Introduction

Tunga penetrans is a hematophagous insect parasitizing man and various animals. Pregnant females of T. penetrans penetrate the skin of its host, leading to the clinical picture of tungiasis, whereas males and non-fertilized females live as ectoparasites without penetrating permanently. The flea is of about 1 mm in length (Hopkins and Rothschild 1953) and is not only parasitizing man, but also uses a wide range of domestic animals as reservoirs (Heukelbach et al. 2004). Once penetrated, the flea is looking for nourishment by puncturing the blood vessels. Its tail is oriented towards the surface, keeping contact to the outside with a sore, which provides air for breathing and a passage for both excretion and eggs. The sore is an entry point for pathogenic microorganisms (Feldmeier et al. 2002) and is also associated with the risk of tetanus complications (Greco et al. 2001; Brothers and Heckmann 1980). Usually the flea infests the area of the toes, heels and soles, but can also be found on nearly every part of the body (Heukelbach et al. 2002). Of the ten current known species of the genus Tunga, only two are supposed to be parasites of man (Fioravanti et al. 2003). The host spectrum of the other eight species is supposed to be restricted to a few genera of wild mammals (e.g. rodents), which are an important reservoir for the parasite (Heukelbach et al. 2004). Tungiasis is mainly a problem of populations living in poverty (Heukelbach et al. 2001). In resource-poor communities in the endemic area, severe infestation with more than a 100 parasites occurs, and high morbidity is common (Feldmeier et al. 2003). Until now, ten different species of the genus Tunga have been described, but only two (T. penetrans and T. trimamillata) parasitizing man. The genus and species determination is generally based on a variety of morphological criteria like shape of abdomen, presence or absence of eyes or even size of the flea. In an approach to study the genetic diversity within the genus Tunga, we amplified and sequenced a partial sequence of mitochondrial 16S rDNA of 24 isolates of Tunga. To compare results with another species of Tunga, we also sequenced 16S rDNA from three isolates of the recently described T. trimamillata (Pampiglione et al. 2002). The amplified region of rDNA contains a hypervariable segment of approximately 200 bp, in which sequence comparison is useful (Parker and Kornfield 1996).

Materials and methods

Parasites

All parasite material was collected in poor urban and rural communities in the State Ceará (northeast Brazil), where tungiasis is hyperendemic (Table 1). T. trimamillata specimens from Ecuador were provided by Prof. Dr. Pampiglione, Prof. Dr. Trentini and colleagues (Bologna).

Table 1 List of isolates examined in this study
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Preparation of genomic DNA

Genomic DNA was isolated using the DNeasy Tissue Kit (Qiagen, Hilden, Germany). One flea individual was frozen in liquid nitrogen and pulverized in a steel mortar. The powder was then mixed with lysis buffer containing Proteinase K and incubated for 4 h. Genomic DNA preparation from the resulting lysate was then performed according to the protocol for animal tissue provided by the manufacturer.

Primers

Sen-mt16S TAC ATA ACA CGA GAA GAC C

Rev-mt16S GTG ATT GCG CTG TTA TCC

Primers were used as previously described (Parker and Kornfield 1996).

DNA amplification

Amplification of DNA of a single flea individual was performed in 100-μl reaction tubes in a total volume of 50 μl using the HotStar Master Mix Kit according to the manufacturer’s instructions (Qiagen). Each reaction mixture contained 0.4 mM of each primer, 2.5 U of Taq polymerase, 200 μM of each dNTP and 2 mM MgCl2. Each of the 30 cycles started with denaturation for 45 s at 94°C, followed by primer annealing at 40°C for 45 s and elongation at 72°C for 45 s.

DNA purification and sequencing

DNA fragments were purified from agarose gels using QIAquick Gel Extraction Kit according to the protocol by the manufacturer (Qiagen). DNA was eluted in 30 μl H2O and sequenced by SEQLAB Sequence Laboratories GmbH (Göttingen, Germany).

Phylogenetic analysis

The multiple sequence alignment program Clustal X version 1.8 was used to obtain a nucleotide sequence alignment file (Thompson et al. 1997). The phylogenetic tree was constructed by neighbour joining method using Kimura 2 parameter distances in MEGA 2.1 software (Kumar et al. 2001). Bootstrap analysis was employed to test the reliability of the phylogenetic tree (1,000 replications).

Light microscopy

The specimens originating from humans (see Fig. 3) were photographed by light microscopy prior to the DNA analysis.

Results

With one exception, all Tunga isolates had a hypervariable section of mitochondrial 16S rDNA of approximately 200 bp. Isolate 23 (collected from an afflicted dog) differed in size with approximately 170 bp (Fig. 1, lane 7).

Fig. 1
figure 1

Amplification products of the hypervariable section of mitochondrial 16S-rDNA region of Tunga isolates. Isolates collected from the same host species showed the same size of amplification product, so that only one is shown representative. 1 isolates 1–5 (collected from cats), 2 isolates 6–12 (collected from humans), 3 isolates 13–15 (T. trimamillata collected from pigs), 4 isolates 16–17 (collected from rats), 5 isolates 18–19 (collected from pigs), 6 isolates 20–22 and 24–26 (collected from dogs), 7 isolate 23 (collected from dog). M 100 bp ladder (Peqlab)

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The PCR products were purified from agarose gel and subsequently sequenced. Aligned sequence data is shown in Fig. 2.

Fig. 2
figure 2

Alignment of the partial sequence of the hypervariable section of the 16S rDNA of Tunga isolates. Sequences of Tunga isolates 1–5 (cats), 20–22 (dogs) and 7–9 (humans) were identical, so that only one is shown representative (named Basic). Sequences of isolates 16–17 and 18–19 were identical in sequence, so that only one is shown representative (named rats and pigs, respectively). Isolates 13–15 were identical in sequence, so that only one is shown representative (named Trima). Congruence in sequence for all isolates is marked grey, a dash indicates a deletion

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Data from Fig. 2 reveals, that the sequence is congruent for all isolates in a smaller region of 6 bp from nucleotide 154–159, and in a larger region of 12 bp from nucleotide 163–174. All isolates show further congruence in seven single positions at nucleotides 116, 122, 129, 134, 137, 150 and 177. In the first 115 bp, not even one position is identical for all isolates.

Sequences from Fig. 1 were used to generate a phylogenetic tree, based on neighbour joining method shown in Fig. 3.

Fig. 3
figure 3

Phylogenetic tree of 27 Tunga isolates based on DNA sequence of hypervariable section of mitochondrial 16S subunit. The tree was generated using Clustal X program for sequence alignment and by neighbour joining method using Kimura’s two-parameter distance (Kimura 1980) in MEGA 2.1 software. Numbers at nodes indicate percent bootstrap values of 1,000 replicates. Numbers in brackets indicate isolate number according to Table 1

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When studying the light micrographs of the fleas obtained from humans, it was seen that three specimens differed from the others. In three specimens (No. 10, 11 and 12) the dorsal side of the head was more rounded (Fig. 4), while in the others the front of the head was somewhat depressed (Fig. 5) giving rise to a curved aspect. However, further characteristics have to be checked in specimens treated with a KOH-solution in order to get further insights.

Fig. 4
figure 4

Flea taken from human skin showing a rounded back side of the head

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Fig. 5
figure 5

Flea originating from human skin showing a curved dorsal side of the head

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Discussion

We investigated genetic diversity of Tunga sp. isolates—the correct species names will be given, when morphological data are also available—by sequencing a hypervariable section of 16S rDNA of 24 isolates all captured in the wild in Brazil, as well as of three isolates of the recently described T. trimamillata (Trentini et al. 2001; Pampiglione et al. 2002, 2003). This region was previously described as AT-rich within holometabolous insects (Whitfield and Cameron 1998). Within T. penetrans of humans as well as in other flea species (e.g. Ctenocephalides felis), a 200-bp sized hypervariable segment has already been reported (Vobis et al. 2004). Both facts were also confirmed for all isolates tested in our study. However, our study revealed a high level of variation in this hypervariable segment within isolates of T. penetrans, that has not even been found between different flea species like C. felis, Ctenocephalides canis, Archeopsylla erinacei or Pulex irritans (Vobis et al. 2004).

The phylogenetic tree that we present here is the first constructed on 16S rDNA sequences for the genus Tunga. Regardless of the hosts where the isolates were collected from, all showed two regions of major congruence, the smaller one of 6-bp size from nucleotide 154–159, and the larger one of 12-bp size from nucleotide 163–174. Apart from these regions, sequence differences within Tunga populations were very high, e.g. 49% between isolates collected from cats or humans compared to isolates collected from rats or pigs. Also isolates collected from dogs only showed significant differences in sequence. These values are even more remarkable, as such differences were not reached in a recent study even between different flea species, e.g. C. felis and A. erinacei (Vobis et al. 2004). It is also noteworthy, that 13 isolates collected from cats, dogs and humans in Brazil seem to be closer related to T. trimamillata from Ecuador, than to other isolates from Tunga collected also from dogs or humans in the same region of Brazil.

As the phylogenetic tree in Fig. 3 shows, the studied isolates can roughly be divided in four clades: one ranging from human isolate 6 to dog isolate 22, two others containing the remaining dogs (isolate 24–27) and human isolates (isolate 10–12), respectively, and a last one containing isolates from pigs and rats. The significant differences in sequences among the isolates were not expected in this dimension, especially in consideration of the small geographical area, where the isolates were collected. Because 13 isolates and four pairs of isolates showed 100% congruence in sequence to each other, these differences cannot been explained by assuming, that individuals of a population are differentiated with the used primer pair Sen-mt16S and Rev-mt16S.

Until now, no detailed morphological studies have been carried out on all isolates, but at least it is mentionable, that one probable clade (human isolates 10, 11 and 12) shows differences in the shape of the head. That this finding is in addition to the presented phylogenetic tree maybe a hint for a necessary subdivision of the species T. penetrans. Further investigations on morphological, biological and genetic traits are needed to address this question. This should include an examination of a different gene region such as the internal transcribed spacer regions 1 and 2 (ITS1/ITS2), that have been proven useful for analysing genetic relationships among species (Baldwin et al. 1995) or also within species (Essig et al. 1999; Marcilla et al. 2002). The construction of phylogenetic trees for T. penetrans isolates based on ITS1 and ITS2 sequences has already been completed and results will soon be published (Vobis et al. 2005).

It should be acknowledged that the relationships of the Tunga populations presented here are based on a mtDNA gene tree that may not necessarily reflect the true species phylogeny. However, the maternal transmission and haploid nature of the mitochondrial genome increases the likelihood of recovering more recent population level history and is more likely to convey a gene tree that is congruent with the species tree (Moore 1995, 1997). But even if true species phylogeny may not be accurately reflected, the existence of one or more new species of Tunga may be presumed—or the studied specimens belong to one of the already described species such as T. caecata, T. bondari or T. travassosi (Barnes and Radovsky 1969; Lewis 1972).