Journal of Parasitology MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF POST-LARVAL PRE-TETRATHYRIDIA OF MESOCESTOIDES SP. (CESTODA: CYCLOPHYLLIDEA) FROM GROUND SKINK, SCINCELLA LATERALIS (SAURIA: SCINCIDAE), FROM SOUTHEASTERN OKLAHOMA, USA --Manuscript Draft-Manuscript Number:
17-178R1
Full Title:
MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF POST-LARVAL PRE-TETRATHYRIDIA OF MESOCESTOIDES SP. (CESTODA: CYCLOPHYLLIDEA) FROM GROUND SKINK, SCINCELLA LATERALIS (SAURIA: SCINCIDAE), FROM SOUTHEASTERN OKLAHOMA, USA
Short Title:
MESOCESTOIDES SP. FROM GROUND SKINK, SCINCELLA LATERALIS
Article Type:
Regular Article
Corresponding Author:
Chris T. McAllister Eastern Oklahoma State College Idabel, OK UNITED STATES
Corresponding Author Secondary Information: Corresponding Author's Institution:
Eastern Oklahoma State College
Corresponding Author's Secondary Institution: First Author:
Chris T. McAllister
First Author Secondary Information: Order of Authors:
Chris T. McAllister Vasyl V Tkach, Ph.D. David Bruce Conn, Ph.D.
Order of Authors Secondary Information: Abstract:
Free pre-tetrathyridia of Mesocestoides sp. are described, for the first time, from samples obtained from the coelomic cavity of a ground skink, Scincella lateralis from Oklahoma, USA. Closer examination of these early stage tapeworms revealed they were transitional metamorphosis stages between a post-hexacanth procercoid form to the full metacestode of Mesocestoides. A series of transitional stages from before the suckers and apical organ are developed, through full development of both structures were found. However, we did not see any fully developed tetrathyridia with classic Mesocestoides morphology but with an atrophied apical sucker. This is the first time that metamorphic pre-tetrathyridial stages of a Mesocestoides sp. have been reported in vivo from a natural infection. These observations corroborate earlier reports of such stages of Mesocestoides vogae developed in vitro, though the previously reported isolate of M. vogae is asexually proliferative and the species from the present study showed no sign of asexual proliferation. The fact that these can occur in a single lizard intermediate host suggests that Mesocestoides spp. might develop through a simple two-host life cycle rather than an obligate three-host cycle that has been speculated to occur by most previous authors. DNA sequence comparisons and phylogenetic analyses based on mitochondrial 12S, cox1 and nad1 genes have demonstrated that our specimens from S. lateralis represent a species clearly distinct from all previously sequenced Mesocestoides, and closely related to two forms from domesric dogs and Channel Island fox in California previously published as Mesocestoides sp. C.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript
Click here to download Manuscript 17-178R1 AP doc 2-518.docx
RH: MCALLISTER ET AL.—PRE-TETRATHYRIDIA OF MESOCESTOIDES SP. MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF POST-LARVAL PRE-TETRATHYRIDIA OF MESOCESTOIDES SP. (CESTODA: CYCLOPHYLLIDEA) FROM GROUND SKINK, SCINCELLA LATERALIS (SAURIA: SCINCIDAE), FROM SOUTHEASTERN OKLAHOMA, USA Chris T. McAllister1, Vasyl V. Tkach2, and David Bruce Conn3 1
Science and Mathematics Division, Eastern Oklahoma State College, Idabel, Oklahoma 74745.
2
Department of Biology, University of North Dakota, Grand Forks, North Dakota 58202.
3
Department of Invertebrate Zoology, Museum of Comparative Zoology, Harvard University,
Cambridge Massachusetts 02138, and Department of Biology and One Health Center, Berry College, Mount Berry, Georgia 30149. Correspondence should be sent to C. T. McAllister at:
[email protected] ABSTRACT: Free pre-tetrathyridia of Mesocestoides sp. are described, for the first time, from samples obtained from the coelomic cavity of a ground skink, Scincella lateralis from Oklahoma. Closer examination of these early-stage tapeworms revealed they were transitional metamorphosis stages between a post-hexacanth procercoid form to the full metacestode of Mesocestoides. A series of transitional stages were found that span the full period of sucker and apical organ development. However, we did not see any fully developed tetrathyridia, i.e., having classic Mesocestoides morphology but with the apical sucker absent following developmental atrophy. This is the first time that metamorphic pre-tetrathyridial stages of a Mesocestoides sp. have been reported in vivo from a natural infection. These observations corroborate earlier reports of such stages of Mesocestoides vogae developed in vitro, though the previously reported isolate of M. vogae is asexually proliferative and the species from the present
1
study showed no sign of asexual proliferation. The fact that these immediately post-hexacanth stages can occur in a single lizard intermediate host may suggest that Mesocestoides spp. might develop through a simple 2-host life cycle rather than an obligate 3-host cycle that has been speculated to occur by most previous authors. DNA sequence comparisons and phylogenetic analyses based on mitochondrial 12S, cox1 and nad1 genes have demonstrated that our specimens from S. lateralis represent a species clearly distinct from all previously sequenced Mesocestoides, and closely related to 2 forms from domestic dogs and Channel Island fox in California previously published as Mesocestoides sp. C. The enigmatic cyclophyllidean cestode genus Mesocestoides Vaillant, 1863 includes parasites of birds and carnivorous mammals, rarely including humans, who obtain infections from eating raw or undercooked meat containing tetrathyridia (Cho et al., 2013). Schmidt (1986) recognized at least 27 species of Mesocestoides but the exact number is far from settled and given their variability, synonymies most likely exist. The life cycle of Mesocestoides spp. has been a continuing enigma; however, Rausch (1994) suggests it requires at least 3 hosts (i.e., a vertebrate definitive host, a supposed arthropod first intermediate host, and a vertebrate second intermediate host). Encapsulated and free tetrathyridia are often found in the body cavity and various organs of amphibians (McAllister and Conn, 1990; McAllister et al., 2014b), reptiles (McAllister et al., 1991) and rodents (Padgett and Boyce, 2004). The supposed role of coprophagic arthropods as first intermediate hosts has been presumed by some authors, including Padgett and Boyce (2005) who detected DNA of Mesocestoides sp. from ants; however, there might be other explanations for that finding, including the possibility that the ants may simply have fed on Mesocestoides proglottids shortly before ants were collected and used for DNA extracts. In addition, there have been experimental trials attempting to infect insects with eggs
2
(Webster, 1949; James, 1968; reviewed by Conn, 1984); however, there is no convincing evidence for this part of the life cycle (Loos-Frank, 1991). Indeed, Mesocestoides remains one of the few genera of common cyclophyllidean tapeworms in which the complete life cycle has not yet been conclusively demonstrated. There are many reports of tetrathyridia of Mesocestoides from amphibians and reptiles around the world (see James and Ulmer, 1967; McAllister and Conn, 1990; McAllister et al., 1991, 1995, 2014b). To our knowledge, nothing has been published on a pre-tetrathyridial form of the parasite from naturally or experimentally infected hosts, though such stages were described in vivo many years ago for the anomalous laboratory strain of Mesocestoides vogae Etges, 1991 (often incorrectly identified as Mesocestoides corti Hoeppli, 1925; see Etges, 1991) (Specht and Voge, 1965; Voge, 1967; Voge and Seidel, 1968). Here, we provide, for the first time, detailed light microscopical and molecular data on the post-larval pre-tetrathyridia metamorphosis forms of Mesocestoides developing in vivo in a naturally infected host. MATERIALS AND METHODS During July 2013, an adult (snout-vent length = 46 mm) ground skink, Scincella lateralis (Say) was collected from the stomach of a recently killed southern black racer, Coluber constrictor priapus (Dunn and Wood) found dead on the road on the campus of Eastern Oklahoma State College, Idabel, McCurtain County, Oklahoma (33°55'14.6352"N, 94°46'37.8228"W). The skink had apparently just been ingested by the snake, was completely intact, and had not yet undergone any noticeable digestion. A midventral incision was made on the skink and the coelomic cavity was examined and it contained many free, 1–2 mm long, premetacestode stages of cestodes. These were fixed in hot water and subsamples were preserved in 10% neutral buffered formalin for morphological study and in 95% DNA grade ethanol.
3
For morphological analysis, numerous formalin-fixed specimens were stained as whole mounts. Specimens were rinsed in 35% ethanol to wash out the formalin, then moved through 50% to 70% ethanol, stained in Ward’s acetocarmine, dehydrated in a graded ethanol series, cleared in methyl salicylate, and mounted on microscope slides using gum Damar as embedding medium. Whole mounts were examined, described, and photographed under brightfield, phasecontrast, and differential interference-contrast (DIC) optics with an Olympus Provis AX-70 compound light microscope (Olympus Corporation of the Americas, Center Valley, Pennsylvania) equipped with planachromatic fluorite objectives. Photographs were taken using Jenoptik ProgRes Gryphax Subra (Jenoptik AG, Jena, Germany) with Jenoptik Gryphax Image Capture Software. Measurements of 18 specimens on 3 slides were made with an ocular micrometer. Voucher specimens of the Mesocestoides described in this study were accessioned into the Harvard University Museum of Comparative Zoology (MCZ), Cambridge, Massachusetts (MCZ Catalog Number 145972). The host voucher specimen was deposited in the Arkansas State University Herpetological Collection (ASUMZ), State University, Arkansas. Genomic DNA for molecular analysis was extracted from 2 Mesocestoides specimens from S. lateralis following the protocol described by Tkach and Pawlowski (1989). DNA was extracted from 2 different stages, namely the form 2 (with evidence of forming scolex) and form 4 (with tetra-acetabulate scolex). Three different gene fragments were amplified and sequenced to enable phylogenetic analysis and future genetic comparisons. Approximately 350-base-pairlong fragments of the mitochondrial ribosomal 12S gene (12S), approximately 425-base-pairlong fragments of the mitochondrial cytochrome oxidase 1 gene (cox1) and approximately 730base-pair-long fragments of the mitochondrial NAD(P)H dehydrogenase 1 gene (nad1) were
4
amplified by PCR on an BioRad T100 thermal cycler using OneTaq Quick-Load Master Mix from New England Biolabs (Ipswich, Massachusetts). Forward primer 60for (5’TTAAGATATATGTGGTACAGGATTAGATACCC-3’) and reverse primer 375rev (5’AACCGAGGGTGACGGGCGGTGTGTACC-3’) published by Nickisch-Rosenegk et al. (1999) were used for 12S amplification; forward primer Jb3 (5’-TTTTTTGGGCATCCTGAGGTTTAT3’) from Bowles et al. (1992) and reverse primer Jb5 (5’ AGCACCTAAACTTAAAACATAATGAAA-3’) from Derycke et al. (2005) were used for cox1 amplification; degenerate forward primer nad1f (5’-GGNTATTSTCARTNTCGTAAGGG-30’) and degenerate reverse primer trnNR (50’-TTCYTGAAGTTAACAGCATCA-3’) were used for nad1 amplification (primers from Littlewood et al., 2008). The thermocycling protocols followed the recommendations of the manufacturer of DNA polymerase. Forty PCR cycles with the same annealing temperature of 45 C was used for all 3 genes with annealing time ranging from 30 to 45 sec depending on the DNA concentration in the individual extracts. PCR products were purified using ExoSap PCR clean-up enzymatic kit from Affymetrix (Santa Clara, California), cycle-sequenced directly using ABI BigDye (Applied Biosystems, Foster City, California) chemistry, ethanol-precipitated, and sequenced directly on an ABI Prism 3100 automated capillary sequencer. DNA products were sequenced using the original PCR primers for all 3 genes. Contiguous sequences were assembled and edited using Sequencher ver. 4 (GeneCodes Corp., Ann Arbor, Michigan) and submitted to GenBank under accession numbers MG323570 – MG323571 (12S), MG323572– MG323573 (cox1), MG323574– MG323575 (nad1). Only 12S sequences were used in phylogenetic analyses because this gene has been previously sequenced from the broadest diversity of North American Mesocestoides as well as
5
samples from other continents. Newly obtained sequences of Mesocestoides in combination with previously published sequences of Mesocestoides from GenBank were used in phylogenetic analyses. Because of the lack of intraspecific variability, only 2 sequences of specimens from S. lateralis were included in each analysis, to indicate a clade. A sequence of Echinococcus granulosus (GenBank AF297617) was used as an outgroup as a phylogenetically close taxon (V. V. Tkach, unpubl. data) and to be compatible with previously published phylogenies. Sequences were aligned using MEGA ver. 7 software (Kumar et al., 2016). Pairwise sequence comparison was done using BioEdit software, version 7.0.1. (Hall, 1999). Phylogenetic analysis was carried out using Bayesian inference (BI) as implemented in the MrBayes program version 3.1 (Ronquist and Huelsenbeck, 2003) and maximum likelihood algorithm as implemented in MEGA 7. The nucleotide substitution model was selected using JModelTest version 0.1.1 (Posada, 2008). The TPM1uf+I+G model was selected for alignments of all 3 mitochondrial genes with the following parameters: lset nst=6, rates = invgamma ngammacat=4, prset revmatpr = fixed, pinvarpr = fixed, shapepr = fixed. Markov chain Monte Carlo (MCMC) chains were run for 3,000,000 generations, log-likelihood scores plotted and only the final 75% of trees were used to produce the consensus trees by setting the “burnin” parameter at 750,000 generations. The resulting phylogenetic trees and posterior probability values were visualized using FigTree version 1.4 software (Rambaut, 2012). ML analysis was run in MEGA 7 with 1,000 bootstrap replicates. RESULTS Morphological analysis The living cestodes recently removed from the host exhibited typical translucent whitish color, with extensive muscular motility of the body (Fig. 1). Later stages of the unfixed unstained
6
cestodes (Fig. 2) exhibited incipient tetrathyridial morphology with a well-formed posterior excretory pore in the solid hindbody, a constricted neck region, but with a distinct apical sucker occupying the rostellar region of the tetra-acetabulate scolex. All specimens contained numerous calcareous corpuscles distributed uniformly throughout the parenchyma of both scolex and hindbody, and were absent only in the sucker and rostellar regions (Fig. 2). Detailed examination of stained whole mounts revealed that all cestodes were post-larval (i.e., post-hexacanth) metamorphic transitional stages. All features of the hexacanth larvae had been lost through development but the fully formed tetrathyridium metacestode morphology had not been attained, as fully developed tetrathyridia lack any apical sucker or other rostellar organ. Thus, no residual oncospheral hooks were present in any specimen and no cercomer typically associated with the early metamorphosis of cestodes was present in any specimen. Specimens encompassed a broad range of direct metamorphosis progression, with apparently no discrete stages. However, we divided the progression for convenience of description into 4 successive transitional morphologies: 1) the smallest forms exhibiting no specific features other than a slightly elongating axis; 2) slightly larger forms (Fig. 3) exhibiting dense areas anteriorly making up the early histogenesis and organogenesis of the scolex, but without an apical sucker; 3) larger forms (Figs. 2, 4) exhibiting further organization of the scolex, with a muscular apical sucker in the larger specimens, and a single excretory bladder with excretory pore posteriorly; and 4) the largest forms (Fig. 5) exhibiting a fully formed tetra-acetabulate scolex with well-differentiated muscular suckers and with the well-formed muscular apical sucker defining the rostellar region still present, and with well-developed excretory bladder and pore posteriorly. Most of our specimens were of this larger more developed form.
7
Viewed under high magnification with DIC optics, all forms, at all morphogenetic points, demonstrated formation of a well-defined tegument, with basal withdrawn cytons, and subdivision of the parenchyma into cortical and medullary regions, consistent with metamorphosis from the hexacanth’s larval features to the somatic features of the metacestode (juvenile) and adult. Most specimens were fully extended, but a few had the anterior (scolex) end invaginated into the hindbody; examination of whole mounts did not allow determination of scolex or apical sucker development for the few invaginated forms. No sign of asexual proliferation or malignant transformation was seen in any specimen. Measurements of the cestodes after fixation and staining are shown in Table I. Molecular phylogenetic analysis Upon trimming to the length of the shortest sequence available in the GenBank, the 12S alignment was 297 nucleotides long with the sequence length varying from 285 nucleotides in Mesocestoides sp. form C from Canis (lupus) familiaris collected in California (see Fig. 6) to 294 nucleotides in Mesocestoides sp. from S. lateralis reported in the present study. Sequences of the specimens from S. lateralis formed a strongly supported (BI posterior probabilities 0.95, ML bootstrap values 99%) clade that clustered together, with strong branch support, with previously published sequences of Mesocestoides sp., form C (see Padgett et al., 2005), from California (Fig. 6). Interestingly, the Mesocestoides sp. C formed 2 very strongly supported subclades, 1 uniting samples from the domestic dog (C. l. familiaris) in California and Oregon and including Mesocestoides from the Channel Island fox (Urocyon littoralis) from San Miguel Island, California. Elsewhere in the phylogenetic tree, all species-level clades were generally characterized by high branch support values (Fig. 6). However, interrelationships between species remained mostly unresolved with exception of the cluster of M. vogae represented by
8
specimens from a variety of definitive and intermediate hosts, and Mesocestoides sp. A published by Padgett et al. (2005). Pairwise sequence comparison has shown that Mesocestoides sp. from S. lateralis differs by 7.5–8.2% of nucleotide positions from Mesocestoides sp. C from dogs and by 8.5–8.9% from Mesocestoides sp. C from Channel Island fox. DISCUSSION In all cestodes, embryonic development culminates in the production of an oncosphere, comprising a definitive larval stage, the hexacanth, enclosed by embryonic envelopes developing from embryonic macromeres (Conn, 2005; Conn and Świderski, 2008). When eaten by an appropriate intermediate host, the hexacanth undergoes metamorphosis into a juvenile stage, known as a metacestode (Conn, 2000). This metacestode juvenile (often incorrectly referred to as a larva) is completely post-metamorphic, possessing none of the features of the larva, but all the somatic features of the adult worm (tegument, parenchyma, excretory system, nervous system, adult scolex, etc.), except for the reproductive systems, which will develop within each of a chain of identical budded proglottids (Conn, 2005). All of these stages have been described in detail for some cestodes, and there is considerable variety among cestode taxa in developmental pattern, morphology, and periods of arrested post-larval development of the metacestode throughout the sequence of hosts in their respective life cycles (Chervy, 2002). Mesocestoides is a very common and cosmopolitan genus that infects a broad range of hosts both in the adult and the tetrathyridium metacestode stages (Conn et al., 2011; McAllister et al., 2014b; Skirnisson et al., 2016). Interestingly, McAllister et al. (2014a) examined a large sample of S. lateralis (n = 75) from Arkansas and Oklahoma for helminths and did not find Mesocestoides in any specimen. Most notably, 20 of these S. lateralis were collected in the same
9
county in Oklahoma where the infected ground skink noted herein was collected, so overall prevalence appears to be quite low for this Mesocestoides in this host species. Within the paruterine organ of the adult stage, embryonic development (Conn et al., 1984; Conn, 1988a) and parauterine organ organogenesis (Conn 1988c), have been described in detail. However, little is known about the hexacanth larvae, and nothing is known of the infectivity of hexacanths, or in what host(s) they might develop to the next stage. We report here, for the first time, the post-larval pre-tetrathyridium stages developing in vivo in an infected host. The only prior reports of these developmental stages were from experimental in vitro studies conducted by Voge (1967) and Voge and Seidel (1968). Our reported features of in vivo metamorphosis stages from a naturally infected lizard closely parallel the stages described from those earlier in vitro reports. Taken together, there can be no doubt that the unsubstantiated report of Soldatova (1944) that Mesocestoides possesses cysticercoid stages infecting mites was in error; that report was always questionable, as Soldatova's work included no experimental verification that the cysticercoids illustrated were Mesocestoides, rather than from some other cestode inhabiting the area in that uncontrolled study of wild-collected mites. The occurrence of transitional metamorphosis stages within the body cavity of intermediate host lizards, which also are known to harbor fully formed tetrathyridia, may lend support to the possibility that Mesocestoides spp. might be able to develop through a 2-host life cycle. It has often been assumed that the life cycle must involve 3 hosts (James, 1968), with the first intermediate host presumably being an arthropod that ingests the oncospheres, and then is eaten by one of many possible second intermediate hosts, which are known to include every class of tetrapod vertebrates (Conn et al., 2011; McAllister et al., 2014b; Skirnisson et al., 2016). Although we did not find stages in the lizard that retained hexacanth larval characteristics, the
10
early post-hexacanth nature of the earliest forms we described here, with small size and no scolex development, is consistent with very early post-hexacanth stages that develop in other cestode species (e.g., see Conn, 1985). It seems that a small post-larval stage that lacks both the hooks and penetration glands of the hexacanth from which it developed, and the scolex of the metacestode into which it is developing, would lack the mechanisms necessary to invade the body cavity of a lizard that had eaten a putative first-intermediate-host arthropod. Thus, it is imperative that future attempts to elucidate the life cycle of Mesocestoides include consideration of direct infection of lizards and other vertebrate intermediate hosts by ingested oncospheres. This should include ingestion of intact proglottids, as the eggs of Mesocestoides lack thickened protective shells (Conn, 1988a), and it seems likely that the function of the paruterine organ is the protection of oncospheres and mass-infection of the intermediate host (Conn et al., 1984; Conn, 1988c). Several authors have attempted to infect known vertebrate hosts as well as various invertebrates, but without success (reviewed by Conn, 1984; Padgett and Boyce, 2004). Nevertheless, the present results argue for maintaining a comprehensive approach to considering life cycle possibilities. Another unusual feature of Mesocestoides as a genus, is that whereas most tetrathyridia do not reproduce asexually (Conn, 1990; McAllister and Conn, 1990; McAllister et al., 1995, 2014b), there are some Mesocestoides species or strains that do so prolifically (see discussion by Conn et al., 2010). Whether this is an aberrancy common to the genus, or a common reproductive strategy, has been widely debated (Conn et al., 2011). However, the propensity for asexual reproduction in some Mesocestoides isolates seems to vary in mechanism, with some reported to divide at the scolex from an apical massif (Hess, 1980) which does not occur in nonproliferative forms (Conn, 1988b). In contrast, other tetrathyridia produce anomalous buds in the
11
hindbody, apparently associated with aberrant neoplastic interactions between abnormally dilated excretory ducts and extensively branched abnormal infoldings of the tegument (Conn et al., 2010). Recently, such cases have been characterized as being cancerous, resulting from malignant transformation of tegumental and excretory epithelia rather than constituting a normal mode of asexual reproduction (Conn, 2016). None of the stages described in the present study, from early post-larval stages to nearly fully developed tetrathyridium juvenile, possessed any feature that could be interpreted as leading to asexual proliferative ability. It is possible that the apical sucker described here could transform into the apical massif referenced by Hess (1980), but no apical sucker has been reported from any fully developed tetrathyridium, and fully developed non-proliferative tetrathyridia lack both an apical massif and an apical sucker (Conn, 1988b). We have obtained, for the first time, sequence data for Mesocestoides from a lizard. We sequenced 2 different stages, one corresponding to form 2 with evidence of forming scolex and the other corresponding to the form 4 possessing tetra-acetabulate scolex. Pairwise sequence comparison did not show any intraspecific differences among the sequenced specimens in any of the 3 sequenced mitochondrial genes. Despite the variable nature of the target genes, the lack of variability is not surprising because the tetrathyridia came from a single lizard most likely infected from a single source. The sequenced 12S fragment is currently by far the best represented for Mesocestoides in GenBank. While the interrelationships among the majority of species-level lineages of Mesocestoides are overall poorly resolved, the tree clearly delineates species and contains some well-supported groups of species. In the phylogenetic tree (Fig. 6), our specimens from S. lateralis clustered with 2 other well-supported species lineages, Mesocestoides sp. C from dogs
12
and Channel Island foxes, all collected in California (Fig. 6). The group of these 3 species is well supported by 95% posterior probability. The level of the genetic divergence in 12S gene between specimens from S. lateralis and Mesocestoides sp. C from dogs (7.5-8.2%) and Channel Island foxes (8.5-8.9%) provides strong evidence that our material from S. lateralis represents a species that has not yet been sequenced (Table II). This conclusion is strongly supported by the comparison of the above inter-clade levels of variability with intra-specific differences within all currently sequenced species and species-level lineages of Mesocestoides (Table II) which range from 0–2.1%. It should be mentioned that intraspecific variability in almost all species does not exceed 1.1% with the exception of M. vogae in which variability rose to 2.1% because of a single sequence which deviated from the general pattern. Noteworthy, sequences of Mesocestoides sp. C from dogs and Channel Island foxes formed 2 independent, strongly supported clades. Even though all Mesocestoides sp. C specimens were collected in California, the form from Channel Island foxes clustered together with sequences of geographically distant Mesocestoides sp. from S. lateralis. This suggests that the sequences previously published as Mesocestoides sp. C (Padgett et al., 2005) and the form from Scincella reported in the present study most likely represent 3 different, albeit closely related, species. Although a fairly large number of Mesocestoides samples have been sequenced previously from a variety of hosts and geographic localities in the United States (Crosbie et al., 2000; Padgett et al., 2005), the species found in S. lateralis has not been sequenced before. It remains unclear, however, whether it may represent an undescribed species with a yet unknown definitive host or 1 of the Mesocestoides species that were missing from the previous molecular studies. Future molecular and morphological studies of Mesocestoides with denser taxonomic
13
and geographical sampling of tapeworms from definitive hosts, particularly from Oklahoma and neighboring states, will likely provide an answer to this intriguing question. ACKNOWLEDGMENTS We thank Dr. Stanley E. Trauth (ASUMZ) for expert curatorial assistance and Nikolas H. McAllister (Lukfata Elementary School) for help in collecting. We are also grateful to Roy Nelson (University of North Dakota) for assistance with specimen processing for molecular analysis. The Oklahoma Department of Wildlife Conservation issued a Scientific Collecting Permit to CTM. LITERATURE CITED Bowles, J., D. Blair, and D. P. McManus. 1992. Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Molecular and Biochemical Parasitology 54: 165– 173. Chervy, L. 2002. The terminology of larval cestodes or metacestodes. Systematic Parasitology 52: 1–33. Cho, S. H., T. S. Kim, Y. Kong, B. K. Na, and W. M. Sohn. 2013. Tetrathyridia of Mesocestoides lineatus in Chinese snakes and their adults recovered from experimental animals. Korean Journal of Parasitology 51: 531–536. Conn, D. B. 1984. Studies on the structure, development and transmission of Mesocestoides lineatus, Mesocestoides corti and Oochoristica anolis (Cestoda: Cyclophyllidea). Ph.D. Dissertation. University of Cincinnati, Cincinnati, Ohio, 149 p. Conn, D. B. 1985. Life-cycle and postembryonic development of Oochoristica anolis (Cestoda: Linstowiidae). Journal of Parasitology 71: 10–16.
14
Conn, D. B. 1988a. Development of the embryonic envelopes of Mesocestoides lineatus (Cestoda: Cyclophyllidea). International Journal of Invertebrate Reproduction and Development 14: 119–130. Conn, D. B. 1988b. Fine structure of the tegument of Mesocestoides lineatus tetrathyridia (Cestoda: Cyclophyllidea). International Journal for Parasitology 18: 133–135. Conn, D. B. 1988c. The role of cellular parenchyma and extracellular matrix in the histogenesis of the paruterine organ of Mesocestoides lineatus (Platyhelminthes: Cestoda). Journal of Morphology 197: 303–314. Conn, D. B. 1990. The rarity of asexual reproduction among Mesocestoides tetrathyridia (Cestoda). Journal of Parasitology 76: 453–455. Conn, D. B. 2000. Atlas of invertebrate reproduction and development, Second Edition. John Wiley & Sons, Inc., New York, New York, 315 p. Conn, D. B. 2005. Glossary of cestode embryonic and larval structures. Wiadomości Parazytologiczne 51: 266 Conn, D. B. 2016. Letter to the editor: Malignant transformation of Hymenolepis nana in a human host. New England Journal of Medicine 374: 1293–1294. Conn, D. B., F. J. Etges, and R. A. Sidner. 1984. Fine structure of the gravid paruterine organ and embryonic envelopes of Mesocestoides lineatus (Cestoda). Journal of Parasitology 70: 68– 77. Conn, D. B., M.-T. Galán-Puchades, and M. V. Fuentes. 2010. Interactions between anomalous excretory and tegumental epithelia in aberrant Mesocestoides tetrathyridia from Apodemus sylvaticus in Spain. Parasitology Research 96: 1109–1115.
15
Conn, D. B., M.-T. Galán-Puchades, and M. V. Fuentes. 2011. Normal and aberrant Mesocestoides tetrathyridia from Crocidura spp. (Soricimorpha) in Corsica and Spain. Journal of Parasitology 97: 915–919. Conn, D. B., and Z. Świderski. 2008. A standardised terminology of the embryonic envelopes and associated developmental stages of tapeworms (Platyhelminthes: Cestoda). Folia Parasitologica 55: 42–52. Crosbie, P. R, S. A. Nadler, E. G. Platzer, C. Kerner, J. Mariaux, and W. M. Boyce. 2000. Molecular systematics of Mesocestoides spp. (Cestoda: Mesocestoididae) from domestic dogs (Canis familiaris) and coyotes (Canis latrans). Journal of Parasitology 86: 350–357. Derycke, S, T. Remerie, A. Vierstraete, T. Backeljau, and J. Vanfleteren. 2005. Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina. Marine Ecology Progress Series 300: 91–103. Etges, F. J. 1991. The proliferative tetrathyridium of Mesocestoides vogae sp. n. (Cestoda). Journal of the Helminthological Society of Washington 58: 181–185. Hall, T. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98. Hess, E. 1980. Ultrastructural study of the tetrathyridium of Mesocestoides corti Hoeppli, 1925: Tegument and parenchyma. Zeitschrift für Parasitenkunde 61: 135–159. James, H. A. 1968. Studies on the genus Mesocestoides (Cestoda: Cyclophyllidea). Ph.D. Dissertation. Iowa State University, Ames, Iowa, 255 p. James, H. A., and M. J. Ulmer. 1967. New amphibian host records for Mesocestoides sp. (Cestoda; Cyclophyllidea). Journal of Parasitology 53: 59.
16
Kumar, S., G. Stecher, and K. Tamura. 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution 33: 1870–1874. Littlewood, D., A. Waeschenbach, and P. Nikolov. 2008. In search of mitochondrial markers for resolving the phylogeny of cyclophyllidean tapeworms (Platyhelminthes, Cestoda) – a test study with Davaineidae. Acta Parasitologica 53: 133–144. Loos-Frank, B. 1991. One or two intermediate hosts in the life cycle of Mesocestoides (Cyclophyllidea: Mesocestoididae)? Parasitology Research 77: 726–728. McAllister, C. T., A. M. Bauer, and A. P. Russell. 1995. Nonproliferous tetrathyridia of Mesocestoides sp. (Eucestoda: Mesocestoididea) in Ptenopus garrulus maculatus (Sauria: Gekkonidae) from Namibia, South West Africa, with a summary of the genus from Old World lizards. Journal of the Helminthological Society of Washington 62: 94–98. McAllister, C. T., C. R. Bursey, M. B. Connior, L. A. Durden, and H. W. Robison. 2014a. Helminth and arthropod parasites of the ground skink, Scincella lateralis (Sauria: Scincidae), from Arkansas and Oklahoma. Comparative Parasitology 81: 210–219. McAllister, C. T., and D. B. Conn. 1990. Occurrence of Mesocestoides sp. tetrathyridia (Cestoidea: Cyclophyllidea) in North American anurans (Amphibia). Journal of Wildlife Diseases 26: 540−543. McAllister, C. T., D. B. Conn, P. S. Freed, and D. A. Burdick. 1991. A new host and locality record for Mesocestoides sp. tetrathyridia (Cestoidea: Cyclophyllidea), with a summary of the genus from snakes of the world. Journal of Parasitology 77: 329–331. McAllister, C. T., M. B. Connior, C. R. Bursey, S. E. Trauth, H. W. Robison, and D. B. Conn. 2014b. Six new host records for Mesocestoides sp. tetrathyridia (Cestoidea: Cyclophyllidea) from amphibians and reptiles of Arkansas, U.S.A. Comparative Parasitology 81: 278–283.
17
Nickisch-Rosenegk, M., R. Lucius, and B. Loos-Frank. 1999. Contributions to the phylogeny of the Cyclophyllidea (Cestoda) inferred from mitochondrial 12S rDNA. Journal of Molecular Evolution 48: 586–596. Padgett, K. A., and W. M. Boyce. 2004. Life-history studies on two molecular strains of Mesocestoides (Cestoda: Mesocestoididae): Identification of sylvatic hosts and infectivity of immature life stages. Journal of Parasitology 90: 108–113. Padgett, K. A., and W. M. Boyce. 2005. Ants as first intermediate hosts of Mesocestoides on San Miguel Island, USA. Journal of Helminthology 79: 67–73. Padgett, K. A., S. A. Nadler, L. Munson, B. Sacks, and W. M. Boyce. 2005. Systematics of Mesocestoides (Cestoda: Mesocestoididae): Evaluation of molecular and morphological variation among isolates. Journal of Parasitology 91: 1435–1443. Posada, D. 2008. jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution 25: 1253–1256. Rambaut, A. 2012. Molecular evolution, phylogenetics and epidemiology. FigTree ver. 1.4 software. Available at: http://tree.bio.ed.ac.uk/software/figtree/. Rausch, R. L. 1994. Family Mesocestoididae Fuhrmann, 1907. In Keys to the cestode parasites of vertebrates, L. F. Khalil, A. Jones, and R. A. Bray (eds.). CAB International, Wallingford, Oxon, U.K., p. 309–314. Ronquist, F., and J. P. Huelsenbeck. 2003. Bayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Schmidt, G. D. 1986. Handbook of tapeworm identification. CRC Press Inc, Boca Raton, Florida, 675 p.
18
Skirnisson, K., Ó. G. Sigurðardóttir, and Ó. K. Nielsen. 2016. Morphological characteristics of Mesocestoides canislagopodis (Krabbe 1865) tetrathyridia found in rock ptarmigan (Lagopus muta) in Iceland. Parasitology Research 115: 3099–3106. Soldatova, A. P. 1944. A contribution to the study of the development cycle in cestode Mesocestoides lineatus (Goeze, 1782), parasitic of carnivorous mammals. Doklady Akademii Nauk SSSR 45: 310–312. Specht, D., and M. Voge. 1965. Asexual multiplication of Mesocestoides tetrathyridia in laboratory animals. Journal of Parasitology 51: 268–272. Tkach, V., and J. Pawlowski. 1999. A new method of DNA extraction from the ethanol-fixed parasitic worms. Acta Parasitologica 44: 147–148. Voge, M. 1955. North American cestodes of the genus Mesocestoides. University of California Publications in Zoology 59: 125–156. Voge, M. 1967. Development in vitro of Mesocestoides (Cestoda) from oncosphere to young tetrathyridium. Journal of Parasitology 53: 78–82 Voge, M., and J. S. Seidel. 1968. Continuous growth in vitro of Mesocestoides (Cestoda) from oncosphere to fully developed tetrathyridium. Journal of Parasitology 54: 269–271. Webster, J. D. 1949. Fragmentary studies on the life history of the cestode Mesocestoides latus. Journal of Parasitology 35: 83–90.
Figures 1–5. Light micrographs of Mesocestoides sp. metamorphic stages removed from body cavity of Scincella lateralis; unstained living (1-2) and fixed acetocarmine-stained (3-5) preparations. (1) Stereoscope image of living cestodes in physiological saline. Note variable sizes and muscular contractions of body. (2) Brightfield image of living transitional stage
19
showing numerous granular calcareous corpuscles in the parenchyma of the hindbody (H) and scolex. Also note incomplete development of suckers (S) and excretory pore (E), and early formation of the apical sucker (S). (3) Differential interference contrast image of early metamorphic stage showing early development of suckers (S) anteriorly and excretory complex (E) posteriorly, no apical sucker, and poor delineation of hindbody (H) from presumptive scolex region. (4) Differential interference contrast image of anterior end of later metamorphic stage showing advanced but incomplete development of the scolex suckers (S) and complete development of the apical sucker (A). (5) Brightfield micrograph of a late developed metamorphic stage with greatly elongated hindbody (H), fully formed excretory complex with posterior pore (E), and definitive scolex with fully developed suckers (S) and apical sucker (A). Color version available online. Figure 6. Phylogenetic tree resulting from BI and ML analysis of 12S alignment of Mesocestoides showing the position of the specimens found in Scincella lateralis. Branch support values are shown at internodes as posterior probabilities/bootstrap percentages. Only posterior probabilities greater than 0.75 and bootstraps greater than 50% are shown.
20
Figs 1-5
Click here to download Figure Figs 1-5 revised_gray.tif
Figs 1-5 (color)
Click here to download Figure Figs 1-5 revised.tif
Fig 6
Click here to download Figure Fig 6 revised (1).tif
Table I
Click here to download Table Table I.docx
Table I. Measurements of stained, mounted transitional stages. Mean (range) dimensions in micrometers (µm). Transitional Form (total n=18) No Suckers (n = 2) Developing scolex with apical sucker (n = 3) Fully developed scolex with apical sucker (n = 13)
Total length 357.7 (351.4– 364.0) 476.9 (464.3– 502.2) 757.8 (577.3– 916.2)
Total width 169.4 (163.2– 175.7) 290.8 (251.0– 320.0) 302.3 (200.8– 464.4)
Scolex width 113.0 (100.4– 125.5) 184.1 (175.7– 188.3) 157.9 (131.8– 188.3)
Table II
Click here to download Table Table II(revised).docx
Table II. Pairwise DNA sequence variability among all species level lineages represented in phylogenetic analysis (Fig. 6). Number represent percentage of variable sites in the sequenced fragment of mitochondrial 12S gene.
Mesocestoides sp. C
Mesocestoides sp. C
U. littoralis
C. familiaris
Mesocestoides sp.
M. litteratus
M. lineatus
S. lateralis
M. vogae
Mesocestoides
(= sp. B)
sp. A
0–0.4
–
–
–
–
–
–
7.3–8.0
0–0.7
–
–
–
–
–
8.5–8.9
7.5–8.2
0
–
–
–
–
M. litteratus
15.7–16.4
12.9–14.3
15.6–16.3
0–1.1
–
–
–
M. lineatus
17.8–18.1
16.4
18.0
14.3–15.0
0
–
–
M. vogae (= sp. B)
13.2–15.0
12.5–13.9
13.6–15.0
11.5–13.6
11.9–13.2
1.0–2.1
–
Mesocestoides sp. A
17.4–18.1
14.3–15.3
17.3–18.0
14.6–15.3
14.6–15.3
8.4–10.5
0.4–1.1
Mesocestoides sp. C from U. littoralis Mesocestoides sp. C from C. familiaris Mesocestoides sp. from S. lateralis
Copyright Form1
Click here to access/download
Copyright Form Scan0001.jpg
Copyright Form2
Click here to access/download
Copyright Form Scan0002.jpg
Cover Letter
Click here to access/download
Cover Letter 1Nov2017LETTER.docx
