HeadLice.Org - Evidence that Head Lice and Body Lice belong to the same species

The debate about whether head and body lice belong to the same species rages on.
Here's two articles from both sides on the issue:

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SHORT COMMUNICATION Evidence from Mitochondrial DNA That Head Lice and Body Lice of Humans (Phthiraptera: Pediculidae) are Conspecific N. P. LEO,1,2 N.J.H. CAMPBELL,2 X. YANG,3 K. MUMCUOGLU,4 AND S. C. BARKER2 J. Med. Entomol. 39(4): 662Ð666 (2002) ABSTRACT The speciÞc status of the head and body lice of humans has been debated for more than 200 yr. To clarify the speciÞc status of head and body lice, we sequenced 524 base pairs (bp) of the cytochrome oxidase I (COI) gene of 28 head and 28 body lice from nine countries. Ten haplotypes that differed by 1Ð5 bp at 11 nucleotide positions were identiÞed. A phylogeny of these sequences indicates that these head and body lice are not from reciprocally monophyletic lineages. Indeed, head and body lice share three of the 10 haplotypes we found. FST values and exact tests of haplotype frequencies showed signiÞcant differences between head and body lice. However, the same tests also showed signiÞcant differences among lice from different countries. Indeed, more of the variation in haplotype frequencies was explained by differences among lice from different countries than by differences between head and body lice. Our results indicate the following: (1) head and body lice do not represent reciprocally monophyletic lineages and are conspeciÞc; (2) gene ßow among populations of lice from different countries is limited; and (3) frequencies of COI haplotypes can be used to study maternal gene ßow among populations of head and body lice and thus transmission of lice among their human hosts. KEY WORDS Pediculus humanus, Pediculus capitis, lice, Phthiraptera, Pediculidae, cytochrome oxidase I ALTHOUGH THE HEAD and body lice of humans are very similar morphologically, investigators have reported consistent morphological differences between the two types (Busvine 1978, Schaefer 1978, Tarasevich et al. 1988). Head and body lice interbreed readily in the laboratory, yet one study of their morphology suggested they do not interbreed in nature (Busvine 1978). However, whether or not these lice are conspeciÞc remains controversial (Durden and Musser 1994, Burgess 1995, Khudobin 1995). We studied a fragment of the cytochrome oxidase I (COI) gene of mitochondrial DNA (mtDNA) from lice collected from nine countries. Our aim was to collect evidence that would clarify the speciÞc status of head and body lice. If they are different species, we would expect head and body lice to be reciprocally monophyletic lineages; whereas if they are conspeciÞc, we would expect that these two types of lice would not be in reciprocally monophyletic lineages (clades). We also 1 E-mail address: n.leo@imb.uq.edu.au. 2 Department of Microbiology & Parasitology, and Institute for 3 Molecular Biosciences and ARC Special ResearchCentre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland, 4072, Australia. Animal Medical Department, Inner Mongolia Agricultural University, Huhehot, 010018, China. 4 Department of Parasitology, Hebrew University, HadassahMedical School (P.O. Box 12272), and the Department of Dermatology, HadassahUniversity Hospital (P.O. Box 12000), 91120 Jerusalem, Israel. 0022-2585/02/0662Ð0666$02.00/0 2002 Entomological Society of America examined genetic differentiation among different populations of these lice using FST values and exact tests of haplotype frequency differences. Materials and Methods Lice from nine countries were studied: Australia, China, Hungary, Israel, Japan, Kenya, New Zealand, Papua New Guinea, and the United States (28 head and 28 body lice, Table 1). These included four lice from Inner Mongolia Province in China that were from two hosts infested withbothh ead lice and body lice. We sampled one head louse and one body louse from each of these two hosts. DNA was extracted from lice by either a phenolchloroform method (Sambrook et al. 1989) or with chelex beads (Bio-Rad, Hercules, CA). The latter involved crushing a louse with a micropestle in a tissue grinding tube withliquid nitrogen. One milliliter of boiling 5% chelex beads in 1TE buffer withRNaseA (1 l of 25 mg/ml RNaseA for every 100 ml of chelex in 1 TE buffer) was added to the tube and then put in boiling water for 15 min. Tubes were cooled for 10 min at 20C and then spun in a microcentrifuge at 12,000g for 10 min. Three microliters of the top layer was used in each25 l polymerase chain reaction (PCR). The insect-speciÞc primer C1-J-1718 (forward: 5-GGAGGATTTGGAAATTGATTAGTTCC- 3) (Simon et al. 1994) and a primer speciÞc to Pediculus humanus (designed by N.L.) C1-N-2191 July 2002 Table 1. Location and hosts of the lice used in this study Country Australia China Hungary Israel Japan Kenya New Zealand Papua New Guinea Auckland Askaroff, Lake Murray Hole in the wall village, Madang United States of America Orlando, Florida & Washington DC (reverse:5-CCAGGAAGAATAAGAATATAAACTTC- 3) were used to amplify 524 bp of the mitochondrial COIgene. Primer names refer to where they anneal by gene, strand and 3 base position relative to the Drosophila yakuba sequence (after Simon et al. 1994). PCR reactions contained 1Ð3 l of DNA template, 2.5 l of Reaction Buffer IV at 10 concentration (AB Gene, Epsom, UK), 2.25 l of MgCl2 (25 mM), 1.1 l of dNTPs (5 mM), 0.3 l of eachprimer (10 M), 0.2 l of Red Hot Taq polymerase (AB Gene), and MilliQ water to a Þnal volume of 25 l. The cycling conditions were 94C for 1 min; 35 cycles of 30 s at 94C, 30 s at 55C, and 40 s at 68C; and a Þnal extension time of 5 min at 68C. PCR products were visualized under UV light after electrophoresis in an ethidium bromidestained agarose gel. If insufÞcient DNA was ampliÞed Locality Townsville, Queensland Brisbane, Queensland Cele, Cele County, Xinjiang Province Yiliqi, Hotan County, Xinjiang Province Huhehot, Inner Mongolia Province Longxi County, Ganshu Province Budapest Bet-Shemesh Sapporo Tokyo Nairobi Head Head Body Body Body Head Head Head Body Body Head Body Mountain village, Inner Mongolia Province Body, Head Body, Head Head Head Head Head Body Body Head Body Head Head Head Body Table 2. Variable sites in the 524 bp region of the cytochrome oxidase I (COI) gene of Pediculus humanus, and the haplotypes found in each country 91 106 116 120 150 255 264 291 345 357 453 T T G C C A T G A A T A . . . . . . . . . . A . . . T . . . . G C 3 A . . . . G . . . . . A . . . T . C . T G . A . . . T . . . . G . A . . T . . . . . . . A . . . T . . A . G . A . A . . . . . . . . A A . . . . . . . . . 10 Sites are numbered according to the alignment of sequences. A dot indicates identity with the sequence of haplotype 1. AUS, Australia; CHI, China; HUN, Hungary; ISR, Israel; JAP, colony originally from Japan; KEN, Kenya; NZ, New Zealand; PNG, Papua New Guinea; USA, colony originally from the United States of America; H, head lice; B, body lice. No. of lice from eachh aplotype from eachlocality Base position HUN ISR JAP CHI Haplotype AUS Total H H B H H B H B H H B 1 1 2 1 7 3 1 1 2 3 3 1 9 1 3 2 1 4 1 5 6 7 8 9 1 1 3 3 27 10 6 3 4 2 1 1 1 2 29 1 3 5 6 3 4 3 56 1 3 1 NZ PNG USA KEN 3 1 LEO ET AL.: MTDNA FROM THE HEAD AND BODY LICE OF HUMANS Type for sequencing, hot-start PCR was attempted with a fresh sample of DNA from that louse. The conditions for hot-start PCR were 5 min at 94C, polymerase added, 2 min at 94C; 35 cycles of 30 s at 94C, 30 s at 55C and 40 s at 68C; and then 5 min at 68C. PCR products were puriÞed withQiaquick columns (QIAGEN, Venlo, The Netherlands) and sequenced directly (DyeDeoxy Terminator; PE Applied Biosystems, Foster City, CA) withth e PCR primers (above), by an ABI 377 gene sequencer. The Þrst 24 lice were sequenced withbothforward and reverse primers. This revealed haplotypes 1Ð5. The rest of the lice were sequenced with the forward primer only. If the sequence data indicated a new haplotype, or if the identity of a nucleotide was ambiguous, the gene was then sequenced withth e reverse primer. Haplotype (s) 2 4 2, 2, 2 3, 3, 3 3, 3, 10 2, 2 2, 2 2, 2, 3 1, 3 3, 3, 3, 2 6 6, 7 6, 6 7 8 2 2, 4, 9 5, 5, 5 2, 2 2, 2, 2 2, 2, 2 4, 4, 4 2, 2, 4 2 2, 2, 2 663 Host 1 louse from 1 host 1 louse from 1 host 3 lice from 1 host 3 lice from 1 host 3 lice from 1 host 2 lice from 1 host 2 lice from 1 host 3 head lice from 3 separate hosts 2 lice from 1 host 3 body lice from 3 separate hosts 1 louse from 1 host 1 louse from 1 host 2 lice from a double infestation 2 lice from a double infestation 1 louse from 1 host 1 louse from 1 host 1 louse from 1 host 3 lice from 3 separate hosts 3 lice from a laboratory colony 2 lice from 1 host 3 lice pooled from 3 hosts 3 lice pooled from 3 hosts 3 lice pooled from 5 hosts 3 lice from 3 separate hosts 1 louse from 1 host 3 lice from a laboratory colony JOURNAL OF MEDICAL ENTOMOLOGY 664 Fig. 1. The single most parsimonious (shortest) unrooted tree (11 steps) of the 10 haplotypes of head and body lice from a branchand bound searchin PAUP. There was only one internal branchin this tree; it had a bootstrap support of 86%. Numbers in circles show the haplotype identiÞcation number (refer to Table 2). The size of the circles indicates the frequency of the haplotypes. Shaded areas indicate the proportion of body lice; nonshaded areas indicate the proportion of head lice. Sequences were aligned by eye in Sequencher 3.1.1 (GeneCodes Corporation,AnnArbor, MI), then comparedwithCOI sequences of other insects inGenBank to check that the COI had been ampliÞed. We executed a branchand bound searchin PAUP 4.0b3a (Swofford 1998) to Þnd the maximum parsimony tree(s) for the sequences, and then tested the robust- Vol. 39, no. 4 ness of these relationships with 1,000 cycles of bootstrap resampling. Arlequin 2.000 (Schneider et al. 2000) was used to calculate FST values and to execute an exact test of sample differentiation from haplotype frequencies, for differences between all head and body lice tested, and for differences among lice from different countries. LEO ET AL.: MTDNA FROM THE HEAD AND BODY LICE OF HUMANS July 2002 Results There was little nucleotide variation among the lice we studied: we found 10 COI haplotypes in the 56 lice from nine countries. These haplotypes differed from eachoth er by 1Ð5 base substitutions (0.2Ð1.1%) at 11 variable sites in the 524 bp fragment (Table 2). Eight of the substitutions were conservative (silent) transitions at the third codon position. The other three substitutions were, relative to the most common haplotype, nonconservative transversions in haplotypes 1 and 10 at the Þrst codon position, and in haplotype 9 at the second codon position (sites 91, 106, and 116, respectively, Table 2). Haplotype 2 was the most common and widespread haplotype overall; it was found in 27 of the 56 lice and in lice from all countries except New Zealand (GenBank accession number AF320286). This haplotype was the most common haplotype in both head lice (16 of 28 lice) and body lice (11 of 28 lice) (Table 2). The single most parsimonious tree of the COI haplotypes had 11 steps and only one internal branch; this branch had bootstrap support of 86% (Fig. 1). This internal branchdivided the lice into two clades: one withbothh ead and body lice from all nine countries (haplotypes 1, 2, 4, 7, 9, and 10) and the other with head and body lice from China and Japan (haplotypes 3, 5, 6, and 8). Haplotypes from bothof these clades were found in a head louse (haplotype 7) and a body louse (haplotype 6) from one host in Inner Mongolia Province, in China. Both lice from the other host infested withtwo types of lice (also from Inner Mongolia Province) had haplotype 6. Bothth e FST values and the exact test of haplotype frequencies showed signiÞcant differences between head and body lice (FST 0.09, P 0.00880; exact P value 0.00000). However, bothtests also showed signiÞcant differencesamonglice from different countries (FST0.23, P0.00098; exact P value0.00236). Some infestations (lice from one host) had more than one haplotype (Table 1). Of nine hosts, from which two to three lice were studied, three hosts had lice withtwo different haplotypes: (1) a person from Xinjiang Province in China had two body lice with haplotype 3, and one body louse with haplotype 10; (2) another person from Xinjiang Province in China had one body louse with haplotype 1 and another body louse withh aplotype 3; and (3) a girl from a remote mountain village in Inner Mongolia Province in China had a body louse with haplotype 6 and a head louse withh aplotype 7. Discussion We examined 28 head and 28 body lice from nine countries. Ten COI haplotypes were identiÞed. Phylogenetic analysis revealed a single tree withone internal branch that separated haplotypes 1, 2, 4, 7, 9, and 10 from 3, 5, 6, and 8. Bothof these clades contained head and body lice; therefore, the phylogeny of the COI sequences indicates that head and body lice do not come from reciprocally monophyletic lineages. 665 This is evidence that head and body lice are conspeciÞc, however, we note that the phylogeny of a single gene does not necessarily indicate the true phylogeny. But it is noteworthy that head and body lice share three of 10 haplotypes. These shared haplotypes may be ancestral Pediculus humanus haplotypes that have been retained by bothtypes of lice after divergence, or the shared haplotypes may be evidence of conspeciÞcity. Wepropose that haplotype 2 is anancestral P. humanus haplotype, because it iscommonand widespread. Haplotypes 3 and 6 were common in the two provinces in China, Xinjiang Province and Inner Mongolia Province, respectively, but were not found in any other locations, so they are probably not ancestral haplotypes. Therefore, the simplest explanation for the presence of haplotypes 3 and 6 in both head and body lice is that head and body lice are conspeciÞc. We looked for differences between head and body lice by comparing haplotype frequencies. The FST and exact tests showed signiÞcant differences between the haplotype frequencies of head and body lice. However, comparison of lice from different countries explained even more of the variation in haplotype frequencies; this is further evidence for conspeciÞcity. Our results from COI provide evidence that the head and body lice of humans belong to the same species. The COI sequences from the head and body lice that we studied did not come from reciprocally monophyletic lineages. Indeed, the head and body lice shared three of the 10 haplotypes we found, which is evidence for conspeciÞcity. Moreover, analysis of haplotype frequencies showed that although there were signiÞcant differences between the head and body lice we studied, more of the variation was explained by differences among lice from different countries than by differences between head and body lice. Further analyses of COI should reveal much about transmission and maternal gene ßow among populations of lice on global, local and individual host levels. Acknowledgments We thank the following for their help in collecting and donating the head and body lice used in this study: Wen Chao, Mutsuo Kobayashi, James Opiyo Ochanda, Ricardo Palma, Lajos Rozsa, Renfu Shao, and Richard Speare. References Cited Burgess, I. F. 1995. Human lice and their management. Adv. Parasitol. 36: 271Ð342. Busvine, J. R. 1978. Evidence from double infestations for the speciÞc status of human head and body lice (Anoplura). Syst. Entomol. 3: 1Ð8. Durden, L. A., and G. G. Musser. 1994. The sucking lice (Insecta: Anoplura) of the world: A taxonomic checklist with records of mammalian hosts and geographical distributions. Bull. Am. Mus. Natl. Hist. 218: 1Ð90. Khudobin, V. V. 1995. The adaptive potentials of human head and clothes lice when parasitizing on man. Medskaya. Parazitol. 1: 23Ð25. JOURNAL OF MEDICAL ENTOMOLOGY 666 Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, New York. Schaefer, C. W. 1978. Ecological separation of the human head and body lice (Anoplura: Pediculidae). Trans. R. Soc. Trop. Med. Hyg. 72: 669. Schneider, S., D. Roessli, and L. Excoffier. 2000. Arlequin.A software for population genetics data analysis, version 2.000. Genetics and Biometry Laboratory, University of Geneva, Switzerland. Simon, C., F. Frati, A. Beckenbach, B. Crespi, H. Liu, and P. Flook. 1994. Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compila- Vol. 39, no. 4 tion of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87: 651Ð701. Swofford, D. L. 1998. PAU, p. 4.0-phylogenetic analysis using parsimony computer program, version 4. Sinauer, Sunderland, MA. Tarasevich, I. V., A. A. Zemskaya, and V. V. Khudobin. 1988. Diagnostics of the species of lice genus Pediculus. Medskaya. Parazitol. 1: 48Ð52. Received for publication 18 December 2000; accepted 2 October 2001.