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Open Access Research article
The sex ratio distortion in the human head louse is conserved over
time
M Alejandra Perotti†1, Silvia S Catalá†2, Analía del V Ormeńo2,
Monika Zelazowska3, Szczepan M Bilinski3 and Henk R Braig*1
Address: 1School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, UK, 2Fundación Barceló Crilar, Mendoza y Entre Ríos,
5301 Anillaco, La Rioja, Argentina and 3Department of Systematic Zoology, Institute of Zoology, Jagiellonian University, R. Ingardena 6, PL-30-
060 Kraków, Poland
Email: M Alejandra Perotti - bssc13@bangor.ac.uk; Silvia S Catalá - scatala@crilar-conicet.com.ar; Analía
del V Ormeńo - lia414ville@yahoo.com; Monika Zelazowska - zawadz@zuk.iz.uj.edu.pl; Szczepan M Bilinski - sbili@zuk.iz.uj.edu.pl;
Henk R Braig* - h.braig@bangor.ac.uk
* Corresponding author †Equal contributors
Published: 12 May 2004 Received: 14 April 2004
Accepted: 12 May 2004
BMC Genetics 2004, 5:10
This article is available from: http://www.biomedcentral.com/1471-2156/5/10
© 2004 Perotti et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
Abstract
Background: At the turn of the 19th century the first observations of a female-biased sex ratio in
broods and populations of the head louse, Pediculus humanus capitis, had been reported. A study by
Buxton in 1940 on the sex ratio of lice on prisoners in Ceylon is still today the subject of reanalyses.
This sex ratio distortion had been detected in ten different countries. In the last sixty years no new
data have been collected, especially on scalp infestations under economically and socially more
developed conditions.
Results: Here we report a female bias of head lice in a survey of 480 school children in Argentina.
This bias is independent of the intensity of the pediculosis, which makes local mate competition
highly unlikely as the source of the aberrant sex ratio; however, other possible adaptive
mechanisms cannot be discounted. These lice as well as lice from pupils in Britain were carrying
several strains of the endosymbiotic bacterium Wolbachia pipientis, one of the most wide spread
intracellular sex ratio distorters. Similar Wolbachia strains are also present in the pig louse,
Haematopinus suis, suggesting that this endosymbiont might have a marked influence on the biology
of the whole order. The presence of a related obligate nutritional bacterium in lice prevents the
investigation of a causal link between sex ratio and endosymbionts.
Conclusions: Regardless of its origin, this sex ratio distortion in head lice that has been reported
world wide, is stable over time and is a remarkable deviation from the stability of frequencydependent
selection of Fisher's sex ratio. A female bias first reported in 1898 is still present over
a hundred years and a thousand generations later.
Background
The early but comprehensive literature on human head
lice, Pediculus humanus capitis, reveals a surprisingly
homogeneous female bias of the sex ratio world wide [1-
3]. The first reported observation of more female than
male lice on human heads reaches back to Harding in
1898 in the USA [1]. Records of sex ratios in human head
lice in Britain [2,3], Kenya [3,4], Tanzania, Colombia,
Australia [3], Nigeria, Ceylon, Palestine [4], India [4,5],
and North America [6] show ~65% of the adult lice to be
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female. Reanalyses of data collected in 1938 are still
employed to deduce life history traits of human head lice
[4,7,8]. The female bias of natural populations on heads
is conserved in the sex ratio of individual broods of experimental
infestations [2,6,7]. Many of the people sampled
in these studies had been afflicted by poverty, confinement
or war. However, in the last sixty years no data have
been collected, especially on more normal levels of infestations.
Local and temporary deviations from equality of
the operational sex ratio should be expected as natural
fluctuations. We wanted to find out whether this deviation
is also maintained over time. Roughly one hundred
years after the original surveys, we determined the sex
ratio of adult head lice on pupils from 6 different schools
in Argentina. This cohort is different from earlier studies
in that it does no longer show any extremely high infestations
due to an increase in general living standards. Nevertheless,
a female bias is still preserved.
Sex ratio distortions in arthropods frequently originate
from cytoplasmic or extrachromosomal factors and parasites
of the host and are most often associated with the
endosymbiont Wolbachia pipientis. Wolbachia has been
shown to induce parthenogenesis in haplodiploid
Hymenoptera and diplodiploid Collembola, feminisation
in terrestrial isopods, and male killing in many insect
species [9-13]. A new bacterium from the Bacteroidetes
group (Cytophaga-Flavobacterium-Bacteroides, CFB) has
been linked with feminisation and parthenogenesis in
mites and insects [14-16]. We screened human lice from
Argentina and Britain and pig lice form Poland for the
presence of Wolbachia and CFB bacteria.
Results and discussion
A total of 480 heads were screened for adult lice along 6
schools exhibiting a prevalence of 29.8% (infested heads
with at least 1 adult louse/head). 10.5% of the infested
heads (15 heads, 94 lice) showed a male bias, 16.8% of
the infested heads (24 heads, 32 lice) had only males,
14.7% of the infested heads (21 heads, 72 lice) had an
equal sex ratio, 28.0% (40 heads, 344 lice) had a female
bias and an additional 30.0% of the children (43 heads,
55 lice) did not carry any male lice, Fig. 1. This translates
into an overall female bias of 59.6% (1.48) and a relative
sex ratio of 0.40 (males/(males + females)) in all the populations
based on 356 females and 241 males. The regression
line for the distribution of female and male lice
differs from an equal sex ratio distribution by a power of
p < 0.01 using Fisher's exact test (Fig. 1). There is no difference
between boys and girls. This is also the first survey
of sex ratio in human head lice for South America. Applying
the standard deviation of the data in Fig. 1, Fig. 2
shows the frequencies of heads that deviate from a normal
distribution of sex ratio. Each of the six schools shows
individually a female bias and replicate the bias of all the
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populations, Fig. 3. There is no difference between urban
and rural schools. These results in female bias correspond
with the data by Buxton and others [5-7]. The probability
of a sampling error is p < 0.05; the difference of the actual
percentages from 50 is 3.34 times the standard error of
this difference [7,18]. Fig. 4 shows that this bias is independent
of the intensity of infestation confirming earlier
studies by Lang and others [6]. This suggests that the
female bias is both conserved in space and time.
Pediculid lice are diploid amphimictic and exhibit an
extra mitotic division following meiosis during spermatogenesis.
Pediculus are sexually dimorphic with a small
proportion of hermaphrodites. Siblings are not gregarious,
males are abundant, both sexes eclose at similar
times, mating starts within one day after eclosure or
encounter, males are very promiscuous, females do not
seem to store sperm, frequent copulation is a requirement
for laying fertile eggs and both sexes show active migratory
behaviour. At the moment it is difficult to determine
the sex of immature stages and the sex-specific mortality
of immature stages directly. However, single-pair matings
sometimes produce all of one sex. Differential mortality
of nymphs does not account for the unusual sex ratio
under non-crowding conditions. P. capitis does not
exhibit parthenogenesis or delayed fertilisation. The sex
ratio does not change with increasing age of the mother.
No evidence has been found that any environmental factor
has any effect upon the sex ratio. Only in a single case
under very crowded conditions, a male bias has been
observed on some heads by Buxton [7,17]. He attributes
this to a severely reduced life span of females [7]. The
shortened life expectancy of females is ascribed to copulation
prior to full sclerotisation leading to injury and death
[6]. A selective reduction in life expectancy of females
under crowded condition has also been observed in other
insect taxa, e.g., tsetse flies. However, highly infested
heads with a maximum of 1,434 lice in a study by Roy and
Ghosh still showed a female bias [5].
Focusing on the data which Buxton obtained in the
Colombo prison in Sri Lanka from 1934 to 1936 that also
showed a female bias, Rózsa suggests that the prison environment
could increase inbreeding in lice. He argues that
the transmission rate was likely to be low simply because
the prison host population consisted of adult males only
and because the prisoners couldn't get rid of their lice;
these infrapopulations would subsist through several generations.
These arguments were employed by Rózsa as
indications for local mate competition leading to an adaptive
sex-ratio manipulation by the lice [8]. This is neither
supported by our data nor the data of Buxton, Roy and
Ghosh, or Lang [5-7]. There is no evidence that female lice
can manipulate the sex ratio of their offspring. Rózsa
claims that both, individual heads of the men in jail and
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30
25
20
15
10
5
0
10 5 0
Males / head
Figure 1 Sex ratio of human head lice of school children in Argentina
Sex ratio of human head lice of school children in Argentina. The graph shows the distribution and regression of female and
male lice per head with many data points overlapping.
the prison itself constitute viscous populations in Hamilton's
sense – that is to say, one where the individual louse
can mate only with a rather permanent set of neighbours
who tend also to be his relatives [19]. Whoever has or has
had school children to look after can confirm that individual
heads of pupils do not constitute a viscous population.
Exchange of adult lice between heads is constantly
ongoing and is a major reason for the continuous spread
of lice in our society. Our data show that the female bias
is present on individual heads as well as in all six schools
surveyed. All the schools are coeducational.
Hamilton has proposed local mate competition as an
explanation of female biased sex ratios in insect and mite
populations. All the arthropod populations listed by
Hamilton are characterised by inbreeding and haplodiploidy
[19]. An ideal for an extreme biofacies may be
described by eight properties. One, the primary sex ratio is
spanandrous – that is, females greatly preponderate. In
human lice, the operational sex ratio is close to equality.
The sex ratio determined by dissecting second instar
nymphs and the sex ratio at eclosion of imagines of experimental
broods is not more spanandrous than the operational
sex ratio. Broods of individual female lice exhibit
the whole spectrum from male only offspring to female
only offspring. Two, reproduction is arrhenotokous. Lice
are diplodiploid and reproduction is amphimictic. Three,
there is at least one male in every batch of offspring. Egg
laying in the human head louse starts with the first blood
meal regardless of insemination; it is more continuous
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y = 1,3139 x + 0,2752
r = 0,74, n=143 p<0,00
30 25 20 15
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Number of heads
Figure 2 Number of male lice per female louse per head
Number of male lice per female louse per head. The columns show the frequency with which on individual heads female lice
were associated with no males, less than one male per female, equality, and more than one male per female. The boundaries of
equality include one standard deviation (SD) on each side, the columns 'less than 1' and 'more than 1' are minus one SD.
given sufficient food than in batches. The complete brood
of a female might contain only males, only females or any
ratio in between. Four, there is gregarious development, as
a group of siblings, from egg to adult. Unlike the body
louse where the female tends to return to the same spot
for oviposition and which will lead to a clustering of eggs,
siblings of head lice are practically not gregarious. Five,
adult males eclose first and can mate many times. Both
sexes of head lice eclose at similar times but males are very
promiscuous. Six, mating takes place immediately after
(or even before) eclosure of adult females. Mating continues
to take place at any time of the day or night and during
the whole adult life span of lice. Mating happens as much
before as after dispersal and both sexes show the same
active migratory behaviour. Seven, males are disinclined,
or unable, to emigrate from the batch. Male lice disperse
as eagerly as females. This is very obvious in our data in
the number of heads carrying male lice only (Fig. 2).
Eight, females can store sperm; one insemination serves to
fertilise the whole egg production. Female lice are stated
to have no receptaculum seminiis (spermatheca) and
therefore are considered not to be capable of storing
sperm. Nevertheless, Bacot reported in 1916 a record
observation for a female being able of laying fertile eggs
twelve days after the removal of the male; however, the
maximum ever observed by Nuttall in 1917 is only five
days, which is in line with the assumption of limited or no
sperm storage capability. These eight reproductive properties
of lice make local mate competition as the cause of a
female-biased sex ratio completely impossible. Further
work on Hamilton's hypothesis has shown that within
each local group, even if founded by a single female, the
Number of males per female
45
40
35
30
25
20
15
10
5
0
less than 1 0 males
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only males more than 1 1
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Figure 3 Female bias of head lice per school
Female bias of head lice per school. An infested head might be considered as a metapopulation, a school as a population, all six
BMC Genetics 2004, 5
schools replicate the female bias; there is no significant difference between the schools, Chi square 2.44, p = 0.78, n = 6.
sex ratio favoured by individual selection is equality [20-
23]. However, groups founded by female-biased genotypes
contribute more individuals to the population as a
whole than groups founded by unbiased genotypes [24].
This requires the ability of kin recognition and selection.
However, in the female-biased parasitoid wasp Nasonia
vitripennis, which is often used in textbooks as an example
of local mate competition, mothers cannot discriminate
between kin [25]. In many cases in which the female bias
could not be attributed to local mate competition, local
resource competition among females or female-biased
dispersal have been put forward as alternative explanations
[eg., [26,27]]. Local resource competition and differential
dispersal can be ruled out in head lice for the same
reasons discussed earlier for local mate competition. Why,
for example, parasitoid wasps like Melittobia digitata that
seemingly fit all the requirements set out by Hamilton fail
to meet the sex ratio prediction of the local mate competition
hypothesis remains unsolved [28]. Orzack emphasises
that the occurrence of local mate competition is not
documented for most species whose sex ratios have been
explained by it [29]. He also documents that the original
evidence presented by Hamilton in support of local mate
competition is not as clear as originally assumed. This suggests
that major adaptive and parasitic sex ratio effecting
mechanisms still remain to be discovered.
Hamilton himself detected a maternally inherited microorganism,
later identified as the endosymbiont Wolbachia,
in four species of parasitoid wasps belonging to the genus
Trichogramma, a genus originally listed as an example of
an extreme biofacies leading to local mate competition.
Removal of the bacterium changed reproduction from
female only thelytoky to biparental arrhenotoky [30]. The
possibility that maternally inherited bacteria might bias
the sex ratio in favour of females has long been predicted
[31]. Parthenogenesis-inducing Wolbachia are quite common
in parasitoid wasp species [32]. However, in some
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Proportion of females (mean)
Figure 4 The sex ratio of human head lice is independent of the infestation level
The sex ratio of human head lice is independent of the infestation level. Number of means = 10, r = 0.07, p = 0.43; number of
heads = 143, r = 0.00, p = 0.98. N: number of heads; the horizontal line indicates equality.
species of female-biased parasitoid wasps Wolbachia does
not influence the sex ratio directly. In double infected
Nasonia for example, two strains of Wolbachia induce cytoplasmic
incompatibility, in Asobara tabida, one of the
three Wolbachia strains of the triple infection is essential
for oocyte maturation, and in Melittobia australica, the
effect of Wolbachia remains to be determined [33-35].
A sex ratio bias is often more likely related to the physiological
state of its host and is consequently not directly
controlled by the mother [36]. The physiological state
includes the manipulation by symbiotic microorganisms.
In the analysis and interpretation of sex ratio deviations,
the discrimination between primary sex ratio and functional
sex ratio is pivotal [37]. This allows the identification
of symbiotic sex ratio distorters.
Using the polymerase chain reaction and specific primers
for 16S rDNA and the Wolbachia outer surface protein
gene wsp, we demonstrated the presence of Wolbachia pipientis
in two blood-sucking Anoplura species, the human
louse Pediculus humanus capitis, Pediculidae, and the pig
louse, Haematopinus suis, Haematopinidae. Primers for
insect-like CFB bacteria did not amplify any product;
primers for mite-like CFB bacteria resulted only in unrelated
lice sequences indicative for the absence of a specific
target sequence. The highest amounts of Wolbachia were
detected in thorax and head. All lice tested of both species
harboured Wolbachia. The Wolbachia infection is likely
close to fixation, which suggests either a relatively young
infection showing no signs of host resistance or indicates
an essential function for Wolbachia in the development
and survival of its lice hosts. The fact that two phylogenetically
very different lice species with two distinct hosts
show similar high levels of penetrance of Wolbachia infection
on two different continents leads us to expect Wolbachia
infections to be widespread in many lice species.
The wsp gene of Wolbachia is one of the fastest evolving.
Wsp sequences of Wolbachia from human and animal lice
overlap. No Wolbachia lineage specific to a louse species or
Adults per head
n=53
n=14
n=14
n=23
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0 1 2 3 4 5 6 7 8 4 - 15 9-14 9 10011
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n=6 n=3 n=7
n=7
n=9
n=7
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wPcap Pediculus humanus wArg1
wRi Pediculus humanus wWal1
wRi Pediculus humanus wWal2
A
wRi Haematopinus suis wPol1
wRi Drosophila simulans
wMel Pediculus humanus wArg2
wMel Drosophila melanogaster
wPap Phlebotomus papatasi AB
wCon Haematopinus suis wPol2
wCon Orius strigicollis wStr1 B
wCon Tribolium confusum
Tunga penetrans
Brugia malayi D
C Onchocerca ochengi
Re Figure 5 lationship of Wolbachia strains from lice to each other and to Wolbachia strains from other insects and filarial worms
Relationship of Wolbachia strains from lice to each other and to Wolbachia strains from other insects and filarial worms. Parsimony
cladogram showing the relationship of Wolbachia strains detected in lice (bold) and their nearest known relatives based
on the wsp gene and constructed with branch-and-bound. From right to left, the Wolbachia group is noted, where known, followed
by the name of the host and the designation for the individual Wolbachia strain; after the bar the Wolbachia supergroup
is indicated. Phlebotomus papatasi is a sand fly (Diptera), Orius strigicollis is a bug (Hemiptera), Tribolium confusum is a beetle
(Coleoptera), Tunga penetrans is a flea (Siphonaptera), and Brugia malayi and Onchocerca ochengi are filarial nematodes.
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Lo Figure 6 calization of microorganisms within reproductive organs of the piglouse, Haematopinus suis
Localization of microorganisms within reproductive organs of the piglouse, Haematopinus suis. A, Intraovarian mycetome consists
of several mycetocytes containing rod-shaped microorganisms (arrows). The mycetome is located in contact with lateral
oviduct cells (lo). Fluorescence microscope, DAPI; scale bar = 10 µm. B, Posterior pole of the oocyte, mycetome located in a
depression of the posterior pole of the oocyte (asterisk). Light microscope, methylene blue; scale bar = 10 µm. C, Mycetome
microorganisms (asterisks) are located extracellularly, in contact with oocyte microvilli (mv); oocyte (o). TEM; scale bar = 1
µm. D, Mycetome microorganisms visualized in SEM; scale bar = 1 µm. E, Intracellular microorganisms (arrow) within the
oocyte cytoplasm. Fluorescence microscope, DAPI; scale bar = 10 µm.
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showed double infections with two different Wolbachia
strains based on wsp sequence. Human head lice from two
continents have been analysed. Head lice from South
to a louse host could be identified. Comparison of the wsp
sequences revealed no special position of lice Wolbachia
among other insect Wolbachia strains (Fig. 5). All lice
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No lice 52
Only empty nits 17 5.70%
Only live nits 92
Live nits and mobiles
Only mobiles 31%
35 15 -5
Number of Heads
Figure 7 Occurrence of eggs, larvae and adults of human head lice
Occurrence of eggs, larvae and adults of human head lice. Distribution of various stages on heads; larvae and adults are combined
as mobiles. Numbers inside the columns denote children's heads followed by the corresponding percentage.
America, Argentina, exhibit infections of two different
strains of Wolbachia from supergroup A, wMel and a new
group, which we propose to call wPcap, while from
Europe, Wales, only two similar strains of supergroup A
(wRi) have been detected. In contrast, pig lice harbour
Wolbachia from supergroup A (wRi) and supergroup B
(wCon closest to strain wStr1). While double infections
with two A supergroup Wolbachia might reflect recombination
events or in-host diversification of Wolbachia, double
infections with A and B supergroup bacteria are likely
to originate from repeated episodes of horizontal transmission.
The diversity of A and B supergroup Wolbachia
make it seem possible that lice have received recent horizontal
transfer from a wide spectrum of Wolbachia donors.
An infectious form of Wolbachia is not known. It is always
vertically transmitted within a species. Lice are one of very
few insect taxa that are not attacked by parasitoid wasps
and endoparasitic Strepsiptera. Parasitoid wasps are the
only so far confirmed vectors for horizontal Wolbachia
transmission; the role of Strepsiptera in interspecies transmission
is still controversal [38]. Interestingly, we were
unable to find any report about lice-specific mites or nemhttp://
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137
17.30%
55
30%
95
45.60%
155 135 115 75
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Proportion
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Figure 8 Influence of social environment on sex ratio of lice
Influence of social environment on sex ratio of lice. Comparison of a school in a deprived neighbourhood (A) with a school in
an affluent neighbourhood (B).
atodes, theorised vectors of Wolbachia. The literature is
limited to the protozoan gut parasite of body lice,
'Crithidia' or Herpetomonas pediculi and omniphagous
steinernematid and heterorhabditid nematodes, which do
not carry Wolbachia [39-42]. This lack of parasites might
be due in part to a deficiency of research; however, it
requires at present to postulate a new route for the horizontal
transmission of Wolbachia to and within lice
species.
Microscopic analysis shows that intracellular Wolbachialike
bacteria are not associated with the well described but
yet unidentified nutritional lice endosymbionts found in
mycetomes (Fig. 6). While the bacteria originating from
the stomach disc mycetome migrate to the ovaries and
enter the developing oocyte at a specialised invagination
forming the future mycetome [43-45], intracellular Wolbachia-
like bacteria are already present in the egg cell and
are dispersed in the cytoplasm of the egg. Due to the difficulty
of cutting thin sections of the yolk part of eggs, Wolbachia
infections are easily missed in ultrastructural
investigations.
The sex ratio bias reported here for the human head louse
does not seem to be an isolated phenomenon. Unusual
sex ratios have also been reported for the human body
louse, Pediculus humanus humanus (P. corporis, P.
vestimenti), the human pubic louse, Pthirus pubis, the cattle
biting louse Bovicola bovis, the shortnosed cattle louse,
Haematopinus eurysternus, the longnosed cattle louse,
Linognathus vituli, and the little blue cattle louse, Solenopotes
capillatus [3,46-49]. The biting louse B. bovis also
exhibits parthenogenetic reproduction. The search for
Wolbachia in many biting and sucking lice (Anoplura,
Mallophaga) is ongoing [50].
Human head lice show a female biased sex ratio that has
been conserved for over 100 years with a global distribution
which represents a remarkable deviation from the
stability of frequency-dependent selection of Fisher's sex
Live nits
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School A
School B
Adults Larva
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160
140
120
100
80
60
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Mobiles per head
40
20
0
-20
-200 0
Figure 9 The highest mortality of head lice is at the egg stage
The highest mortality of head lice is at the egg stage. Correlation between live eggs and mobiles (nymphs and adults) per head.
Pearson, n = 229, p < 0.001.
ratio [1,51]. The association of this sex ratio distortion
with a sex-ratio distorting endosymbiont is intriguing. A
direct treatment of lice with antibiotics to cure them of
Wolbachia is not feasible at the moment because of the
lethal effect on mobile stages of lice, as it eliminates the
Gram-negative obligate lice endosymbionts as well [52].
Male killing is one of several possible mechanisms leading
to distorted sex ratios. In other insects, male-killing Wolbachia
act at the earliest stages of development and manifest
as eggs that fail to hatch [53]. Indeed, Buxton
identified the failure of eggs to hatch as an important
cause of mortality in Pediculus humanus corresponding to
what would be expected with low-level male killing as it is
well known, for example in lady bird beetles [54,55]. Mortality
data have not been reported for head lice. In a new
study we determined the number of empty eggs, alive
eggs, nymphs, and adults of 300 children of two coeducational
schools at an age between 6 and 8 years, Fig. 7. Per
head, alive eggs represent 90% of the accountable metapopulation,
nymphs and adults, which can be combined as
mobiles, account for the further 10%. This relationship is
not affected by the social status of the pupils. The living
standard of the children might be used as a proxy of environmental
influences on the lice. In Fig. 8, we compared
the data from a school (A, 161 children) in a deprived
neighbourhood with the data from a school in an affluent
surrounding (B, 139 children). The pattern is identical.
The correlation between living eggs and mobiles is significant
(p < 0.01) and suggests a high mortality at the egg
stage, Fig. 9, the origin of which remains to be
determined.
Live nits per head
Mobiles = .734 + .12254 * live nits
Correlation: r = .92833
400 200
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1200 1000 800 600
Conclusions
Adaptive sex ratio distortions fluctuate over time and are
often specific to local habitats and environmental conditions.
Mechanisms such as local mate competition, local
resource competition or differential dispersal can lead to
female bias populations. The female bias in the human
head louse cannot be ascribed to any of these three mechanisms.
This bias is found on 4 continents. It is stable over
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more than one century. This makes it one of the best
documented deviations from the stability of frequencydependent
selection of Fisher's equal sex ratio.
Methods
Lice
The surveys and collections of head lice have been conducted
between October 2000 and August 2003. In
Argentina, the schools are situated in urban and rural
parts of the provinces of La Rioja and Córdoba. The lice
were collected by the authors [AVO and SSC]. The hair
was wetted and treated with conditioner. With the help of
a metal comb, eggs, nits and adults were collected under
water and immediately transferred into 95% alcohol. This
method of collecting lice preludes any sampling bias
favouring larger females over smaller males as suggested
by Nuttall and Marshall [37]. Eggs and empty egg cases
were distinguished and the sexes of imagines were determined
using a light microscope. The lice samples from
Britain were collected by their parents in Llandudno,
Conwy, and Bangor, Gwynedd, both in northern Wales,
United Kingdom. The pig lice were collected from domestic
pigs kept on a small farm near Kraków, Poland.
Wolbachia
Total genomic DNA was extracted from single lice. Males
and females were sectioned in two parts: head-thorax and
abdomen and DNA was extracted separately for both
sections. QIAGEN DNAeasy™ Tissue Kit was used for DNA
extraction following the DNeasy Protocol for Animal Tissues.
The DNA obtained was purified with 10% CHELEX®
100 at 60°C for 2 h. The samples were centrifuged at
8,000 rpm for 5 min and the supernatant was stored at -
20°C. PCR was performed with specific primers for Wolbachia
16S rDNA, 99F and 994R [56], Wolbachia outer surface
protein, wsp81F and wsp691R [57] and universal
primers for 28S rDNA, 28Sa and 28Sb [58] as control. CFB
bacteria were detected with primers for 16S rDNA. Primers
for insect-like CFB bacteria were based on sequences
derived from the parasitoid wasps of the genus Encarsia,
EPS-f and EPS-r [15], mite-like CFB bacteria were designed
with the help of the sequence from the feminised mite
Brevipalpus phoenicis, GenBank accession AF350221,
CFBmite16S-F 5'-CCT GCG GGG GCT CTT GA-3' and
CFBmite16S-R 5'-GGG TTT CGC TCG TTA TAG GAC TTA-
3' amplifying a 644 bp fragment in B. phoenicis. Relative
amounts were estimated with serial dilutions of the template
before amplifications. The products were ligated
into QUIAGEN pDrive Cloning Vectors and 8 positive colonies
per ligation were sequenced in both directions. The
sequences are deposited in GenBank with accession numbers
AY596781-AY596786.
http://www.biomedcentral.com/1471-2156/5/10
Authors' contributions
MAP carried out the molecular work. AO collected the
human lice in Argentina. MZ and SMB collected the pig
lice and performed the microscopic work. SSC, MAP and
HRB analysed the data. MAP and HRB wrote a first draft
of the manuscript. All authors contributed to the final
analyses and writing of the manuscript.
Acknowledgements
Field and laboratory work in Argentina (SSC and AO) have been supported
by Fundación Barceló. MAP and HRB are supported by funding from the
Natural Environment Research Council (GR3/13199) and HRB acknowledges
funding by the European Commission (EUWOL, QLRT-2000-01079).
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