA methylation.

We demonstrated that disrupting DNA methylation with 5-aza-dC did not ablate the facultative sex allocation response, but instead found that sex ratios were shifted in the direction of the paternal optimum (sex ratios became more less female-biased; [1]).

This result was consistent with predictions from genomic conflict over sex allocation theory [5], suggesting that unmanipulated sex ratios are closer to the optimum for maternally-inherited alleles (Figure 1).

Ellers et al. ( 2019), in their valuable comment on this study, raised the valid point that the efficacy of 5-aza-dC in insect systems should be tested, and have suggested from their own experimentation that 5-aza-dC does not disrupt methylation in Nasonia vitripennis.

To fully address their comments, we will begin by briefly reviewing the mechanism by which 5-aza-dC brings about the demethylation of DNA, before showing that assessing the role of 5-az-dC requires a whole genome approach.


Originally synthesized in 1964 by Piskala and Sorm (and also known as 5-AZA-CdR, DAC, decitabine, ZdCyD and AzaD amongst other names), 5-aza-dC is an analog of the natural nucleoside 2′-deoxycytidine (the carbon at the 5-position of the pyrimidine ring is replaced by a nitrogen) and was repurposed as a demethylating agent in 1980 [6].

Much of the current attention surrounding 5-aza-dC is attributable to its success in the treatment of cancers, particularly acute myeloid leukaemia and myelodysplastic syndrome [7][8][9].

In mammals, DNA methylation acts as a transcriptional silencer and the clinical efficacy of 5-aza-dC results from its ability to reactivate tumor suppressor genes silenced by aberrant DNA methylation [10].

DNA methylation for the vast majority of cases occurs at the C5 position of the cytosine ring in CpG dinucleotides and is catalysed by DNA methyltransferase enzymes (Dnmts), of which 3 types are known in eukaryotes.

Dnmt3 establishes DNA methylation patterns de novo, Dnmt1 maintains these patterns, and Dnmt2 is involved in tRNA methylation [11].

DNA methyltransferase genes have been found across the Insecta, varying in presence/absence and copy number across species.

The Nasonia vitripennis genome encodes a full DNA methylation toolkit including 3 copies of Dnmt1 [12].

DNA methylation itself is also present across the Insecta and is largely restricted to the transcribed regions of genes, in contrast to DNA methylation in mammals, which is found throughout the genome [13][14].

In addition, genomic levels of DNA methylation in insects is much lower (0-14%; [13]) as compared to 60-90% in mammals [14].

Regardless of the level of DNA methylation present within a genome, or whether methylation signals transcriptional suppression (as in mammals) or acts to maintain transcript integrity and/or initiate/regulate splicing (as has been suggested for the insects: reviewed [14]), the epigenetic modification itself is, to our knowledge, chemically identical.


The introduction of 5-aza-dC to a cell results in a cascade of chemical reactions to cause an eventual reduction in DNA methylation.

Upon its introduction to a cell 5-aza-dC is activated in situ via phosphorylation by deoxycytidine kinase, producing the biologically active triphosphate form of 5-aza-dC, 5-aza-dCTP [15].

This active form is an excellent substrate for DNA polymerase and is readily incorporated into DNA during the S-phase of the cell cycle [16][17][18][19].

In the resulting daughter strands produced from a DNA replication event, hemi-methylated CpG sites are remethylated by Dnmt1 to fully restore the patterns of methylation present in the parent strand.

However, where 5-aza-dCTP is incorporated into the daughter DNA strand, a covalent bond is formed between 5-aza-dCTP and a cysteine residue in the active site of Dnmt1.

Since the carbon at the 5-position in dCTP is replaced with a nitrogen in 5-aza-dCTP, the methyl transfer reaction cannot take place and Dnmt1 becomes irreversibly bound to the DNA [15][20][21].

Thus, treatment with 5-aza-dC results in a diminishing pool of Dnmt1, resulting in the passive loss of DNA methylation: this is the key effect of 5-aza-dC. Importantly, there is evidence that the effects of 5-aza-dC are unlikely to be random across methylated CpGs in a given genome, as genomic context can influence the extent to which its action is effective.

DNA sequence context plays a role, with demethylation of CpGs outside of CpG islands occurring at a higher frequency [22][23].

The distribution of transcription factor binding sites is also influential with, for example, “demethylation-sensitive” genes enriched for Forkhead box (Fox) binding sites and “demethylation-resistant” genes enriched for basic Helix-Loop-Helix (bHLH) binding sites.

Indeed, some genes are never demethylated and these are enriched for binding motifs not present in demethylated genes.

More broadly, 5-aza-dC activity appears to be targeted to transcriptionally active euchromatin in mammalian cells [24].

Given that the mode of action of 5-aza-dC is passive, these results indicate that specific loci remain demethylated after replication, whereas others at first appear “resistant” because they become preferentially remethylated by the remaining pool of DNA methyltransferase, leading to non-random patterns of demethylation [22].


We agree with Ellers et al. ( 2019) that quantifying the demethylation efficacy of orally-administered 5-aza-dC in N. vitripennis will permit a more complete understanding of the relationship between DNA methylation and the sex allocation phenotype in Nasonia (see [1]).

More broadly, this will allow us to determine the extent to which genome-wide demethylation can be altered with 5-aza-dC in insect systems.

In their analysis of the involvement of DNA methylation in the photoperiodic diapause response in Nasonia, Pegoraro et al. ( 2016) demonstrated that consumption of 5-aza-dC led to demethylation at specified CpG sites on a small scale.

Ellers et al. ( 2019) have examined the demethylating effect of 5-aza-dC at 155 CpG sites spread across 9 candidate genes in a slightly more comprehensive analysis.

The authors found no significantly differentially methylated CpGs at any of the 155 sites in association with 5-aza-dC exposure and conclude from this result that 5-aza-dC has no demethylating effect.

To our knowledge, there is no evidence to suggest that the candidate genes chosen by Ellers et al. ( 2019) are more (or less) likely to be influenced by 5-aza-dC – i.e. to have genomic contexts which favor or disfavor its action.

It would also be the first time that 5-aza-dC would have been shown to be inert in an organism in terms of influencing DNA methylation.

As such, to get a more complete picture and to more accurately quantify the demethylation efficacy of 5-aza-dC, we suggest that a whole-genome approach is required.


In the first instance, it is important to note that genomic DNA methylation in Nasonia is relatively low, with estimates ranging from 0.63% to 1.6 % of all CpG sites methylated [25][26].

These estimates are comparable with other hymenopterans such as the honeybee, which has approximately 0.7% methylated CpGs [27].

Therefore, a demethylation effect will be inherently difficult to detect just by looking across a small number of CpGs.

Ellers et al. ( 2019) examined fragments of 9 genes to examine for evidence of demethylation due to 5-aza-dC treatment.

Two genes were chosen because of a heavy methylation pattern (aa and rnapol) and five (acc, fabd, fasn1, fasn3, and mcd) are part of the fatty acid metabolic pathway, purported to be epigenetically regulated.

Two additional genes were selected from the study of Pegoraro et al. ( 2016; perq and wdr36) based on the incorrect assumption that these genes are heavily methylated under the conditions tested by Ellers et al. In fact, perq and wdr36 display elevated methylation only under long photoperiods.

Whilst these genes may be heavily methylated under certain conditions and/or epigenetically regulated, there is no confirmed evidence in insects that the genomic context of these particular genes predisposes them to demethylation by 5-aza-dC. As outlined above, demethylation via 5-aza-dC occurs due to a lack of functional Dnmt1 to restore methylation to CpG sites post-replication.

Given the contextual nature of the action of 5-aza-dC, there is a risk of false negatives in terms of assessing its genome-wide action.

Therefore, a lack of demethylation in the gene fragments examined by Ellers et al. ( 2019) cannot be used to conclude that 5-aza-dC does not affect DNA methylation across the genome more broadly.


Quantifying the efficacy of 5-aza-dC Recently we carried out a genome-wide analysis of the demethylating capacity of 5-aza-dC in Nasonia vitripennis [28].

Briefly, female wasps were exposed to one of three 5-aza-dC exposure regimes: 1) 20% sucrose for 24h, 2) 20% sucrose supplemented with 10 µM 5-aza-dC for 24h and 3) 20% sucrose supplemented with 10 µM 5-aza-dC for 48h.

For each of these three regimes, females were harvested at 0, 24 and 48h post-exposure and, prior to DNA extraction, heads were excised from the bodies as a nod in the direction of a tissue-specific analysis.

Whole-genome sequencing of bisulphite-treated DNA samples was carried out to determine the methylation status of individual CpG sites across the genome (proportion of methylated reads/total reads per CpG).

Median coverage depth across the whole experiment was approximately 900X across 4 million CpGs after quality filtering.

To examine the data as a whole, we summed proportion of methylated reads for each CpG by gene, to broadly account for contextual similarity of CpGs within the same genic region.

Data were available for over 11,000 genes comprising 85% of the protein-coding genes in the Nasonia vitripennis genome.

Distinct clustering of samples was associated with 5-aza-dC exposure regime, clearly demonstrating that 5-aza-dC exposure has a quantifiable effect on DNA methylation status on a genome-wide scale (Figure 2A).

To explore DNA methylation changes at the gene level, we used a generalized linear modelling approach to determine which genes had significantly different proportions of methylation across the gene with respect to 5-aza-dC exposure regime, time-point after exposure, and tissue.

To encapsulate these results, we found that approximately 8500 genes showed significantly altered methylation in response to treatment with 5-aza-dC. Interestingly, it appears that methylation in some genes initially increases in response to treatment with 5-aza-dC, an effect that is noticeable after 24h of exposure and then less prevalent after 48h of exposure (Figure 2B).

In our data, two of the genes tested by Ellers et al., aa and rnapol, do not exhibit significant differential methylation in association with “feeding regime” in our experiments.

However, four of their tested genes (perq, acc, fasn1, and fasn3) exhibit significantly altered methylation levels in association with an overall effect of “feeding regime”.

Interestingly a decrease in methylation levels of these genes (relative to controls) is noted in wasps that were exposed to 5-aza-dC for a 48-hour period but not in wasps exposed to 5-aza-dC for a 24-hour period.

This indicates that 5-aza-dC may take longer to exert its effects on these four genes.

We also note that Ellers et al. have used newly emerged N. vitripennis females, whereas we used two-day old mated females; the effects of 5-aza-dC are known to differ with life-stage (see below).

We are unable to draw comparisons between our analysis and that of Ellers et al. for their remaining three genes; for wdr36 and fabd we do not find any CpGs in the Ensembl Nasonia vitripennis annotation (v 2.1), and mcd was excluded from our analysis as genes with zero coverage for all samples in any particular treatment group were removed prior to statistical analysis.

Hyper-methylation has been observed previously in response to treatment with 5-aza-dC. Using “amplification of intermethylated sites” (AIMS) in honeybees, Amarasinghe et al. ( 2014) observed hypomethylation in 10 and hypermethylation in 9 of 62 loci in 5-aza-dC exposed individuals relative to controls.

Also, it looks as though Ellers et al. ( 2019), using bisulphite amplicon sequencing, may have uncovered some CpGs that exhibit hypermethylation in 5-aza-dC treated samples, particularly in perq (Ellers et al. 2019: Figure 3).

It is also noteworthy that two of the nine genes reported by Ellers et al. ( 2019; in their Table 3 and Figure 3) show marginally significant Treatment X Site interaction (fabd, p=0.055; fasn1, p=0.079).

This reflects the fact that 5-aza-dC affects CpG sites in a context-specific manner (and possibly in different directions), an effect which is likely masked in their data by other sites that do not differ.

The mechanism by which hypermethylation is brought about by 5-aza-dC is as yet unclear.

Current understanding of the DNA methylation machinery suggests that there are a range of Dnmt-including complexes and that there is cross-talk between them in terms of their roles in de novo and maintenance methylation [29].

It is possible that drug-induced demethylation in one region of the genome may have altered the regulation of the DNA methylation machinery itself, such that hyper-methylation occurs.

Nonetheless, it is clear that 5-aza-dC alters methylation patterns in the N. vitripennis genome and that this is associated with the allocation of offspring sex ratio [28][1].


Possible confounding effects Ellers et al. ( 2019) raised the valid concern that the anti-metabolic activity of 5-aza-dC could result in a decline in the physical condition of Nasonia vitripennis females, indirectly producing observed phenotypic differences through a mechanism independent of DNA demethylation.

Whilst we have shown that 5-aza-dC at the dosage used in our original experiment [1] produces altered methylation patterns (above; [28]), the possibility of confounding side-effects should not be ignored.

We do note, however, that an effect of condition-dependent sex allocation has yet to be elucidated in Nasonia vitripennis, although pathological side-effects of any given chemical exposure cannot be discounted.

We also note that sub-lethal exposure to neurotoxic neonicotinoid pesticides does also influence sex allocation, albeit in the opposite direction to that seen with 5-aza-dC; i.e. more female-biased sex ratios are seen than expected: [30][31].

In humans, the major side effect of 5-aza-dC treatment is myelosuppression [10], a condition in which bone marrow activity is decreased resulting in fewer red and white blood cells and platelets.

It is difficult of course to extrapolate to what the major side effect of the drug in insects might be, if any.

In mammals, high-dose schedules result in an increased presence of DNA-Dnmt1 adducts which impede DNA polymerase function, leading to growth arrest and apoptosis.

However, low-dose schedules are known to capitalize on the hypomethylating capability of the drug and minimize these additional cytotoxic effects [6][32][33][34].

In trialling the cancer drug 5-aza-dC as a tool for molecular ecology and evolution studies, researchers have also used low doses to achieve DNA demethylation [35][36][1][37][38].

In their 2014 study, examining DNA methylation differences between different reproductive worker castes in Bombus terrestris, Amarasinghe et al. used 5-aza-dC (<10µM) on both callows (bees less than one day old) and adults (bees more than one day old).

This is less than half of the lowest concentration (25µM) used to test for genotoxic effects of 5-aza-dC in Drosophila melanogaster at a range of concentrations [39].

Their experimentation showed no effect of 5-aza-dC on the adult bees in contrast to the callows, which developed larger ovaries, became more aggressive, and showed altered methylation patterns (at 19 of 62 tested loci) relative to controls of the same age.

The authors hypothesized that cells in adult bees may be largely post-mitotic thus decreasing the efficacy of 5-aza-dC in adults vs callows.

Importantly, neither callows nor adults showed reduced activity or reproduction, indeed callows became more reproductive.

If the effects of 5-aza-dC were merely toxic it is reasonable to assume that reduced fitness would be observed in both age groups even if altered methylation patterns occurred only in callow bees.

Ellers et al. quantified the lipid reserves of both control wasps and those treated with 5-aza-dC as a general measure of body condition.

The authors showed that lipid reserves were significantly lower as a result of 5-aza-dC treatment and that reserves in both treatment and control groups declined over the duration of the experiment.

The authors conclude that these differences in lipid reserves can only be attributed to a detrimental effect of 5-aza-dC on wasp health given that they observed no alterations in DNA methylation in a total of 6 genes involved in fatty acid metabolism.

Given demonstrations that 5-aza-dC does produce altered methylation patterns [36][37][28] it is possible that hypo- or hyper-methylation in genes untested in their experiment could have induced a decrease in lipid reserves; the six genes tested cannot be regarded as representative of the entire fatty acid metabolism pathway.

Interestingly, Amarasinghe et al. ( 2014) noted increased reproductive capability as a result of 5-aza-dC so it is possible that the observed reduction in lipid reserves is due to the diversion of resources elsewhere.

It is also unclear whether the decline in lipid reserves was sufficient to produce a true reduction in fitness in terms of offspring production or longevity as this information is not available.


5-aza-dC and evolutionary ecology Research has determined that 5-aza-dC acts to alter methylation patterns in insects and has assisted evolutionary biologists in elucidating a role for epigenetics in a range of phenotypic traits, including sex allocation, worker reproduction, and diapause [28][37][36].

However, a greater understanding of how 5-aza-dC brings about altered methylation could improve the precision of its use in evolutionary ecology studies.

As a starting point, careful selection of the life-stage of experimental individuals can prove important.

5-aza-dC had no demonstrable effect on DNA methylation when administered to bees more than one day old but resulted in significant hypo- and hyper-methylation in younger bees [36].

This may be due to the passive mode of action of 5-aza-dC and a higher percentage of cells in adult bees being post-mitotic.

This effect is also likely to be dose-dependent and species-specific.

Therefore, if no effect of 5-aza-dC is initially observed in a given species, it is worthwhile investigating the efficacy at different life-stages.

Upon introduction to the cell, 5-aza-dC is converted to 5-aza-dCTP but, in addition, 5-aza-dC is also subject to deamination resulting in an inactive form of the drug being produced alongside the active form.

This is due to very high levels of cytidine deaminase in the human liver and spleen [40][41].

Ellers et al. ( 2019) called attention to the fact that cytidine deaminase is also present in insects.

Indeed, there is a “cytidine deaminase-like” gene in the Nasonia vitripennis genome (NCBI: LOC107980937) which may act to degrade some of the introduced 5-aza-dC to its inactive form.

To counteract this effect, the use of an inhibitor of cytidine deaminase, such as tetrahydrouridine or zebularine has been suggested [42].

When administered in combination with 5-aza-dC in preclinical trials, the cytidine deaminase inhibitor zebularine was shown to increase the clinical efficacy of 5-aza-dC in cell lines by reducing the degradation of 5-aza-dC [43].

Pilot experiments to determine whether the use of a cytidine deaminase inhibitor increases the efficacy of 5-aza-dC in insect study systems would be beneficial.


Concluding remarks Here, we have discussed the mechanisms by which the demethylating agent 5-aza-dC exerts its effect and illustrated that a whole-genome approach is required to determine the efficacy of this agent in Nasonia vitripennis.

Having used drug-induced demethylation to validate a role for epigenetic regulation in sex allocation [1][28], the desirable next step would be to ascertain which CpGs are involved in the expression of the sex allocation phenotype; i.e. the causal CpGs.

As outlined above, research has shown that drug-induced demethylation is non-random and reproducible.

Experimentation is warranted to determine the reproducibility and the genomic context underlying the methylation patterns produced by 5-aza-dC in N. vitripennis and other insect systems.


Behavioral traits of waterfowl have led to various hypotheses about the location and degree of popu- lation genetic structure.

First, in most waterfowl species females exhibit greater natal and breeding site fidelity than males (Greenwood 1980, Anderson et al. 1992).

Over sufficient periods of time and spatial scale, natal philopatry can lead to genetic differenti- ation, especially at maternally inherited molecular loci such as mtDNA.

Second, both sexes may exhibit winter site fidelity for a variety of ecological and genetic reasons, including re-establishment of breed- ing pairs as mate choice is thought to occur on the wintering grounds (reviewed by Robertson and Cooke 1999).

In either case adult nest or winter site fidelity might also serve to differentiate populations, but only at larger scales where populations are separated at distances greater than all dispersal events.

However, even at these larger scales, genetic differentiation can be limited by historical demo- graphic processes and large effective population sizes, which reduces the diversifying forces of mutation and drift (reviewed by Avise 2004).


In Harlequin Ducks, molt-site fidelity would only lead to demographic and population genetic structure if molting flocks remain as distinct units throughout the entire year.

Regehr et al. ( 2001) observed that Harlequin Duck broods may accompany adult females to molting sites.

However, it is not known if juvenile Harlequin Ducks, especially females, return to these same molting areas as adults in subsequent years.

Tracking natal dispersal of birds via mark-recapture methods is very difficult be- cause efforts must be made to recapture previous- ly marked individuals across a wide range of possi- ble dispersal distances.

This difficulty is compound- ed among sea duck species, which typically do not breed until their second or third year.

Thus, data from multiple years and wide geographical efforts would be needed to discern the range of possible dispersal events (Lebreton et. al. 2003).


However, if dispersal is male-biased, one might predict higher levels of population differentiation using maternally inherited mtDNA in contrast to nuclear DNA, which tracks both maternal and paternal lineages.

Lanctot et al. ( 1999) observed precisely this pattern, with slightly elevated levels of differentiation for mtDNA in comparison to nuclear microsatellite data among regional groupings of female Harlequin Ducks.

Still, the level of mtDNA differentiation observed by Lanctot et al. ( 1999) was low (W ST 5 0.05) suggesting some female gene flow, but at a reduced level compared to males.

Cooke et al. ( 2000) viewed the genetic data of Lanctot et al. ( 1999) as validation of their conclu- sions of male-mediated gene flow, but Iverson et al. ( 2004) found the genetic data unable to ''indicate'' the fine-scale population structure suggested by the homing rate.


First, little is known about the breeding area origins of molting Harlequin Ducks in Prince William Sound.

Genetic data would only indicate fine-scale structure if each molting flock remained isolated throughout the year and over thousands of generations.

A more likely scenario is that each molting flock is composed of birds from multiple breeding areas.

Second, consider the genetic implica- tions of demographic independence on such a fine scale.

If juveniles follow females to molting sites each year and then both sexes continually return to those sites each year at a homing rate of 0.95-1.0 (Iverson et al. 2004), two immediate predictions for these flocks are: (1) elevated levels of inbreeding within each ''demographically independent'' molting flock, and (2) substantially higher levels of mtDNA population differentiation due to the smaller effective population size of mtDNA compared to nuclear loci and the small size of each molting flock (n , 30), compounding genetic drift.

Neither of these predic- tions is borne out by Lanctot et al. ( 1999).

Genotype data from five microsatellite loci showed no signif- icant heterozygote deficiencies, inbreeding coeffi- cients did not differ significantly from zero, and levels of population differentiation estimated using mtDNA were low.


Other hypotheses concerning the population ge- netics of wintering Harlequin Ducks in Prince William Sound include: (1) a lack of power among genetic markers to distinguish wintering groups, (2) insufficient time since populations expanded into northern latitudes for site fidelity patterns to accrue genetic differences, and (3) molt-site fidelity is a complex behavioral process.


A lack of power seems unlikely because fewer genetic loci have shown population differentiation in other waterfowl that exhibit high levels of adult site fidelity (Pearce et al. 2000).

Harlequin Ducks have likely experienced historical population growth and expansion since the last glacial maximum.


Consistently low levels of nucleotide diversity (near zero) across populations, as observed by Lanctot et al. ( 1999), are indicative of sudden population growth and expected if time is sufficient for recovery of haplotype variation via mutation, but too short for the accumulation of differences among sequences (reviewed by Avise 2000).


Lastly, Lanctot et al. ( 1999) correctly ac- knowledged that movements of subadult birds among molting sites before patterns of adult fidelity are established might nullify the effects of high adult fidelity to these same sites (as observed in other species; Cabe 1999, Arsenault et al. 2005).

No increase in DNA samples or genetic loci would recover a signal of population differentiation if natal and adult dispersal occur among these molting groups.

Such movements would also erode any demographic independence among these same groups.


The hypothesis of molt or winter site fidelity leading to demographic and genetic structure de- serves further examination because anthropogenic effects on sea duck populations often occur on the wintering grounds (Flint et al. 1999, Esler et al. 2000, Camphuysen et al. 2002).

However, recent work suggests little genetic evidence for population struc- ture among wintering sea ducks at either regional (Lanctot et al. 1999) or continental scales (Pearce et al. 2004).

Additionally, the literature presented by Iverson et al. ( 2004) does not support a hypothesis that strong affiliations to nonbreeding areas are common among sea duck species.

Papers by Alison (1974) and Savard (1985) are each based upon a single marked bird, and Limpert (1980) estimated a homing rate of 39%.

Of 26 male and two female Harlequin Ducks banded by Breault and Savard (1999), nine males were seen at the same location in the following year, while two males were seen at an adjacent molting site.

Thus, this and other literature (Cooke et al. 2000, Flint et al. 2000, Hatton and Marquiss 2004, Mehl et al. 2004) suggests that annual affiliations to molting areas by sea ducks are quite variable.

Lastly, we view molt-site fidelity as a less than robust measure of population structure without evidence that molting flocks originate from distinct breeding areas.

Under the scenario that molting flocks are composed of birds from a variety of breeding areas, fidelity is instead an intriguing behavioral pattern and not a measure of demographic independence.


Conservation plans often seek to delineate geo- graphic or taxonomic units as distinct population segments to effectively monitor status and trends (U.S. Fish and Wildlife Service and National Marine Fisheries Service 1996).

Defining such units relies upon a wide array of criteria that for migratory birds may include morphological or plumage characteris- tics, demographic patterns quantified by banding or radio-telemetry, molecular genetic data, or geograph- ic separation of population segments during the annual cycle.

However, focusing on only one type of data to define such units misdirects valuable research and conservation efforts (Zink et al. 2000) and tends to promote population structure as a binary condi- tion: populations are either structured or they are not (Crandall et al. 2000).

A larger set of direct and indirect markers are now available for assessing movement patterns (reviewed by Webster et al. 2002, Kendall and Nichols 2004), as well as novel analytical methods that estimate levels, directionality, and sources of variation in dispersal rather than simply testing dispersal as a binary condition (Pritchard et al. 2000, Hey and Nielsen 2004, Kendall and Nichols 2004).

Thus, we wish to stress that behavioral patterns among avian populations are intricate and idiosyncratic (Avise et al. 1992, Zink et al. 2003, Coltman 2004), and efforts should focus on the robust quantification of these multifaceted processes with as much information as possible.


For example, a number of avian genetic studies discuss the impact of historical demographic pro- cesses on levels of population differentiation and the need for other, nongenetic data to verify or challenge conclusions (Bossart and Prowell 1998, Kimura et al. 2002, Pearce et al. 2005).

Similarly, banding studies have called for DNA-based estimates of movement after recognizing the difficulty of detecting and quantifying long-distance dispersal (Koenig et al. 2000, Arsenault et al. 2005).

In a joint analysis of banding and genetic data for the Lesser Snow Goose (Anser caerulescens), Avise et al. ( 1992:1094) argued appropriately that ''both evolutionary (genetic) and contemporary (behavioral) perspectives are required for a full appreciation of the geographic population structure of a species.''


An examination of the table of contents of recent ornithological and molecular ecology journals reveals the current focus on demographic parameters (e.g., nesting success, productivity, survival, dispersal, and colonization history) and their role in population status and trends.

Yet a common misconception is that genetic data are a panacea for inferring population structure.

Instead, molecular genetic markers should be viewed as offering a singular but multifaceted perspective on population differentia- tion and demography.

Regardless of whether genetic or nongenetic methods are used, we encourage researchers to use multiple data types when they are available.


Even in cases where genetic data suggest no differentiation among sampling locales, such as among molting groups of Harlequin Ducks, there is still a wealth of information that can be inferred from the molecular information, such as historical popu- lation trends (Emerson et al. 2001), geographic variation in genetic diversity (Busch et al. 2000, Zink et al. 2000), relative levels of female natal philopatry and male dispersal (Pearce et al. 2005), or evidence COMMENTARY 477 Downloaded From: https://bioone.org/journals/The-Condor on 21 Sep 2025 Terms of Use: https://bioone.org/terms-of-use for gene flow via dispersal that is difficult to assess with localized banding data (Arsenault et al. 2005).

Because inferences from multiple markers reveal the weaknesses and strengths of each method, compar- isons among data types provide a competing model framework to reassess predictions, evaluate temporal and spatial scales, and best infer population patterns.


We thank D. Esler and S. Iverson for the numerous amicable and engaging discussions on topics presented here.

C. Ely, D. Derksen, P. Flint, M. Lindberg, K. McCracken, J. Schmidt, D. Dobkin, T. Sachtleben, and one anonymous reviewer offered comments on earlier drafts of the paper.


Iverson et al. ( 2004) used estimates of the homing rate for molting adult Harlequin Ducks (Histrionicus histrionicus) in Alaska to draw inferences about population structure.

Homing rates, defined as one minus the ratio of birds recaptured elsewhere to those recaptured at the original banding site, were high (0.95–1.00) for males and females.

Iverson et al. ( 2004) concluded that these high rates of homing are indicative of demographic independence among molting groups separated by small distances (tens to hundreds of kilometers) and that conservation efforts should recognize this fine-scale population structure.


We re-examined their use of the homing rate, because their assumption of equal detection probability across a wide sampling area could have led to an upward bias in their estimates of site fidelity.

As a result, we are hesitant to agree with their conclusion of high adult homing to molting areas and that molt-site fidelity is evidence for demographic independence.

Our hesitancy stems from the fact that little is known about juvenile and adult movements within and among years, breeding area origins, and the variation of demographic parameters (e.g., survival and productivity) among molting groups.


Furthermore, population genetic data of these molting groups suggest gene flow at both nuclear and mitochondrial loci.

Such mixed messages between demographic (i.e., banding) and genetic data are increasingly common in ornithological studies and offer unique opportunities to reassess predictions and make more robust inferences about population structure across broad temporal and spatial scales.

Thus, we stress that it is this broader scale perspective, which combines both demography and genetics, that biologists should seek to quantify and conservation efforts should seek to recognize.


In a recent paper on Harlequin Duck (Histrionicus histrionicus) interannual site fidelity [1], we concluded that wintering populations were demographically structured at a finer geographic scale than that at which genetic differentiation was observed and that conservation efforts should recognize this degree of demographic independence.

In a critique of our study, Pearce and Talbot [2] contend that our measures of fidelity were not robust and imply that in the face of 'mixed messages' we failed to appreciate the role of genetic data in defining population units.

We recognize, as we did in our original paper, that our methods for quantifying site fidelity have some limitations; however, the patterns in our data are consistent with a considerable body of literature indicating high winter site fidelity in Harlequin Ducks.

Moreover, we do not consider differences in the scales at which genetic and demographic structure are expressed to be 'mixed messages,' given the different spatial and temporal scales at which genetic and contemporary demographic processes operate.

We emphasize that a lack of genetic differentiation does not necessarily preclude the existence of contemporary demographic structure with relevance for conservation.

Population management is clearly enhanced by knowledge of the demographic structure of animal populations, i.e., the scale at which changes in numbers of animals in one area are largely independent of changes occurring in another area.

Quantifying the rate and scale of movements among putative population subunits is critical for this understanding [3], [4].

In simple terms, in situations where animals move frequently and far, population dynamics are affected by immigration and emigration at relatively large geographic scales.

Conversely, for animals that show strong site fidelity, population dynamics at relatively small scales are driven primarily by the intrinsic demographic properties of survival and productivity, i.e., they are demographically independent from other areas.

Understanding the degree and scale of demographic independence is critical for identification of appropriate population subunits for conservation.

Definition of appropriate management units is difficult [5], [6], particularly when applied to migratory animals [7], [8].


We [1] conducted a study of winter site fidelity of Harlequin Ducks (Histrionicus histrionicus) in Prince William Sound, Alaska to evaluate the degree and scale of interannual dispersal and consider the subsequent conservation implications.

We found high homing rates (>90%) to nonbreeding sites at a scale of kilometers to tens of kilometers and concluded that demographically independent population subunits exist at relatively small scales.

Further, we noted that the scale of demographic independence indicated by our data was much smaller than the scale at which genetic differentiation was observed [9], and that genetic panmixia does not necessarily imply nonexistence of relevant demographic population structuring.

Pearce and Talbot [2] have two primary criticisms of our paper [1].

First, they suggest our approach may not have adequately measured site fidelity.

Second, they consider mechanisms by which the observed 'mixed message' (different patterns and scales of genetic versus demographic population structuring) may have arisen, and imply that we did not fully appreciate the value derived from considering multiple markers when evaluating population structure.

We address their criticisms below, primarily in relation to the latter concern, as we feel this issue has broad relevance for wildlife conservation.


Good science starts with a clear definition of the question being asked.

Confusion expressed over definition of population subunits for conservation, as well as the appropriateness of tools that can be used to evaluate population structure, may have arisen from imprecise questions.

Specifically, managers and researchers need to communicate the spatial and temporal scales over which they wish to quantify population structure, as well as the underlying mechanisms and patterns that they wish to describe.

Some conservation efforts are explicitly concerned with preservation of genetic diversity; this is particularly true in cases where populations are extremely small or where population subunits are highly isolated [10].

However, we argue that the majority of wildlife conservation issues are concerned with understanding changes in animal numbers, across a range of spatial scales, at time frames measured in years to decades.

In other words, the issues are primarily demographic and contemporary in nature.

Under this scenario, we contend that conservation units are most appropriately defined by identifying the scale at which population dynamics of putative subunits are demographically independent.

There are many methods for inferring the degree of contemporary demographic independence among areas, each with associated advantages and shortcomings.

Genetic data have been used for many taxa; advantages of this approach include the opportunity to gather samples across broad geographic scales, and the ease of sample processing and analysis relative to large-scale mark-recapture programs.

However, one limitation of genetic data is that the patterns observed are the product of both historical and contemporary population processes.

As such, one cannot necessarily infer patterns of contemporary demographic isolation from patterns in genetic data.

This lack of a one-to-one relationship between contemporary demographic structure and genetic structure has often been treated as a 'mixed message' with unclear interpretation.

We appreciate that there is considerable scientific interest in population structure beyond definition of appropriate population subunits for conservation, and that the field of population genetics encompasses a broad suite of interesting and important questions.

However, our specific interest, as stated in our original paper, was to consider the conservation implications of observed levels of demographic independence, and to contrast our findings with inferences one might be tempted to draw from existing genetic data [9].

For that particular purpose, we believe there is common misunderstanding of the inferences one can draw from different data types, which could be interpreted as 'mixed messages' without a full appreciation of the spatial and temporal scales at which different markers indicate population structure.


We measured homing rates of molting Harlequin Ducks, defined as the proportion of marked ducks that were recaptured at their original capture site, and used the results to infer the degree of interannual fidelity to nonbreeding sites.

We found that over 90% of recaptured individuals were in the same locations as their original captures and that detected movements were on the scale of kilometers to tens of kilometers.

From these findings, we concluded that site fidelity was high, with implications for the scale of demographic independence among nonbreeding population units.

We recognize that our measure of homing rate has limitations and assumptions and explicitly acknowledged these in our original paper.

Pearce and Talbot [2] correctly indicated that our measure of homing rate would be an overestimate of site fidelity if recapture efforts were lower in areas outside the primary study area.

To evaluate whether this was likely to have strong influences on our estimates, we conducted captures at numerous supplementary sites that held concentrations of molting Harlequin Ducks and represented a range of distances (8–62 km) from the primary study area.

Although we did not sample at every possible location within this range, none of the birds marked at the primary study site were captured at supplementary sites; we suggest this result would be very unlikely if there were demographically relevant movements from our primary study site at this scale.

We also acknowledge that dispersal beyond a 62 km radius from our primary study site was possible; however, given the lack of evidence for movement at smaller scales, we suspect that demographically relevant movement at larger scales was unlikely.

Our findings and conclusions are corroborated by several studies on Harlequin Duck dispersal and site fidelity [11], [12], [13], [14].

Collectively, these studies provide a strong basis for concluding that fidelity to nonbreeding sites is quite high, and that this degree of site fidelity likely leads to demographic independence among nonbreeding sites at relatively small scales.


Pearce and Talbot [2] suggested that, because our data were collected on adult Harlequin Ducks, high levels of juvenile dispersal might increase the scale of demographic independence and therefore might explain the 'mixed message' observed between genetic and demographic population structure.

However, the available data do not indicate this is the case.

Regehr et al. [ 15] found that at least some juvenile Harlequin Ducks accompany their mothers to her wintering area, and Regehr [16] further discovered that juvenile dispersal during the first winter was on a scale of only tens of kilometers.

Also, Iverson and Esler [14] found that intra-annual winter site fidelity was similar among all age cohorts of females tracked by radio-telemetry.

Finally, juveniles constitute a small portion of the overall Harlequin Duck population; therefore, movement patterns of adults have a highly disproportionate effect on the degree of demographic independence.

We do not dispute that some dispersal of adult and juvenile Harlequin Ducks occurs; in fact, we have documented this at a range of spatial scales [1], [14].

However, we have suggested [1] and quantitatively demonstrated [14] that the observed rates of movement are low and lead to demographic independence of wintering aggregations at relatively small spatial scales.


We agree with Pearce and Talbot [2] that dispersal may lead to homogenization of gene frequencies observed at regional scales [9]; however, this absolutely does not preclude the possibility that populations are structured demographically at much smaller scales.

In addition, we disagree with Pearce and Talbot’s [2] assertion that high fidelity to nonbreeding sites would lead to demographic and genetic structure only if nonbreeding population units remained as distinct units during the rest of the annual cycle.

A number of mechanisms could maintain genetic distinctiveness even when different population units co-occur (e.g., assortative mating, pair formation at a different annual stage).

More important for this discussion is that demographic independence can be relevant for conservation at any stage of the annual cycle.

Our conservation concerns were framed as an issue on nonbreeding areas, and therefore it does not matter whether Harlequin Ducks from demographically distinct wintering areas co-occur during the breeding season.

As long as inter- and intra-annual winter site fidelity are high, numerical trends of nonbreeding subunits will be driven by intrinsic survival rates, without opportunity for demographic rescue through dispersal from other subunits.


Pearce and Talbot [2] recognize that genetic and demographic population structure are expressed at different spatial and temporal scales.

However, rather than acknowledging that this may explain the 'mixed message' observed for Harlequin Ducks, they seem to suggest that methodological problems with our demographic data are the cause.

Our data, and those in the accumulated literature on non-breeding site fidelity of Harlequin Ducks, strongly support the existence of demographic structuring at much smaller spatial scales than those at which genetic structure exists.

We see this neither as conflicting data nor as a mixed message, but rather as a reasonable outcome of measurements of processes that occur at different spatial and temporal scales.

These are not merely academic issues, but have important implications for wildlife conservation.

Our study site included areas affected by the 1989 Exxon Valdez oil spill and Harlequin Ducks were one of the species showing delayed recovery [17].

Understanding the geographic scale at which putative subpopulations are demographically linked by dispersal allows clearer insights into the constraints on population recovery.

We have determined [1], [14] that rates of Harlequin Duck movement between oiled and unoiled areas of Prince William Sound are low, thus the contribution of immigration to population recovery in oiled areas will be correspondingly low.

Therefore, to be effective, management and restoration activities to ameliorate effects of the oil spill will need to be targeted to the specific areas and population subunits in the oil spill zone.


To reiterate, this population management issue for Harlequin Ducks, like most wildlife conservation problems, is concerned with population dynamics at a contemporary time scale and, as such, requires a clear understanding of contemporary demographic population structure.

Our primary message in our original paper and this reply is that researchers and managers should not necessarily expect genetic population structure and contemporary demographic structure to show similar patterns.

We want to be clear that we are not saying that genetic data are useless for inferring contemporary demographic population structure.

In fact, they could be quite valuable; if genetic data indicate structure at a given scale, then there is certainly demographic structure at that scale.

Genetics may be a particularly efficient way of evaluating demographic structure at a scale that may be of interest to managers.

However, if no genetic structure is evident, one cannot infer that there is demographic panmixia at the same scale.

In other words, lack of genetic structure does not provide any indication whether population subunits are demographically discrete at the contemporary time scale at which population management generally operates.

We agree with Pearce and Talbot [2] that consideration of more than one type of marker can lead to additional insights into questions of interest.

We simply caution that genetic and demographic data should not be expected to necessarily lead to the same inferences about population structure relevant for conservation.

We hope this exchange leads to critical thinking and additional research on the topic.


Nicotine is a major driver of tobacco use and the chemical that makes it so frustratingly difficult for smokers to successfully quit.

1 In 2017, the US Food and Drug Administration (FDA) announced a shift in its approach to tobacco regulation, with a key focus on reducing nicotine in cigarettes to levels low enough that cigar- ettes would be minimally or non-addictive.

2,3 If implemented, this standard would allow only very low nicotine content (VLNC) cigar- ettes to be sold in the United States.

Clinical research has found that VLNC cigarettes lead to lower dependence, fewer cigarettes smoked per day, and increased quit attempts compared to standard nicotine level cigarettes.

4-9 The FDA has not stated whether a nicotine re- duction standard would be implemented in one large reduction or gradually phased in over a number of years.

Models of population- level effects of a nicotine reduction product standard in the United States suggest that a large reduction of nicotine would lead to 5 mil- lion smokers quitting in the first year alone, and tens of millions of life-years saved over the next few decades.

10 Toxicologically, VLNC cigarettes would retain all or nearly all of the same carcinogens and other toxins in current cigarettes and would therefore not be mean- ingfully safer to smoke.


However, 80% of the public believes nicotine is the carcinogenic substance in cigarettes, 11 and a national survey shows nearly half of smokers believe VLNC cigarettes are less carcinogenic to smoke than current cigarettes, 12 in line with findings from clinical trials.

13,14 This misunderstanding is associated with a lower motivation to quit smoking under a VLNC standard.

12 Thus, it is critical to study how to announce a nicotine reduction standard to the public in a way that does not exacerbate misperceptions.

The public needs to understand what the standard does (greatly reduces nicotine and addictiveness) and what it does not do (make cigarettes safe to smoke).


In the era of social media, in which facts and rumors can spread rapidly, framing plays an increasingly important role in how the public receives new health information.

For example, re- commendations for HPV vaccination and changes in breast cancer screening in the United States generated substantial confusion, misinformation, and backlash that effective message framing may have at least lessened. 15-17 Preliminarily testing of messages to communicate a new nicotine standard is vital to preparing for a smooth implementation.


Message testing is also important to help the FDA clearly and consistently communicate to the public, amid opposing communications from the tobacco industry, which has deliberately misled the public for decades.

18 In formal responses to possible FDA nicotine reduction, the industry has written long lists of concerns.

19,20 An important question is how to explain to the public what a nicotine reduction standard would do in terms of the nicotine level in cigarettes.

The scientific standard (potentially a maximal limit of 0.4 mg nicotine per gram of tobacco) would likely be hard for the public to interpret.

Alternatives such as "nicotine- free" would not be entirely accurate and could be legally prob- lematic because the FDA is not authorized to reduce the nicotine content in cigarettes to zero.

21 In public announcements, the FDA has used phrases such as "lowering nicotine in cigarettes to non-addictive levels" 3 and "lowering nicotine in combustible cigarettes to minimally or non-addictive levels." 22 In studies of the public's understanding of VLNC cigarettes researchers have also used various descriptions.

These include describing cigarettes that have "a very low nicotine level, compared to most cigarettes available in the United States" 14 or are "lower in nicotine... than regular cigarettes."


Both for the potential rollout of an FDA nicotine reduction policy and for research studies that involve describing VLNC cig- arettes, there is a need to examine how people interpret different phrasings and how the FDA can most effectively convey nicotine reduction information.

The FDA can use these findings to inform wording used in press releases and in mass media communication.

Drawing from risk communication techniques, we sought to learn how different ways of describing the nicotine level of VLNC cigar- ettes affects the accuracy of perceived nicotine content, perceived addictiveness, and perceived cancer risk of VLNC cigarettes com- pared to current cigarettes.


Participants were a national convenience sample of US adults (ages 18+) recruited in April 2018 through Amazon Mechanical Turk (MTurk), a platform often used for scientific research recruitment.

24 The inclusion criteria were being aged 18 or older and residing in the United States.

Experiments using MTurk and probability-based samples generate the same general findings.

25 We have previously reported additional details on the recruitment and other methods for our study.


Participants provided informed consent before taking the survey.

In a between-subjects online experiment, we randomized participants to view 1 of 6 descriptions of a potential FDA product standard mandating lower nicotine levels in cigarettes or a control description (Table 1).

We developed the descriptions by applying evidence-based risk communication principles.

28 The first description used concise language: "Imagine if tobacco companies were required to reduce the nicotine in cigarettes." This follows the finding that communica- tions are more effective if they are brief and devoid of extraneous in- formation.

29 The second description provided numeric information as a percentage: "Imagine if tobacco companies were required to remove 95% of the nicotine from cigarettes." Research shows that people have more accurate perceptions when given a number, rather than a qualitative word such as "common" or "low." 30 The third de- scription provided an interpretation of nicotine reduction: "Imagine if tobacco companies were required to make cigarettes nearly nicotine-free." This interpretative language could make it easier for readers to gain the meaning of a message.

31,32 The fourth descrip- tion combined a percentage and interpretation: "Imagine if tobacco companies were required to remove 95% of the nicotine from cig- arettes.

This would make cigarettes nearly nicotine-free." The fifth description combined the percentage and pictographs.

Pictographs show the numerator and denominator to help a reader visualize a percentage.

33 The sixth description combined the percentage, inter- pretation, and pictographs.

The control description used language from FDA's wording in press releases to describe the reduction in nicotine 22 : "Imagine if tobacco companies were required to lower the nicotine in cigarettes to a minimally or non-addictive level." On the same webpage where participants saw their randomly assigned description of the potential new cigarette standard, participants an- swered questions about the description.

Participants received $2.20 for taking the survey.

The institutional review board at the University of North Carolina approved the study.


The survey presented the three outcome measures in a random order for each participant.

The perceived nicotine content item read, "Compared to current cigarettes, how much nicotine do you think the changed cigarettes would have?" The perceived addictiveness item read, "Compared to current cigarettes, how addictive do you think the changed cigarettes would be?" The perceived cancer risk item read, "Compared to smoking current cigarettes, smoking these changed cigarettes for 30 years would have what risk of lung cancer?" The perceived cancer risk item included recommended practices for perceived risk questions 34 and emphasized that the item was about risk related to continuing to actively smoke VLNC cigar- ettes.

All items used the same five-point response scale: "Much, much less" (scored as 1), "Much less" (2), "Somewhat less" (3), "A little less" (4), and "The same" (5).

In previous research using a broader scale, we found that few people responded that risks of VLNC cig- arettes would be higher than current cigarettes.

12 We dichotomized responses as accurate (1) or inaccurate (0).

On the basis of cur- rent scientific understanding, 35 nicotine content ratings of 1 or 2, addictiveness ratings of 1 or 2, and perceived risk ratings of 4 or 5 were scored as being accurate.


We defined current smoking as having smoked at least 100 cig- arettes in one's lifetime and now smoking some days or every day.

36 The survey also assessed standard demographics and tobacco use.


The analytic sample included 1353 participants with complete data on our outcomes.

Analyses were conducted using SAS 9.4 (Cary, NC) and a critical alpha of .05.

We first conducted chi-square tests to check for demographic differences among the seven description conditions and found none.


We next put the description conditions in rank order by per- ceived nicotine content and then determined whether the difference ' associations with each other.


Overall, 22% of participants were current smokers (Table 2).

Participants' mean age was 37 years (SD: 12), and 52% were male, 47% female, and 1% transgender.

The sample was 82% white, 9% black, 5% Asian, and 4% other or multiracial; 9% were Hispanic.

In addition, 36% did not have a college degree, and 49% reported a household income below $50 000.


Around 49% of participants provided with the control description accurately understood that VLNC cigarettes would have much less nicotine than current cigarettes (Figure 1).

The concise language de- scription elicited less accurate perceived nicotine content from par- ticipants (14% accurate) than the control description (p < .001).

The interpretation description (58%) did not differ from control (p = .10).

The percentage and interpretation description (74%) elicited more accuracy than the interpretation description (p < .001) and along with the remaining descriptions offered the highest ac- curacy: percentage (76%, p = .60 vs. percentage and interpretation); percentage and pictograph (81%, p = .23 vs. percentage); percentage, interpretation, and pictograph (87%, p = .12 vs. percentage and pictograph).


About a third (34%) of participants provided with the control description accurately understood that VLNC cigarettes would be much less addictive than current cigarettes.

Again, the concise language description elicited less accurate perceived addictiveness from participants (17% accurate) than the control description (p < .001).

The interpretation description (36%) did not differ from con- trol (p = .67).

The percentage and interpretation description (48%) elicited more accuracy than the interpretation description (p = .01) and along with the remaining descriptions offered the highest ac- curacy: percentage (44%, p = .36 vs. percentage and interpretation); percentage and pictograph (52%, p = .13 vs. percentage); percentage, interpretation, and pictograph (48%, p = .51 vs. percentage and pictograph).


Around 68% of participants provided with the control description accurately understood that VLNC cigarettes would be about as carcinogenic as current cigarettes if smoked.

In contrast with the prior two outcomes, the concise language description elicited more accurate perceived cancer risk from participants (77% accurate) than the control description (p = .03).

The interpretation descrip- tion (68%) did not differ from control (p = .91).

In contrast to the previous two outcomes, the percentage and interpretation descrip- tion (57%) elicited less accuracy than the interpretation descrip- tion (p = .03) and along with the remaining descriptions offered the lowest accuracy: percentage (56%, p =.83 vs. percentage and inter- pretation); percentage and pictograph (48%, p = .13 vs. percentage); percentage, interpretation, and pictograph (55%, p = .20 vs. per- centage and pictograph).


The three outcomes were all associated (all p <.001).

Higher perceived nicotine content was strongly associated with higher per- ceived addictiveness (r = .59).

Higher perceived cancer risk was mod- estly associated with higher perceived nicotine content (r = .27) and higher perceived addictiveness (r = .34).


Using risk communication best practices for describing the nico- tine level of VLNC cigarettes to the public yielded consistent find- ings.

A substantial percentage of participants had misperceptions of nicotine content, addictiveness, and cancer risk.

As descriptions elicited more accurate perceptions of the low nicotine content and low addictiveness of VLNC cigarettes, they elicited less accurate perceptions of the high cancer risk of these cigarettes.

For com- munications or research studies that are only concerned with con- veying that VLNC cigarettes have much less nicotine than current


Our findings reinforce previous studies' findings that many people incorrectly believe nicotine is the main harmful chemical or carcinogen in cigarettes.

11,37-39 For example, people perceive cigar- ettes such as Quest that are advertised as "low" nicotine to be less harmful than other cigarettes.

39,40 Researchers have also shown that participants trying VLNC cigarettes 14 or participants asked about "very low" nicotine cigarettes perceive them to be less harmful.

23 Our findings indicate that not only are VLNC cigarettes perceived as less harmful to smoke but also that this problem becomes exacerbated as people are provided more detailed information about the amount of nicotine reduction.

This highlights the need for more research on how to communicate nicotine reduction clearly without causing po- tentially harmful misperceptions about risk.

Communication about a VLNC standard could include a disclaimer that "The new cigar- ettes will not be safer to smoke than current cigarettes." However, disclaimers are often ineffective, 41-45 and additionally, the disclaimers would not necessarily be repeated in media coverage, social media discussions, or interpersonal communications.

Another possible solution could be explaining a VLNC standard not by the nicotine content but by saying that cigarettes would be "changed so that they no longer relieved your cravings." 46 Popova and colleagues found that this language led to more accurate perceived risk than other scenarios in a study of what smokers said they would do in response to a VLNC policy.

We are currently developing and testing messages to reduce the misperception that VLNC cigarettes are less carcinogenic.


For press releases, mass media campaigns, and other commu- nications, the FDA will have to make important decisions about what the public needs to know about a reduced nicotine standard.

Is there a legal or ethical obligation that smokers be fully in- formed about the great reduction in the amount of nicotine and addictiveness? If so, more communication research is needed to learn how to do this while simultaneously conveying the high cancer risk of continuing to smoke VLNC cigarettes.

In addition, it will be important to consider how other actors, such as the news media, the tobacco industry, and opponents on social media will frame nicotine reduction.


19 As our study surveyed a convenience sample, a limitation is that the generalizability to the US adult population remains to be established.

Our point estimates are likely to differ from those of probability samples, but our experimental findings are likely to be replicable across representative samples.


25 Second, a single survey item assessed risk beliefs, and because it was asked of both nonsmokers and smokers, it did not specifically ask about the participant's own cancer risk.

Multi-item risk perception scales about own risk can yield stronger associations with behavior.


34 Third, the response scale offered responses that ranged from "much, much less" to "the same," and some participants could have wanted to respond that VLNC cigarettes would have more nicotine, be more addictive, or be more carcinogenic.

However, in previous work, we found that few (~5%) people used this end of the response scale.


12 Fourth, if the FDA chooses a gradual nicotine reduction ap- proach, the phrasings suggested here should be revised accordingly and re-tested.


Our study found that introducing VLNC cigarettes by describing their percentage reduction in nicotine (95%) led to accurate percep- tions of nicotine content and addictiveness.

However, we also found that as descriptions of VLNC cigarettes conveyed more accurate perceptions about the nicotine content and addictiveness of VLNC cigarettes, they elicited less accurate perceived cancer risk.

Future research can examine how to educate the public about the continued risk of smoking cigarettes under a VLNC standard.


In 2017, the US Food and Drug Administration (FDA) proposed lowering the amount of nicotine in combusted cigarettes to minimally addictive levels [1].

The Family Smoking Prevention and Tobacco Control Act gives FDA the authority to limit the maximum nicotine content in tobacco products [2].

This approach differs from reductions in nicotine delivery in mainstream smoke, which resulted in tobacco companies marketing “light” and “ultralight” cigarettes [3].


Previous studies have shown that use of nonnicotine cigarettes can reduce craving because of sensory cues [4-6], but the reward value of these cues is extinguished over time when the reinforcing effects of nicotine are removed [7, 8].

Reducing nicotine in cigarettes is an unprecedented tobacco control strategy that could save millions of lives [9].

A simulation model estimated that this policy could result in approximately 5 million additional smokers quitting within a year of the policy’s implementation [9].


However, public reactions to such a policy are hard to anticipate, and there may be unintended consequences, such as misperceptions that very low nicotine cigarettes (VLNCs) are less harmful than regular cigarettes [10-12].

This study investigated effects of different framings of reduced nicotine in combusted cigarettes.

Framing the nicotine tobacco product standard as “cigarettes no longer relieved cravings” resulted in the highest proportion of smokers reporting they intend to quit in response to this standard, lowest proportions reporting anticipated intentions to continue using combusted tobacco products, and lowest proportion believing that VLNCs are less harmful than regular cigarettes.

This framing might be a promising way to communicate to the public the practical impact of the nicotine tobacco product standard.


The US Food and Drug Administration (FDA) is considering a very low nicotine content (VLNC) product standard to substantially reduce nicotine in cigarettes.

We examined whether learning about a potential VLNC standard increased smokers’ interest in illicit purchases of cigarettes with regular nicotine content if such a standard were adopted.

Participants were a national convenience sample of 1712 US adult smokers.

In an online experiment, we randomly assigned smokers to view information about a new VLNC standard (experimental condition) or no information (control condition).

The experimental condition explained that a VLNC standard would remove 95% of the nicotine in cigarettes and would require stores to only sell VLNC cigarettes.

Then, the survey assessed smokers’ interest in purchasing regular cigarettes from three illicit sources.

Smokers who learned about the VLNC standard were more likely to be very or extremely interested in purchasing regular cigarettes illicitly from a Web site compared to smokers in the control group (24% vs. 16%, p < .001).

They were also more interested in illicitly buying cigarettes from a street vendor (19% vs. 13%, p < .001) and a store on an Indian reservation (28% vs. 22%, p < .05), compared to the control.

The impact of learning about the VLNC standard on interest in illicit purchases did not differ by smoking frequency or current e-cigarette use.

A VLNC standard could increase smokers’ interest in illicit purchases of regular nicotine cigarettes.

To prevent VLNC-induced illicit trade from undermining public health, FDA should consider proven measures such as track and trace for these products.

Little is known about how a VLNC cigarette standard would affect consumer interest in regular content cigarettes purchased from illicit sources (eg, the Internet).

We found that smokers informed about a potential VLNC product standard had greater interest in illicit cigarette purchases, compared to controls.

This suggests the importance of proactive measures accompanying a VLNC standard, such as track-and-trace cigarette packaging regulations and communication campaigns, in order to maximize the standard’s public health impact.


In July 2017, the US Food and Drug Administration (FDA) announced a comprehensive plan for tobacco regulation that included a very low nicotine content (VLNC) standard [1].

This VLNC standard, if implemented, would reduce nicotine in cigarettes to minimally addictive or nonaddictive levels.

Clinical trials have shown that using VLNC cigarettes has led smokers to smoke fewer cigarettes and make more quit attempts [2][3][4].

In addition, a recent simulation model estimated that a VLNC standard in the United States would prevent 2.8 million tobacco-related deaths by 2060 [5].

If FDA implemented a VLNC standard, however, some smokers might seek banned regular nicotine content cigarettes [6].

Such increased illicit cigarette trade could undermine some of the public health benefits of a VLNC standard.

Illicitly purchased cigarettes, which tend to be substantially cheaper than cigarettes purchased legally [7][8][9], account for between 7% and 21% of the total US cigarette market [10].

Illicit purchases in the United States arise from many sources, including sales from the Internet, tribal lands, and street vendors [10].

Previous studies have shown an uptick in online searches for illicit products after the 2009 federal tax increase [11] and modest increases in illicit sales after cigarette excise tax increases [12][13].

However, the extent to which a VLNC standard could increase illicit tobacco trade is unknown.

Clinical trials that state that participants should only smoke VLNC cigarettes during the trial have found substantial nonadherence [3][14], suggesting that, under a VLNC standard, smokers’ interest in banned regular nicotine content cigarettes would likely increase.

However, studies have not explored smokers’ interest in illicit purchases of cigarettes in response to a VLNC standard.

Our study aimed to examine whether learning about a potential VLNC standard would increase smokers’ interest in illicitly buying cigarettes with regular nicotine content if such a standard were adopted.

We predicted that informing smokers of a potential VLNC standard would increase interest in illicit purchases of cigarettes.


In August 2018, we recruited a convenience sample of US adults aged 18 years or older as part of a larger online study about e-cigarette health messages [15].

Online convenience samples are a quick and low-cost way to study health behavior and can yield highly generalizable findings for experiments [16].

Participants in this study were current smokers (defined as having smoked at least 100 cigarettes and now smoking every day or some days) [17][18].

Recruitment occurred through Prime Panels (www.turkprime.com), an online platform with access to over 20 million participants for behavioral research.

Participants provided informed consent before taking the survey.

We randomly assigned participants to one of two conditions.

Participants in the experimental condition viewed the following prompt: “Imagine a new law requiring tobacco companies to remove 95% of the nicotine in cigarettes.

Stores could legally sell only these new very low nicotine cigarettes, but not cigarettes that have regular amounts of nicotine.

” Participants in the control condition did not see a prompt.

Then, participants responded to questions assessing purchase interest as described later.

Participants received incentives in cash, gift cards, or reward points from Prime Panels.

The University of North Carolina institutional review board approved the study procedures.

Prior to data collection, we preregistered the study on AsPredicted.org (https://aspredicted.org/sp8s5.pdf).