RESEARCH Open Access

Description of Hymenolepis microstoma(Nottingham strain) a classical tapewormmodel for research in the genomic eraLucas J Cunningham Peter D Olson

Abstract

Background Hymenolepis microstoma (Dujardin 1845) Blanchard 1891 the mouse bile duct tapeworm is arodentbeetle-hosted laboratory model that has been used in research and teaching since its domestication in the1950s Recent characterization of its genome has prompted us to describe the specific strain that underpins thesedata anchoring its identity and bringing the 150+ year-old original description up-to-date

Results Morphometric and ultrastructural analyses were carried out on laboratory-reared specimens of thelsquoNottinghamrsquo strain of Hymenolepis microstoma used for genome characterization A contemporary description ofthe species is provided including detailed illustration of adult anatomy and elucidation of its taxonomy and thehistory of the specific laboratory isolate

Conclusions Our work acts to anchor the specific strain from which the H microstoma genome has beencharacterized and provides an anatomical reference for researchers needing to employ a model tapeworm systemthat enables easy access to all stages of the life cycle We review its classification life history and development andbriefly discuss the genome and other model systems being employed at the beginning of a genomic era incestodology

BackgroundSpecies of Hymenolepis Weinland 1858 (Platyhel-minthes Cestoda Cyclophyllidea) have been used astapeworm models in research and teaching since the1950s when they were first domesticated in the labora-tory of Clark P Read [1] Adult parasites of rodentswith beetle intermediate hosts they benefit from easyculture in vivo using natural hosts that are themselvesmodel organisms (eg Mus musculus L Tribolium con-fusum Jacquelin du Val) Research on Hymenolepis andespecially H diminuta (Rudolphi 1819) H nana (vonSiebold 1852) and H microstoma is underpinned by anextensive literature that includes much of our classicalknowledge of tapeworm biology [eg [2]] A recentlyinitiated effort sponsored by The Wellcome Trust San-ger Institute to characterize the genome and adultand larval transcriptomes of H microstoma httpwwwsangeracuksequencingHymenolepismicrostoma has

brought this classical model into the genomic eragreatly advancing its utility for researchers interested inemploying a practical tapeworm system that allowsaccess to all life cycle stages In light of this develop-ment and the fact that laboratory isolates can vary infeatures of their biology [3] it is desirable to have adescription of the exact strain on which the genome isbased and to thus anchor the data to a well-definedentityHymenolepis microstoma was first described from the

bile ducts of mice in 1845 by Dujardin [4] who placed itin the genus Taenia L 1758 which housed all tape-worms known at that time In 1891 Blanchard [5] trans-ferred the species to the genus Hymenolepis andprovided an expanded description of the speciesAlthough Bear and Tenora [6] suggested synonymybetween H microstoma and H straminea (Goeze 1782)species status of H microstoma historically has beenwidely accepted and molecular data have shown bothspecies to represent independent albeit closely relatedlineages [78] In contrast the genus Hymenolepis has

Correspondence POlsonnhmacukDepartment of Zoology The Natural History Museum Cromwell RoadLondon SW7 5BD UK

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

copy 2010 Cunningham and Olson licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution andreproduction in any medium provided the original work is properly cited

itself been overhauled on several occasions and its mem-bership and internal structure remain controversial Forexample whereas Hughes [910] accepted the genericassignment H microstoma by Blanchard Spasskii [11]subdivided the genus and transferred H microstoma tothe genus Rodentolepis Spasskii 1954 which he erectedto house the rodent-hosted species of Hymenolepis witharmed rostella At the same time Spasskii erected thegenus Vampirolepis Spasskii 1954 which Schmidt sub-sequently considered a senior synonym of Rodentolepisthus resulting in the new combination Vampirolepismicrostoma (Dujardin 1854) Schmidt 1986 [12] Thegenus Rodentolepis was retained by Czaplinski andVaucher [13] in the most recent synoptic treatment oftapeworms [14] but this work did not consider specieslevel taxa and therefore did not arbitrate on the genericassignment of H microstoma Thus although Vampirole-pis microstoma [12] represents the most recent formaltaxonomic assignment of the species few investigatorshave adopted this name and most reports refer to it aseither a member of the genus Hymenolepis or with lessfrequency Rodentolepis In our view a natural circum-scription of hymenolepid species will not be attainedwithout the application of molecular data [15]To this end Haukisalmi et al [8] recently used 28S

rDNA to analyze phylogenetic relationships among 32hymenolepidid species from rodents shrews and batsshowing that both Hymenolepis and Rodentolepis repre-sented paraphyletic assemblages Although their workassigned H microstoma to a lsquoRodentolepisrsquo clade the lackof resolution and widespread paraphyly of the taxa in theiranalyses indicate that greater taxonomic representationand more robust data are needed before such nomencla-tural circumscriptions can be made reliably We thereforefollow Blanchard [5] in recognizing the mouse bile ducttapeworm as a member of the genus Hymenolepis employ-ing the most common name in usage whilst appreciatingthat a more comprehensive understanding of hymenlepididinterrelationships is likely to warrant generic reassignmentHere we provide a description of a lsquoNottinghamrsquo strain

of H microstoma based on light and scanning electronmicroscopy of laboratory-reared specimens from thesame culture used to characterize the genome Historyof the isolate dating back to the laboratory of C PRead [1] suggests that it represents a model that hasbeen widely employed and disseminated within the para-sitological community for over 50 years making thegenome data directly relevant to a significant pre-exist-ing literature on its biology

ResultsDescription of Hymenolepis microstoma (Nottingham strain)Hymenolepis microstoma (Dujardin 1845) Blanchard1891

Recorded synonymsTaenia microstoma Dujardin 1845 Cercocystis tenebrio-nis Villot 1882 Cysticercus tenebrionis (Villot 1882)Leuckart 1886 Cysticercus taenia-microstomae Dolly1894 Cysticercoides tenebrionis (Villot 1882) Braun1898 Scolex (= Onchoscolex) decipiens (Diesing 1853)Joyeux and Kobozieff 1928 Rodentolepis microstoma(Dujardin 1845) Spasskii 1954 Vampirolepis micro-stoma (Dujardin 1845) Schmidt 1986Common namemouse bile duct tapewormLaboratory strain designationrsquoNottinghamrsquoLaboratory strain history2005-present The Natural History Museum London(PDO) 1977-2005 University of Nottingham UK (ProfJerzy Behnke) 1964-1977 University of Glasgow UK(Prof Adrian Hopkins) before 1964 Texas Rice Univer-sity USA (Prof Clark P Read)Laboratory hostsflour beetles (Tribolium confusum) and BKW outbredconventional mice (Mus musculus)Voucher specimens20 whole-mounted specimens (BMNH 20101281-20)22 slides of histological sections of adult worms (scolexand neck BMNH 201012821-30 immature strobilaBMNH 201012831-36 mature strobila BMNH201012837-42) and 12 whole and partial specimensprepared for SEM retained by the correspondingauthorNo chromosomes12 diploid all acrocentric [1617]Genome size~140 Mb (haploid)Genome datahttpwwwsangeracukresourcesdownloadshel-minthshymenolepis-microstomahtmlDescription(based on 14-16 day old in vivo laboratory-reared speci-mens 20 whole-mounted 2 sectioned and 12 speci-mens prepared for SEM Figures 1-2 all measurementsare given as length times width in μm except where noted)worms anapolytic weakly craspedote 47 (25-81) cmlong with 659 (291-1087) total segments (Figure 1A)scolex 138 (116-157) times 232 (204-284) with four muscu-lar suckers 102 (79-129) times 96 (76-113) (Figure 1B) Ros-tellum 38 (26-52) times 71 (51-75) with an irregular surfacelacking microtriches (Figures 2A B) armed with 25(22-26) hooks retractable into contractile rostellarpouch 104 (83-139) times 101 (79-140) (Figure 1B) Hookscricetoid a = 139 b = 123 g = 6 grsquo = 44 (Figure 1C)Width at level of neck 175 (94-225) Immature segments62 (38-83) times 404 (437-463) mature segments 117 (70-167) times 729 (360-887) gravid terminal segments 164

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 2 of 9

u

rp

r

C

g

bh

D

A

Et

ov

tt

docvoc sr

va

esv cisvcs

docncvoc

lmcm

F

tt t

ov

sr

esv

isvu

Bα β

γ

γrsquo

ehoc

esebpf

Figure 1 Illustrations of adult Hymenolepis microstoma (Nottingham strain) A Whole worm B Hook showing measurement vectors CEgg D Scolex E Mature proglottide F Cross section of mature proglottide Abbreviations b blade c cirrus cs cirrus sac doc dorsalosmoregulatory canal eb embryophore eh embryonic hooks es eggshell esv external seminal vesicle g guard h handle isv internal seminalvesicle nc nerve cord o ovary oc oncosphere pf polar filaments r rostellum rp rostellar bulb s shell sr seminal receptacle t testis u uterusva vagina voc ventral osmoregulatory canal Scale bars A = 1 mm B = 10 μm C = 50 μm D-F = 100 μm

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 3 of 9

Figure 2 Scanning electron micrographs of adult Hymenolepis microstoma (Nottingham strain) A Scolex and rostellum B Rostellar hooksC Microtriches on the scolex D Internal view of gravid strobila E Seminal receptacle with spermatozoa surrounded by eggs F Three-day oldtransforming oncosphere showing larval hooks (arrows and insets) Scale bars A = 50 μm B = 5 μm C = 2 μm D = 100 μm E-F = 20 μm(insets = 2 μm)

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 4 of 9

(131-262) times 1160 (454-1426) Paired osmoregulatoryvessels and longitudinal nerve cords lateral (Figure 1EF) Entire worm covered by short (~2 um) uniform anddensely packed filiform microtriches [18] (Figure 2C)Male system consisting of three spherical to oval

testes 72 (51-114) times 78 (61-115) arranged one poraland two aporal (occasionally reversed in individual pro-glottides) Vas deferens expands to form an externalseminal vesicle 105 (73-195) times 56 (38-93) (Figure 1E)Cirrus pouch ovoid 58 (45-99) times 153 (97- 302) enclos-ing coiled cirrus and internal seminal vesicle 52 (38-93) times 94 (72-195) Female system consisting of a centrallobed ovary 63 (34-103) times 234 (130-360) partially over-lapping a compact vitellarium 38 (41-94) times 56 (32-68)Seminal receptacle median Vagina 299 (197-431) times 13(9-18) situated ventral to male system (Figure 1F) Com-mon genital pore dextral unilateral near mid-point ofmargin Eggs thin-shelled (Figure 2E) enclosing embryo-phore with 3 polar filaments and oncosphere withembryonic hooks arranged in parallel (Figure 1D)

RemarksHymenolepis species exhibit the well-documentedlsquocrowding effectrsquo in which overall size and egg produc-tion are inversely related to the intensity of infection[19] Consequently size is dependent not only on theage of the worms but on the number of worms presentin the host and cannot be used diagnostically [20]Crowding in H microstoma has been shown to decreaselinear growth egg production and the rate of proglottideformation [21] Moreover we chose to document gravidadult specimens at an age and size most useful forlaboratory manipulations in which larger worms poseunnecessary practical problems (eg assays involvingwhole mount in situ hybridization or in vitro culture)de Rycke [22] showed that H microstoma is in rapidstate of growth starting around 12-14 days post-infec-tion in Mus musculus and whereas our length measure-ments correspond to those reported by de Rycke for therelevant age class (see Table 1) they are obviously lesscomparable to reports based on older specimens suchas those stemming from natural infections

DiscussionLife historyHymenolepis microstoma is most probably cosmopolitanin distribution [20] and is not known from human infec-tions outside of a single report in which mixed infec-tions of H nana and H microstoma were identified infour individuals from a remote region of WesternAustralia [23] Reported natural definitive hosts includea large range of rodent genera that include mice (egApodemus Kaup Dendromus Smith Leggada GrayMastomys Thomas Mus L) gerbils (Meriones Illiger)

and voles (Microtus Schrank) [91224] Infections in ratsis controversial whereas Joyeux and Kobozieff [25]reported successful infection of laboratory rats Dvoraket al [20] found rats to be refractory to H microstomaand Litchford [24] showed that rats became refractorywith age Similarly although infections can be estab-lished in golden hamsters (Mesocricetus Nehring) theyresult in underdeveloped worms and cause severepathology to the host [2024] Dvorak et al [20] demon-strated that mice could not be infected via eggs as isthe case with H nana (ie auto-infection) [26] Howeverin congenitally athymic mice Andreassen et al [27]found that autoinfection was possible showing thatoncospheres penetrated the intestinal tissues and devel-oped into cysticeroids that subsequently excysted anddeveloped normally in the bile duct and duodenum in amanner similar to the direct cycle of H nana Autoin-fection of BALBc mice was also implied by the detec-tion of stage-specific antigens [28]The life history of H microstoma (Figure 3) has been

described in detail previously [202529] and is typical ofother hymenolepid species save its unusual location inthe bile duct of the mammalian host In brief eggs con-taining patent oncospheres are expelled with faeces intothe environment and may be ingested by either theadult or larval stage of an appropriate beetle host (egTribolium confusum T castaneum Tenebrio molitorand Oryzaephilus surinamensis) Oncospheral larvae(~20 μm Figure 1D Figure 2F) are released from theirthin shells (Figure 2E nb appearing as a lsquohymenrsquo vialight microscopy and the eponym of the genus) throughthe action of the host mouthparts and after ingestionuse their three pairs of hooks and proteolytic secretions[30] to enter the haemocoel There they undergo a com-plete metamorphosis reconstituting their bodies intocycsticeroid larvae [31] in approximately seven days thephases of which have been documented by both Voge[32] and Goodchild and Stullken [33] Upon infection ofthe definitive host the combination of pepsin and HClin the stomach act to dissolve the larval membranesand juvenile worms are then activated in the duodenumin response to trypsin and bile salts de Rycke [22]described adult growth and organogenesis in Mus mus-culus (summarized in Table 1) in the first three daysthe juveniles move anteriorly in the upper 20 of thesmall intestine and duodenum before establishing per-manently in the bile duct where they commence strobi-lation Within approximately 14 days terminal segmentsare gravid and most of their strobila extends outside ofthe bile duct and into the duodenum Thus the entirelife cycle from egg to gravid adult can be completed inthe laboratory in only three weeks Although the germi-native (rsquoneckrsquo) region of tapeworms has the potential forlsquoimmortalityrsquo as demonstrated in H diminuta by

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 5 of 9

Read [34] infections of H microstoma in mice persistfor an average of six months whereas those in the inter-mediate host can remain infective for the life of the bee-tle (gt one year)

The Hymenolepis genomeThrough collaboration with The Wellcome Trust SangerInstitute a draft genome of H microstoma derived fromthe cultures described herein is now publically availablehttpwwwsangeracukresourcesdownloadshelminthshymenolepis-microstomahtml The latest assembly(October 2010) includes more than 40times coverage of theestimated 140 Mb haploid genome and is based on dataproduced by a combination of Roche 454 and IlluminaSolexa next-generation sequencing technologies Geneannotation is presently being conducted using a combi-nation of RNA-Seq [35] and automated gene predictiontools revealing intron-exon structures and other aspectsof their genomic organization and additional tools arebeing used to characterize non-coding regions (M Zaro-wiecki and M Berriman pers comm)Hymenolepis microstoma is one of four tapeworm spe-

cies to have complete genomes characterized a referencegenome of Echinococcus multilocularis Leukart 1863 anddraft genome of E granulosus (Batsch 1786) have beenproduced by the Sanger Institute (available from httpwwwsangeracukresourcesdownloadshelminths) incollaboration with Profs Klaus Brehm and CeceliaFernandez respectively and a consortium in Mexico arecurrently working to characterize the genome of Taeniasolium L 1758 [36] These data herald the beginning ofthe genomic era in cestodology and are already accelerat-ing advances in our understanding of tapeworm biologyand infection At present the only published platyhel-minth genome is that of the human blood fluke Schisto-soma mansoni Sambon 1907 [37] However genomedata for Schistosoma Weinland 1858 and EchinococcusRudolphi 1801 as well as the free-living flatworm

Table 1 Growth of Hymenolepis microstoma in Mus musculus (summarized from de Rycke [22])

Days pi Avg length (mm) Development and position in gut

1-2 025-050 no external segmentation or genital anlagen worms localized in the first 10-20 cm of the intestine

3 158 some internal segmentation appearance of genital anlagen worms localized in the first 10 cm of the intestine

4-5 340-385 external segmentation and male amp female genital anlagen discernable worms localized in the bile duct

6 585 testes in few segments

7 915 testes mature

8 1350 early-mature to mature proglottides

9-10 17-2050 all proglottides mature

11 27 disappearance of female glands few pre-oncospheres

12 36 pre-oncospheres no hooks

13 465 semi-gravid proglottides

14 625 near gravid proglottides

15-16 94-129 gravid proglottides

Figure 3 Life cycle of Hymenolepis microstoma Infected adult orlarval beetles (eg Tribolium confusum) are consumed by rodents(eg Mus musculus) releasing the cysticercoids which excyst andlocate in the bile duct before commencing strobilation Gravid adultworms develop in 12-14 days in vivo and release embryonatedeggs in the duodenum that are expelled with the host faecesOncosphereal larvae are released when the eggs are consumed bybeetles allowing them penetrate the gut wall and metamorphoseinto patent cysticercoids in the haemocoel (apx one week)Illustration adapted from Olsen [63]

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 6 of 9

Figure 2 Scanning electron micrographs of adult Hymenolepis microstoma (Nottingham strain) A Scolex and rostellum B Rostellar hooksC Microtriches on the scolex D Internal view of gravid strobila E Seminal receptacle with spermatozoa surrounded by eggs F Three-day oldtransforming oncosphere showing larval hooks (arrows and insets) Scale bars A = 50 μm B = 5 μm C = 2 μm D = 100 μm E-F = 20 μm(insets = 2 μm)

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 4 of 9

(131-262) times 1160 (454-1426) Paired osmoregulatoryvessels and longitudinal nerve cords lateral (Figure 1EF) Entire worm covered by short (~2 um) uniform anddensely packed filiform microtriches [18] (Figure 2C)Male system consisting of three spherical to oval

testes 72 (51-114) times 78 (61-115) arranged one poraland two aporal (occasionally reversed in individual pro-glottides) Vas deferens expands to form an externalseminal vesicle 105 (73-195) times 56 (38-93) (Figure 1E)Cirrus pouch ovoid 58 (45-99) times 153 (97- 302) enclos-ing coiled cirrus and internal seminal vesicle 52 (38-93) times 94 (72-195) Female system consisting of a centrallobed ovary 63 (34-103) times 234 (130-360) partially over-lapping a compact vitellarium 38 (41-94) times 56 (32-68)Seminal receptacle median Vagina 299 (197-431) times 13(9-18) situated ventral to male system (Figure 1F) Com-mon genital pore dextral unilateral near mid-point ofmargin Eggs thin-shelled (Figure 2E) enclosing embryo-phore with 3 polar filaments and oncosphere withembryonic hooks arranged in parallel (Figure 1D)

RemarksHymenolepis species exhibit the well-documentedlsquocrowding effectrsquo in which overall size and egg produc-tion are inversely related to the intensity of infection[19] Consequently size is dependent not only on theage of the worms but on the number of worms presentin the host and cannot be used diagnostically [20]Crowding in H microstoma has been shown to decreaselinear growth egg production and the rate of proglottideformation [21] Moreover we chose to document gravidadult specimens at an age and size most useful forlaboratory manipulations in which larger worms poseunnecessary practical problems (eg assays involvingwhole mount in situ hybridization or in vitro culture)de Rycke [22] showed that H microstoma is in rapidstate of growth starting around 12-14 days post-infec-tion in Mus musculus and whereas our length measure-ments correspond to those reported by de Rycke for therelevant age class (see Table 1) they are obviously lesscomparable to reports based on older specimens suchas those stemming from natural infections

DiscussionLife historyHymenolepis microstoma is most probably cosmopolitanin distribution [20] and is not known from human infec-tions outside of a single report in which mixed infec-tions of H nana and H microstoma were identified infour individuals from a remote region of WesternAustralia [23] Reported natural definitive hosts includea large range of rodent genera that include mice (egApodemus Kaup Dendromus Smith Leggada GrayMastomys Thomas Mus L) gerbils (Meriones Illiger)

and voles (Microtus Schrank) [91224] Infections in ratsis controversial whereas Joyeux and Kobozieff [25]reported successful infection of laboratory rats Dvoraket al [20] found rats to be refractory to H microstomaand Litchford [24] showed that rats became refractorywith age Similarly although infections can be estab-lished in golden hamsters (Mesocricetus Nehring) theyresult in underdeveloped worms and cause severepathology to the host [2024] Dvorak et al [20] demon-strated that mice could not be infected via eggs as isthe case with H nana (ie auto-infection) [26] Howeverin congenitally athymic mice Andreassen et al [27]found that autoinfection was possible showing thatoncospheres penetrated the intestinal tissues and devel-oped into cysticeroids that subsequently excysted anddeveloped normally in the bile duct and duodenum in amanner similar to the direct cycle of H nana Autoin-fection of BALBc mice was also implied by the detec-tion of stage-specific antigens [28]The life history of H microstoma (Figure 3) has been

described in detail previously [202529] and is typical ofother hymenolepid species save its unusual location inthe bile duct of the mammalian host In brief eggs con-taining patent oncospheres are expelled with faeces intothe environment and may be ingested by either theadult or larval stage of an appropriate beetle host (egTribolium confusum T castaneum Tenebrio molitorand Oryzaephilus surinamensis) Oncospheral larvae(~20 μm Figure 1D Figure 2F) are released from theirthin shells (Figure 2E nb appearing as a lsquohymenrsquo vialight microscopy and the eponym of the genus) throughthe action of the host mouthparts and after ingestionuse their three pairs of hooks and proteolytic secretions[30] to enter the haemocoel There they undergo a com-plete metamorphosis reconstituting their bodies intocycsticeroid larvae [31] in approximately seven days thephases of which have been documented by both Voge[32] and Goodchild and Stullken [33] Upon infection ofthe definitive host the combination of pepsin and HClin the stomach act to dissolve the larval membranesand juvenile worms are then activated in the duodenumin response to trypsin and bile salts de Rycke [22]described adult growth and organogenesis in Mus mus-culus (summarized in Table 1) in the first three daysthe juveniles move anteriorly in the upper 20 of thesmall intestine and duodenum before establishing per-manently in the bile duct where they commence strobi-lation Within approximately 14 days terminal segmentsare gravid and most of their strobila extends outside ofthe bile duct and into the duodenum Thus the entirelife cycle from egg to gravid adult can be completed inthe laboratory in only three weeks Although the germi-native (rsquoneckrsquo) region of tapeworms has the potential forlsquoimmortalityrsquo as demonstrated in H diminuta by

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 5 of 9

Read [34] infections of H microstoma in mice persistfor an average of six months whereas those in the inter-mediate host can remain infective for the life of the bee-tle (gt one year)

The Hymenolepis genomeThrough collaboration with The Wellcome Trust SangerInstitute a draft genome of H microstoma derived fromthe cultures described herein is now publically availablehttpwwwsangeracukresourcesdownloadshelminthshymenolepis-microstomahtml The latest assembly(October 2010) includes more than 40times coverage of theestimated 140 Mb haploid genome and is based on dataproduced by a combination of Roche 454 and IlluminaSolexa next-generation sequencing technologies Geneannotation is presently being conducted using a combi-nation of RNA-Seq [35] and automated gene predictiontools revealing intron-exon structures and other aspectsof their genomic organization and additional tools arebeing used to characterize non-coding regions (M Zaro-wiecki and M Berriman pers comm)Hymenolepis microstoma is one of four tapeworm spe-

cies to have complete genomes characterized a referencegenome of Echinococcus multilocularis Leukart 1863 anddraft genome of E granulosus (Batsch 1786) have beenproduced by the Sanger Institute (available from httpwwwsangeracukresourcesdownloadshelminths) incollaboration with Profs Klaus Brehm and CeceliaFernandez respectively and a consortium in Mexico arecurrently working to characterize the genome of Taeniasolium L 1758 [36] These data herald the beginning ofthe genomic era in cestodology and are already accelerat-ing advances in our understanding of tapeworm biologyand infection At present the only published platyhel-minth genome is that of the human blood fluke Schisto-soma mansoni Sambon 1907 [37] However genomedata for Schistosoma Weinland 1858 and EchinococcusRudolphi 1801 as well as the free-living flatworm

Table 1 Growth of Hymenolepis microstoma in Mus musculus (summarized from de Rycke [22])

Days pi Avg length (mm) Development and position in gut

1-2 025-050 no external segmentation or genital anlagen worms localized in the first 10-20 cm of the intestine

3 158 some internal segmentation appearance of genital anlagen worms localized in the first 10 cm of the intestine

4-5 340-385 external segmentation and male amp female genital anlagen discernable worms localized in the bile duct

6 585 testes in few segments

7 915 testes mature

8 1350 early-mature to mature proglottides

9-10 17-2050 all proglottides mature

11 27 disappearance of female glands few pre-oncospheres

12 36 pre-oncospheres no hooks

13 465 semi-gravid proglottides

14 625 near gravid proglottides

15-16 94-129 gravid proglottides

Figure 3 Life cycle of Hymenolepis microstoma Infected adult orlarval beetles (eg Tribolium confusum) are consumed by rodents(eg Mus musculus) releasing the cysticercoids which excyst andlocate in the bile duct before commencing strobilation Gravid adultworms develop in 12-14 days in vivo and release embryonatedeggs in the duodenum that are expelled with the host faecesOncosphereal larvae are released when the eggs are consumed bybeetles allowing them penetrate the gut wall and metamorphoseinto patent cysticercoids in the haemocoel (apx one week)Illustration adapted from Olsen [63]

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 6 of 9

(131-262) times 1160 (454-1426) Paired osmoregulatoryvessels and longitudinal nerve cords lateral (Figure 1EF) Entire worm covered by short (~2 um) uniform anddensely packed filiform microtriches [18] (Figure 2C)Male system consisting of three spherical to oval

testes 72 (51-114) times 78 (61-115) arranged one poraland two aporal (occasionally reversed in individual pro-glottides) Vas deferens expands to form an externalseminal vesicle 105 (73-195) times 56 (38-93) (Figure 1E)Cirrus pouch ovoid 58 (45-99) times 153 (97- 302) enclos-ing coiled cirrus and internal seminal vesicle 52 (38-93) times 94 (72-195) Female system consisting of a centrallobed ovary 63 (34-103) times 234 (130-360) partially over-lapping a compact vitellarium 38 (41-94) times 56 (32-68)Seminal receptacle median Vagina 299 (197-431) times 13(9-18) situated ventral to male system (Figure 1F) Com-mon genital pore dextral unilateral near mid-point ofmargin Eggs thin-shelled (Figure 2E) enclosing embryo-phore with 3 polar filaments and oncosphere withembryonic hooks arranged in parallel (Figure 1D)

RemarksHymenolepis species exhibit the well-documentedlsquocrowding effectrsquo in which overall size and egg produc-tion are inversely related to the intensity of infection[19] Consequently size is dependent not only on theage of the worms but on the number of worms presentin the host and cannot be used diagnostically [20]Crowding in H microstoma has been shown to decreaselinear growth egg production and the rate of proglottideformation [21] Moreover we chose to document gravidadult specimens at an age and size most useful forlaboratory manipulations in which larger worms poseunnecessary practical problems (eg assays involvingwhole mount in situ hybridization or in vitro culture)de Rycke [22] showed that H microstoma is in rapidstate of growth starting around 12-14 days post-infec-tion in Mus musculus and whereas our length measure-ments correspond to those reported by de Rycke for therelevant age class (see Table 1) they are obviously lesscomparable to reports based on older specimens suchas those stemming from natural infections

DiscussionLife historyHymenolepis microstoma is most probably cosmopolitanin distribution [20] and is not known from human infec-tions outside of a single report in which mixed infec-tions of H nana and H microstoma were identified infour individuals from a remote region of WesternAustralia [23] Reported natural definitive hosts includea large range of rodent genera that include mice (egApodemus Kaup Dendromus Smith Leggada GrayMastomys Thomas Mus L) gerbils (Meriones Illiger)

and voles (Microtus Schrank) [91224] Infections in ratsis controversial whereas Joyeux and Kobozieff [25]reported successful infection of laboratory rats Dvoraket al [20] found rats to be refractory to H microstomaand Litchford [24] showed that rats became refractorywith age Similarly although infections can be estab-lished in golden hamsters (Mesocricetus Nehring) theyresult in underdeveloped worms and cause severepathology to the host [2024] Dvorak et al [20] demon-strated that mice could not be infected via eggs as isthe case with H nana (ie auto-infection) [26] Howeverin congenitally athymic mice Andreassen et al [27]found that autoinfection was possible showing thatoncospheres penetrated the intestinal tissues and devel-oped into cysticeroids that subsequently excysted anddeveloped normally in the bile duct and duodenum in amanner similar to the direct cycle of H nana Autoin-fection of BALBc mice was also implied by the detec-tion of stage-specific antigens [28]The life history of H microstoma (Figure 3) has been

described in detail previously [202529] and is typical ofother hymenolepid species save its unusual location inthe bile duct of the mammalian host In brief eggs con-taining patent oncospheres are expelled with faeces intothe environment and may be ingested by either theadult or larval stage of an appropriate beetle host (egTribolium confusum T castaneum Tenebrio molitorand Oryzaephilus surinamensis) Oncospheral larvae(~20 μm Figure 1D Figure 2F) are released from theirthin shells (Figure 2E nb appearing as a lsquohymenrsquo vialight microscopy and the eponym of the genus) throughthe action of the host mouthparts and after ingestionuse their three pairs of hooks and proteolytic secretions[30] to enter the haemocoel There they undergo a com-plete metamorphosis reconstituting their bodies intocycsticeroid larvae [31] in approximately seven days thephases of which have been documented by both Voge[32] and Goodchild and Stullken [33] Upon infection ofthe definitive host the combination of pepsin and HClin the stomach act to dissolve the larval membranesand juvenile worms are then activated in the duodenumin response to trypsin and bile salts de Rycke [22]described adult growth and organogenesis in Mus mus-culus (summarized in Table 1) in the first three daysthe juveniles move anteriorly in the upper 20 of thesmall intestine and duodenum before establishing per-manently in the bile duct where they commence strobi-lation Within approximately 14 days terminal segmentsare gravid and most of their strobila extends outside ofthe bile duct and into the duodenum Thus the entirelife cycle from egg to gravid adult can be completed inthe laboratory in only three weeks Although the germi-native (rsquoneckrsquo) region of tapeworms has the potential forlsquoimmortalityrsquo as demonstrated in H diminuta by

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 5 of 9

Read [34] infections of H microstoma in mice persistfor an average of six months whereas those in the inter-mediate host can remain infective for the life of the bee-tle (gt one year)

The Hymenolepis genomeThrough collaboration with The Wellcome Trust SangerInstitute a draft genome of H microstoma derived fromthe cultures described herein is now publically availablehttpwwwsangeracukresourcesdownloadshelminthshymenolepis-microstomahtml The latest assembly(October 2010) includes more than 40times coverage of theestimated 140 Mb haploid genome and is based on dataproduced by a combination of Roche 454 and IlluminaSolexa next-generation sequencing technologies Geneannotation is presently being conducted using a combi-nation of RNA-Seq [35] and automated gene predictiontools revealing intron-exon structures and other aspectsof their genomic organization and additional tools arebeing used to characterize non-coding regions (M Zaro-wiecki and M Berriman pers comm)Hymenolepis microstoma is one of four tapeworm spe-

cies to have complete genomes characterized a referencegenome of Echinococcus multilocularis Leukart 1863 anddraft genome of E granulosus (Batsch 1786) have beenproduced by the Sanger Institute (available from httpwwwsangeracukresourcesdownloadshelminths) incollaboration with Profs Klaus Brehm and CeceliaFernandez respectively and a consortium in Mexico arecurrently working to characterize the genome of Taeniasolium L 1758 [36] These data herald the beginning ofthe genomic era in cestodology and are already accelerat-ing advances in our understanding of tapeworm biologyand infection At present the only published platyhel-minth genome is that of the human blood fluke Schisto-soma mansoni Sambon 1907 [37] However genomedata for Schistosoma Weinland 1858 and EchinococcusRudolphi 1801 as well as the free-living flatworm

Table 1 Growth of Hymenolepis microstoma in Mus musculus (summarized from de Rycke [22])

Days pi Avg length (mm) Development and position in gut

1-2 025-050 no external segmentation or genital anlagen worms localized in the first 10-20 cm of the intestine

3 158 some internal segmentation appearance of genital anlagen worms localized in the first 10 cm of the intestine

4-5 340-385 external segmentation and male amp female genital anlagen discernable worms localized in the bile duct

6 585 testes in few segments

7 915 testes mature

8 1350 early-mature to mature proglottides

9-10 17-2050 all proglottides mature

11 27 disappearance of female glands few pre-oncospheres

12 36 pre-oncospheres no hooks

13 465 semi-gravid proglottides

14 625 near gravid proglottides

15-16 94-129 gravid proglottides

Figure 3 Life cycle of Hymenolepis microstoma Infected adult orlarval beetles (eg Tribolium confusum) are consumed by rodents(eg Mus musculus) releasing the cysticercoids which excyst andlocate in the bile duct before commencing strobilation Gravid adultworms develop in 12-14 days in vivo and release embryonatedeggs in the duodenum that are expelled with the host faecesOncosphereal larvae are released when the eggs are consumed bybeetles allowing them penetrate the gut wall and metamorphoseinto patent cysticercoids in the haemocoel (apx one week)Illustration adapted from Olsen [63]

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 6 of 9

Read [34] infections of H microstoma in mice persistfor an average of six months whereas those in the inter-mediate host can remain infective for the life of the bee-tle (gt one year)

The Hymenolepis genomeThrough collaboration with The Wellcome Trust SangerInstitute a draft genome of H microstoma derived fromthe cultures described herein is now publically availablehttpwwwsangeracukresourcesdownloadshelminthshymenolepis-microstomahtml The latest assembly(October 2010) includes more than 40times coverage of theestimated 140 Mb haploid genome and is based on dataproduced by a combination of Roche 454 and IlluminaSolexa next-generation sequencing technologies Geneannotation is presently being conducted using a combi-nation of RNA-Seq [35] and automated gene predictiontools revealing intron-exon structures and other aspectsof their genomic organization and additional tools arebeing used to characterize non-coding regions (M Zaro-wiecki and M Berriman pers comm)Hymenolepis microstoma is one of four tapeworm spe-

cies to have complete genomes characterized a referencegenome of Echinococcus multilocularis Leukart 1863 anddraft genome of E granulosus (Batsch 1786) have beenproduced by the Sanger Institute (available from httpwwwsangeracukresourcesdownloadshelminths) incollaboration with Profs Klaus Brehm and CeceliaFernandez respectively and a consortium in Mexico arecurrently working to characterize the genome of Taeniasolium L 1758 [36] These data herald the beginning ofthe genomic era in cestodology and are already accelerat-ing advances in our understanding of tapeworm biologyand infection At present the only published platyhel-minth genome is that of the human blood fluke Schisto-soma mansoni Sambon 1907 [37] However genomedata for Schistosoma Weinland 1858 and EchinococcusRudolphi 1801 as well as the free-living flatworm

Table 1 Growth of Hymenolepis microstoma in Mus musculus (summarized from de Rycke [22])

Days pi Avg length (mm) Development and position in gut

1-2 025-050 no external segmentation or genital anlagen worms localized in the first 10-20 cm of the intestine

3 158 some internal segmentation appearance of genital anlagen worms localized in the first 10 cm of the intestine

4-5 340-385 external segmentation and male amp female genital anlagen discernable worms localized in the bile duct

6 585 testes in few segments

7 915 testes mature

8 1350 early-mature to mature proglottides

9-10 17-2050 all proglottides mature

11 27 disappearance of female glands few pre-oncospheres

12 36 pre-oncospheres no hooks

13 465 semi-gravid proglottides

14 625 near gravid proglottides

15-16 94-129 gravid proglottides

Figure 3 Life cycle of Hymenolepis microstoma Infected adult orlarval beetles (eg Tribolium confusum) are consumed by rodents(eg Mus musculus) releasing the cysticercoids which excyst andlocate in the bile duct before commencing strobilation Gravid adultworms develop in 12-14 days in vivo and release embryonatedeggs in the duodenum that are expelled with the host faecesOncosphereal larvae are released when the eggs are consumed bybeetles allowing them penetrate the gut wall and metamorphoseinto patent cysticercoids in the haemocoel (apx one week)Illustration adapted from Olsen [63]

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 6 of 9

models Schmidtea mediterranea Benazzi Baguna Balles-ter Puccinelli and Del Papa 1975 [38] and Macrostomumlignano Ladurner Scharer Salvenmoser and Rieger 2005(httpwwwmacgenomeorg) have been available forsome time and full reports on the characteristics of all ofthese genomes including that of H microstoma areexpected soon

Model systems in the genomic era of cestodologyOf the three Hymenolepis species that have beenemployed in laboratory research most literature con-cerns the rat tapeworm H diminuta followed by themedically important dwarf tapeworm H nana andfinally by the mouse bile duct tapeworm H microstomaAs a model for research in the genomic age howeverH microstoma has advantages over both of these alter-native systems For example compared to H diminutait is both smaller and mouse-hosted enabling smallerand thus less expensive assay sizes (eg for RNAi) aswell as less expensive animal costs whereas the mouse-hosted H nana is both a human pathogen (albeit con-troversy persists regarding the conspecficity of humanand mouse strains) and capable of infecting otherlaboratory animals through faecal contamination via itsdirect life cycle [26] Moreover whereas H nana sur-vives only weeks in the mouse host [39] H microstomapersist for ~6 months and thus require less frequentpassage Although the smaller size of H nana would bepreferable for assays on balance H microstoma providesthe best practical solution for contemporary researchprogrammes that wish to employ a tapeworm modelproviding easy access to all stages of their life cycle atminimal expense and risk to human and animal healthCompletion of the H microstoma life cycle in vitro

from egg to gravid adult was demonstrated in the 1960sand 70s by De Rycke and Berntzen [40] Evans [4142]and Seidel [4344] but to our knowledge no report ofresearch employing these techniques has been publishedsubsequently Our initial attempts to follow these proto-cols for the cultivation of adult worms resulted in onlylimited growth (3times increase in length) without the onsetsegmentation (unpub data) However as many of thereported media used by previous authors are no longercommercially available more work is needed to developcontemporary protocols for in vitro culture Among themost advanced in vitro systems available for tapewormresearch today has been developed by Brehm and collea-gues for Echinococcus [45-48] the genus on which mostof our understanding of tapeworm molecular biology isbased [49] Development of an axenic culture system ofthe hydatid stage of E multilocularis has allowed themto introduce transgenic and functional genomic techni-ques (eg RNAi) to cestodology and their system is cur-rently being used to pioneer research on stem-cells and

developmental biology in parasitic flatworms [4550]Although not yet supported by genome characterizationanother currently employed in vitro system is that ofMesocestoides Vaillant 1863 [eg [51]] which are readilymaintained in the larval tetrathyridial stage [31] and canincrease their numbers in culture via asexual fission [52]Adult worms have also been grown in vitro and inducedto strobilate through the addition of bile salts [53] How-ever as with species of Echinoccocus and Taenia in vivodevelopment of strobilar stages of Mesocestoides is pro-hibited by the legalities and expense of maintaining largevertebrate hosts in the laboratory Rodent hosted Hyme-nolepis species therefore remain the most convenient sys-tems for research on the biology of adult tapeworms andfor this reason we have been developing H microstomaas a model to study the development and evolution oftapeworm segmentation [54]Although the basic framework of cestode evolution

has been revealed by previous molecular studies [55-58]and the interrelationships of select groups are now wellresolved [59-61] there has yet to be a comprehensivemolecular phylogenetic study of the largest and mostimportant group of tapeworms with regard to humanand animal health the Cyclophyllidea All of the tape-worm species for which genomes have been character-ized thus far belong to this order and thus it isespecially important that we elucidate the relative phylo-genetic positions of the 350+ described genera [14]Such knowledge will provide an evolutionary underpin-ning for comparative genomic studies within the groupand allow us to identify the sister lineages whose gen-omes share the closest evolutionary histories to the spe-cies for which full genome data are now available

MethodsA seed culture of Hymenolepis microstoma infected bee-tles was obtained from Nottingham University in 2005courtesy of Prof Jerzy Behnke and subsequently main-tained in vivo at the Natural History Museum (London)using flour beetles (Tribolium confusum) and BKWoutbred conventional mice (full protocols can found athttpwwwolsonlabcom please contact the correspond-ing author to enquire about seed cultures) Gravid14-16 day old specimens were removed from the bileducts and duodenum of mice and quickly swirled innear-boiling 085 saline for ~4 secs to fully extend theworms prior to fixation in cold 4 paraformaldehydeovernight at -4 C Whole-mounted specimens weredehydrated in a graded ethanol series stained usingGillrsquos haematoxylon or left unstained cleared in beach-wood creosote and mounted in Canada balsam Sectionswere prepared by paraffin embedding using standardhistological techniques and stained with Mayerrsquos Hae-malum [62] Measurements and illustrations were made

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 7 of 9

under differential interference contrast on a LeicaDM5000B compound microscope equipped with a cam-era lucida and digital documentation system Specimensused for SEM were dehydrated as above critically-pointdried sputter-coated with goldpalladium and viewed ona JEOL XL30 scanning electron microscope Internalstructures were imaged by SEM by cutting worms cru-dely using a razor blade

AcknowledgementsWe thank especially Jerzy Behnke for providing a seed culture of Hmicrostoma and for assistance in tracking the history of the laboratoryisolate Special thanks also to Matt Berriman Magdalena Zarowiecki andAlejandro Sanchez-Flores for leading the genome initiative at the WellcomeTrust Sanger Institute Thanks to Jayne King and Natasha Pouchkina-Stantcheva for assistance with maintenance of the model to Dave Cooperfor histological sectioning and to Lauren Howard and Alex Ball forassistance with SEM Thanks also to Rod Bray for commenting on an earlierdraft of the manuscript This work was supported in part by a BBSRC grantto PDO (BBG0038151)This work is dedicated to the memory of Clark P Read father of theHymenolepis model and a scientist who was in his time ldquoParasitologyrsquosambassador to the fields of physiology biochemistry and molecular biologyrdquo[1]

Authorsrsquo contributionsPDO designed the study and drafted the manuscript LC carried outresearch Both authors read and approved the final manuscript

Competing interestsThe authors declare that they have no competing interests

Received 10 November 2010 Accepted 31 December 2010Published 31 December 2010

References1 Stewart GL Lumsden RD Fisher FM The contributions of Clark P Read on

ecology of the vertebrate gut and its parasites Bios 1975 463-212 Arai HP Biology of the tapeworm Hymenolepis diminuta New York

Academic Press 19803 Pappas PW Leiby DA Variation in the sizes of eggs and oncospheres

and the numbers and distributions of testes in the tapewormHymenolepis diminuta J Parasitol 1986 72383-391

4 Dujardin MF Histoire Naturelle des Helminthes ou vers intestinaux 18451-680

5 Blanchard R Histoire Zoologique et Meacutedicale des Teacuteniadeacutes du genreHymenolepis Weinland Paris 1891

6 Bear JG Tenora F Some species of Hymenolepis (Cestoidea) from rodentsand from primates Acta Scientarium Naturalium Academiae ScientariumBohemoslovacaendashBrno 1970 41-32

7 Casanova JC Santalla F Durand P Vaucher C Feliu C Renaud FMorphological and genetic differentiation of Rodentolepis straminea(Goeze 1752) and Rodentolepis microstoma (Dujardin 1845)(Hymenolepididae) Parasitol Res 2001 87439-444

8 Haukisalmi V Hardman LM Foronda P Feliu C Laakkonen J Niemimaa JLehtonen JT Henttonen H Systematic relationships of hymenolepididcestodes of rodents and shrews inferred from squences of 28Sribosomal RNA Zool Scr 2010 39631-641

9 Hughes RC The genus Hymenolepis Weinland 1858 Oklahoma Agriculturaland Mechanical College Agricultural Experiment Station 1940 81-42

10 Hughes RC A key to the species of tapeworms in Hymenolepis Trans AmMicrosc Soc 1941 60378-414

11 Spasskii AA Classification of Hymenolepididae from mammals TrGelrsquomintol Lab 1954 7120-167

12 Schmidt GD Handbook of Tapeworm Identification Boca Raton FloridaCRC Press 1986

13 Czaplinski B Vaucher C Family Hymenolepididae Ariola 1899 In Keys tothe Cestode Parasites of Vertebrates Edited by Khalil LF Jones A Bray RAWallingford UK CAB International 1994595-663

14 Khalil LF Jones A Bray RA eds Keys to the Cestode Parasites ofVertebrates Wallingford CAB International 1994

15 Olson PD Tkach VV Advances and trends in the molecular systematics ofthe parasitic Platyhelminthes Adv Parasitol 2005 60165-243

16 Hossain MM Jones AW The chromosomes of Hymenolepis microstoma(Dujardin 1845) J Parasitol 1963 49305-307

17 Proffitt MR Jones AW Chromosome analysis of Hymenolepis microstomaExp Parasitol 1969 2572-84

18 Chervy L Unified terminology for cestode microtriches a proposal fromthe International Workshops on Cestode Systematics in 2002-2008 FoliaParasitol 2009 56199-230

19 Read CP The ldquoCrowding Effectrdquo in tapeworm infections J Parasitol 195137174-178

20 Dvorak JA Jones AW Kuhlman HH Studies on the biology of Hymenolepismicrostoma (Dujardin 1845) J Parasitol 1961 47833-838

21 Jones AW Tan BD Effect of crowding upon the growth and fecundity inthe mouse bile duct tapeworm Hymenolepis microstoma J Parasitol 19715788-93

22 de Rycke PH Development of the cestode Hymenolepis microstoma inMus musculus Zeitschrift fur Parasitenkunde 1966 27350-354

23 Macnish MG Ryan UM Behnke JM Thompson RCA Detection of therodent tapeworm Hymenolepis (= Rodentolepis) microstoma in humans anew zoonosis Int J Parasitol 2003 331079-1085

24 Litchford RG Observations on Hymenolepis microstoma in threelaboratory hosts Mesocricetus auratus Mus musculus and Rattusnovegicus J Parasitol 1963 49403-410

25 Joyeux C Kobozieff NI Recherches sur lrsquoHymenolepis microstoma(Dujardin 1845) Annales de Parasitologie 1928 659-79

26 Heyneman D Auto-reinfection in white mice resulting from infection byHymenolepis nana J Parasitol 1953 3928

27 Andreassen J Ito A Ito M Nakao M Nakaya K Hymenolepis microstomadirect life cycle in immunodeficient mice J Helminthol 2004 781-5

28 Ito A Itoh M Andreassen J Onitake K Stage-specific antigens ofHymenolepis microstoma recognized in BALBc mice Parasite Immunol1989 11453-462

29 Hickman JL The biology of Hymenolepis microstoma (Dujardin) Pap ProcR Soc Tasman 1964 9873-77

30 Fairweather I Threadgold LT Hymenolepis nana the fine structure of thelsquopenetration glandrsquo and nerve cells within the oncosphere Parasitology1981 82445-458

31 Chervy L The terminology of larval cestodes or metacestodes SystParasitol 2002 521-33

32 Voge M Development of Hymenolepis microstoma (CestodaCyclophyllidea) in the intermediate host Tribolium confusum J Parasitol1964 5077-80

33 Goodchild CG Stullken RE Hymenolepis microstoma cysticercoidmorphologenesis Trans Am Microsc Soc 1970 89224-229

34 Read CP Longevity of the tapeworm Hymenolepis diminuta J Parasitol1967 531055

35 Wang Z Gerstein M Snyder M RNA-Seq a revolutionary tool fortranscriptomics Nat Rev Genet 2009 1057-63

36 Santamariacutea RI Soberoacuten X de la Torre P Valdeacutes V Yaacutenez J The Taeniasolium genome Parasitol Int 2005 55S127-S130

37 Berriman M Haas BJ Loverde PT Wilson RA Dillon GP Cerqueira GCMashiyama ST Al-Lazikani B Andrade LF Ashton PD et al The genome ofthe blood fluke Schistosoma mansoni Nature 2009 460352-358

38 Saacutenchez Alvarado A Newmark PA Robb SMC Juste R The Schmidteamediterranea database as a molecular resource for studyingplatyhelminthes stem cells and regeneration Development 20021295659-5665

39 Ito A Smyth JD Adult cestodes In Immune responses in parasitic infectionsimmunology immunopathology and immunoprophilaxis Volume 2 Editedby Soulsby EJL Boca Raton CRC Press 1987115-163

40 de Rycke PH Berntzen AK Maintenance and growth of Hymenolepismicrostoma (Cestoda Cyclophyllidea) in vitro J Parasitol 1967 53352-354

41 Evans WS The in vitro cultivation of Hymenolepis microstoma fromcysticercoid to egg-producing adult Can J Zool 1970 481135-1137

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 8 of 9

42 Evans WS The cultivation of Hymenolepis in vitro In Biology of thetapeworm Hymenolepis diminuta Edited by Arai HP New York AcademicPress 1980425-448

43 Seidel JS Hemin as a requirement in the development in vitro ofHymenolepis microstoma (Cestoda Cyclophyllidea) J Parasitol 197157566-570

44 Seidel JS The life cycle in vitro of Hymenolepis microstoma (Cestoda) JParasitol 1975 61677-681

45 Brehm K Echinococcus multilocularis as a model in stem cell researchand molecular host-parasite interaction Parasitology 2010 137537-555

46 Brehm K Spiliotis M Recent advances in the in vitro cultivation andgenetic manipulation of Echinococcus multilocularis metacestodes andgerminal cells Exp Parasitol 2008 119506-515

47 Spiliotis M Lechner S Tappe D Scheller C Krohne G Brehm K Transienttransfection of Echinococcus multilocularis primary cells and complete invitro regeneration of metacestode vesicles Int J Parasitol 2008381025-1039

48 Spiliotis M Mizukami C Oku Y Kiss F Brehm K Gottstein B Echinococcusmultilocularis primary cells Improved isolation small-scale cultivationand RNA interference Mol Biochem Parasitol 2010 17483-87

49 Hemphill A Kern P Special issue Experimental studies in EchinococcusExp Parasitol 2008 119437-438

50 Brehm K The role of evolutionary conserved signalling systems inEchinococcus multilocularis development and host-parasite interactionMed Microbiol Immunol 2010 199247-259

51 Koziol U Dominguez MF Marin M Kun A Castillo E Stem cell proliferationduring in vitro development of the model cestode Mesocestoides cortifrom larva to adult worm Front Zool 2010 71-12

52 Sprecht D Voge M Asexual multiplication of Mesocestoides tetrathyrdidiain laboratory animals J Parasitol 1965 51268-272

53 Markoski MM Bizarro CV Farias S Espinoza I Galanti N Zaha A Ferreira HBIn vitro segmentation induction of Mesocestoides corti (Cestoda)tetrathyridia J Parasitol 2003 8927-34

54 Olson PD Hox genes and the parasitic flatworms New opportunitieschallenges and lessons from the free-living Parasitol Int 2008 578-17

55 Olson PD Caira JN Evolution of the major lineages of tapeworms(Platyhelminthes Cestoidea) inferred from 18S ribosomal DNA andelongation factor-1α J Parasitol 1999 851134-1159

56 Olson PD Littlewood DTJ Bray RA Mariaux J Interrelationships andevolution of the tapeworms (Platyhelminthes Cestoda) Mol PhylogenetEvol 2001 19443-467

57 Olson PD Poddubnaya LG Littlewood DTJ Scholz T On the position ofArchigetes and its bearing on the early evolution of the tapeworms JParasitol 2008 94898-904

58 Waeschenbach A Webster BL Bray RA Littlewood DTJ Added resolutionamong ordinal level relationships of tapeworms (PlatyhelminthesCestoda) with complete small and large subunit nuclear ribosomal RNAgenes Mol Phylogenet Evol 2007 45311-325

59 Healy CJ Caira JN Jensen K Webster B Littlewood DTJ Proposal for a newtapeworm order Rhinebothriidea Int J Parasitol 2009 39497-511

60 Kuchta R Scholz T Brabec J Bray RA Suppression of the tapeworm orderPseudophyllidea (Platyhelminthes Eucestoda) and the proposal of twonew orders Bothriocephalidea and Diphyllobothriidea Int J Parasitol2008 3849-55

61 Olson PD Caira JN Jensen K Overstreet RM Palm HW Beveridge IEvolution of the trypanorhynch tapeworms parasite phylogeny supportsindependent lineages of sharks and rays Int J Parasitol 2010 40223-242

62 Cooper D The preparation of serial sections of platyhelminth parasiteswith details of the materials and facilities required Syst Parasitol 198812211-229

63 Olsen OW Animal parasites their biology and life cycles MinneapolisBurgess Publishing Co 1962

doi1011861756-3305-3-123Cite this article as Cunningham and Olson Description of Hymenolepismicrostoma (Nottingham strain) a classical tapeworm model forresearch in the genomic era Parasites amp Vectors 2010 3123

Submit your next manuscript to BioMed Centraland take full advantage of

bull Convenient online submission

bull Thorough peer review

bull No space constraints or color figure charges

bull Immediate publication on acceptance

bull Inclusion in PubMed CAS Scopus and Google Scholar

bull Research which is freely available for redistribution

Submit your manuscript at wwwbiomedcentralcomsubmit

Cunningham and Olson Parasites amp Vectors 2010 3123httpwwwparasitesandvectorscomcontent31123

Page 9 of 9