Microb Ecol (2010) 60:373–380
DOI 10.1007/s00248-010-9716-4

HOST MICROBE INTERACTIONSMicroorganisms Associated with Feathers of Barn Swallows
in Radioactively Contaminated Areas Around Chernobyl
Gábor Árpád Czirják & Anders Pape Møller &Timothy A. Mousseau & Philipp Heeb

Received: 25 November 2009 / Accepted: 23 June 2010 / Published online: 17 July 2010
# Springer Science+Business Media, LLC 2010

Abstract The Chernobyl catastrophe provides a rareopportunity to study the ecological and evolutionary
consequences of low-level, environmental radiation onliving organisms. Despite some recent studies about
negative effects of environmental radiation on macroorganisms, there is little knowledge about the effect ofradioactive contamination on diversity and abundanceof microorganisms. We examined abundance patterns of
total cultivable bacteria and fungi and the abundanceof feather-degrading bacterial subset present on feathersof barn swallows (Hirundo rustica), a colonial migratory
passerine, around Chernobyl in relation to levels ofground level environmental radiation. After controllingfor confounding variables, total cultivable bacterial loads
were negatively correlated with environmental radioactivity, whereas abundance of fungi and feather-degrading
bacteria was not significantly related to contamination
G. Á. Czirják : P. Heeb
Laboratoire Évolution et Diversité Biologique (EDB),
UMR 5174 Centre National de la Recherche Scientifique (CNRS),
Université Paul Sabatier,
118 Route de Narbonne,
31062 Toulouse, France
G. Á. Czirják (*)
Department of Infectious Diseases,
Faculty of Veterinary Medicine, University of Agricultural
Sciences and Veterinary Medicine,Mănăştur street 3–5,
400372 Cluj-Napoca, Romania
e-mail: czirjakgabor@yahoo.com
A. P. Møller
Laboratoire d’Ecologie, Systématique et Evolution,
UMR 8079 CNRS, Université Paris-Sud,
Bâtiment 362,
91405 Orsay Cedex, France

levels. Abundance of both total and feather-degrading
bacteria increased with barn swallow colony size,
showing a potential cost of sociality. Males had lower
abundance of feather-degrading bacteria than females.
Our results show the detrimental effects of low-level
environmental radiation on total cultivable bacterial
assemblage on feathers, while the abundance of othermicroorganism groups living on barn swallow feathers,
such as feather-degrading bacteria, are shaped by otherfactors like host sociality or host sex. These data lead us
to conclude that the ecological effects of Chernobyl may
be more general than previously assumed and may havelong-term implications for host–microbe interactions andoverall ecosystem functioning.
Keywords Bacteria . Radiation . Chernobyl . Fungi .
Sociality

A. P. Møller
Center for Advanced Study,
Drammensveien 78,
0271 Oslo, Norway

T. A. Mousseau
Department of Biological Sciences, University of South Carolina,
Columbia, SC 29208, USA

Present Address:
G. Á. Czirják
Leibniz Institute for Zoo and Wildlife Research,
Alfred-Kowalke-Strasse 17,10315 Berlin, Germany

374

Introduction
The natural levels of radiation vary considerably on thesurface of the Earth, implying that living organisms canexperience some levels of radiation. Besides the naturally
occurring high levels of radiation (e.g. in Evolution Canyon,
Israel or in the Andean Altiplano) [11], another very
important sources of anthropogenic radiation, with both
long- and short-term effects on living organisms, are theradiation resulting from the nuclear disaster at Chernobyl,
Ukraine or the testing and contamination from nuclear
weapons [27]. The negative effect of both low- and highlevel radiation is well known [40, 41], but information islacking on the ecological or evolutionary consequences ofhuman-induced and naturally occurring radiation [34].
The meltdown and subsequent explosion of one of thereactors at Chernobyl Nuclear Power Plant on 26 April
1986 released >80 PBq of radioactive materials affecting
200,000 km2 area in Europe [34]. The nuclear accident at
Chernobyl provides a rare experimental framework to study
long-term ecological and evolutionary effects of low-level
radiation on different organisms. The detrimental effects ofbackground radiation on genetics, ecology and evolution
have been found for a wide range of organisms [34, 56].
There have been several studies showing genetic andphenotypic modifications in plants [23], insects [29] ordifferent vertebrates [14, 33, 54] associated with higher
doses of environmental radiation due to the Chernobyl
accident. It has been suggested that these modifications inenvironmental radiation affect reproduction and survival of
the organisms [34, 38]. Accordingly, population sizes anddiversity of several taxonomic and ecological groups
decreased [35–37] with increasing levels of radiation with
the Chernobyl population acting as an ecological sink [32].
However, to date, there is little knowledge about theeffects of radioactive contamination on diversity andabundance of microorganisms. Around Chernobyl, filamentous fungal species diversity has declined with increasing
radioactive contamination [13]. Highly resistant melanized[58] and genetically adapted [27] species are favoured inradioactive areas, and it has been suggested that somefungal lineages may be able to use ionizing radiation as anenergy in their life cycles [10, 11]. Using cultivable soil
bacteria, Romanovskaya et al. [46, 47] showed thatanthropogenic radiation had a negative effect on abundanceand diversity of bacteria. Numerous functional bacterialgroups (nitrifying, sulphate-reducing, nitrogen-fixing,
cellulose-fermenting bacteria) disappeared or reduced significantly in contaminated soils, severely affecting soil's
ecological balance [46]. The most abundant isolated species
from contaminated soils were found to be resistant to different
stress factors (γ-radiation, desiccation, UV, H2O2, X-rays).
Therefore, radioactive contamination was consideredG. Á. Czirják et al.

to be acting as a selective force on the bacterial soil
community [44, 45, 57].
Avian plumage is a diverse microbial ecosystem harbouring a variety of fungi [19, 42] and bacteria [2, 6, 49], sharing
at least one important functional group, feather-degrading
bacteria, with the soil [24], which is considered to be an
important source in acquisition and colonization of feathers
by microorganisms [2, 6]. It has been shown that host
behaviour and ecology such as social structure [31],reproductive investment [25], migration [3] or foragingstrategies [6] affect the bacterial assemblage on feathers.
Besides host-associated traits, geographic location and local
environmental factors (humidity, temperature, radiationlevel) may drive the composition of the plumage-associatedmicrobial communities [2, 7]. Therefore, the objective of ourstudy was to investigate if the effects of radioactive
contamination in areas around Chernobyl on soil microbiota
can be detected via changes in the abundance of cultivablemicroorganisms on feathers of barn swallows, Hirundo
rustica, a colonial migratory passerine.
Methods
Study Area and Populations
As part of an ongoing long-term project on barn swallows
breeding in Ukraine and Belarus, feathers from randomly
chosen individuals captured in 2007 and 2008 from ten
farms were included in this study (Fig. 1). Our study farms
are surrounded by open farmland and scattered trees andplantations, presenting similar climate and habitat type, butdifferent environmental radiation levels [38]. In these
populations, environmental radiation is the main factor
affecting different life-history traits of the barn swallows[38]. We took care to have equilibrated sex ratios and weworked with the following sample sizes: N2007 =31 females,
29 males; N2008 =58 females, 56 males.
Field Procedures
We measured radiation at each farm and cross-validatedwith measurements by the Ukrainian Ministry of Emergencies [52], recording α, β and γ radiation at groundlevel using a hand-held dosimeter (Model: Inspector, SEInternational, Inc., Summertown, TN, USA); both measures were strongly correlated with each other (linear
regression on log–log-transformed data: F=165.9, r2 =0.49, P<0.0001, slope (SE)=0.445 (0.034)).
Barn swallows were captured at windows and doors of farmbuildings using mist nests. This method has been shown to beunbiased and highly efficient during the breeding seasons [39].
Birds were captured after finishing their nest construction,and most were incubating eggs during capture sessions.

Abundance of Feather-Associated Microorganisms from Chernobyl

375

Figure 1 Location of the study
sites and levels of radioactive
contamination in Ukraine,
Russia and Belarus. Adaptedfrom the European Union, 1998
[15]

Wearing sterile gloves immediately after capture a small
number of feathers (five to ten) from the centre of the back,the red throat patch and the centre of the breast were
removed and placed in a sealed plastic bag before being
stored ~6 months in a freezer in the dark until microbiological analysis. Storage type and duration can affectbacterial viability and diversity [1]. Our samples werestored at −20°C without any cryoprotectant, but previous
studies showed that this does not lead to significant changes
in community composition [1]. Furthermore, since all
samples were stored under the same condition, there is noobjective reason to believe that our storage would havebiased our results.
Birds were then ringed; morphological characters were
measured, including tarsus and length of outermost tailfeathers (with a ruler to the nearest millimetres), and bodymass was recorded (with a Pesola spring balance to thenearest 0.1 g). Previous studies of barn swallows in
Chernobyl have shown that capture probability of adults
is very high [38]. We used the total number of occupied
nests at each site as an estimate of colony size.
Microbiological Analyses
Microbiological analyses were performed under sterile
conditions on five randomly selected body feathers, using

the method described by Møller et al. [31]. Briefly, inorder to obtain both free-living and attached microorganisms, feathers were sonicated for 15 min in three repeats(5 min each with 5 min pause between the repeats) in
0.8 ml of sterile (0.90%, w/v) saline solution. Aftersonication, samples were vortexed for 20 s and bacterial
suspensions were transferred to a sterile 1.5 ml Eppendorf
tube. Feathers were re-suspended in 0.5 ml sterile saline
solution and vortexed again for 20 s. The supernatant wastransferred to a similar sterile Eppendorf, resulting in
a ~1.3-ml solution. After obtaining the bacterial solution,the ‘clean’ feathers from individual birds were placedin sterile Eppendorf, dried for 24 h at 70°C and weighedto the nearest 1 μg on an analytical balance (AT1
Comparator, Mettler Toledo).
To quantify different groups of microorganisms, we used
three different growth media. First, tryptic soy agar
(TSA; #22091, Fluka) is a rich medium on which bothheterotrophic bacteria and fungi grow, thus enablingassessment of the cultivable microorganism load of feathers. Second, we inhibited fungal growth and quantified
overall bacterial load, including most of the featherdegrading bacteria and other bacterial biota, by adding
100 mgml−1 of cycloheximide (#01810, Fluka) to TSA(TSAcy) [12, 53]. Third, for the samples from 2008, wequantified feather-degrading bacterial load using feather

376

G. Á. Czirják et al.

meal agar (FMA) containing 15 gl−1 feather meal, 0.5 gl−1
NaCl, 0.30 gl−1 K2HPO4, 0.40 gl−1 KH2PO4, 15 gl−1 agar
and 0.1 mgl−1 cycloheximide [31, 48, 55]. FMA was used
specifically to estimate abundance of the feather-degrading
bacterial subset, as it contains feather keratin as the only
carbon source [48, 55]. Therefore, any bacterium growing
on it must be able to secrete keratinases in order to digest
feather and thus could be considered ‘feather-degraders’[51]. Previous studies of bird–bacteria interactions using
FMA for feather-degrading bacterial load quantification
have shown that FMA is a selective medium for bacterial
species able to use keratin as unique carbon source [17, 31,
51].
To measure bacterial counts, we spread 100 μl of the
microbial solution on different growth media, and the plates
were incubated at 25°C, for 3 days in the case of TSA andTSAcy and for 14 days in the case of FMA [51]. Afterincubation, the numbers of visible colony-forming units(CFU) on each plate were counted. Counts were thencorrected for initial volume of the suspension and feather
mass. The microbial concentration for each medium typewas expressed as CFU per milligram feather. We used only
one dilution because an earlier study on another barnswallow population showed that counts were always
between 0 and 150 CFU per plate [31]. All counts were
performed blindly by one of us (GAC). The fungal load offeathers was estimated indirectly by calculating the difference between total microorganism counts and total bacterial
counts (TSA−TSAcy).
Since the total cultivable bacterial load is composed inpart of feather-degrading bacteria and in part of otherfunctional bacterial groups [51], for the samples from 2008,
we calculated the abundance of cultivable bacteria ‘otherthan feather-degrading bacteria’ as the total abundance ofbacteria minus the abundance of feather-degrading bacteria(TSAcy−FMA) [31]. In this paper, we limit our analyses
and discussion to the results for the total cultivable bacteria
because we had data for 2 years for this bacterial group and
we found the same results and effects when using thecounts for the total cultivable and the ‘other than featherdegrading bacteria’ (see Table 3).
Statistical Analyses
The abundance of total and other cultivable bacteria,
radiation levels, and colony size was log10-transformed inorder to normalize the data. Because we were unable toobtain normally distributed data for the abundance of fungi
and feather-degrading bacteria, we used non-parametric
Spearman rank correlation tests when examining thesevariables. The relationship between total cultivable bacterial
abundance and radiation was examined in a model which
included year and colony size as potentially confounding
variables and host-related parameters (sex, body mass,
tarsus length and tail length) as covariates. The nonsignificant (P>0.05) terms (sex, body weight, tarsus, tail lengthand interactions) were removed from the final model. For
the non-parametric analyses, an index of condition was
calculated using residuals from the linear regression ofbody mass on tarsus length [18, 21, 51]. The conditionindex was not correlated with wing length (rs =−0.02, P=0.87), assuring that our estimate was free of allometriceffects [16]. Except for one recapture, all the birds were
measured once. Sampling sites were classified as low(background radiation range 0.02–0.11 μSv/h), intermediate (background radiation range 0.45–0.46 μSv/h) or highlevel (background radiation 2.9 μSv/h) [4], and Tukey–
Kramer post hoc test was made to determine differences inbacterial abundances between different radiation levels. Allanalyses were performed using the software JMP 5.0.1[22].

Results
The abundance of microorganisms associated with feathersof barn swallows from radioactively contaminated areas
around Chernobyl was highly variable (Table 1). Most

Table 1 Summary statistics for abundance of different groups of microorganism isolated from feathers of adult barn swallows from areas around
Chernobyl
Microorganism

Mean

SE

MedianRange

N

Total cultivable bacteria
Fungi
Feather-degrading bacteria
Cultivable bacteria ‘other than feather-degrading bacteria’

50.09
14.03
15.09
42.85

5.67
1.03
1.66
7.49

25.98
10.25
9.93
16.9

0–550.63
0–65.85
0–87.54
0–505.3

60; 11460; 114–; 114–; 113

Values were back-transformed from log10-transformed values in the case of total cultivable bacteria and other cultivable bacteria, while values are
CFUs for fungi and feather-degrading bacteria. N represents the sample sizes for the two sampling years (2007, 2008)

Abundance of Feather-Associated Microorganisms from Chernobyl

surface microorganisms are harmless or even beneficial,
and thus, host individuals with lower levels of total bacteria
or fungi do not necessarily imply better condition. In
contrast, increased feather-degrading bacterial load appears
to have detrimental effects on host fitness [17]. There
were positive correlations between the four categories of
microorganisms (Table 2).
After removing the nonsignificant host-related parameters (sex, body weight, tarsus, tail length and interactions)from the model and controlling for the effect of colony size,
the abundance of total cultivable bacteria of barn swallow
feathers was negatively correlated with the level ofbackground radiation (Table 3; Fig. 2). Total cultivablebacterial load of barn swallows living in areas with low andintermediate environmental radiation did not differ significantly (Tukey–Kramer post hoc test, P>0.05), while there
was a significant difference between areas with lower
radiation levels and highly contaminated areas (Tukey–
Kramer post hoc test, all P<0.05). In contrast with results
on total bacterial loads, the abundances of featherdegrading bacteria and fungi were not significantly correlated with background radiation (feather-degrading bacteria:
rs =−0.11, P=0.26; fungi: rs =−0.08, P=0.30).
Swallows breeding in larger colonies had higher
loads of total cultivable bacteria than swallows breedingin smaller colonies (Table 3). The interaction between
background radiation and colony size was not significant
for total cultivable bacteria (Table 3). Feather-degrading
bacteria loads tended to increase with colony size (rs =0.18, P=0.055), whereas the abundance of fungi was
not significantly correlated with colony size (rs =0.11,
P=0.15).
Males had significantly fewer feather-degrading bacteria than females (feather-degrading bacteria on males:
median (range)=6.91 (0–71.20); feather-degrading bacteria of females: median (range) = 12.87 (0–87.55);
z=−2.00, P=0.045). In contrast, there were no significant
sex differences in abundance of total cultivable bacteria(F1, 143 =0.019, P=0.887) or fungi (z=−0.41, P=0.68).
There were no significant correlations between abundanceof total cultivable bacteria and body mass, tarsus length or

377

abundance of fungi, feather-degrading bacteria and condition index (all P>0.40).

Discussion
The main finding of this study was that the abundance of
total cultivable bacteria on barn swallow feathers was lower
in populations with higher levels of background radiation,
while that was not the case for fungi or feather-degrading
bacteria. Furthermore, the abundance of feather-degrading
and total cultivable bacteria increased with colony size,reflecting a potential cost of sociality. Finally, male barnswallows breeding in the Chernobyl area had significantly
fewer feather-degrading bacteria than females.
In accordance with previous studies on soil bacteria[46, 47], the abundance of total cultivable bacteria of barnswallow feathers was lower in the most contaminated areas.
Besides differences in environmental radioactive contamination level, our study sites had similar microclimates [38].
Thus, we could expect similar microbial load since biogeographic factors and habitat quality are important factors
shaping feather bacterial assemblages [2]. Romanovskaya et
al. [46, 47] have reported reduced abundance of soil bacteria
in highly contaminated areas around Chernobyl, and theirfindings could be due to the fact that soil bacteria arecontinuously exposed to background radiation. Our results
suggest that bacteria present in radioactive environments
contaminate barn swallow feathers either when at the nest
and/or when occasionally landing on the ground to collectmud for nest construction.
Two mechanisms may explain why there are fewer
cultivable bacteria on feathers of barn swallows in the mostcontaminated areas. Either direct effects of radiation on
bacteria could account for the association or radiation may
have an indirect effect on host defences. First, the dosedependent detrimental effects of radiation on bacterial
survival [8] may reduce the abundance of total cultivablebacteria. A test of this hypothesis will require thatexperimental exposure to radiation reduces the abundanceof bacteria. Second, barn swallows breeding around

Table 2 Correlations between the four categories of microorganisms isolated from feathers of adult barn swallows from areas around Chernobyl

Total cultivable bacteria and feather-degrading bacteria
Total cultivable bacteria and cultivable bacteria ‘other than feather-degrading bacteria’
Feather-degrading bacteria and cultivable bacteria ‘other than feather-degrading bacteria’
Fungi and total cultivable bacteria
Fungi and feather-degrading bacteria
Fungi and cultivable bacteria ‘other than feather-degrading bacteria’
rs = Spearman

rs

P

0.64
0.94
0.51
0.35
0.21
0.23

<0.0001<0.00001<0.0001<0.0001
0.02
0.02

378
Table 3 Relationship between
abundance of total cultivablebacteria and cultivable bacteria‘other than feather-degrading
bacteria’ with year, backgroundradiation and colony size

G. Á. Czirják et al.
Variable

Sum of squares

dfFAbundance of total cultivable bacteria
Year
0.31
1
1.32
Radiation
1.22
1
5.17
Colony size
1.40
1
5.90
Radiation × colony size
0.09
1
0.37
Abundance of cultivable bacteria ‘other than feather-degrading bacteria’Radiation
1.53
1
5.45
Colony size
1.29
1
4.62
Radiation × colony size
0.13
1
0.47

Chernobyl have reduced reproductive success if they
reproduce at all [38], and this may cause such individuals
to allocate more time to self-maintenance and preening
behaviours. Thus, reduced abundance of total and othercultivable bacteria may arise as a consequence of greater
investment in defence. In starlings, parent birds that raise an
experimentally increased number of nestlings have lesstime for feather maintenance than controls, causing an
increase in the abundance of free living, but not of attachedbacteria [25]. We believe that this mechanism is unlikely as
Møller et al. have shown that investment in the uropygial
gland and its anti-microbial secretions primarily reduces theabundance of feather-degrading bacteria, but not theabundance of other bacteria or fungi [31].
The abundance of cultivable fungi and feather-degrading
bacteria was not affected by environmental radioactivity.
There are several reasons that may account for this fact.
Firstly, the stability of the microbial communities can be
explained by the interactions between different bacterial
and fungal species, as a result of microbial interference [5,12, 31]. The lack of negative relationship between theabundance of our measured microbial groups suggests thatthe stability of microbial communities in Chernobyl may be

Figure 2 Abundance of total cultivable bacteria (CFU per milligramfeather) in relation to environmental radiation (microsieverts per hour)
around Chernobyl. The line is the linear regression line on logtransformed values

P

Slope (SE)

0.25
0.024
0.016
0.54

−0.046 (0.040)−0.156 (0.068)
0.666 (0.274)−0.311 (0.509)

0.021
0.033
0.49

−0.233 (0.099)
0.866 (0.402)−0.733 (1.071)

explained by other mechanisms and not by interference.
However, further in vitro tests and bacterial identification
are needed to test this hypothesis.
In addition, the abundance of microorganisms can beaffected by anti-microbial defences of the hosts. Birds
possess diverse behavioural and anatomical anti-microbial
defence mechanisms that can affect the abundance offeather-associated microorganisms [9, 20, 31, 43, 50].
These mechanisms could act as a filter favouring establishment of beneficial microorganisms whilst excluding harmless ones or selecting for specific resistant bacterial andfungal species. In contaminated areas around Chernobyl,
the additive effects of environmental radiation and hostdefences could have selected for resistant bacteria andfungi. We believe that this hypothesis would explain thestability of the fungal and feather-degrading bacterial loads.
The fungal species associated with plumage are predominantly dark-spored melanized fungi [42] (Czirják et al.,personal observations). Several species of fungi isolated
from the plumage of birds [19, 42] were found to be present
within and around the Chernobyl nuclear reactor [13].
These species show genetic adaptations like melanincontent of the cell wall that enable them to cope with andpotentially exploit the radioactive environment [10, 27, 58].
Using culture-based surveys, spore-forming, featherdegrading bacterial species from the genus Bacillus
predominate on feathers [49]. These species are present in
the soil around Chernobyl and are highly resistant todifferent stressors [44, 45]. Such defence mechanisms
could explain why the abundance of fungi and featherdegrading bacteria did not covary with levels of radioactive
contamination.
We have shown that the abundance of total cultivablebacteria was positively related to colony size, while theabundance of feather-degrading bacteria was marginally
positively related to colony size. These findings are
consistent with predictions concerning parasitism as a costof sociality [30] and with a previous study of barn swallows
from Denmark which found the same positive correlation[31].

Abundance of Feather-Associated Microorganisms from Chernobyl

In our study, male barn swallows breeding in Chernobyl
had significantly fewer feather-degrading bacteria thanfemales, while there was no sex difference for theabundance of total cultivable bacteria or fungi. A similar
result was also found by Møller et al. in Denmark [31] andcould be explained by female barn swallows investing morein reproduction than males [28], as well as the likelihoodthat females are more exposed to feather-degrading bacteria
from soil [24] when incubating and brooding.
In this study, we were only able to quantify theabundance of cultivable microorganisms, which imposes
limitations on our interpretations. Extending our results towhole bacterial communities using culture-independent
techniques [2, 26, 49] and identifying the bacterial andfungal species that are affected or are resistant to theenvironmental radiation would certainly be promising steps
towards an understanding of the ecological and evolutionary effects of low-level radiation on microbiota.

Conclusion
In this study, we found that abundance of different
cultivable microorganisms on feathers of barn swallows
breeding around Chernobyl was differentially affected byboth ecological factors and host-associated traits. Previous
research has demonstrated significant negative effect ofChernobyl-related fallout on abundance and diversity ofsoil bacteria and fungi, with highly resistant and genetically
adapted species being favoured in highly contaminatedareas [13, 46]. Here, we generalize these findings, documenting similar patterns for feather-associated cultivablemicroorganisms of an aerial, migratory bird. Extensive
reduction in the abundance of overall bacterial assemblage
on feathers of barn swallows with increasing levels ofradiation around Chernobyl contrasted with opportunistic
pathogens, such as feather-degrading bacteria, which wereunrelated to environmental radiation. These effects may
have long-term implications for host–microbe interactions
and overall ecosystem functioning and reinforce the notionthat the Chernobyl region and its surroundings are a
perturbed ecosystem.
Acknowledgements We are grateful to Emese and Sára Czirják,
Mathieu Giraudeau, Alex R. Gunderson, Pascal Mirleau and Matthew
D. Shawkey for their help. This project was supported in part by aFrench research grant from ANR to PH and Marcel Lambrechts(ANR-05, NT05-3_42075). GÁC was supported by a Ph.D. scholarship of the Romanian Ministry of Education and Research and by an
Eiffel grant from the French Ministry of Foreign Affairs. APM andTAM gratefully acknowledge support from the CNRS (France), the
US National Science Foundation, the National Geographic Society
and the Samuel Freeman Charitable Trust. Three anonymous
reviewers kindly provided constructive criticism on an earlier versionof the manuscript.

379

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