Variable Microsatellite Loci for Population Genetics Studies of a Sucking (Pedicinus badii) found on Old World Monkeys

Katlyn M. Scholl Florida Museum of Natural History

Abstract:

Sucking lice are permanent, obligate ectoparasites and cannot survive in absence of their host. Pedicinus badii is a type of blood feeding louse that parasitizes an endangered old world , the red colobus monkey (Piliocolobus rufomitratus).

This paper describes 8 microsatellite markers that can be used for population genetic studies of P.badii. These markers were tested on a sample of 73 lice collected from 26 individual red colobus living in Kibale National Park, Uganda. Preliminary analysis of another 16 louse individuals finds evidence for two species of lice found on this population of red colobus.

Introduction:

When most people think of lice, they think of elementary school outbreaks of itchy scalps, but the lice that live on human heads are just one of many species in suborder Anoplura, the classification of sucking lice. Humans act as host to three distinct types of lice: the head louse (Pediculus humanus capitis), the clothing louse

(Pediculus humanus humanus) and the pubic louse ( pubis). These lice have been widely studied because of their importance as a disease vector for relapsing fever and epidemic typhus (Buxton 1939).

Sucking lice are obligate ectoparasites--they cannot survive if removed from their host. They have adapted to spend the entirety of their life cycle on the host. Female lice lay their eggs, called nits, on the host. The eggs are cemented to the host hair shaft with a very strong adhesive substance of unknown origin (Buxton 1939).

When the juvenile lice, called nymphs, emerge from the egg they are able to grip and move along the hair of their host. Their morphology is adapted to life on the host: their claws enable them to hold tightly and travel easily through hair, and their piercing mouthparts enable them to penetrate the skin and feed directly from the blood vessels of their host (Light et al. 2010).

Approximately one fifth of all mammalian species are parasitized by these blood-feeding ectoparasites (Light et al. 2010). Some species of lice are found on more than one type of mammal, while others are so host specific in that they can only survive on one host species (Johnson et al. 2002). Because lice are dependent upon their hosts for survival, over long periods of time the host and parasite change together. The coevolution of lice and their hosts means that relationships between different species of lice can be used to understand the relationships between their hosts. It has long been recognized that more closely related species of mammals tend to host more closely related lice (Buxton 1939). Lice can only move between hosts through direct host-to-host contact. Because of this, in humans and other , sucking lice have been examined to understand not only long term evolutionary history, but also to answer questions about behavior of host populations (Reed et al. 2007).

Pedicinus badii is a that parasitizes red colobus monkeys

(Piliocolobus rufomitratus), an lineage that is found along equatorial Africa in rainforest habitats (Struhsaker 1975). P. badii has not been widely studied, and little is known about how often the parasites move between red colobus hosts. Migration is necessary to have gene flow between different populations and prevent inbreeding, but dangerous because the lice can only live on certain species, and cannot survive in absence of a host. This means that if a louse attempts to move to a new host and is unsuccessful, it will likely not survive.

Direct physical contact between host individuals is necessary for lice to move from one monkey to another. It is possible that the lice are transmitted vertically, moving from mother to offspring during nursing. An alternative possibility is that the lice are transferred horizontally, moving between unrelated monkeys who come into contact through social interactions, such as grooming (Clayton & Tompkins

1994).

We have developed microsatellites to use for population genetic studies of P. badii. A microsatellite is a stretch of DNA sequence that is comprised of a short sequence, between one and six base pairs, which is repeated a number of times.

Microsatellites are useful for population genetics because they are highly polymorphic; individuals within a population vary in the number of repeats they carry (Zane et al. 2002). Using these markers we can distinguish between individuals and look for evidence of gene flow between parasite populations.

The lice for this project were collected from two troops of red colobus monkeys that live in Kibale National Park in Uganda. The red colobus monkey is listed as endangered by the International Union for Conservation of Nature (IUCN), and habitat destruction as well as predation by chimpanzees are contributing factors to their continued decline. Kibale National Park is home to thirteen different species of primates, including the largest population of red colobus in the world.

Observational and genetic studies of these monkeys has shown that while they behave as two separate groups, individual monkeys are often moving between the troops and breeding. The red colobus monkeys in Kibale represent a single genetic population (Allen et al. in prep).

While there have been many studies of lice that parasitize humans and the great , those that parasitize old world monkeys have been neglected. Studies of these lice are critical because of their importance as a disease vector, and the potential to learn more about their hosts. Here we present 8 microsatellite loci that are valid for population genetic studies of these lice.

Materials and Methods:

Initially, we used the program MSAT COMMANDER (Faircloth 2008) to search for tetranucleotide microsatellites within the genome of the human (Pediculus humanus humanus) and used PRIMER3 (Rozen and Skaletsky 2000) to design primers for 96 loci. I amplified each locus using in eight individuals, and used gel electrophoresis to scan for variability. Next the PCR products were cloned and sequenced to verify that the primers were in fact amplifying microsatellites, since the primers were created from a genome of a related species and not Pedicinus badii. We found that these primers designed from the pediculus genome could not be used for cross species amplification with Pedicinus.

The next step was to search through the P. badii DNA sequence directly for microsatellites. Sequence data was obtained using 454 sequencing, a type of high- throughput sequencing technology. I searched through the 454 reads for di- and tri-

nucleotide microsatellites, identified 72 loci with good flanking regions for

designing PCR primers. PCR was then used to amplify each locus in eight

individuals; again the products were cloned and sequenced to verify that the

primers were amplifying the intended sequence. We found that the primers

designed from the 454 sequences were amplifying the microsatellites. These 72 loci

were screened for variability in an initial sample of 8 lice; I identified 15 loci that

appeared to be polymorphic. Thus far 8 of these 15 have been successfully amplified

genotyped in the larger sample. The primer sequences, annealing temperatures and

size ranges for these loci are presented in Table 1.

Table 1 Primer pairs that amplify microsatellites in P. badii with annealing temperatures and size Ann. T. Size range Locus Forward primers Reverse primers (°C)

PbTri1 TGTCAATGGTAATAAAACAGAATCAA CAGGCTCAAGTGCGGAATAC 52 158-170 PbTri2 GCTTTCTTTTTCAAAGCAATTT CGTACTCGACCCGGAGTAAA 55 214-223 PbTri3 AATCGAAGCCGTTAATTTGC TTGGATAAGGTCAATTGATTCG 52 143-170 PbTri4 AGCGTGAAGCCATACAACTTT TGCAATTAATTGATCGATAGGTTG 52 155-167 PbTri5 GGGTGAGAGAGAAAGAAAATGC TCAAGCATAACACCATCACTAACA 50 177-192 PbDi1 AATTCACACTACGGCTCACG TTGGTTTTCCACCTTTCACC 55 347-375 PbDi2 AAATGAAAAACTTTGCCAGGAA GTAACTGGGAAAACGCAAGG 55 ~180 PbDi3 TTTGTGCCACATTGAAAATGA TGCACAATAGTGGTGGGAGATGCACAATAGTGGTGGGAGA 54 ~220

The total sample is comprised of 89 lice collected from 26 individual

monkeys across two monkey troops in Kibale. I also included 3 lice collected from a

different species of Red Colobus (Procolobus badius) and 1 louse collected from a

Black-and-White Colobus (Colobus guereza), all from the Ivory Coast to serve as an

outgroup. DNA was extracted from the lice using a QiAmp DNA micro kit (Cat. No 56304). I used two methods to extract DNA from the lice: some of the lice were crushed, while some were cut in half. Following extraction, the two halves of the cut lice remain intact and can be mounted on a microscope slide for future morphological studies.

For each louse extract, PCR was used to amplify the mitochondrial gene CO1.

The final volume for this reaction was 25 ul, containing 11 ul water, 10 ul

HotMasterMix (5PRIME), 1 ul each of forward and reverse primer, and 2 ul of DNA.

The steps for the PCR were: denaturation at 94°C for 10 min, followed by 4 cycles of

94°C for 1 min, 48°C for 1 min, 65°C for 2 min, and then 29 cycles of 94°C for 1 min,

52°C for 1 min, 65°C for 2 min, followed by 65°C for 10 min.

COI sequences for all of the Kibale and Ivory Coast lice were aligned in the program Seaview, and PAUP* (Version 4.0) was used to create a neighbor-joining gene tree using the default parameters (Fig. 1).

The microsatellite loci were amplified by PCR, in a reaction with a total volume of 15 ul containing 7.5 ul of Type-it microsatellite PCR mix (Quiagen), 5.38 ul of water, 0.06 ul of forward primer (5uM), .06 ul of reverse primer (5uM), 1 ul of dye (10uM) and 1 ul of DNA (about 10 uM). The 5’ end of each forward primer was modified to include a M-13 or CAG tag, so that a florescent dye label could be incorporated into the product.

The steps for the microsatellite PCR amplification were: initial denaturation at 95°C for 5 min, followed by 10 cycles of 94° for 30 s, a locus specific annealing temp (listed above in table 1) for 45 s and 72° for 45 s, followed by 25 cycles of 94° for 30 s, 55° for 45 s and 72° for 45 s, followed by 72°for 10 min. I used the program Genemarker (Version 1.6) to score the alleles for the microsatellite loci. The program STRUCTURE (Pritchard et al. 2000) was used to determine the number of populations of lice present in the entire sample based on the microsatellite data (Fig. 2). For the larger of the two louse populations from

Kibale, which is comprised of 73 lice, I tested for Hardy-Weinberg equilibrium using the software CERVUS (Kalinowski et al. 2007) and assessed the possibility of scoring error due using Microchecker (van Oosterhout et al. 2004; Table 2).

Results and Discussion:

Table 2 Analysis alleles for 8 loci describing number of lice (N), number of alleles, observed and expected heterozygosity (Ho and He respectively), Hardy- Weinberg Equilibrium, and potential for null alleles and scoring error. Null Alleles Scoring Locus N # Alleles Ho He HWE Error

PbTri1 69 2 0.217 0.218 NS N N

PbTri2 73 4 0.329 0.407 NS N N

PbTri3 65 3 0.000 0.090 ND Y Y

PbTri4 69 2 0.000 0.029 ND Y N

PbTri5 72 2 0.431 0.461 NS N N

PbDi1 54 5 0.593 0.613 NS N N

PbDi2 73 5 0.096 0.144 ND Y N

PbDi3 66 4 0.030 0.060 ND Y N

The 8 microsatellites that have been analyzed thus far are polymorphic, with number of alleles ranging from 2 to 5, and length of the fragments ranging from 143 to 361 base pairs. The mean expected heterozygosity (He) was 0.253 and the mean observed heterozygosity (Ho) was 0.212. Two of the loci, PbTri3 and PbTri4, were exclusively homozygous in the individuals tested. Cervus could not test HWE for these loci, and for PbDi2 and PbDi3 due to an excess of homozygotes. Genechecker also detected possible existence of null alleles, alleles that fail to amplify, for these 4 loci. At one locus, PbTri3, Genechecker found evidence for error in allele scoring due to stuttering, or peaks in the data one or two base pairs off from the true allele that are scored as alleles.

The genetic data suggests that there are two distinct populations of lice on the Red Colobus monkeys in Kibale. Interestingly, the two populations do not correspond with the two troops of monkeys. Lice from both populations are found in both troops. In some cases, lice from both populations were collected from the same individual monkey.

A neighbor-joining tree created from the mitochondrial gene sequences (Fig.

1) shows that the Kibale lice are split into two distinct groups (red and blue) with the Ivory Coast sample (green) falling between them. Analysis of the microsatellites produces similar results in STRUCTURE (Fig. 2). The two populations of Kibale lice do not share any haplotypes, and there is no indication of gene flow based on the microsatellite data. These results suggest that the two Kibale populations are likely two distinct species of lice that do not interbreed.

Figure1. A neighbor-joining tree created from the mitochondrial CO1 sequences. The Kibale sample is divided into two distinct groups (red and blue) with no shared haplotypes. The Ivory coast sample (green ) is also a separate group.

Figure 2 The results from structure output K=3 show there are two distinct populations within the Kibale sample (red and blue) while the Ivory Coast sample is a third population (green).

Conclusions:

Returning to the original question of whether lice are moving between monkeys, within the larger population of Kibale lice (N=73), the same alleles are found in lice collected from both troops of monkeys. This indicates that the lice are able to move between individual monkeys and between troops. This is an indication that the monkeys are moving often and coming into close contact with monkeys from other troops. Both species of lice are found in both troops of monkeys.

It is not uncommon to find more than one species of louse on one host. Often the different species will partition the host, as is the case with the human head, clothing and pubic lice; each is restricted to a different part of the host. The two species of lice from Kibale may live on separate parts of the monkeys, and the protocol used to collect the lice could have covered an area of the host where the two louse species are found adjacent to one another. Whether the different species of parasite are restricted to specific parts of the host is not yet clear.

It is possible that one of the two species of lice is able to move onto different host species, while the other is specific to red colobus. Lice of the family Pedicinidae, to which P. badii belongs, can be found on many old world primate lineages. There are 12 other species of primate living within Kibale national park that may come into contact with the red colobus population. The 8 loci presented in this paper work for at least two species of lice, and may not only be useful for studies of P. badii, but also could amplify microsatellites in other Pedicinus. It is interesting that the primers designed from the Pediculus genome did not cross amplify with Pedicinus because primers designed to amplify microsatellites in the red colobus monkey work on humans. The parasites’ sequences are less similar than those of their hosts.

These eight microsatellites prove useful for population studies, but 15-20 polymorphic loci are necessary to obtain reliable results (Lowe et al. 2004). I have primers designed for another 7 microsatellite loci that need to be amplified and genotyped for all individuals. The next step for this project is to increase both the number of lice sampled and the number of loci examined.

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