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.

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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.


However, as a worst-case scenario, the model was parametrized with a much smaller estimate of the probit slope, 1.095, an estimate obtained by Saeglitz et al. ( 2006a,b) for O. nubilalis over a number of exper- iments.

This compares reasonably well with estimates made by Farinós et al. ( 2004), which for O. nubilalis were in the range 1.33-3.15, and for S. nonagrioides were in the range 0.92-2.96.

The value 1.095 was used through- out for all three species considered.

Since the slope is invariant under the change of scale for probit analysis, the estimated slope applies equally both to concentrations expressed in units of pollen grains per square centimetre leaf area and to doses in units of nanogram of truncated toxin used by Saeglitz et al. ( 2006a,b).


Hence, if p B represents the proportion of I. io and V. atalanta individuals that suffer mortality as a result of a concentration, d, expressed as the number of maize MON810 pollen grains cm 22 , then probitð p B Þ ¼ À4:121 þ 1:095 log 10 d; and if p M represents the proportion of the P. xylostella indi- viduals that suffer mortality, then probitð p M Þ ¼ À3:898 þ 1:095 log 10 d:


Working with logits rather than probits yields an exact solution for expressions involving p. The above probit- regression relationships are well approximated by the following logit-regression relationships:


The concentration, d, of pollen grains adhering to the leaves of host plants declines rapidly with increasing dis- tance in metres, E, from the maize source (Eastham & Sweet 2002; Jarosz et al. 2004; Devos et al. 2005).

This decline in pollen deposition within the margins of maize fields was modelled through a linear regression of log 10 d on E, using the extensive data of Wraight et al. ( 2000, especially fig. 2).

Wraight et al. ( 2000) measured maize pollen falling on microscope slides covered with a coat of petroleum jelly.

The slides provided an accurate measure of total pollen deposition but probably overesti- mated the amount of pollen retained on foliage by about threefold (Pleasants et al. 2001; Lang et al. 2004).

Allowance for this results in the following relationship:


This relationship is consistent with results of Jesse & Obrycki (2000) from a smaller set of data, and is appro- priate for P. xylostella larvae feeding on Brassicaceae.

However, for larvae of I. io and V. atalanta, the results of Darvas et al. ( 2004) suggest that about 2.85 times as much pollen adheres to the hairy leaves of its host plant U. dioica, suggesting that the relationship be amended to log 10 d ¼ 2:346 À 0:145E:


For larvae of I. io and V. atalanta, equations (2.1) and (2.4) may be combined to express mortality within the field margin directly in terms of distance from the edge of the crop, thus yielding the parameter denoted g(E) above:


Note that at the very edge of a maize MON810 crop, where E ¼ 0, the estimated mortality rate is g(0) ¼ p B ¼ 0.0292 (equivalent to one individual in 34.25), and that 2 m into the margin, this rate itself is approximately halved.

Numerical integration shows that average mor- tality within a 2 m band within the margin is 0.0209 (cf.

with the value of 0.075 for the corresponding par- ameter conjectured by the EFSA GMO Panel; EFSA 2009).


For larvae of P. xylostella, equations (2.2) and (2.3) may be combined similarly to yield


Estimates of the parameter h may be derived by noting that pollen deposition within a maize crop is approxi- mately 2.7 times that at the edge ( Jesse & Obrycki 2000; Wraight et al. 2000; Pleasants et al. 2001).

For larvae of I. io and V. atalanta, the adjusted equations (2.1) and (2.4) are combined to give h ¼ 0:0805; ð2:7Þ and for larvae of P. xylostella a value of h ¼ 0:0486:


The expected proportion of all the larvae in a margin that suffer worst-case mortality, before allowing for effects that reduce exposure, is denoted by m ¼ m(D), and is obtained by averaging the value of g(E) over the margin.

In practice, m is obtained by the numerical integration of equation (2.5) or (2.6), between the values E ¼ 0 and E ¼ D. Values of m are shown for various margin 1420 J. N. Perry et al. Modelling exposure of Lepidoptera Proc.

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B (2010) widths, for the butterflies and the moth, in figure 1.

For all species considered, the parameter m(D) is about double the value for small values of D (very narrow mar- gins) than for D ¼ 5, and there is a further approximate halving of m(D) at D ¼ 10.

Estimates of ax are typically about one-third, so the estimated actual within-crop mortality of individual larvae is about 2- 3% for I. io and V. atalanta and about 1 -2% for P. xylostella, and the estimated actual within-margin mortality of individual larvae, for any width of margin, is always less than 1 per cent for I. io and V. atalanta and less than 0.6 per cent for P. xylostella.


For an assumed specific square field of size C (ha) with a margin of D (m), the within-margin area is approximately 400D p C (m 2 ), so the expected number of host plants within the field margins is 400Df p C. The expected number of host plants within the crop is 10 000Ce.

Then, of the population that is potentially exposed within the crop and field margins, an approximate pro- portion (10 000Ceh þ 400fD p Cm)/(10 000Ce þ 400fD p C) suffers mortality, which may be simplified to (25eh p C þ fDm)/(25e p C þ fD).

After allowance for large-scale demo- graphic effects, physical effects, temporal overlap and spatial overlap, the estimated proportion of the population that suffers mortality is


For regions where margins have a bimodal distribution for which there is a probability of q that a field will have no margin at all, then this proportion becomes


Values of parameters other than g, m, and h were chosen by the authors separately for each region mod- elled.

Information on the parameter v for maize MON810 is only available from the larger Bt-maize culti- vation areas in Spain, where it has reached 0.65 in some regions.

Hence, for the regions modelled in Germany, Hungary and Italy, the maximum limit for Bt-maize (v ¼ 0.8) was chosen (based on current insect resistance management requirements for lepidopteran target pests, requiring 20% non-Bt-maize refuge areas), to ensure that a worst-case scenario was modelled that would yield relatively large estimates of mortality.

A summary of all chosen parameter values is given in table 1.


While it is generally agreed that densities of pollen grains decline rapidly with increasing distance from the maize source (Eastham & Sweet 2002; Jarosz et al. 2004; Devos et al. 2005), few studies have measured the varia- bility of pollen concentrations.

Pollen deposition may vary greatly spatially depending upon weather conditions (Jarosz et al. 2004; Hofmann 2009).

Owing to vertical wind movements or gusts, particularly in thundery con- ditions on summer afternoons, a particular small area may experience a larger than average concentration of pollen, even many tens of metres away from a maize field (Vogler et al. 2009).

However, such larger than aver- age pollen concentrations are balanced by smaller than average values elsewhere, where effects are diluted.


The frequency distributions given in table 2 of Pleasants et al. ( 2001) demonstrate that this variability may be con- siderable.

This effect has been quantified in fig. 4 of Hofmann (2007), where log 10 d is shown to decline line- arly with log 10 E, as expected, but with variability about the regression line that may be represented by a normal random variable with variance about 0.175, so that the 99% confidence interval around the line encompasses about 12-fold variation in concentration, at any given dis- tance, in both directions.

For linear systems, the average expected effect of such variation would be zero, but the model considered here is highly nonlinear; therefore, it is necessary to assess what is the effect of the measured variability on the deterministic estimates of mortality.


This was done by simulating, for each of a range of dis- tances E, a thousand random variables N, where N is a normal random variable with zero mean and variance 0.175, and computing log 10 d ¼ 1.891 - 0.145E þ N, the stochastic analogue of equation (2.4) and, in separate simulations, log 10 d ¼ 2.346 - 0.145E, the stochastic ana- logue of equation (2.6).

From that set of values, equations (2.1) and (2.2) were used to derive estimates of mortality that allowed for stochastic variability in pollen deposition, for comparison with predictions from the deterministic case derived in equations (2.5) and (2.6).


Modelling exposure of Lepidoptera J. N. Perry et al. 1421 Proc.

R. Soc.

B (2010) et al. 2004; Lang & Vojtech 2006) is defined as a reduction in larval weight gain, then observations in Spain on the lepidopteran pests O. nubilalis and S. nonagrioides indicate that mortality rates caused by maize MON810 pollen of about 25 and 10 per cent correspond to sublethality rates of about 100 and 50 per cent, respectively.

These values are broadly consistent with the data of Lang & Vojtech (2006) and Felke et al. ( 2002).

Therefore, sub- lethal effects were modelled by assuming that, for any given width of margin D, and the average worst-case mor- tality rate within the margin of m(D), the proportion of larvae suffering sublethal effects, before allowance for any effects that reduced exposure, was 4m(D).

Similarly, within the crop, worst-case sublethality rates before allow- ance for other effects were assumed to be four times worst-case mortality rates, i.e. 0.322 for larvae of I. io/ V. atalanta and 0.194 for larvae of P. xylostella.

A key effect of Cry toxins on lepidopteran larvae is reduced feeding, which can lead to a greatly reduced rate of devel- opment and a level of functional mortality much greater than estimates of direct mortality measured in laboratory assays over short periods.

Under a worst-case scenario, all larvae suffering sublethal effects would be assumed to die without completing their development, although this would most probably greatly overestimate the actual mortality.


Estimates of population mortality and sublethality rates are given for all species and regions considered in table 2.

For the two butterflies, estimates for Spain were zero because for those regions, at the time of maize polli- nation, there are almost no host plants for larvae to feed on in the fields or field margins, since weed control and local irrigation culture suppress weed and field margin vegetation strongly.

Indeed, intensive field surveys in 2009 recorded no U. dioica plants and only one species, C. draba (L.), of Cruciferae, with no lepidopteran larvae feeding on this weed (G. P. Farinó s 2009, personal com- munication).

For each species, the minimum, maximum and median rates, excluding Spain, were computed over the regions considered.

The mean of the stochastic ana- logues of the parameter g(E ), calculated using the variability described in Hofmann (2007), was, for I. io and V. atalanta, between 1.53 (for E ¼ 0) and 1.63 (for E ¼ 4.5) times greater than the deterministic value shown in figure 1.

For P. xylostella, the mean of the sto- chastic analogue was between 1.71 (for E ¼ 0) and 1.79 (for E ¼ 4.5) times greater than the deterministic value.

After allowance for all the effects modelled, the estimated median stochastic mortality rate over regions (excluding Spain) for both I. io and V. atalanta was 2.66 Â 10 24 (1.33 times the corresponding deterministic value), and for P. xylostella was 2.67 Â 10 24 (1.17 times the corresponding deterministic value).


The conclusions from this modelling exercise are that a full exposure assessment is possible for several non- target lepidopteran species exposed to Cry1Ab expressing pollen in or near maize MON810 fields, but it requires many factors to be taken into account, some of which have had to be modelled with restricted available data.

However, we believe that the predictions made here are relatively robust for the following reasons.

First, the esti- mates for larvae of non-target Lepidoptera reported here represent worst-case scenarios, in which any assumptions would tend towards overestimation, rather than underes- timation of mortality.

The estimates were probably most sensitive to the variable measuring the rate of change of mortality with concentration, i.e. the slope in the probit analysis.

Indeed, had the estimate of Felke et al. ( in press) been used instead of that of Saeglitz et al. ( 2006a,b) the mortality rates reported would have been about 8 Â 10 27 smaller.

Furthermore, while future utiliz- ation rates measured by parameter v, cannot now be known accurately, the assumed rate of 0.8 is most unlikely to be achieved even if cultivation were unrestricted.

Therefore, population mortality rates would most prob- ably be even smaller than reported here.


Second, the stochastic simulations suggested that the increase in mor- tality rates owing to aggregation of pollen deposition into heterogeneous clumps will be no more than one-third of those reported in table 2.


Third, for P. xylostella, while there can be marked variance in the sensitivity of different unselected populations to Cry toxins and while the species, cultivar and age of the leaf of the brassica used can also significantly influence the toxicity in bioassays, the sensitivity of the population used by Felke & Langenbruch (2005), from which our data derives, was relatively high.


Fourth, the model used here suggests that population sublethality rates would be likely to be proportionally larger than population mortality rates by the same multiplicative factor that sublethality was assumed to exceed mortality for individual larvae.

In that case, future estimates of population sublethality might usefully be based on estimates for individuals from laboratory studies.


Predicted environmental impact on the studied non- target lepidopteran larvae owing to exposure to potentially harmful amounts of pollen deposited on host plants in or near maize MON810 fields was low.

In all regions: Aachen, Berkatal, Bonn, Grebbin, Oderbruch and the Upper Rhine Valley from Germany; the Po Valley (central) and the Po Valley (coastal) from Italy; Tolna County in Hungary; and the Ebro Valley and Madrid from Spain, the calculated mortality rate was less than 6.36 Â 10 24 (one individual in every 1572) for the butterflies I. io and V. atalanta and 2.55 Â 10 23 (one individual in every 392) for the moth P. xylostella.

The median (typical) rates across all regions excluding Spain were 2 Â 10 24 (one individual in every 5000) for the but- terflies and 2.29 Â 10 24 (one individual in every 4366) for the moth.

Our results suggest that previous estimates (EFSA 2009), using similar techniques but relying on experts' estimates for parameters g(E) and h rather than calculated values, were overly cautious and that mortality and sublethality are about four times less than they estimated.


To place the above results into a quantified population- dynamic context, it would be necessary to predict the precise effects of mortality owing to Bt-maize MON810 in a particular generation(s) on All modelling exercises are subject to uncertainties; as with any ecological model, further data would refine the estimates reported here.

The variability in our reported estimates arises from: (i) natural variation between areas, reflecting expected agronomic and environmental heterogeneity, such as those relating to parameters C, D, e, f, etc.; ( ii) differences between experts' interpretation in parameters other than g(E) and h; and (iii) uncertain- ties arising from incomplete availability of data, particularly regarding sublethal effects.


We thank Stefan Rauschen, Ralf Wilhelm, Gema P. Farinó s, two referees and an anonymous colleague for helpful discussions.

We thank the European Food Safety Authority for paying page charges.


Perry et al. [ 1] developed a model to simulate the field exposure and adverse effects for three European non-target Lepidoptera species (Inachis io L., Vanessa atalanta L., and Plutella xylostella L.) to pollen of the Bacillus thuringiensis (Bt) maize MON810 containing lepidopteran-targeting Cry1Ab toxin.

Perry et al. explicitly modelled the worst case scenario and came to hard quantitative predictions.

However, the incomplete and uncertain input data cause a higher uncertainty than Perry et al. indicate, and we are specifically concerned with the possibility that the effects might be worse than they predict.

Here, we specify this uncertainty by addressing some of the basic model assumptions and input data regarding the toxic effects of Cry1Ab to lepidopteran larvae.

We do not address the hypotheses of the model regarding maize pollen dispersal and deposition, or population-dynamic effects.


A key problem of the modelling study of Perry et al. is that virtually no studies exist in the peer-reviewed literature reporting on the dose–response effects of MON810 pollen to European non-target Lepidoptera [2].

In consequence, the authors had to substitute data obtained from experiments with another transgenic event expressing Cry1Ab, the Bt176 maize [3,4].

In our view, the use of these studies is problematic for several reasons.


First, the authors set the toxicity of MON810 pollen to 31-fold less than Bt176 maize pollen, but did not provide data supporting the implied assumption of a linear relationship between Cry1Ab dose and adverse effects.

The dose–effect relation may be nonlinear, possibly resulting in a disproportionally higher effect of low Cry1Ab concentrations [5,6].


Second, the assumption of a 31-fold difference in Cry1Ab concentration of pollen of MON810 and Bt176 is an average value, which Perry et al. seemingly derived from four papers (but three of them refer to the same source, the US Environmental Protection Agency).

However, Cry1Ab content in pollen varies substantially even within the same event depending on the year, the site, the plant and possibly the cultivar [7].

For example, Cry1Ab concentration has been recorded to be as low as 389 ng g21 in Bt176 pollen and as high as 97 ng g21 in MON810 pollen, which is only a four-fold difference [8].


Third, Perry et al. claim that larvae of the red admiral, Vanessa atalanta, are equally susceptible to Cry1Ab as those of Inachis io.

The evidence cited [9] did not test or report Cry1Ab susceptibility of V. atalanta, and indeed, no toxicological tests with Cry1Ab are published for this species so far.

Susceptibility to Bt can vary greatly among lepidopteran species, e.g. 10-fold between close relatives within a genus, making a prediction of the sensitivity to Bt for any given species difficult [3,10], and extrapolating model data from one species to another may be inappropriate [11,12].


Fourth, the values for mortality incorporated into the model are likely to underestimate the real values owing to two experimental limitations in the studies used: Felke & Langenbruch [3] and Felke et al. [ 4] exposed the lepidopteran larvae to Bt maize pollen for 2 days and terminated the trials after 7 days.

Exposure in the field is likely to last longer as maize fields shed pollen over a prolonged period, on average 8 days or even longer [13].

Also, the larvae will continue to suffer lethal and sublethal effects in later stages (older than 7 days) owing to long-term effects following a short acute dose of Cry1Ab [6].

Both will result in lower LC/EC50 values than applied in the model.

Several other conditions of the above laboratory studies also do not reflect realistic field situations, which have the potential to underestimate a Cry1Ab effect [2], e.g. using cut leaf disks instead of whole host plants, providing ample food to the larvae, keeping larvae under favourable abiotic conditions and excluding multiple environmental stressors exacerbating a Bt effect, e.g. bacterial infections of lepidopteran larvae [14].


Acute toxicity may not be a reliable predictor of sub-lethal effects [15].

Owing to lack of data, Perry et al. assume sublethality rate to be four times the mortality rate.

The justification of this assumed relationship remains to be clarified as the papers ‘broadly consistent’ with this assumption [4,6] do not allow deriving such a ratio of sub-lethal effects to mortality.

For example, a Bt maize pollen density of 18.7 grains cm22 caused a significant average reduction in body mass of caterpillars of the common swallowtail (Papilio machaon), while the same pollen density caused no increased mortality [6].

The notion of the authors that the reduction of larval body mass would be representative for all other types of sublethal parameters seems to lack conclusive support.

Especially, effects on fecundity can be as important as effects on survival [16].


Any Cry1Ab effect will be moderated in the field by variation of exposure.

However, all publications cited in Perry et al. in support of a possible reduction in exposure through behaviour of the larvae refer to Danaus plexippus, the American monarch butterfly, but none to the three modelled European species which show different (feeding) behaviours.

Likewise, several other parameters were also set by personal (unreferenced) estimates.

These include the proportion and density of host plants in and near maize fields, or the temporal overlap of larval occurrence and Bt-maize cultivation (see parameters in table 1 of Perry et al. [ 1]).


Perry et al. provided the first model study for Bt-maize effects on Lepidoptera.

The assessment of such effects is complex, thus modelling approaches are welcomed.

Model studies are especially helpful to better identify and understand complex interactions of key parameters and basic processes.


However, risk assessment of genetically modified organisms is a sensitive area, and any quantitative conclusions should be drawn and published with greatest care, as these could have significant policy and regulatory implications.


As specified above, the claim of Perry et al. to have simulated the worst-case scenario must be challenged.

Moreover, their model study involves considerable uncertainty in values of key ingredients, and their chosen approach is not sufficiently cautious to reflect this uncertainty.


A full uncertainty/sensitivity analysis would need to be performed before making detailed quantitative predictions, and certainly for predictions outside the original application.


We respond to the Comment of Lang et al. [ 1] regarding our mathematical model [2] of exposure of non-target Lepidoptera to Bt-maize pollen expressing Cry1Ab within Europe.

Lang et al. remark on the degree to which the model was subject to uncertainty.

Perry et al. [ 2] did indeed emphasize precaution: they made four separate decisions to model worst-case scenarios; identified six distinct sources of variability to which their results might be sensitive; and emphasized six different bases for the uncertainty of predictions.


Lang et al. rightly emphasize the importance of identifying to which parameters the results of a model are most sensitive; Perry et al. should perhaps have emphasized more that the parameter to which their estimates of mortality were most sensitive was undoubtedly the variable measuring the rate of change of mortality with concentration/dose of the Cry1Ab protein (i.e. the slope of the probit/logit regression line; see below).


Regarding the relationship between the toxicity of MON810 and Bt176 pollen, Lang et al. imply that the relationship between mortality and dose may be nonlinear.

Regressions from bioassay should always be checked for nonlinearity, but there was no evidence of this in any of the extensive number of regressions of Saeglitz et al. [ 3,4] upon which our slope estimate was based.

Of course, the standard transformation of mortality to probits (or logits) and the logarithmic transformation of concentration [5,6] are designed to achieve a linear regression; both papers cited by Lang et al. use this method.

Data from one of these papers [7] were tested for nonlinearity; none was found, and no disproportionately higher mortality at low Cry1Ab concentrations could be verified (figure 1).


Lang et al. are correct that Perry et al. used the range of published [8,9] values (12.2 – 78.9) to derive an average of the ratio of the concentration of the Cry1Ab protein expressed in pollen of maize Bt176 relative to that in maize MON810 for which a 31.05-fold difference was assumed.

This is already likely to be a worst-case underestimate because, as Sears et al. [ 8] noted, the value of expression for MON810 was near the current level of detection by immunoassay.


We do not agree with Lang et al. ’s interpretation of the data from Nguyen [10]: they compared the smallest Bt176 value from 2002 to the largest MON810 value from 2003.

The within-year ratio of Bt176 : MON810 was 64.8 in 2002 and 30.5 in 2003.

The latter value, very close to that adopted by Perry et al., leads to larger mortality estimates for MON810 than the former, a further example of Perry et al. ’s use of worst-case scenarios.


Figure 2 shows the importance of the distinction between the intercept and slope of the probit/logit line in this issue.

The conclusions of the model for risk management depend on the degree of estimated mortality.

The conclusions are clearly highly sensitive to assumptions concerning the slope.

They are sensitive, but much less so, to the intercept; it is governed by the Bt176 : MON810 ratio.

Figure 2 demonstrates the effect of choosing an alternative worst-case value of the ratio (12.2) at the end of the range.


Regarding the assumption of equal susceptibility for the butterflies Inachis io and Vanessa atalanta, we apologize for the incorrect citation given by Perry et al. We acknowledge that data to compare the sensitivities of I. io and V. atalanta are very limited.

We are aware of no evidence that the sensitivities differ; unpublished data from a single field experiment appear to suggest that they may not.

Both Perry et al. and Lang et al. remarked on the need for further data on European Lepidoptera of conservation concern.


Lang et al. argue that the experimental methodology of Felke et al. [ 11] was likely to give results that underestimated true mortality.

However, in many experiments with MON810, larvae have been exposed to longer time periods than those of Felke et al. [ 11]: 10 days [12]; 14 – 22 days [13]; 7 days [14]; 10 – 14 days [15].


These and other experiments with MON810 pollen have shown no negative effects when lepidopteran larvae were exposed to MON810 pollen alone.

Furthermore, susceptibility to Bt toxin declines with age in older instars (e.g. [16]), so any potential for negative impacts of Bt pollen is reduced as the larvae develop.


Lang et al. claim that fig. 1 of [7] demonstrates long-term effects (longer than 7 days) following a short acute dose of Bt.

However, comparison of treatment and control in that figure appears to contradict rather than support their claim.


In the period between 7 and 27 days, the mortality for a dose of 2.5 mg of Bt is only marginally (approx. 2%) greater than that of the control, while the mortalities for all other doses (1, 5, 7.5, 10, 20, 30 mg) are at least 5 per cent less than that of the control.


We fully agree with Lang et al. that sublethal effects should encompass fecundity and other parameters.

However, many studies neglect to test parameters other than larval or pupal weight (e.g. [7]).

Perry et al. emphasized that ‘our methods are subject to considerable uncertainty’, a caveat repeated in the final sentence of the Discussion.


Lang et al. stated that ‘all publications cited in Perry et al. in support of a possible reduction in exposure through behaviour of the larvae refer to Danaus plexippus’, but have perhaps overlooked Perry et al. ’s text: ‘Both species [V. atalanta and I. io] are somewhat protected from pollen deposition; the former species creates “leaf bags”, the latter builds webs (e.g. [17])’.

We agree that the extent to which exposure is reduced through such behaviour is variable but it is surely not contentious to state that there is evidence for this for neonate larvae within Europe (see e.g. http://tristram.squarespace.com/home/2009/6/9/peacock-butterfly-caterpillars.html).


Regarding sensitivity analysis, it is important to allow for the fact that depositions of pollen in the field occur at far lesser concentrations than the LC50s for the three species considered by Perry et al. In consequence, as shown in figure 2, differing assumptions for the slope of the assumed probit (or logit) line will have little effect on the results for concentrations close to the LC50, but result in very large differences at concentrations around those expected within the crop or in the margin.

Within the crop, the estimated mortality using the slope estimated by Felke et al. [ 11] is vanishingly small and such a value would be impossible to measure in field conditions.

Even a doubling of the Saeglitz et al. [ 3,4] slope of close to 1.1 to the moderately small average 2.25 estimated by Farino´s et al. [ 18] would result in roughly 10-fold decrease in estimated mortality.


It is for these reasons that we consider that the consequence of this sensitivity completely outweighs any of the several effects claimed by Lang et al. to engender uncertainty and affect mortality estimates.

We regard them as minor compared with the ‘safety margin’ factor of 8 x 10^-7 by which Perry et al. inflated the estimated mortality through deliberate choice of a small value of the logit slope, designed to give worst-case mortality.


The value of the Perry et al. model is that it provides a transparent, structured and simple approach to exposure analysis that may be followed for other species and taxa if sufficient data become available.

Further, in other settings, it offers the opportunity for accurate estimation of mortality – dose relationships to supplement relatively inaccurate determinations of mortality in the field.

We agree with Lang et al. that species’ sensitivity to particular GM events that express different forms of Cry1 proteins is an important determinant of mortality; also that further data would be welcome on the mortality – dose relationships (particularly regarding the slopes) for a range of species, especially those of conservation concern.

However, we disagree that we have been incautious regarding the implications of our results for conclusions regarding regulatory policy.

We therefore reaffirm the robustness of our conclusion from our model that, after accounting for large-scale exposure effects, the ‘estimated environmental impact of MON810 pollen on non-target Lepidoptera is low’.


We describe the clinical progression in this unique case supplemented with video and imaging findings in the form of magnetic resonance imaging and brain single photon emission computed tomography.

Final diagnosis is via pathological analysis at autopsy.


We present an elderly gentleman who manifested a clinical syndrome consisting of subacute onset of chorea that at presentation was distinctly unilateral and a frontal behavioral syndrome in the setting of mild thrombocytopenia and elevated anticardiolipin antibodies.

Positive antiphospholipid antibodies resulted in an initial antemortem diagnosis of primary antiphospholipid syndrome as a cause of his chorea.

Longitudinal follow-up over 5 years demonstrated a progression of clinical features with hemi-motor impersistence/chorea, disinhibition and impulsivity, and eventually corticospinal distribution weakness on the initially affected side.

He required nursing home care and falls necessitated wheelchair use.

Postmortem neuropathological study revealed a diagnosis of frontotemporal lobar degeneration-tau, PSP-PNLA.


This case broadens the phenotype of PSP-PNLA and to our knowledge is the only case presenting with unilateral chorea.


Pathologically, progressive supranuclear palsy (PSP) is considered a tauopathy because of the presence of abnormally phosphory- lated tau in the neurons and glia in subcortical and cortical areas.

1 The substantia nigra, subthalamic nucleus, and globus pallidus consistently contain tau pathology.

PSP-pallido-nigro-luysian atrophy (PNLA) is a rare neurodegenerative disease defined by bilateral degeneration of these same structures.

Although PNLA and PSP share prominent degeneration of the pallidonigroluysian region, PNLA has less widespread degeneration, few or absent neurofibrillary changes, absent or rare tufted astrocytes, and fewer/shorter argyrophilic threads compared with PSP.

Thus, important pathological differences exist.


There have been cases described that fulfill the criteria for both PSP and PNLA, and these are called PSP-PNLA.


Pathologically the same types of lesions are observed in PSP and PSP-PNLA but with differences in the distribution and density of tau pathology, with less tau in the motor cortex, striatum, pontine nuclei, and cerebellum in PSP-PNLA.

2 Clinical presentation of PSP-PNLA includes a younger onset age and longer disease duration than patients with PSP (57.1 AE 3.1 vs. 65.5 AE 1.9 years of age at onset, respectively, and disease durations of 11.5 AE 1.6 vs. 7.6 AE 0.5 years, respectively), 2 and with early handwriting and gait abnormalities, such as freez- ing.

Falls, dysphagia, and vertical gaze palsy were late features of PSP-PNLA in contrast to PSP-Richardson syndrome in which these clinical findings are pathognomonic and occur early on.

Although cases of PSP-PNLA are extremely rare and there is limited clinical information available, uniformly all cases are clini- cally characterized by akinesia and parkinsonism.


We describe a case of PSP-PNLA confirmed pathologically with a clinical presentation of hemichorea/ballism, spasticity, progressive hemiparesis, and a frontal behavioral syndrome with relative cognitive sparing early on.

This clinical presentation in the setting of laboratory findings resulted in an antemortem diag- nosis of primary antiphospholipid syndrome (aPS).

Longitudinal clinical evaluation and laboratory, neuropsychological, and neu- roimaging investigations serve to document this rare presentation of PSP-PNLA.


A 79-year-old right-handed Palestinian man developed 1 year of right-hand clumsiness and involuntary movements.

He noted difficulties including writing and dropping objects from his right hand.

He experienced falls, tripping over his cane with the right leg.

Involuntary, low-amplitude movements of his right arm and leg presented insidiously.

There were word-finding difficulties, but he continued to work as an international consultant.


His previous medical history included coronary artery disease, hypertension, hypercholesterolemia, and lumbar degenerative disc disease.

He had no history of autoimmune conditions, can- cer, or nonatherosclerotic thrombotic events.

There was no exposure to antipsychotics or antiemetics.

There was no relevant family history.


Vital signs and general examination were normal.

Mini- Mental State Examination was 28/30.

He behaved inappropri- ately, talking loudly and seeming overly familiar; he touched the examiner's shoes because he "liked them." He was short- tempered with his wife.

This was out of character according to the family.


Extraocular movements were full with saccadic pur- suit.

There was occasional blinking with horizontal saccades.

His face was symmetric, and the tongue was midline with no motor impersistence or orobuccal dyskinesia.

There was a positive gla- bellar tap and bilateral palmomental reflexes.

Muscle bulk, tone, and power were normal.

Reflexes were exaggerated in the right arm and leg (3+) and normal on the left (2+), with bilateral flexor plantar responses.

There was no ataxia.

Primary and sec- ondary sensory modalities were intact.


Chorea was present at rest and increased with action in the right arm and leg, sometimes with a ballistic quality.

He easily stood up from a chair and had a narrow gait with chorea in his right hand and leg while walking.

The chorea in his leg sometimes impaired his walking and bal- ance (Video 1, segment S1).


The following year, chorea increased in amplitude but remained unilateral.

He became more irritable, impulsive, and unpredictable in his behavior, for example, tipping a musician excessively and increased interest in sexual activity with his wife discordant with her wishes.

There was no paranoia, hallucina- tions, or delusions.

Cognitively, his family noted distractibility and short-term memory problems.

However, he remained intact in his instrumental activities of daily living.

Efforts to reduce cho- rea were unsuccessful: tetrabenazine caused sedation, and risperi- done did not help.


He scored within normal limits on the Mattis Dementia Rat- ing Scale, although a formal neuropsychological assessment rev- ealed mild to moderate impairment in areas of speeded information processing, verbal working memory, complex visuo- spatial construction, effortful encoding, and retrieval of verbal information, suggesting frontosubcortical and mild left temporal dysfunction.

Frontal Behavioral Inventory score was 33 (cutoff score ≥ 30).


Initial investigations including brain magnetic resonance imag- ing revealed mild generalized atrophy and microangiopathic changes consistent with age, but with no strokes or other pathol- ogy in the subthalamic nuclei or basal ganglia to account for the hemichorea (Fig. 1).

Blood work, including electrolytes, ESR, AST/ALT, B12, ANA, CPK, C3/C4, INR/PTT, and thyroid functions, was normal.


Further investigations were pursued, including brain single photon emission computed tomography showing increased perfusion in the basal ganglia bilaterally com- pared with matched normal controls (patient vs. control average percentage increase: left, +12%; right, +18%; see Fig 1E).

Serum paraneoplastic antibody panel was negative.

Huntington's disease and SCA 1, 2, 3, 6, 7, 8, and 17 genetic testing were negative.

CBC was normal except for thrombocytopenia (platelets 120).

Lupus anticoagulant was negative; however, anticardiolipin immunoglobulin G (IgG) antibody level was 54 (repeated 3 months later = 66; normal, 0-18), with an immunoglobulin M (IgM) value of 33 (repeated 3 months later = 18; normal, 0-10).

Because of these results in conjunction with his clinical presentation, he was reviewed by a rheumatologist who suggested a diagnosis of aPS.


He received a 3-day course of methylprednisolone 1000 mg intravenously daily followed by longer term immunosuppression with methotrexate.

Despite improvement in his antibody param- eters, he continued to progress clinically.


He had motor imper- sistence of the right arm resulting in difficulty maintaining any posture in addition to choreic/ballistic movements exacerbated with action.

There was truncal chorea and subtle left leg chorea with action (Video 1, segment S1).

Reflexes were exaggerated bilaterally (right > left), and gait was unsteady with lunging and ballistic movements.


A neurodegenerative disorder was suspected.

Given the asymmetry at presentation, atypical cor- ticobasal syndrome was considered, but he did not meet the clin- ical criteria.

Apraxia was hard to assess given the chorea, but he lacked any cortical sensory loss, and his neuropsychological assessment was also not consistent with that diagnosis.


Symptom- atic treatment with flupenthixol depot at 10 mg intramuscularly every 3 weeks was added to both dampen movements and improve behavior.

This reduced the chorea; however, he contin- ued to have progressive loss of use of his right arm with associ- ated 4+ corticospinal distribution weakness and superimposed motor impersistence.


Treatment with intravenous immunoglob- ulin at a dose of 1 g/kg/day for 2 days was instituted without benefit.


During the next 3 years he became progressively disabled with spastic right hemiplegia and increasing chorea/ballism on his left.

He was admitted to a nursing home, had frequent falls, and became wheelchair bound.

He became dysarthric to the point of incomprehensibility, and in the last months of life he had vertical gaze palsy and severe pseudobulbar dysfunction.

He died of sepsis secondary to an infected sacral ulcer, approxi- mately 5 years after symptom onset.

His brain was submitted for postmortem examination after informed consent was obtained.


The brain was examined and sampled for microscopy according to a standard neurodegenerative neuropathology blocking proto- col.

Formalin-fixed, paraffin-embedded blocks were cut into 4-μm thick sections, mounted on glass slides, and stained with hematoxylin-eosin/luxol fast blue, Gallyas, and the immunohis- tochemistry antibodies listed in Table 1.


The brain weighed 1040 grams.

There was mild symmetrical atrophy of the frontal and temporal lobes bilaterally.

The mid- brain and pons were small in caliber.

There was mild dark discol- oration of the putamen and nucleus of Luys bilaterally, and the substantia nigra appeared pale (Fig. 2A).


Microscopic examination revealed a unique glioneuronal tauopathy with atrophy of the corticospinal tracts (left greater than right; Fig. 2F) and subthalamic nuclei and sparing of the inferior olivary and dentate nuclei.

The tau-positive inclusions strongly stained with Gallyas and were predominantly glial and cytoplasmic; the oligodendroglial inclusions were mostly coiled bodies, and the astrocytic inclusions often had a "bushy" mor- phology.

Neurofibrillary tangles were a relatively minor component.


Examination of the deep gray structures showed numerous oligodendroglial tau-positive inclusions, less frequent bushy astrocytic tau-positive inclusions, no tufted astrocytes, some neu- ronal cytoplasmic tau-positive inclusions in the putamen (Fig. 2D), a normal anterior striatum, neuronal dropout and gliosis of the pallidum, and severe neuronal loss, vacuolation, glial inclusions, axonal spheroids (Fig. 2C, red arrows) and iron deposition in the subthalamic nuclei (Fig. 2B,C).

The substantia nigra showed neuronal loss, gliosis, and coiled bodies on tau immunolabeling.

The remainder of the brainstem and cerebel- lum appeared normal without tau inclusions, including the olivary and dentate nuclei.


Neocortical pathology was mild and included slight cortical neuronal loss with scattered bushy tau- immunopositive astrocytic inclusions (Fig. 2E).

Only rare cortical ballooned neurons were present, and no glial plaques were noted.

In the amygdala and hippocampus, there were neuronal pretangles and glial tau-positive inclusions.

There was no evi- dence of hippocampal sclerosis.


Transactive response DNA bind- ing protein 43 kDa (TDP-43), α-synuclein, and β-amyloid staining were negative, and p62 highlighted the tau pathology only.

There was no significant cerebrovascular pathology includ- ing lacunes or thrombotic microangiopathy.


This case features a man with a frontal behavioral syndrome and hemichorea in the setting of positive antiphospholipid antibodies that resulted in an initial diagnosis of aPS, although subsequent clinical evolution led to the suspicion of an atypical neurodegenerative disorder, ultimately confirmed at postmortem study to be PSP-PNLA.


Chorea, particularly unilateral, without an obvious explana- tion is exceedingly rare.

3 The working diagnosis of aPS was rea- sonable considering the clinical presentation and laboratory findings.

aPS is a rare but well-established cause of chorea,


Postmortem neuropathological study revealed atrophy and tau immunopathology in the pallidum, substantia nigra, and most severely, the subthalamic nuclei, with unilateral corticospinal tract degeneration.

In contrast, the areas usually found to be affected in conventional PSP such as the inferior olives and den- tate nuclei were largely spared, and tufted astrocytes, a feature commonly found in PSP, were mostly absent.

PSP-PNLA has pathological findings with a similar distribution to our case.

However, of the published cases of PSP-PNLA, none presented with unilateral chorea to our knowledge.

Wong et al 5 describe the case of a 70-year-old man who presented with slurred speech and went on to develop gait difficulties and left-sided chorea.

At autopsy, this patient was found to have right-predominant degeneration in the globus pallidus and subthalamic nucleus.

TDP-43 staining was positive in the hippocampus, amygdala, cerebellum, and frontoparietal cortices.

Immunohistochemistry revealed p62-positive, tau-negative, thread-like profiles in glial cells.

There were no tau-positive glial inclusions, and the patient was diagnosed with PNLA.

Chorea is described in PNLA and can be seen in up to 20% of cases, but it is a relatively uncom- mon symptom in adult neurodegenerative diseases and when present should raise the question of PNLA or dentatorubral- pallidoluysian atrophy.


Despite the presence of nigral pathology, our case had no parkinso- nian features as described in PSP and PNLA.

Supranuclear gaze and pseudobulbar palsies, clues to the PSP phenotype, only appeared near the end when neurological functions were severely impaired.

The prominent degeneration and tauopathy affecting the subthalamic nucleus (STN) likely accounted for the chorea/ballism.

The relative sparing of the dentate and olives, the dominant "bushy" morphology to the astrocytic inclusions, and corticospinal tract degeneration are atypical of PSP.

7 This correlated poorly with the clinical phenotype marked by hemichorea, hemiparesis, and lack of parkinsonism.

Some of the observed pathologic findings are reminiscent of those described in globular glial tauopathies (GGT), 8 including the involvement of the corticospinal tract.

8,9 However, the present case contained more abundant coiled bodies then usually encountered in GGT, and some astrocytic inclusions stained positively with Gallyas.


The laboratory findings of aPS were a red herring.

Anti-immu- noglobulin-like cell adhesion molecule 5 (IgLON5) antibodies were not described at the time of this case and so are unavailable, but the neuropathology is not consistent with this condition.

10 Other diagnoses that have a similar tau burden and distribution to our case include some cases of frontotemporal dementia- parkinsonism linked to chromosome 17 associated with MAPT gene mutations (FTDP-17T).

Microtubule-associated protein tau (MAPT) mutation screening was negative in our patient, although he had the MAPT H1/H1 genotype, a common genetic risk factor for PSP and corticobasal degeneration.

11 This case broadens the phenotype of PSP-PNLA and is the only case to our knowledge presenting with unilateral chorea.


(1) Manuscript Preparation: A. Writing of the First Draft, B. Review and Critique.


This work was embedded within the Sunnybrook Dementia Study (SDS) (ClinicalTrials.

gov: NCT01800214) - a prospective observational study of patients with neurodegenerative dementia and movement disor- ders.

It is approved by the local Research Ethics Board at Sun- nybrook Health Sciences Centre and written informed consent was obtained from the participant and their surrogate caregivers according to the Declaration of Helsinki.

A separate written con- sent was also obtained from the patient for the purposes of case report preparation.

All authors have read and complied with the Journal's Ethical Publication Guidelines.

We confirm that we have read the Journal's position on issues involved in ethical pub- lication and affirm that this work is consistent with those guidelines.


No specific funding was received for this work.

The authors declare that there are no conflicts of interest relevant to this work.

Financial Disclosures for the Previous 12 Months: The authors declare that there are no additional disclosures to report.


I read with interest Galit Kleiner and colleagues’ report of a 79-year-old man suffering from progressive hemichorea, spasticity, and a frontal behavioral syndrome, with thrombocytopenia and positive antiphospholipid antibodies [1].

Rationally, antiphospholipid antibody syndrome (aPS) was the antemortem diagnosis.

However, brain autopsy revealed a pattern of tau aggregation diagnostic of the pallidonigroluysian atrophy (PNLA) variant of progressive supranuclear palsy (PSP).

The authors concluded that aPS must have been a red herring.


Is neuropathology always the final diagnosis? If so, was chorea also a red herring? Will any entity ever cease to expand if held to a pathology gold standard? Will the differential between hyper- and hypokinetic movement disorders remain negotiable pending a pathology report?


As a clinician, I struggle with these questions.

We recently evaluated a 60-year-old woman with a 7-year history of a progressive ataxic-dystonia syndrome, an unusual jerky head tremor, and supranuclear vertical gaze palsy we suspected to be Niemann-Pick C [2].

There were no mutations in NPC1 or NPC2.

To our surprise, the neuropathological evaluation revealed tau deposition in a pattern diagnostic of PSP.

As we considered reporting this case as an “expansion of PSP phenotype,” genetic studies demonstrated a pathogenic mutation in TGM6, which codes for transglutaminase 6 (TG6).

TG6 has a role in polyaminating proteins and fostering isopeptide bonds between them, supporting microtubular stability [3].

With low TG6, tau crosslinking is affected and axon microtubules become compromised.

Plausibly, this enzymatic deficiency affecting the structure of tau could have caused this patient’s disease; the 4-repeat tau deposition was its consequence.


A similar dilemma is at play in Kleiner et al. ’s case.

As anticardiolipin antibodies can be elevated among healthy elderly and microangiopathy was absent on autopsy, aCL may have been a red herring.

On the other hand, aCL was associated with thrombocytopenia, raising the odds for an underlying autoimmune disorder [4].

Advances in immunology have revealed disorders never before considered antibody-mediated, such as “atypical PSP” due to anti-immunoglobulin-like cell adhesion molecule 5 (IgLON5) disease.

Anti-IgLON5 antibodies disrupt the neuronal cytoskeleton and induce neurodegeneration.

The authors argue that IgLON5 antibodies were unlikely because of insufficiently supportive pathology.

Even then, would it not be reasonable to speculate that an autoantibody could have caused (or contributed to) the tau deposition in a man with aPS and thrombocytopenia?


Toward supporting the notion that pathology is a secondary expression of many diseases, Kleiner et al. ’s case exhibited nigral pathology without parkinsonism.

The authors helpfully referenced a previously reported case (Wong et al. [ 5]) reminiscent of theirs: progressive hemichorea and gait difficulties with autopsy-diagnosed PNLA, although with TDP-43 instead of tau pathology.

Hemichorea, then, could be thought of as a non-specific manifestation of a PNLA pattern of degeneration, regardless of the associated proteinopathy.


The value of clinicopathologic correlations notwithstanding, the weight of human observations suggests that protein aggregation is not the epicenter of neurotoxicity but a reactive phenomenon to a broad range of cellular pathobiologies [5].

If we continue to enshrine pathology as the final diagnosis, would not we continue to find paradoxes, red herrings, and “expansions of the phenotype”?


We read with interest Dr. Espay’s viewpoint regarding the place of pathology in defining disease [1].

He describes a fascinating case challenging how we ultimately classify disease—a patient with a progressive dystonia-ataxia syndrome and a supranuclear gaze palsy who fulfilled pathological diagnostic criteria for PSP, but was shown to harbor a variant in the TGM6 gene that they postulate ultimately led to the tau pathology [2].

Medicine, and the way in which we come to a medical diagnosis, is ever evolving.

As a neurologist one typically starts by localizing the clinical problem, and then considers pathologies known to affect this area.

The available paraclinical tests, antibodies and genetic analyses are ever growing.

Which of these gets the ultimate say on the diagnosis is subject to revision as knowledge expands.


Our case demonstrated clear clinical-pathological correlation over a decade of follow-up [3].

The right sided chorea correlated with pathological findings in the STN.

The Wallerian degeneration and atrophy of the left > right corticospinal tract correlated with the eventual development of right hemiplegia.

Both clinical presentation and pathology are relevant to nosology.

Dr Espay asks: could an autoantibody have caused or contributed to the visualized tau deposition in a man with aPL antibodies and thrombocytopenia?

Indeed, given the chorea and antiphospholipid (aPL) antibodies without an obvious alternate cause, we elected to treat empirically with pulsed IV steroids, IVIG and methotrexate.

The aPL antibodies and thrombocytopenia normalized but there was ongoing neurological progression, making a causal relationship unlikely.

Furthermore, at autopsy there was no microangiopathy that might have been expected had there been a causal relationship and we know that aPL antibodies are common in the general population [4].


Anti-IgLON5 related tauopathy is a fascinating disorder that makes us think about correlations between neuroinflammation, neurodegeneration and genetics.

This condition has another expanding clinical phenotype, and emerging pathological correlates and genetic associations.

As testing for this antibody was not available when treating our patient, we cannot refute that this as-yet-unknown antibody potentially existed.

There are different pathological distributions of tau between PSP-PNLA and anti-IgLON5 cases [5], both the pathology and its distribution are worthy of consideration.

Furthermore, sequencing of the MAPT gene did not reveal any pathological mutations.

However, he was found to have the MAPT H1/H1 genotype, a known and common genetic risk factor for PSP pathology.


Reporting novel clinical presentations with pathological findings on autopsy remains worthwhile.

Consider how we classify dementias, one could group dementias based on site of lesion, histopathology, clinical features, or etiology—reliance on one alone would cause error but an integrated nosology brings both clinical and experimental benefits [6].

As our understanding of disease pathways evolves, so our nosology will have to also.

We thank Dr Espay for his thoughtful comments.


Information processing is optimized by predicting not only the location or identity of upcom- ing events but also their timing and allocating resources accordingly [1-6].

Understanding the mechanisms of temporal prediction is thus central for models of cognitive and neural process- ing.

One ubiquitous source for temporal predictions is regularity in the stimulus stream, mainly when events appear rhythmically, as in speech, music, or biological motion [3,4,6-9].

It has been suggested that in such cases, a costly "vigilance mode" is replaced by a metabolically efficient "rhythmic mode" by entraining low-frequency neural oscillations to the external rhythm [10,11].

In this study, we tease apart the neural manifestations of oscillatory entrain- ment from rhythm-independent mechanisms of temporal predictions.

This is done by com- paring temporal prediction based on rhythms to predictions based on memory, a critical comparison that was so far missing.


Neural excitability fluctuates in low frequencies from sub-delta to alpha [12,13], with appar- ent optimal phases for performance in these frequencies, as indicated by findings of an associa- tion between phase and performance [3,7,14,15].

According to entrainment models, such endogenous oscillations can be entrained (i.e., undergo period correction and phase align- ment) to an external rhythmic stream such that optimal phases align with on-beat times of rhythmic stimuli.

Consistent with this idea, behavioral performance is facilitated for rhythm on-beat compared to off-beat times [9,16-20].

In addition, electrophysiological recordings found that forming temporal predictions based on rhythmic streams is associated with phase concentration (PC) of delta-band activity in task-relevant sensory regions [6,21,22], such that the optimal phase for performance is observed at the time of rhythmic stimuli [7] (for a review see [23]).

This pattern is consistent with entrainment and is often treated as a direct indicator for this mechanism (e.g., [3,4,7,21,22,24].


Arguably, entrainment should be most applicable and efficient when the sequence is periodic.

Conversely, entrainment should become less applicable and less efficient as the stimulus stream becomes less periodic (i.e., the stream inter- vals are more jittered), because stable alignment of endogenous oscillations with the stream is not possible.

However, temporal predictions can also be formed without periodicity, or any continuous stimulus stream, when the interval between an expected event and a preceding event is known based on previous experience [1,25,26] (for a review see [27]).

Such predictions presumably operate through dedicated interval-timing circuits [28,29], forming temporal memory traces to which new intervals are compared [30,31].

This mechanism can operate regardless of the periodicity of the stimulus stream as long as there is knowledge regarding the temporal contin- gency.


This raises the question whether temporal predictions in rhythmic scenarios are medi- ated by oscillatory entrainment, by "memory-based" mechanisms based on the repeated exposure to the interval [32], or by both.

Recent findings in a speeded discrimination task sug- gest that while any type of temporal prediction improves perceptual accuracy, only rhythms facilitate response speed, seemingly in line with the latter alternative [33].

However, other studies found that response speed can also be facilitated by memory-based predictions [25,26,34].


To date, no study has directly compared the neural expressions of these two prediction modes, namely, memory-based and rhythm\entrainment-based.

Electrophysiological studies have associated rhythm-based predictions with phase adjustments of delta-band activity [3,6,7,21,22] and of the envelope of alpha activity (amplitude reduction prior to on-beat times [34,35]).

However, as these were not tested in memory-based predictions, it is impossible to determine that they are unique to rhythm entrainment.

Rhythm-based predictions are also associated with the contingent negative variation (CNV) potential peaking at on-beat times and resolve immediately after [9,34] and with earlier latency of the P3 potential [8,36].

How- ever, separate studies found CNV and P3 effects for memory-based predictions as well [26,37,38], and in the absence of direct comparison, it is impossible to determine the unique contribution of rhythmicity to these effects.


In the current study, we identify the unique mechanisms of rhythm-based processing by directly comparing the behavioral and electroencephalogram (EEG) expressions of temporal predictions in a perfect periodic condition and in two less periodic conditions, one that enabled formation of memory-based temporal predictions and one that provided no temporal information for prediction.

We show that the patterns of delta-band PC, and also of alpha- band envelope and target-evoked event-related potentials (ERPs), are identical in the rhyth- mic- and memory-based conditions.

We also show that the delta PC pattern is inconsistent with a computational model of oscillatory entrainment, which predicts weaker entrainment for the less periodic stimulation of the memory-based condition.

However, we also find evi- dence for rhythm-unique neural patterns: violation of predictions results in immediate obliga- tory resource withdrawal and larger behavioral costs when rhythm-based, while memory- based predictions are more flexible, in line with the concept of resonance that is predicted by oscillatory entrainment.


To test the unique signatures of rhythm-based predictions, participants (n = 21) responded to targets that followed a sequence of visual stimuli and a warning signal (WS, Fig 1A) in three different conditions.

In the Rhythmic condition, the sequence was perfectly periodic (i.e., the sequence stimuli appeared with identical stimulus onset asynchrony [SOA]), and the target fol- lowing the WS had high probability to appear on-beat, making its timing predictable.

In the Repeated-Interval condition, every odd interval was identical to the SOA of the Rhythmic con- dition, whereas even intervals were jittered around a different interval duration and were never identical to the rhythm interval or an integer multiple of it.

To equate the information Mechanisms of rhythm- and memory-based temporal prediction PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 3 / 30 PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 4 / 30 provided by the Rhythmic and Repeated-Interval conditions, the number of repeated fixed SOA intervals in the Repeated-Interval was equal to the number of overall intervals in the Rhythmic condition.

The target had high probability to follow the WS at the interval of the fixed SOA, making its timing predictable.


In the Random condition, the sequence SOAs were jittered around the SOA of the Rhythmic condition, and the target SOA (interval between the WS onset and the target onset) was drawn from a wider distribution that was not centered on the rhythm SOA orthogonally to the sequence mean SOA.

Thus, the sequence provided no information regarding the timing of the upcoming target.

The Random and Repeated-Interval conditions were, therefore, much less periodic than the Rhythmic condition, as is indicated by (1) SOA variance: zero in the Rhythmic conditions, larger in the other conditions (Fig 1A); (2) frequency domain representation: strong amplitudes in the delta frequency in the Rhythmic condition (its base frequency and harmonics), lower amplitude in the other conditions (Fig 1B); and (3) autocorrelation function: high autocorrelation in the Rhythmic condition at equal lags, minimal autocorrelation in the other conditions (Fig 1C).

We surmised that because of this variability, low-frequency oscillations would be less efficiently entrained to the stimulus stream in the Random and Repeated-Interval conditions.

However, unlike the Random condi- tion, the Repeated-Interval condition enables accurate temporal prediction because of the repeated presentation of the WS-Target SOA.

Thus, any effects that are not different between the Rhythmic and Repeated-Interval conditions reflect temporal predictability, which is not driven by periodicity in the stream.


Our design allowed us to separately test two indicators of forming temporal predictions, namely, benefit and cost, using planned contrasts.

First, we could examine whether having a context affording temporal predictions leads to a benefit relative to an unpredictable context and whether the benefit is different between rhythm- and memory-based predictions.

For this purpose, we compared response times (RTs) of valid trials, in which the temporal predic- tion was fulfilled (i.e., targets appeared at the expected time), between the three experimental conditions (for analysis purposes, "valid" trials were defined in the Random condition as those in which cue and target SOAs were drawn from the same distributions; see Materials and methods for more details).

Second, we could examine whether violating a temporal pre- diction leads to a cost relative to an unpredictable context and whether the cost is different between rhythm- and memory-based predictions.

For this purpose, we compared RTs of invalid trials, in which targets appeared at an unexpected time, between the three experimen- tal conditions.


RTs to targets were first analyzed using an omnibus three-way ANOVA with factors condi- tion X target SOA X validity.

RTs were overall shorter to valid compared to invalid trials (F (1,20) = 76.37, p = 2.9 × 10 −8 , ω 2 p = 0.64, Fig 1D), but the validity effect differed across condi- tions (interaction F (2,40) = 18.69, p = 1.9 × 10 −6 , ω 2 p = 0.22).

For valid trials, RTs were faster rel- ative to the Random condition both in the Rhythmic (t (20) = 5.88, p = 9 × 10 −6 , Cohen's d = 1.28) and the Repeated-Interval (t (20) = 5.8, p = 1 × 10 −6 , d = 1.27) conditions with no differ- ence between the two (t (20) = 0.12, uncorrected p = 0.91, 95% confidence interval [CI] for the difference −6.23 to 6.99 ms).

Thus, while both rhythm-based and memory-based predictions lead to a benefit relative to no prediction, there is no advantage to one of them over the other.


Unlike for valid trials, responses to invalid trials were slower in the Rhythmic relative to the Repeated-Interval condition, (t (20) = 3.14, p = 0.005, d = 0.69).

Responses were also slower in weakest in the Repeated-Interval condition.

( C) Autocorrelation functions of the stimulus sequence in the three experimental conditions, calculated on the same modeled stimulus sequences that were used to create the spectral representations in B. The Rhythmic condition has high autocorrelation in fixed lags because of the perfect periodicity; the other conditions have relatively low autocorrelation at varying lags.

( D) Mean reaction times in each combination of SOA and cue validity in the three experimental conditions.

Error bars represent standard errors of the validity effect within each SOA and condition. * p < 0.05.

Mechanisms of rhythm- and memory-based temporal prediction PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 5 / 30 the Rhythmic relative to the Random condition (t (20) = 1.88, p = 0.037, one-tailed, d = 0.41), while the Repeated-Interval condition did not differ from the Random condition (t (20) = 1.16, p = 0.26).

Thus, unlike their similar benefits, the cost of violation of rhythm-based prediction was larger than that of memory-based predictions.

Finally, there was a main effect of condition (F (2,40) = 13.01, p = 4.4 × 10 −5 , ω 2 p = 0.28) and a condition X SOA interaction (F (2,40) = 6.5, p = 0.0036, ω 2 p = 0.08).

This was because of faster responses to long than short SOA targets (foreperiod effect, [27]) only in the Random condition (F (1,20) = 8.04, p = 0.01, ω 2 p = 0.14).


The data of two participants were rejected from all EEG analyses because of excessive artifacts, leaving 19 datasets.

Analysis of ERPs during the presentation of the sequence was focused on the CNV, a negative deflection that builds up over fronto-central sites in the interval between a WS and an impending event and resolves (towards baseline) when the latter has occurred [39,40].

We first examined the predictive conditions to test whether a CNV is observed during the presentation of the stream prior to the WS, only after WS, or in both time ranges.


If the CNV reflects oscillatory entrainment, it could be expected to be elicited repeatedly in the Rhythmic condition during the entire stream.

We observed a typi- cal CNV after the WS in both the Rhythmic and Repeated-interval conditions (Fig 2A, top and middle rows, respectively) but no repeating CNV or any oscillatory pattern prior to the WS in the Rhythmic condition or the Repeated-Interval condition.

Furthermore, no CNV was observed when locking to the final pair prior to the WS in the Repeated-Interval condition (Fig 2A, bottom row), confirming that the lack of CNV was not due to averaging across the temporally jittered stimuli.


We repeated this analysis in an occipital electrode cluster to test for similar modulations or oscillatory pattern at the sensory level.

Notably, no oscillatory pattern or any repeating deflection was observed in occipital electrodes during any of the conditions (Fig 2B).

Instead, there were typical stimulus-evoked visual responses and a positive deflection following the WS that changed trajectory to a buildup of negativity till the time of the target.


Importantly, locking to the final pair prior to the WS in the Repeated-Interval condition revealed no trace of a slow oscillation elicited by the S1 or S2 stimuli of the sequence (Fig 2B, bottom row).

Thus, the CNV was not driven automatically by the rhythm when subjects were exposed to the stream and used it to form a temporal prediction but was only elicited when preparing for the target.


We compared two parameters of the CNV between the two sources of prediction: the rate of CNV buildup and its resolution after omission of a temporally expected event (Fig 2C).


First, the CNV characteristically builds up faster as the expected interval is shorter [9,34,38].

We tested whether rhythm-based temporal prediction is associated with a distinct modulation pattern of the CNV buildup compared to the modulation that is associated with memory- based temporal prediction.


To quantify this effect, we averaged the CNV amplitude in a time window preceding the short SOA target.

We expected to find more negative waveforms in this window when the target was expected in the short compared to long SOA both in the Rhyth- mic and Repeated-Interval conditions and compared this effect between conditions.


We found more negative amplitude of the CNV buildup when expecting the target at short compared to long SOAs in the Rhythmic (t (18) = 2.81, p = 0.012, d = 0.64) condition as well as in the Repeated-Interval (t (18) = 2.81, p = 0.012, d = 0.64) condition.


Importantly, there was no differ- ence between the effects of the expected SOA in the two conditions (interaction between the effects of SOA and condition: F (1,18) = 0.18, p = 0.68, 95% CI for the difference −1.46 to 2.19 μV, Fig 2D).


For illustration, we also present the Random condition, in which there was no temporal prediction, and, indeed, the SOA had no significant effect (t (18) = 0.36, p = 0.72).


Thus, the modulation of CNV buildup was affected by temporal predictions but did not show a rhythm-specific pattern.


Another characteristic of the CNV is that it resolves when an expected interval elapses even when the impending event is delayed [9,37,40].

In our data, this occurred in the Rhythmic and Repeated-Interval conditions when a target was expected at the short SOA but omitted (i.e., it unexpectedly appeared in the long SOA; Fig 3A).

We examined whether the CNV resolution is different, in magnitude or latency, when the temporal prediction is based on rhythm com- pared to when it is based on memory.

To quantify the CNV resolution, we fitted the CNV waveform (averaged across subjects) in the Rhythmic and Repeated-Interval conditions with a continuous model that included the latencies of buildup initiation, slope, and termination, and resolution initiation and slope as free parameters (Fig 3B).

This approach is superior to quanti- fying the CNV resolution in a predefined interval [9] or using fixed length bins [37], which could miss the actual latencies of different CNV stages and distort the estimated slope if the trajectory is not linear during the entire interval.


Comparison of the estimated parameters between the two predictive conditions revealed that the time of initiation (T 0 ), slope (S 1 ), and termination (T 1 ) of the CNV buildup and the slope of its resolution (S 2 ) were not different between the Rhythmic and Repeated-Interval Mechanisms of rhythm- and memory-based temporal prediction PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 8 / 30 conditions (t (18) = 0.34, 0.37, 0.03, 1.62, respectively, all p > 0.05).

However, the start point of the CNV's return to baseline (T 2 ) occurred later in the Repeated-Interval (1,152 ms) than in the Rhythmic condition (767 ms, t (18) = 3.73, p = 0.0015, d = 0.86, Fig 3E and 3F).

Further- more, there was a significant gap between T 1 and T 2 in the Repeated-Interval condition (t (18) = 2.17, p = 0.043, d = 0.5) but not in the Rhythmic condition (t (18) = 0.09, p = 0.93).