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).


The survey assessed interest in illicit purchases of cigarettes from three sources: the Internet, street vendors, and retailers on tribal lands.

We selected these sources because an FDA draft paper predicted that these would be among the most likely sources to sell illicit regular nicotine content cigarettes if a VLNC standard were enacted [19].

The three items for the control group read: “How interested would you be in buying cigarettes from the following places, even if it is not legal? ... A website, A street vendor, A store on an Indian reservation.

” The items for the experimental group were identical except for the addition of the phrase “regular nicotine” as follows: “How interested would you be in buying regular nicotine cigarettes from the following places, even if it is not legal?” The phrase “A store on an Indian reservation” came from the Population Assessment of Tobacco and Health study [20].

The items had a five-point response scale ranging from “not at all interested” (coded as 1) to “extremely interested” (coded as 5).

We averaged the items to form a scale for interest in illicit purchases of cigarettes (α =.83).

Participants then answered items about tobacco use and standard demographic items.

Analyses used Stata/SE version 14.1 with two-tailed tests and a critical α of .05.

The analytic sample included 1712 smokers randomized to the VLNC experiment with complete data on the outcomes (only two smokers had missing data; we excluded them from analyses).

In preliminary analyses, we examined whether randomization created equivalent groups using chi-squared tests for categorical variables and t tests for continuous variables.

We found no differences (Table 1).

We used chi-square tests to determine if the proportion of participants who answered “very interested” or “extremely interested” to each item was higher in the experimental group than the control group (compared to those answering “somewhat interested,” “a little interested,” or “not at all interested”).

In the main analyses, we examined the impact of experimental condition on the interest in illicit purchases scale (aggregated across all three sources) using a t test.

Finally, we examined whether daily smoking status (vs. nondaily) moderated the impact of experimental condition on interest in illicit purchases across the three sources.

This model used linear regression with the continuously measured purchase interest variable as the outcome; predictors were experimental condition, daily smoking status, and an interaction term of experimental condition with daily smoking status.

Using the same approach, we examined two additional moderators: number of cigarettes smoked per day and current e-cigarette use (defined as currently using e-cigarettes some days or every day).

All analyses followed our pre-registration with the exception of moderation by e-cigarette use; we conducted this unplanned exploratory analysis in response to peer reviewers’ feedback.


Smokers’ mean age was 44 years (Table 1).

Most (63%) participants had less than a college education and 49% were low income.

About half of smokers (51%) were also current e-cigarette users.

In the past year, 11% of smokers had bought cigarettes from a Web site, 17% had bought cigarettes from a street vendor, and 23% had bought cigarettes from a retailer on tribal lands.

Smokers who learned about the VLNC standard were more likely to be very or extremely interested in illicit purchases of cigarettes from all three sources (Supplementary Table 1).

Twenty-four percent of smokers who learned about the VLNC standard were interested (ie, answered “very interested” or “extremely interested”) in purchasing cigarettes illicitly from a Web site compared to 16% of smokers in the control group (p < .001; Figure 1).

Similarly, 19% of smokers who learned about a VLNC standard were interested in purchasing cigarettes from a street vendor compared to 13% in the control group (p < .001).

Over a quarter (28%) of smokers who learned about the VLNC standard were interested in purchasing cigarettes illicitly from a retailer on tribal lands compared to 22% in the control group (p < .01).

Among smokers who learned about a potential VLNC standard, 36% were very or extremely interested in illicit cigarettes from any source, compared with 30% in the control group (p < .01).

Next, we looked at interest in purchasing cigarettes illegally as a continuous outcome, averaged across sources.

Learning about a potential VLNC standard increased smokers’ interest in purchasing cigarettes illegally from the three different sources (mean [M] in experimental group = 2.3, standard deviation = 1.2, p < .001 vs. M in control group = 2.0, standard deviation = 1.1).

The impact of learning about the VLNC standard on overall interest in illegal purchases did not differ by daily smoking status (interaction p = .40), smoking frequency (interaction p = .53), or current e-cigarette user status (interaction p = .62).


US smokers who learned about a possible VLNC standard in our brief online experiment were more interested in illicitly purchasing cigarettes, compared to smokers who did not learn about the VLNC standard.

The magnitude of the effect was modest; across the three sources, 36% of smokers who learned about the VLNC standard were interested in illicit cigarettes compared with 30% in the control group.

The finding held for illicit purchases from Web sites, street vendors, and tribal lands.

Moreover, the findings did not differ based on smoking frequency or e-cigarette use.

Smokers in this study were most interested in buying cigarettes from retailers on tribal lands, followed by the Internet and then street vendors.

These findings build on prior research that has demonstrated greater illicit cigarette sales following cigarette excise tax increases [11][12][13].

Assuming a VLNC standard is not accompanied by a tax increase, however, the motivator would likely not be cost savings but rather gaining access to regular nicotine cigarettes banned under a VLNC standard.

Coupled with prior research on tax evasion, our study findings suggest that FDA and other US governing agencies should proactively plan to prevent illicit cigarette trade under a VLNC standard.

If sufficient demand exists for regular content cigarettes under a VLNC standard, it is likely that some illicit product will be available and illicit sales will result unless measures are undertaken to reduce illicit trade.

A recent article suggested several key regulatory actions for controlling the illicit market under a VLNC standard [6].

For instance, the United States could require encrypted tax stamps as part of a “track and trace” system for tobacco products, strengthen and enforce regulations banning online cigarette vendors from processing payments and shipping orders, and strengthen licensing requirements, compliance and enforcement, and illicit trade penalties for tobacco manufacturers and distributers.

These types of measures have previously proven effective in combating illicit cigarette sales due to tax evasion [10][21][22].

Moreover, the United States should ensure that cessation products and less harmful nicotine products (eg, e-cigarettes, Swedish snus) are both widely available and affordable to help smokers handle nicotine cravings and reduce the motivation to seek regular nicotine content cigarettes from illicit sources [6].

Our findings should also be taken in proper context.

Although they suggest greater smoker interest in purchasing illicit regular nicotine products, the magnitude of the observed effect was modest and should not be a deterrent for FDA or any other regulatory agency for implementing a VLNC product standard.

Moreover, if an illicit market grew large, it would not be invisible to enforcement agencies [6].

In addition to these regulatory measures, health communications (eg, mass-media campaigns) could help to maximize the impact of a new VLNC product standard.

These communications could deter smokers from seeking banned regular nicotine content cigarettes by informing them about penalties for illicit purchases and encouraging them to quit smoking altogether.

Campaigns could also encourage smokers to use this change as the time to quit smoking, remind smokers of cessation products and less harmful nicotine products, and educate smokers that VLNC cigarettes are not less harmful than current cigarettes (a common misperception) [23][24][25][26].

Future studies should test various campaign messages’ ability to avert possible unintended consequences of a VLNC standard, including illicit cigarette trade and misperceptions of harm [27][28].


Strengths of our study include the use of an experimental design with successful randomization and the inclusion of a large national sample of smokers.

One limitation is that the outcome of interest in illicit purchases could have overestimated the potential impact of a VLNC standard on actual illicit cigarette purchases given that intentions do not perfectly predict behavior [29].

However, we guarded against this possibility by categorizing smokers as being interested in illicit cigarettes only if they answered “very interested” or “extremely interested.

” The small difference in item wording for the two study conditions could have contributed to differences across conditions.

We did not assess reasons for interest in illicit cigarettes; future studies should consider examining these reasons both quantitatively and qualitatively.

Finally, the use of a convenience sample limits the ability to infer population prevalence estimates, although online convenience samples tend to provide valid results for experiments, accurately estimating the impact of experimentally manipulated variables [16][30][31].

This experiment found that learning about a potential low nicotine content product standard increased US smokers’ interest in illicit purchases of regular nicotine content cigarettes.

These findings suggest the potential for an increased demand for illicit cigarette trade following a VLNC product standard.

FDA and other US government agencies should consider regulatory actions such as track and trace to prevent illicit cigarette sales from weakening the public health impact of a VLNC standard.

They could also develop effective communications to help smokers understand the new standard and provide support to help smokers quit smoking altogether.


Several genetically modified (GM) crops have been devel- oped to provide protection against certain lepidopteran target pests, such as the European corn borer Ostrinia nubilalis (Hü bner) and the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre), by the introduction of a part of a Bacillus thuringiensis (Bt) gene encoding the insecticidal Cry1Ab protein (http://www.agbios.com/ dbase.php).

The Bt protein binds to specific receptors on the epithelial surface of the midgut of lepidopteran species, leading to the death of larvae through pore for- mation, cell burst and septicaemia (Crickmore 2005; Soberó n et al. 2009).

At present, maize MON810 is the only commercial Bt crop grown in the European Union (EU), having been cropped over a significant area since 2003, mainly in Spain (Gó mez-Barbero et al. 2008).


Within Europe, maize is not an important food resource for indigenous lepidopteran larvae, with the exception of few pest species, so exposure to potentially harmful amounts of Bt-containing pollen deposited on host plants in or near maize MON810 fields is the main risk to non-target Lepidoptera, as reviewed in the BEETLE report (2009).


A laboratory assay suggested a hazard to the North American Monarch butterfly (Danaus plexippus L.) larvae that consumed maize Bt11 pollen deposited on milkweed (Asclepias spp., especially common milkweed, Asclepias syriaca L.) leaves compared This journal is q 2010 The Royal Society with those reared on leaves dusted with non-GM maize pollen or on leaves without pollen (Losey et al. 1999).


Subsequently, lethal and sublethal effects of Bt-maize pollen consumption by lepidopteran larvae have been reported for several non-target lepidopteran species under laboratory conditions in the USA ( Jesse & Obrycki 2000; Wraight et al. 2000; Hellmich et al. 2001; Dively et al. 2004; Anderson et al. 2005; Mattila et al. 2005) and within Europe (Felke et al. 2002; Felke & Langenbruch 2005; Lang & Vojtech 2006), the magni- tude of the hazard being dependent on the Bt-maize event, the lepidopteran species and the larval stage, the amount of pollen consumed and amount of Cry1Ab protein ingested.


The hazard reported for the Monarch butterfly in lab- oratory experiments (Losey et al. 1999) led to extensive exposure assessments in the field in the USA, which found that the proportion of Monarch butterfly popu- lations exposed to toxic levels of Bt-pollen is small owing to the limited spatial distribution of maize pollen (Pleasants et al. 2001) and the limited temporal overlap between larval development and pollen shed (Oberhauser et al. 2001).

Exposure to potentially harmful quantities of maize pollen is largely restricted to pollen deposited on milkweed host plants in the area of field margins within 1- 5 m of the edge of maize fields, since the highest pollen concentrations occur in and near maize fields.


A risk assessment model estimated that the average prob- ability of short-duration exposure to Bt-maize pollen within maize fields, for those states and provinces within the corn belt that constitute 50 per cent of the eastern North American Monarch breeding habitat, was less than 0.1 per cent (Sears et al. 2001).

Comparable con- clusions were reached by a similar approach applied to the risk associated with longer term exposure of Monarch butterfly populations (Dively et al. 2004).


Extensive exposure assessment studies similar to those performed in the USA have not been conducted under European environmental conditions, although Darvas et al. ( 2004) and Gathmann et al. ( 2006b) conducted exposure and abundance studies of certain species of non-target Lepidoptera in specific localities in Europe.

Information regarding the scale of the hazard is available from estimates of the number of non-target macrolepi- dopteran species that might theoretically be exposed and thus affected on a regional scale by Bt-maize pollen (Schmitz et al. 2003; Darvas et al. 2004; Traxler et al. 2005).

In addition, other field studies provide some rel- evant data on the exposure of European lepidopteran species in agricultural landscapes on a population level (Lang et al. 2004; Gathmann et al. 2006a,b), but data on some aspects of exposure, particularly plant - insect phenology, pollen consumption and subsequent mortality in field conditions, are rare within Europe.


Although the cultivation of Bt-maize has been ongoing for several years in Spain, data from Spain on effects on non-target Lepidoptera are scarce because their abundance tends to be low at the time when maize is pollinating.

Extrapo- lating observations made on one non-target lepidopteran species to another is problematic because of between- species variability in both acute sensitivity to Cry1Ab protein and plant - insect phenological coincidence (Schmitz et al. 2003).

Moreover, the MON810 event has now been integrated into many commercial maize varieties with differing sowing dates and developmental characteristics, resulting in a range of flowering dates, thus increasing temporal variability in exposure to the Cry1Ab protein (Van Hout et al. 2008).

Aviron et al. ( 2009) also emphasized the practical difficulties of con- ducting experiments to detect small effects, where they exist, on all lepidopteran species that could be potentially exposed to Bt-maize pollen.


Under EU Directive 2001/18/EC on the deliberate release into the environment of GM organisms, there is a legal requirement for an environmental risk assessment of the cultivation of maize MON810 within the EU (EFSA 2009); this must include an assessment of the adverse effects on non-target Lepidoptera resulting from exposure to pollen from this Bt-maize (see Sears et al. 2001; Wolt et al. 2005; Peterson et al. 2006).


This paper describes a mathematical model used to facilitate the quantification of risk assessment.


The model explores possible scenarios for the exposure of three widespread European lepidopteran species in 11 representative maize cultivation regions in four European countries.


For the first time, to our knowledge, the integration of a mortality - dose relationship from the laboratory with a dose - distance relationship from the field allows direct estimates of larval mortality both within the crop and within the field margin as a function of distance from the crop edge.


These estimates were adjusted to allow for physical effects; the lack of temporal coincidence between the susceptible larval stage concerned and the period over which maize MON810 pollen is shed; and seven further exposure parameters concerned with maize agronomy and host-plant ecology.


Sublethal effects were estimated and allowance made for aggregated pollen deposition.


At each stage in the model development, where there was a choice we have endeavoured to model 'worst-case' scenarios, in which any assumptions would tend towards overestimation rather than underestimation of mortality and sublethality.


The model has 11 parameters.

The principal parameters are denoted g and h. The parameter g(E ) represents the worst-case probability that a given larva will suffer mor- tality from ingesting maize MON810 pollen deposited onto its host plant located in the field margin at distance E from the nearest edge of the maize MON810 crop.

Here, the term worst-case refers to potential mortality, as measured in the laboratory or under controlled exper- imental conditions, before allowance for factors such as physical effects and temporal coincidence, which reduce this mortality to realistic values observed in the field (see below).

The parameter g represents an average prob- ability over factors such as whether the margin is on the upwind or downwind side of the field, or time of day.

The parameter h represents the worst-case probability that a given larva will suffer mortality from ingesting maize MON810 pollen deposited onto its host plant located within the maize MON810 crop.


Two parameters model effects that reduce exposure.

The parameter x represents the proportion of larvae that remains exposed, after allowance for a set of physical effects that include: degradation of pollen toxicity 1418 J. N. Perry et al. Modelling exposure of Lepidoptera Proc.

R. Soc.

B (2010) (Griego & Spence 1978; Obrist et al. 2006); rain washing pollen off leaves (Pleasants et al. 2001); larvae feeding on the underside of leaves where pollen densities are smaller (Jesse & Obrycki 2003); larval avoidance of leaf midrib area where pollen densities tend to be aggregated (Pleasants et al. 2001); larvae feeding on lower leaves on which less pollen has been deposited through the shading effect of leaves above them (Pleasants et al. 2001), etc.

The parameter a represents the proportion by which exposure is reduced owing to lack of temporal coinci- dence between the susceptible larval stage concerned and the period over which maize MON810 pollen is shed (Wolt et al. 2003; Lang et al. 2004; Gathmann et al. 2006b).


For simplicity, this model considers a single larval instar.

The quantification of temporal coinci- dence is conceptualized as follows.

Lepidopteran larvae do not enter instars synchronously; it is assumed that there is a bivariate distribution for the days on which larvae enter (t s ) and leave (t l ) the particular susceptible instar modelled.

Regarding anthesis, similarly, the period within the flowering of maize when pollen is shed is asynchronous between individual crop plants, and it is assumed that there is a bivariate distribution for the days on which plants begin (t b ) and end (t e ) shed- ding pollen.

Consider the ellipsoids that represent the 95th percentiles of these two bivariate distributions.

This model assumes that the full exposure of larvae to pollen is reduced proportionately by the degree to which the overlap between these two ellipsoids is incomplete.

Specifically, the parameter a measures the proportion of the ellipsoid that represents the 95th percentile for instar development that is not overlapped by the ellipsoid that represents the 95th percentile for pollen shed.

Note that the expected actual mortality rate for an individual larva is therefore xag(E ) within the field margin and xah within the crop.


The parameters g(E ) and h are specific to the host plant, lepidopteran species and larval stage modelled, but generic across regions; x and a are assumed to be specific to the geographical region.


The following three parameters of the model control large-scale demographic aspects of exposure and are all specific to regions but gen- eric across host plant and species.

The parameter z represents the proportion of arable fields that are cropped with maize in any given year in the defined region.

The parameter v represents the proportion of maize sown within the defined region, that is, the variety MON810 (Gó mez-Barbero et al. 2008).

The parameter y represents the proportion of the lepidopteran host plant that is found within arable crops and in their margins (as opposed to gardens, woodlands, non-arable fields, etc., which are too far from maize MON810 fields for pollen deposition to present any quantifiable risk).

Note that the proportion of the population potentially exposed, after allowance for reduction owing to these large-scale demographic factors, is then yzv.


The next two parameters are specific to regions, host plants and species; parameters e and f measure, respectively, the density of the host plant within the maize crop and in the field margins, in plants m 22 .

For simplicity, it is assumed that host plants occur spatially at random within crops and field margins.


The final two parameters relate to field size and are therefore specific to regions but generic across host plants and species.

For simplicity, it is assumed that maize MON810 fields are square.

The parameter C represents the average size of a maize field in hectares and the par- ameter D represents the average width of a field margin in metres.

When parametrizing the model for specific regions, allowance is made for possible bimodal distri- butions of field margins because fields in a region may often have margins that are several metres wide or have no margins at all.


The model was parametrized for exposed larvae of three non-target lepidopteran species: the peacock butterfly Inachis io L. (Lepidoptera Nymphalidae), the red admiral butterfly Vanessa atalanta L. (Lepidoptera Nymphalidae) and the diamondback moth Plutella xylostella L. (Lepidoptera Plutellidae), for 11 representative European maize cultivation regions: Aachen, Berkatal, Bonn, Grebbin, Oderbruch and the Upper Rhine Valley from Germany; the Po Valley (east and central) and the Po Valley (southern and coastal) in Italy; Tolna County in Hungary; and the Ebro Valley and Madrid from Spain.

For both butterflies, the model was parametrized for the most susceptible first instars (Darvas et al. 2004), and the widespread nettle host plant Urtica dioica L. For the moth, the model was parametrized for fourth instar larvae, which were the most sensitive of the species and instars studied by Felke & Langenbruch (2005), and which were assumed to feed on widespread Brassicaceae host plants such as Lepi- dium draba (L.), Capsella bursa-pastoris (L. Medik.), Thlaspi arvense (L.) and Raphanus raphanistrum (L.).

The two but- terfly species were chosen for their conservation value (Schmitz et al. 2003; Darvas et al. 2004; Traxler et al. 2005), while the moth, a serious pest of cultivated Cruci- ferae, was chosen because it is one of the most susceptible Lepidoptera to the Cry1Ab insecticidal protein (Felke & Langenbruch 2005).

In controlled conditions, unpro- tected larvae of V. atalanta are thought to be of very similar susceptibility to those of I. io (Darvas et al. 2004).

Both species are somewhat protected under field conditions from pollen deposition; the former species creates 'leaf bags', the latter builds webs (e.g. Scott 1986).


In this section: (i) published mortality - dose relation- ships, for individuals of the three lepidopteran species with pollen of the different event maize Bt176, are cali- brated to relate to maize MON810 pollen and expressed in terms of logits; (ii) published pollen deposition - distance relationships for pollen deposited on the different host plant A. syriaca are calibrated to relate to U. dioica and Brassicaceae; and (iii) results from (i) and (ii) are integrated to yield field margin and within-crop mortality - distance relationships for individual larvae of the chosen larval/host-plant combinations.


In no-choice feeding studies with leaf discs coated with maize Bt176 pollen, Felke & Langenbruch (2005) showed that the average lethal concentration value that kills half (LC 50 ) of the fourth instars of P. xylostella was eight pollen grains placed on a 0.071 cm 2 host-plant leaf disc (113.1 pollen grains cm 22 leaf area).

Results for other lepidopteran larvae included O. nubilalis, LC 50 452.6 pollen grains cm 22 leaf area for second instars, and I. io (see especially Felke et al. in press), LC 50 186.8 pollen Modelling exposure of Lepidoptera J. N. Perry et al. 1419 Proc.

R. Soc.

B (2010) grains cm 22 leaf area for first instars.

Since concentrations of the Cry1Ab protein in pollen of maize MON810 are approximately 31-fold less than those in maize Bt176 (Hellmich et al. 2001; Sears et al. 2001; Mendelsohn et al. 2003; Nguyen & Jehle 2007), the LC 50 for MON810 was estimated as 3626 pollen grains cm 22 leaf area for fourth instars of P. xylostella and 5800 pollen grains cm 22 leaf area for first instars of I. io and V. atalanta.


Quantification of the mortality - dose relationship through a probit- or logit-regression relationship requires information concerning not only the intercept (effectively given by the LC 50 ) but also the slope (the rate of change of mortality with change in concentration).

Estimates of slopes are scarce for non-target Lepidoptera, but Felke et al. ( in press) estimated the probit slope to be in excess of 5.0.

Considering that pollen concentrations on host plants through field deposition are likely to be con- siderably smaller than the LC 50 value (Wraight et al. 2000), such a large slope implies very low mortality rates at concentrations typically encountered in or near fields.

Indeed, for the slope estimate given by Felke et al. ( in press), the value of the parameter h for first instars of I. io would be less than 10 29 , and the value of rate g(E) within field margins would be even smaller.

Whether such estimates are realistic in the field is yet to be determined.