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Biodiversity, taxonomy and systematics of new world freshwater leeches (Annelida: Hirudinea) with particular emphasis on glossiphoniid leeches and their bacterial endosymbionts

ProQuest Dissertations and Theses, 2011
Dissertation
Author: Alejandro Francisco Oceguera-Figueroa
Abstract:
The phylum Annelida Lamarck, 1809 includes segmented worms such as leeches, earthworms, lugworms, sandworms and clamworms that inhabit almost all possible environments and places of the world. Leeches (Class Hirudinea) represents only one group of around 680 species out of the approximately 16,500 described species of Annelida. The class Hirudinea has been divided in two groups based on their mouthparts. The order Rhynchobdellida, a paraphyletic assemblage, includes species with a large and eversible proboscis and the order Arhynchobdellida that includes species with a muscular pharynx with or without jaws. Both orders include organisms specialized to feed on vertebrate blood. This study includes the description of eight species of leeches new to science that belong to three families (Glossiphoniidae, Macrobdellidae and Praobdellidae). The phylogenetic relationships of species of three families (Glossiphoniidae, Macrobdellidae and Praobdellidae) and one suborder (Erpobdelliformes) were investigated using molecular and morphological data and a suite of phylogenetic methods (Parsimony, Maximum Likelihood and Bayesian Inference). The description of the new species Tyrannobdella rex (Praobdellidae) and Oxyptychus bora (Macrobdellidae) are discussed in the context of their placement in phylogenies. The phylogenetic study of Erpobdelliformes includes the comparison of alternative classification schemes. Based on the results, the phylogenetic position of the terrestrial and macrophagous Orobdella octonaria (Gastrostomobdellidae) within the Erpobdelliformes is established for the first time. The phylogenetic relationships of the proboscis-bearing species of the genera Haementeria , Helobdella and Placobdella were investigated using a combination of nuclear and mitochondrial markers and Parsimony and Bayesian Inference methods. In addition to the monophyly of Haementeria , Helobdella and Placobdella , the 3 genera formed a monophyletic group notwithstanding their different feeding preferences. The correlation with phylogeny and some morphological traits is shown. These include, eyespot morphology, annulation patterns, shape of the ovisacs, sensory organs on the dorsal surface and presence of bacteriomes. Species of Haementeria and Placobdella have specialized organs called bacteriomes associated with their salivary complex that harbor symbiotic proteobacteria. Using DNA bacterial sequences (16S rRNA), the exclusive association of Haementeria spp. with gammaproteobacteria and Placobdella spp. with alphaproteobacteria is shown. Using pyrosequencing technology, the nucleotide sequences of a DNA sample extracted from the bacteriomes of Placobdella parasitica were analyzed. A total of 1,053,345 DNA fragments were obtained and assembled. Leech and symbiont DNA fragments were separated using Blast tools and 50 bacterial and Helobdella robusta genomes for reference. Finally, the so-called DNA barcoding protocol is discussed and some recommendations were given to increase the information content of the database (Bold system). In addition, DNA barcoding protocol was used to estimate the diversity of species of Helobdella from Mexico.

x T ABLE OF C ONTENTS P AGE ABSTRACT……………………………………………………………………………...iv ACKNOWLEDGMENTS………………………………………………………………..vi LIST OF TABLES…………………………………………………………………….….iv LIST OF FIGURES………………………………………………………………………xi CHAPTER 1: GENERAL CHARACTERISTICS OF THE GROUP.……………………1 CHAPTER 2: BARCODING……………………………………………………………..7 CHAPTER 3: BIODIVERSITY AND SPECIES DESCRIPTION……………………...61 CHAPTER 4: LEECH PHYLOGENY,………………….……………………………..128 CHAPTER 5: BACTERIAL ENDOSYMBIONTS……………………………………197 GENERAL CONCLUSIONS…………………………………………………………..224 DISCLAIMER………………………………………………………………………….227 LITERATURE CITED…………………………………………………………………228 AUTOBIOGRAPHICAL STATEMENT………………………………….…………...257

xi L IST OF T ABLES P AGE 1. List of specimens used in the Hirudo medicinalis case study…………………….…..19 2. Taxa, localities, and GenBank accession numbers for the CO1 sequences/catalog number of leeches of Helobdella spp…………………….……28 3. Taxa used for the phylogenetic analyses of the suborder Erpobdelliformes with collection localities and GenBank accession numbers…….……43 4. Primers used for gene amplification and sequencing for the phylogenetic analyses of Erpobdelliformes. …………………………………...………..46 5. Species and collection locality of leech from Washington state, USA…………..……66 6. Taxa, localities and GenBank accession numbers for the phylogenetic analyses of Haementeria

spp……………………………………………. 132 7. Primers used for gene amplification and sequencing for the phylogenetic analyses of Haementeria

spp. ……………………………………………135 8. Taxa, localities and GenBank accession numbers for the phylogenetic analyses of Macrobdellidae…………………………………………...….153 9. Morphological matrix of taxa used in the phylogenetic analyses of Macrobdellidae………………………………………………………………………157 10. Taxa, localities and GenBank accession numbers for the phylogenetic analyses of Praobdellidae……………………………………………...…180 11. List of the species used for subtractive scaffolding, orthologue recovery and phylogenetic analysis.……………………………………… 202

xii L IST OF F IGURES F IGURE ……………………………………………………………………………...

P AGE 1. Schematics of the process of establishing information content for reference barcodes…………………………………………….13 2. Neighbor-joining tree of the CO1 locus showing three clades representing H. orientalis, H. medicinalis and H. verbana …………….………..22 3. Neighbor-joining tree based on the Kimura two-parameter substitution model of the CO1 locus of representative species of Helobdella …………..32 4. Maximum Parsimony tree of Erpobdelliformes………………………………………51 5. Morphology of selected Erpobdelliformes……………………………………………55 6. Map of Washington showing the 13 collection localities……………………………..65 7. Morphological characteristics of Placobdella kwetlumye, n. sp………………………69 8. Morphological characteristics of Placobdella sophie, n. sp…………………….….…74 9. Glossiphoniids found in Washington state………………………………………..…..79 10. Erpobdellids found in Washington state…………………………………………......84 11, Morphological characteristics of Helobdella virginiae, n. sp………………..……....93 12. Morphological characteristics of Haementeria acuecueyetzin, n. sp…………….…102 13. Morphological characteristics of Placobdella lamothei, n. sp…………………..….112 14. Morphological characteristics of Placobdella ringueleti, n. sp……………….……122 15. Strict consensus tree resulting from parsimony analysis of Haementeria ………....139 16. Bayesian Inference tree resulting from analysis of Haementeria ………………….141 17. External and internal morphology of glossiphoniid leeches…………………….....145 18. Morphological characteristics of Oxyptychus bora, n. sp………………………….161

xiii 19. Map of South America showing the distribution of Oxyptychus spp………………164 20. Phylogenetic relationships of Macrobdellidae……..……………………………….168 21. Mucosally invasive hirudinoid leeches……………………………………….…….176 22. Comparative jaw morphology of hirudinids ……………………………...…….…..185 23. Morphological characteristics of Tyrannobdella rex, n. sp………………………...188 24. Phylogenetic relationships of Praobdellidae……….. ……………………….……..192 25. Main workflow followed in Reichenowia parasitica genome analyses……………206 26. Phylogenetic relationships of Proteobacteria……………………..……………...…210 27. Phylogenetic relationships of leech bacterial symbionts…………………………...221

1 C HAPTER 1 GENERAL CHARACTERISTICS OF THE GROUP

2 G ENERAL CHARACTERISTICS OF THE GROUP The phylum Annelida Lamarck, 1809 (from Latin "anellus" and Greek "eidos" form) includes approximately 16 500 described species, such as leeches, earthworms and tubeworms. Annelids are bilaterial organisms, segmented, schizocelic, protostomes, with complete digestive system, closed circulatory system with pigments such as hemoglobin, clorocruorina and hemerythrin, well-developed nervous system with a dorsal ganglion and longitudinal nerve cords, with metanephridial excretory apparatus, hermaphrodites and several species have trochophore larvae, which is lost secondarily in some groups. Most of the species of the phylum are marine, however some species inhabit terrestrial and freshwater environments. The phylum Annelida includes two classes: Polychaeta and Clitellata, the latter is composed by the subclass Oligochaeta (paraphyletic) containing more than 6 000 species of terrestrial, freshwater and some marine forms, arranged in approximately 25 families and the subclass Hirudinoidea, which includes species with a fixed number of somites annulated superficially, without setae or parapodia or if present, in small number. Species of the group present a clitellum and one or two suckers. The posterior sucker works as an attachment organ and is present in all members of the group. Anterior sucker abscent in one group. Almost all species live in freshwater or marine environments, some are semi-terrestrial, ectoparasites, predators or macrophagous (Brusca and Brusca, 2003). The Subclass Hirudinoidea includes 3 orders: Order Acanthobdellida: Maximum body length 3 cm, freshwater habitat, ectoparasites of fish, body with 30 somites, posterior sucker present, setae restricted to anterior somites, partially reduced coelom, with one species: Acanthobdella pelledina , parasite of salmon and grayling.

3 Order Branchiobdellida: Usually, less than 1 cm long. Ectocommensals or ectoparasites of freshwater crustaceans, the body is composed of 15 somites, with a posterior sucker, without setae, small but spacious coelom. Order Hirudinida: The members of this group are considered "true leeches" (for Sawyer, 1986 this group correspond to the Subclass Euhirudinea). Most of the species are freshwater and marine, some are semi-terrestrial or amphibious, hematophagous ectoparasites or free-living predators or scavengers. Some parasitic forms act as vectors pathogenic protozoa, treamtodes and cestodes. Setae absent, coelom reduced to a complex system of channels (lacuna). Body with 34 somites, of which only 27 are observed externally. The simplest complete somite (midd-body somite) consists of three primary annulli called a1, a2 and a3, the middle ring is the a2, which carries the sensilla or sensory organs and reflects the location of the ganglion. In some species, primary rings are split forming secundary annuli. Most leeches are dorsoventrally flattened (Davies, 1991). Besides gonopores and nefridiopores, leeches may have tubercules, papillae and another sensory organs. The digestive system is complete with mouth and anus at the ends. In members of the families Ozobranchidae, Piscicolidae and Glossiphoniidae, the pharynx is modified to form a muscular and eversible proboscis. Other species have might have a muscular pharynx with or without jaws. Continuing from the pharynx, the crop might be tubular or with 6 to 11 pairs of blind caeca. From there it continues to the intestine, which is a simple tube in Hirudiniforms, but in the species of the family Glossiphoniidae has four pairs of blind caeca. The anus opens dorsally in the somite XXVII. Leeches are hermaphrodites with complex reproductive systems, sexual reproduction and direct development. The male and female gonopores are observable

4 externally on the ventral surfice of somites XI and XII. Male gonopore always anterior. Leeches present ovisacs and testisacs. Testisacs may be spherical or foliaceous. Vas deferens are anteriorly directed and join the epididymis, then the atrial cornua and finally, reaching the common atrium. The female reproductive system consists of a single pair of small and spherical ovisacs confined to somite XII, or might elongated tubes. During egg laying, the clitellum secretes an ootheca that protects embryos. The circulatory system consists of dorsal and ventral vessels interconnected through lateral vassels. Gas exchange occurs through the body wall. The excretory system is metanephridial and consists of a maximum of 17 pairs of metanephridia. B ACKGROUND Human knowledge of leeches dates back to antiquity, evidence of this can be found in Greek and Roman writings. Most modern and accurate records are found between 1500 to 1750 ( Moquin-Tandon, 1846), but nevertheless it is not until the work of Linnaeus (1758) that the study of this group formally started. Linnaeus divided all the animals into six groups, of which only two were invertebrates: Insecta and Vermes. Leeches were included in group of Vermes. In 1809, Lamarck subdivided Vermes into four independent groups: mollusks, echinoderms and polyps and what remained of the group Vermes. Subsequently, Lamarck splited Vermes again and recognized two large groups with completely different internal morphology. On of the groups included relatively simple worms without internal organs, most of them parasites of vertebrates. The other group included annelids , or segmented worms with complex internal morphological traits. Lamarck recognized the affinities between polychaetes and oligochaetes, but

5 surprisingly, leeches remained grouped with trematodes or flipping between the annelids and trematodes until 1851 when Vogt finally established the relationships between leeches and annelids (Gould, 1999, Brusca and Brusca, 2003). During the first decades of the nineteenth century in France, the taxonomic school of comparative morphology of hirudinea developed. This tradition based their observations on the analysis of annulation patterns. This school continued mainly by Whitman, Oka, Harding, Weber, Pinto and Caballero. Later, J. Percy Moore studied anatomy of both internal and external characters and proposed various terms to refer to types or kinds of structures. Particularly important is the detailed study of the reproduct structures, which constitute the basis of his taxonomic practise (Richardson, 1969). The taxonomic arrangement of the groups has been the subject of recent work. Siddall et al.

(2001) concluded that the sister group of the hirudinea are branchiobdellids, and together, they form the sister group of acanthobdellids. With the development of phylogenetic systematics and the implementation molecular biology techniques, a large number of groups and hypotheses of evolutionary relationships have been tested (Apakupakul et al.,

1999; Borda and Siddall, 2003; Borda et al.,

2005; Light and Siddall, 1999; Oceguera-Figueroa et al.,

2005; Phillips and Siddall, 2005; Siddall, 2002; Siddall and Burreson, 1995, 1998; Trontelj and Sket, 2000) suggesting that some groups recognized by taxonomists are artificial. It is clear that a lot of aspects on the taxonomy and phylogenetic analyses of leeches have been investigated, however several groups remain understudied and the phylogenetic position of several groups remains unresolved.

6 G ENERAL O BJECTIVE The main objective of this work is the study of the biodiversity of freshwater leeches from the New World with emphasis on the members of the family Glossiphoniidae. The first approach to the study of biodiversity included the morphological characterization and taxonomic description of new species. Secondarily, the phylogenetic relationships of several groups were analyzed using a suite of methods, including distance methods and character based methods such as Maximum Parsimony, Maximum Likelihood and Bayesian Inference. Finally, the last approach to understand biodiversity of leeches was the understanding of their symbiotic relationships with bacteria using two approaches: phylogenetics and genomics.

7 CHAPTER 2 Barcoding 2.1 Barcoding, types and the Hirudo files: Using information content to critically evaluate the identity of DNA barcodes (Adapted from: Kvist, S., Oceguera-Figueroa A., Siddall, M. E. and Erséus, C. 2010. Barcoding, types and the Hirudo files: Using information content to critically evaluate the identity of DNA barcodes. Mitochondrial DNA, 21: 198–205.) Text extracts, figures and tables, reproduced by permission from the publishers.

8 I NTRODUCTION The growth and increased frequency of DNA barcoding campaigns emphasizes the importance of validating the databases upon which species identifications are based. In terms of actual practice, relatively little attention is given by submitters and users of barcodes to the layers of validation that would ensure proper identifications. One conspicuous aspect of this shortcoming is that the two main repositories of barcodes [GenBank/NCBI and the Barcode of Life Data System (BOLD)] do not fully recognize the distinction between the reference database entries and subsequently generated barcodes. The former should be predicated on authoritative entries providing a baseline for association with the latter. In other words, the taxonomic identities of reference barcodes need to be disambiguated through information content and critical evaluation prior to any sensible identity inference of query barcodes. This scenario is often overlooked in the growing barcode databases (although somewhat alleviated in BOLD; see below) such that all barcodes are collectively lumped into a single bin upon which subsequent identifications are based. In addition, there is a lack of information annotated in the barcode submissions that, if present, would allow for discrimination of the validity of the taxonomic labels of the barcodes in the bin. This has led to uncritical majority rules in actual barcoding practice; the determination of a result from barcoding is based on the number of high scoring hits that are encountered in the database. This is even more disconcerting given that taxonomic labels from consistently wrongly identified specimens are free to spread throughout the public sequence repositories (Nilsson et al.,

2006; Ross and Murugan, 2006). Had holotypes for all species ever described been sequenced for the barcode region, or at least been provided with a DNA voucher, subsequent barcoding

9 would be greatly improved. Yet, several issues such as multiple copies of CO1 in the mitochondrion, nuclear pseudogenes, or the fact that different species have almost identical CO1 sequences (Williams and Knowlton, 2001; Wiemers and Fiedler, 2007) would still be problematic. A sensible approach to ameliorating the problem of non- sequenceable type specimens would be major barcode repositories retaining and providing information about barcoded non-types regarding georeferenced locality data, collector, identifier of record, dates of collection and identification, as well as morphological and genetic voucher catalogue numbers. Insofar as CO1 sequences (i.e. barcodes) are available for rather few type specimens, considerable care must be taken to provide maximal information content relating to specimens actually being used to generate the reference barcode database. The barcoding process is intended to more rapidly allow specimen identification through comparisons of short DNA sequences without the laborious process of morphological identifications (Hebert and Gregory, 2005). To the extent that reference barcodes are useful for such a rapid procedure, they must actually be predicated on just such a laborious process. It has been rightly argued that DNA barcoding itself is a poor tool for species discovery and delimitation (Will and Rubinoff, 2004; DeSalle et al.,

2005; Ebach and Holdredge, 2005; Will et al.,

2005). Standard delimitation of species requires rigorous morphological analyses and, in some cases, genetic investigations. Typically, this is achieved by comparisons with type material or voucher specimens as well as specialized literature such as original descriptions, monographs, and/or taxonomic keys. Instead, barcoding should be used as a tool for identifying specimens belonging to a species already represented in the database or as an initial, crude way of “flagging” a potentially new species that needs further

10 investigation (DeSalle, 2006). However, the decreased attention to morphological attributes of specimens from which query barcodes are generated strongly increases the need for taxonomic validity of specimens from which the reference sequences were obtained. Furthermore, it increases the need for a connection between the barcode sequence and morphology or other descriptive characteristics (like geography) that are common in standard taxonomy. We recognize the initial need for momentum to generate as many DNA barcodes for as many species as possible for the idea of barcoding to gain traction. For the approach to mature, however, it is now more critical that authoritative barcode reference sequences be created for each species, with specifications as to which pre-existing barcode(s) have the highest informati on content relative to its connection to type material. Here, we contemplate this issue by discussing the scientific value added by the use of information-rich voucher specimens and the inherent need to create barcode reference databases that allow for critical evaluation of their validity. L OCATION , LOCATION , LOCATION Unlike GenBank, which is not designed to aid barcoding initiatives, BOLD does distinguish between “validated” and “unvalidated” barcodes. This distinction might readily give the user a sense of security that some barcoding-based identification of a query sequence carries the weight of scientific authority if only made in reference to the “validated” subset. Such complacency is misplaced as the criteria for inclusion in BOLD’s “validated” barcode database are only that three or more minimum length representatives with the same taxonomic label are included in a cluster and are collectively less than 2% different (Ratnasingham and Hebert, 2007). Given these criteria

11 for validity, the taxonomic label of any consistently misidentified species will remain faulty (e.g. Siddall et al ., 2009). Barcode databases are not heavily policed, nor do any submissions necessarily have the benefit of peer review. Such procedures, while they would allow for an approach to validation, might also severely hamper the progress of barcoding initiatives. One possible counterbalance to the presently unknowable taxonomic validity of even the “validated” BOLD sequences is to ameliorate the scarcity of information content regarding geographical location of a specimen’s collection site, its defining morphological characteristics, as well as collection dates, collector, identifier and the like. In particular, too little attention has been paid to the significance of type localities. A schematic layout of the information content establishment for reference barcodes is presented in Figure 1. The best approach to DNA barcoding would be that a single yardstick for each species would be based on the highest possible information content; that is a barcode from the holotype (or, when applicable, the lectotype or neotype) that would forever represent a species as the reference barcode. Any identity of subsequent sequences, on the other hand, would only be determined based on their similarity to that reference barcode. However, much of the type material in museums or other scientific collections is too old, too degenerate, or stored in media not allowing for DNA sequencing. As such, the second highest level of certainty when creating a reference barcode would come from sequencing specimens from the remainder of the type series (e.g. paratypes, and paralectotypes). If these also prove refractor y to sequencing, it is reasonable to suggest that the third highest level of certainty would be brought through an established morphological and geographical relatedness between the specimen from which the reference barcode is to be obtained and the holotype of the

12 FIGURE 1. Schematics of the process of establishing information content for reference barcodes. The largest certainty in the taxonomic identity of a specimen comes from following the left path (using the holotype), the second largest from following the middle path (using remainder of type series), and the third largest from following the right path (using nontype specimens from the type locality with the addition of voucher specimens). *Information carry-over (morphological, geographic and ecological) from the type series to nontype specimens.

13

14 species. As with any taxonomic survey program, this should include information regarding some correspondence to the holotype itself or its description. That is, beyond being morphologically compatible, information content would be increased if there were some geographic correspondence between specimens from which the reference sequence is obtained and the type specimen. Collecting specimens from the type locality (the collection site of the holotype) has two main benefits. To the extent that both biotic and abiotic factors have shaped species distributions, these factors are information embodied by the type locality. A link between species, their geographic distribution, and a reference sequence would greatly improve the argument that the reference barcode obtained from a specimen from the type locality belongs to the same species as the holotype. Furthermore, in light of the many morphologically cryptic species being uncovered (Bickford et al.,

2007), in large part eventuated by barcoding initiatives themselves, geography can assist in determinations. Present DNA databases largely consist of specimens for which identifications have either not been accurately validated in these ways, or for which such critical evaluation is not possible. This is not to ignore the possibility of several morphologically cryptic species co-occurring at a type locality (Hebert et al..

2004; Bely and Weisblat, 2006) although, in such cases, if viable holotype DNA is absent, neither genetics nor morphology can solve the ambiguity. With a more information-rich database, different levels of certainty (or actual validity) would be discernable from the annotations of individual barcodes. Even without the benefit of peer review for multitudinous reference sequences, users would be empowered to evaluate identifications in more rigorous ways. If no morphological information is available for a barcode submission or if it came from other than the type locality, one need not

15 completely disregard a submission’s identification, putative as it may be. However, any vague association between such a putative reference sequence and the type series should be readily indicated in some way. This more careful strategy for establishing a reference barcode library mirrors the best practices of taxonomy, wherein a holotype has precedence over the remainder of the type series and in which neotypes are best designated from the type locality (International Commission on Zoological Nomenclature 1999, Article 75.3.6). This practice is also followed in comprehensive phylogenetic revisions where the authors spend considerable time and resources collecting specimens of type species of type genera from type localities (e.g. Oceguera-Figueroa et al.,

Full document contains 271 pages
Abstract: The phylum Annelida Lamarck, 1809 includes segmented worms such as leeches, earthworms, lugworms, sandworms and clamworms that inhabit almost all possible environments and places of the world. Leeches (Class Hirudinea) represents only one group of around 680 species out of the approximately 16,500 described species of Annelida. The class Hirudinea has been divided in two groups based on their mouthparts. The order Rhynchobdellida, a paraphyletic assemblage, includes species with a large and eversible proboscis and the order Arhynchobdellida that includes species with a muscular pharynx with or without jaws. Both orders include organisms specialized to feed on vertebrate blood. This study includes the description of eight species of leeches new to science that belong to three families (Glossiphoniidae, Macrobdellidae and Praobdellidae). The phylogenetic relationships of species of three families (Glossiphoniidae, Macrobdellidae and Praobdellidae) and one suborder (Erpobdelliformes) were investigated using molecular and morphological data and a suite of phylogenetic methods (Parsimony, Maximum Likelihood and Bayesian Inference). The description of the new species Tyrannobdella rex (Praobdellidae) and Oxyptychus bora (Macrobdellidae) are discussed in the context of their placement in phylogenies. The phylogenetic study of Erpobdelliformes includes the comparison of alternative classification schemes. Based on the results, the phylogenetic position of the terrestrial and macrophagous Orobdella octonaria (Gastrostomobdellidae) within the Erpobdelliformes is established for the first time. The phylogenetic relationships of the proboscis-bearing species of the genera Haementeria , Helobdella and Placobdella were investigated using a combination of nuclear and mitochondrial markers and Parsimony and Bayesian Inference methods. In addition to the monophyly of Haementeria , Helobdella and Placobdella , the 3 genera formed a monophyletic group notwithstanding their different feeding preferences. The correlation with phylogeny and some morphological traits is shown. These include, eyespot morphology, annulation patterns, shape of the ovisacs, sensory organs on the dorsal surface and presence of bacteriomes. Species of Haementeria and Placobdella have specialized organs called bacteriomes associated with their salivary complex that harbor symbiotic proteobacteria. Using DNA bacterial sequences (16S rRNA), the exclusive association of Haementeria spp. with gammaproteobacteria and Placobdella spp. with alphaproteobacteria is shown. Using pyrosequencing technology, the nucleotide sequences of a DNA sample extracted from the bacteriomes of Placobdella parasitica were analyzed. A total of 1,053,345 DNA fragments were obtained and assembled. Leech and symbiont DNA fragments were separated using Blast tools and 50 bacterial and Helobdella robusta genomes for reference. Finally, the so-called DNA barcoding protocol is discussed and some recommendations were given to increase the information content of the database (Bold system). In addition, DNA barcoding protocol was used to estimate the diversity of species of Helobdella from Mexico.