Tyrannobdella rex N. Gen. N. Sp. and the Evolutionary Origins of Mucosal Leech Infestations

Background Leeches have gained a fearsome reputation by feeding externally on blood, often from human hosts. Orificial hirudiniasis is a condition in which a leech enters a body orifice, most often the nasopharyngeal region, but there are many cases of leeches infesting the eyes, urethra, vagina, or rectum. Several leech species particularly in Africa and Asia are well-known for their propensity to afflict humans. Because there has not previously been any data suggesting a close relationship for such geographically disparate species, this unnerving tendency to be invasive has been regarded only as a loathsome oddity and not a unifying character for a group of related organisms. Principal Findings A new genus and species of leech from Perú was found feeding from the nasopharynx of humans. Unlike any other leech previously described, this new taxon has but a single jaw with very large teeth. Phylogenetic analyses of nuclear and mitochondrial genes using parsimony and Bayesian inference demonstrate that the new species belongs among a larger, global clade of leeches, all of which feed from the mucosal surfaces of mammals. Conclusions This new species, found feeding from the upper respiratory tract of humans in Perú, clarifies an expansion of the family Praobdellidae to include the new species Tyrannobdella rex n. gen. n.sp., along with others in the genera Dinobdella, Myxobdella, Praobdella and Pintobdella. Moreover, the results clarify a single evolutionary origin of a group of leeches that specializes on mucous membranes, thus, posing a distinct threat to human health.


Introduction
Most people realize that they are being parasitized by a leech upon finding the worm attached to their skin. Disturbingly, leeches occasionally enter human orifices-a condition known as mucosal, orifical, vesical, or internal hirudiniasis depending on the localization of the leech (Fig. 1). Whereas most bloodfeeding leeches feed as ecto-parasites for short periods of time, those that feed on mucous membranes have been known to stay in an orifice for days or weeks on end [1,2]. Cases of hirudiniasis are underreported because patients suffering from orificial hirudiniasis may only resort to medical attention if they are personally unsuccessful in extracting the leech [3]. Whereas invasive leeches are usually found in the nasopharyngeal region, there are many cases of leeches infesting various body orifices such as the eyes, urethra, vagina, or rectum [4]. Depending on the exact site of the bite in the nasopharyngeal region, symptoms may include hemorrhaging, hemoptysis, dysphonia, coughing, a tickling sensation, dyspnea or, in extreme cases, severe anemia and death [5,6]. Hemorrhaging from leeches in the urethra, or even in the bladder, also poses a particular problem in that clot formation is inhibited by urine flow [7]. Underlying conditions, such as coagulation disorders or secondary bacterial infections can cause a patient's condition to escalate from relatively minor to lifethreatening very quickly [2,8,7].
Reported cases of human orificial hirudiniasis are most common in rural areas of the Middle East, Africa, and Asia, however cases have been recorded from almost all continents [2]. Domestic and wild mammals in these regions, especially livestock, are at the greatest risk for orificial hirudiniasis in relation to the amount of time such animals spend at leech-inhabited watering holes [1]. Some species are more likely to afflict humans, such as Dinobdella ferox (Blanchard, 1896; literally translated to ''terrible ferocious leech''), or species of Limnatis, Praobdella, and Myxobdella [9].
Leech systematics has been transformed with the addition of comparative sequence data and most of the groups historically recognized by taxonomists have been redefined or eliminated [e.g., 10,11]. That said, Phillips and Siddall expressed reluctance to fully reorganize the systematics of New World hirudinoid leeches under Semiscolescidae in light of the unexpected finding of several interrelated Old World species in that group [10]. Our discovery of a species that is new to science, found feeding from the upper respiratory tract of humans in Perú, leads to a reanalysis of the phylogeny and classification of one clade of hirudinoid leeches, clarifying a single evolutionary origin of a group that specializes on mucous membranes and poses a threat to human health.

Clinical Presentations
In 1997, a previously healthy six-year-old boy was admitted to a health center in Lamas province, department of San Martín, Perú complaining of frontal cephalgia. The patient's history revealed that, prior to admission, he frequently bathed in local lakes and natural streams. The patient reported neither bleeding nor respiratory distress. A 25 mm long leech was removed from the right nostril and preserved in formalin.
Again, in 1997, a 16-month-old boy was admitted to a local heath center in Yochegua province, San Francisco district, department of Ayacucho, Perú also complaining of frontal cephalgia and also without respiratory symptoms. It was ascertained that prior to admission the boy had bathed in small local lakes. A 60 mm leech was removed from the patient's nasal cavity, washed with saline solution and preserved in 10% formalin. Nasal bleeding continued for two days.
In 2007, a nine-year-old girl was admitted to La Merced hospital in Chanchamayo province, department of Junín, Perú following a two-week history of frontal cephalgia and a ''sliding'' sensation inside her nose. The patient's parents noticed a black worm moving inside her right nostril and sought medical attention. No other respiratory symptoms presented. The patient volunteered that she had been traveling in Satipo province, department of Junín, Perú where she frequently bathed in lakes, rivers and streams. Physical examination was remarkable only for nasal pain with hand pressure and a black mass inside the right nasal cavity. With some effort, a 65-70 mm black leech was removed without significant bleeding from the patient's nasal cavity, and was preserved in ethanol.  One dorsal jaw armed with eight large (up to 130 mm high) teeth forming a single (i.e., monostichodont) row (Fig. 2a,c). Two of eight teeth may be sub-cuticular and observable only with compound microscopy (Fig. 2c). Pharynx muscular and tubular. Crop from IX to XXV, first nine cecal pairs in IX through XIX, post-ceca extend blitaterally to XXV. First and second cecal chambers subdivided into two unequal sub-ceca with the larger being posterior, otherwise one cecal pair per somite. Intestine tubular, acecate.
Male and female reproductive organs extremely micromorphic, same size as or smaller than ventral ganglia (Fig. 3c). Penis sheath U-shaped, with initial posterior disposition and subsequent anterior procurrent portion leading to small epidydimis. Ejaculatory bulbs absent. Glandular prostate absent. Vagina present, Ushaped, no common oviduct and oviducts half the size of vagina. Vaginal cecum absent. Ovaries simple, bulbous.
The ZooBank LSID for the species T. rex is urn:lsid:zoobank. org:act:F8C0E97B-F525-4EB3-B11B-B8CBA1CB8F5F. Etymology: Remarks: No other leech species is known to have but a single armed jaw with such large teeth. The reduced number of teeth, a caudal sucker wider than the posterior of the body, and preference for feeding on mucous membranes of mammals all indicate the placement of this new taxon within the family Praobdellidae among the genera Praobdella, Myxobdella, Dinobdella, Limnatis, and Limnobdella. Pintobdella chiapasensis (Caballero, 1957) similarly has few (six) teeth per jaw, albeit for each of three jaws. Tyrannobdella rex n.sp. unique in possessing only one jaw with eight large teeth (e.g., ,five times the height of those in the genus Limnatis).
Members of the genus Limnobdella have two pairs of equal crop ceca in each gastric somite and an extended female reproductive structure. In comparison, T. rex has one pair of crop ceca per somite except in the first two chambers of the crop, which have two unequal crop ceca per somite. Overall, the relatively simple structure of the reproductive system in T. rex resembles that of Limnobdella species, but with considerable differences in size. Tyrannobdella rex is easily distinguished from members of the genus Limnatis by the possession of smooth jaws without salivary papillae, having a velar mouth without a longitudinal furrow in the upper lip, and by the simple minute reproductive structures. Also, species of Limnatis, like Limnobdella, have two equal pairs of crop ceca per somite, whereas T. rex has a single pair per mid-body somite.
Species of Myxobdella and Praobdella are morphologically similar to T. rex in possessing a velar mouth, a reduced number of teeth, and micromorphic reproductive structures. Unlike Myxobdella and Praobdella, each possessing two rows of teeth (i.e. distichodont) and three jaws, T. rex only possesses a single row (i.e. monostichodont) and one jaw. Myxobdella species are distributed throughout Southeast Asia and Africa, whereas Praobdella species are restricted to Africa. Besides differences in jaw armature, the genus Myxobdella is characterized by imperfect annulation and annulation furrows of unequal depth. In contrast, T. rex demonstrates 15 complete fiveannulate mid-body somites with only the three most posterior somites having partially subdivided annuli. Species in the genus Myxobdella have gonopores separated by five or five and a half annuli, whereas T. rex has gonopores separated by 1/2+4+1/2 annuli. Species in the genus Praobdella lack the velar mouth and have at least seven annuli between gonopores.

Phylogenetic analyses
The combined dataset included a total of 5256 molecular characters (18S rDNA: 2041 characters, 28S rDNA: 2189 characters, 12S mt rDNA: 367 characters, COI: 657 characters). Parsimony recovered a single tree with 2725 steps and the two runs of the Bayesian analysis had a harmonic mean of log likelihood values that averaged to-19830. 23. Phylogenetic analyses recovered identical topologies regardless of method (Fig. 4).

Discussion
Hirudinoid leeches that show a preference for mammalian mucosal surfaces all appear to have descended from a common ancestor millions of years ago. Among these, the new species Tyrannobdella rex is the first from South America and one with a particularly unpleasant habit of infesting humans [12]. Another New World orifice-invading leech known from southern Mexico, P. chiapasensis, and sister taxon to T. rex, has only been found to parasitize the nasal passages of tapirs [13]. Limnobdella species from central and northern Mexico are known to be pests of livestock [14]. The consistency with which pain was reported by its victims may relate to the relatively enormous teeth T. rex has on its jaw.
Most of the documented cases of leech infestation are in tropical regions. Such cases are closely related to unsafe drinking water habits and people swimming in natural sources. It is in these situations that these worms enter the rectum, vagina or upper airway and attach to the mucosa [15]. A recent study revealed that the nose is the most common site of infestation (71%), followed by the hypopharynx (14%) [16]. Less often, leech infestations affect the lower airways causing haemoptysis, haematemesis, severe anaemia, airway obstruction or death [17]. While little is known of the symbiotic fauna for praobdellids, species of Aeromonas are known to inhabit the gastric ceca of various hirudinoid leeches [18,19]. Insofar as praobdellids have been reported to remain attached for prolonged periods [1], there may also be a serious risk of bacterial infection to the extent that prophylactic antibiotic treatments is indicated in all cases of orificial hirudiniasis.
Several species of leech are known to invade human orifices, most notably various Old World species in the genera Myxobdella, Praobdella, and Dinobdella. Until now, the family Praobdellidae (sensu Sawyer, 1986) included only those three genera, two representatives of which were monophyletic in our analyses: Myxobdella annandalei and Dinobdella ferox. We found strong support for monophyly of that pair in a broader clade that also includes species of Limnatis, Limnobdella, T. rex and P. chiapasensis. This clade is defined not only by our molecular evidence, but also by three morphological and behavioral synapomorphies: reduced number of teeth-less than 12 in Myxobdella, Dinobdella, Praobdella, Tyrannobdella, and Pintobdella, and less than 40 in Limnatis and Limnobdella; the caudal sucker is wider than the posterior of the body; and a preference for feeding primarily on mammalian mucous membranes. The enlarged caudal sucker seen throughout this family may well be an adaptation that mediates attachment to moist mucous membranes [2]. Only once has a praobdellid been reported feeding opportunistically on amphibians when mammals were not available [20].
The systematics of the family Praobdellidae (sensu Sawyer, 1986) has been plagued by ill-defined groups and by substandard type specimens being the sole representatives for some species [1,2]. The characteristics of the oral sucker, the color pattern, and the location of the gonopores seem to hold the most phylogenetic information among species, but organizing these species within genera has been confused. The Terrible Ferocious Leech, Dinobdella ferox, for example, was initially described as a species of Whitmania, a genus of non-bloodfeeding leeches more closely related to Hirudo [10]. Several morphological similarities have been noted [21] between Praobdella radiata Moore, 1958 and Myxobdella africana Moore, 1939, while Praobdella guineensis Blanchard, 1896, Praobdella buettneri Blanchard, 1896, and Praobdella maculata (Moore, 1939) have each been considered potential synonyms of D. ferox (Moore, 1958). It has generally been agreed that these taxonomic conundra will only be resolved with the addition of fresh specimens [2,21]. Nonetheless, the monophyly in our phylogenetic analyses of the genera Myxobdella, Dinobdella, Limnatis, Limnobdella, Tyrannobdella, and Pintobdella agree with the morphological and behavioral synapomorphies observed throughout the clade suggesting that the family Praobdellidae should be expanded to include them all. In turn, this settles the problem faced by Phillips and Siddall [10], and allows Semiscolecidae Scriban and Autrum, 1934 to retain its traditional scope comprising non-bloodfeeding South American taxa and allows Macrobdellidae Richardson, 1969 to encompass the bloodfeeding genera Macrobdella, Philobdella and Oxyptychus.
Representatives of the genus Praobdella, preferably the type species P. buettneri, are sorely needed to definitively establish the relationships of members of the family Praobdellidae. Praobdella buettneri has not been collected since its description in 1896 (from Bismarksburg, Togoland, now the Togolese Republic) along with P. guineensis, which shares the same type locality [22]. Only external morphology was mentioned in Blanchard's (1896) description and the type specimens are long-since dried out making it difficult to relate them to newly collected material not found at the type locality. Additional species of this family that warrant scrutiny are M. africana, Myxobdella sinanensis Oka, 1925, Myxobdella weberi (Blanchard, 1897), Myxobdella nepalica Nesemann and Sharma, 2001, P. maculata, and P. radiata. Further collection efforts in Africa and Asia may yet successfully provide the required material, though our standard methods of attracting leeches to our exposed selves may prove awkward given their established propensity for particular anatomical feeding sites.

Methods
Specimens of T. rex were collected from two states of Perú in 1997; one from a health center in Lamas province, department of San Martin, Perú, and one from a local heath center in Yochegua province, San Francisco district. Both of these specimens were preserved in formalin. A third specimen collected from a clinic in La Merced Chanchamayo Junin, Perú in 2007, was preserved in ethanol, and was the specimen chosen both for the holotype and for sequencing in these analyses. Specimens of P. chiapasensis were collected from forest streams leading to the lakes of Montebello, State of Chiapas, Mexico between 6 and 18 July, 2008. One M. annandalei was received in December, 2008 from Dharamsala, India. Tissue samples of D. ferox were collected on 13 April, 2008 in Taiwan. Examination of external and internal morphology was accomplished with a Nikon SMZ-U stereo microscope on whole and dissected specimens. Photographs were taken with a SPOT-RT digital camera. Drawings were made by superposition of vector art over images placed in Adobe IllustratorH 10 and Adobe PhotoshopH 7.

DNA sequencing and alignment
Tissue was collected from the caudal sucker in order to avoid contamination from host DNA in gastric or intestinal regions of the leech. DNeasy Tissue Kit (Qiagen Valencia, CA) was used for tissue lysis and DNA purification. Primers for the PCR amplification of nuclear 18S rDNA and 28S rDNA and mitochondrial cytochrome oxidase I (COI) and 12S rDNA gene fragments were adapted from published protocols [23,24,25,26,27] and are listed in Table 1. Amplification reactions of gene fragments were conducted using either Ready-To-Go PCR Beads

Phylogenetic analyses
A total of 17 species comprising 19 terminals were used in the analyses with Hirudo medicinalis specified as the outgroup (Table 1).
Phylogenetic analyses were conducted using two approaches: Parsimony and Bayesian Inference (BI). Parsimony analyses were conducted in TNT v 1.1 [29] using 10 replicates of random taxon addition, sectorial searching, the Ratchet [30], and tree-bisectionreconnection branch swapping for each gene as well as for the combined dataset (18S, 28S, 12S, COI). Bootstrap values for combined analyses were obtained using 10 heuristic pseudoreplicates and the same analytical settings. Bayesian analyses were conducted in MrBayes v. 3.1.2 [31]. The data were partitioned by gene for 18S, 28S, 12S, and by codon position for COI (three partition; 3p). A GTR+I+C model was applied to each unlinked data partition based on the Akaike Information Criterion [via ModelTest v. 3.7; 32, 33]. For the Metropolis-Coupled Markov Chain Monte Carlo (MCMCMC) analyses, default prior distributions of parameters were used twice with one cold chain and three hot chains for 10 million generations and sampled every 1000 th generation. The BI analyses burned-in before 100,000 generations. Split frequencies of the standard deviation of simultaneous BI analyses were well below 0.01. As such, the burn-in was set to discard the first 100,000 generations, leaving 9,900 trees sampled for estimation of posterior probabilities.

Nomenclatural Acts
The electronic version of this document does not represent a published work according to the International Code of Zoological Nomenclature (ICZN), and hence the nomenclatural acts contained in the electronic version are not available under that Code from the electronic edition. Therefore, a separate edition of this document was produced by a method that assures numerous Table 2. Taxa used for the phylogenetic analyses, collection localities, and GenBank accession numbers. identical and durable copies, and those copies were simultaneously obtainable (from the publication date noted on the first page of this article) for the purpose of providing a public and permanent scientific record, in accordance with Article 8.1 of the Code. The separate print-only edition is available on request from PLoS by sending a request to PLoS ONE, 185 Berry Street, Suite 3100, San Francisco, CA 94107, USA along with a check for $10 (to cover printing and postage) payable to ''Public Library of Science''. In addition, this published work and the nomenclatural acts it contains have been registered in ZooBank, the proposed online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information viewed, through any standard web browser by appending the LSID to the prefix ''http://zoobank.org/''. The ZooBank LSID for this publication is: urn:lsid:zoobank.org:pub:8D431ED1-B837-4781-A591-D3886285283A