SPINITECTUS OSORIOI N. SP. (NEMATODA: CYSTIDICOLIDAE) FROM CHIROSTOMA SPP. (OSTEICHTHYES: ATHERINIDAE) IN LAKE PÁTZCUARO, MICHOACÁN, MÉXICO

J. Parasitol., 87(3), 2001, p. 648 655 American Society of Parasitologists 2001 SPINITECTUS OSORIOI N. SP. (NEMATODA: CYSTIDICOLIDAE) FROM CHIROSTOMA SPP. (OSTEICHTHYES: ATHERINIDAE) IN LAKE PÁTZCUARO, MICHOACÁN, MÉXICO Anindo Choudhury and Gerardo Pérez-Ponce de León* U.S.G.S. National Wildlife Health Center, 6006 Schroeder Road, Madison, Wisconsin 53711 ABSTRACT: Spinitectus osorioi (Nematoda: Cystidicolidae) is described from the freshwater atherinids Chirostoma estor and Chirostoma attenuatum from Lake Pátzcuaro in the Mesa Central, Michoacán State, México. This nematode is characterized by a conspicuous protuberance on the ventral surface of the distal end of the long spicule that distinguishes it from its congeners in North America and in the neotropics. In addition, the species can be readily distinguished from 4 of the 5 nominal species of North American freshwater Spinitectus by the absence of either a terminal barb or heel on the short spicule and from Spinitectus mexicanus by the spination. Previous records of Spinitectus carolini from Chirostoma spp. in México (Lakes Pátzcuaro and Zirahuén) refer to S. osorioi, and the species appears to be specific to Chirostoma spp. The geological history of the Mesa Central drainages and the historical biogeography of freshwater atherinids in this region suggest that the origin of S. osorioi may be associated with either the marine history of their hosts or with host-switching from more distantly related freshwater hosts after colonization of freshwater environments by atherinids. The genus Spinitectus Fourment, 1883, comprises a large number of species, most of which are parasites of fishes (Yamaguti, 1935; Skrjabin et al., 1965; Overstreet, 1970; Soota, 1983; Sood, 1989; Boomker and Puylaert, 1994; Moravec, 1994, 1998; Hoffman, 1999). Five species have been described from freshwater fishes in North America, Spinitectus gracilis Ward and Magath, 1917, Spinitectus carolini Holl, 1928, Spinitectus micracanthus Christian, 1972, Spinitectus acipenseri Choudhury and Dick, 1992, and Spinitectus mexicanus Caspeta- Mandujano, Moravec, and Salgado-Maldonado, 2000 (Choudhury and Dick, 1992; Hoffman, 1999; Caspeta-Mandujano et al., 2000). Seven species of Spinitectus have been described from the freshwaters of South America (Moravec [1998] recognizes 5 of these as valid). To our knowledge, no species of Spinitectus has been described or reported from the freshwaters of Central America. Previous records of Spinitectus from freshwater fishes in México include S. carolini, a typical parasite of centrarchids (Mueller and VanCleave, 1932; VanCleave and Mueller, 1934; Hoffman, 1999), from atherinids Chirostoma attenuatum (Meek) and Chirostoma estor Jordan (Osorio-Sarabia et al., 1986; Pérez-Ponce de León et al., 1994, 1996) and a species of Spinitectus (possibly Spinitectus agonostomi Moravec and Barus, 1971) from Agonostomus monticola (Bancroft) in Rio Bobos de Tlapacoyan, Veracruz (Garduño-Lugo et al., 1987). A re-examination of specimens from atherinids, deposited in the Colección Nacional de Helmintos (CNHE), Universidad Nacional Autónoma de México (UNAM), and of specimens collected subsequently from the same hosts in Lake Pátzcuaro, Michoacán, México, indicated that they comprise a distinct species that we describe here. MATERIALS AND METHODS Specimens upon which this decription is based were obtained from the Colección Nacional de Helmintos (CNHE), Universidad Nacional Autónoma de México (UNAM) (specimens from Chirostoma estor and C. attenuatum from Lake Pátzcuaro), CNHE 2261 2265, and from specimens collected from the atherinid, C. attenuatum, from Lake Pátzcuaro on 10 September 1997. Fish were examined within a few hours of capture, and live nematodes, recovered from the intestine of C. at- Received 26 May 2000; revised 17 October 2000; accepted 17 October 2000. * Instituto de Biología, Universidad Autónoma de México, A.P. 70-153, 04510 México D.F., México. tenuatum, were fixed in hot 70% ethanol or hot (steaming) 4% formalin. Specimens fixed in formalin were subsequently transferred to 70% ethanol. Nematodes were cleared by placing them in a mixture of 5% glycerine in 70% ethanol, in an uncovered dish, and allowing the ethanol (and water) to evaporate. Some specimens were processed for scanning electron microscopy (SEM) as follows: the specimens were dehydrated in an ascending series of ethanol, critical-point dried, and sputter coated with gold before being examined using a Hitachi S 2060N SEM unit. Additional species of Spinitectus examined included: S. gracilis: ex. Micropterus dolomieui, Whitemouth Falls, Whitemouth River Winnipeg River confluence, Manitoba, Canada, ex. Ambloplites rupestris, Red River floodway, St. Norbert, Manitoba (authors collection); S. carolini: ex. Micropterus dolomieui, Whitemouth Falls, Whitemouth River Winnipeg River confluence, Manitoba, Canada. (authors collection); S. carolini: CNHE (UNAM) 2263, 2265 ex. C. attenuatum (7 females), ex. C. estor (1 male). (These are in fact S. osorioi, see Remarks section.) Spinitectus micracanthus: ex. Lepomis cyanellus, ex. Algonquin Park, Ontario, Canada (authors collection); S. mexicanus: ex. Heterandria bimaculata. La Basura River, CNHE 3715 and 3716. Spinitectus acipenseri is very similar to S. gracilis (see Choudhury and Dick, 1992) and was not examined. Cleared specimens were mounted in glycerine, on slides, and examined using brightfield and Nomarski differential interference contrast (DIC) settings on an Olympus BX 50 compound microscope. Illustrations were made using an Olympus drawing tube attached to the microscope. CNHE refers to the Colección Nacional de Helmintos (National Helminth Collection), housed at the Universidad Nacional Autónoma de México; USNPC refers to the U.S. National Parasite Collection, Beltsville, Maryland; and HWML refers to the Harold W. Manter Laboratory, University of Nebraska, Lincoln, Nebraska. Measurements are in micrometers and are reported as range, with measurements of male holotype and female allotype in parentheses. DESCRIPTION Spinitectus osorioi n. sp. (Figs. 1 16) General: Body with short anterior unspined region, posterior to this, bearing transverse rows of cuticular spines. Unspined region (head) slightly wider and rounded anteriorly. Rows of spines divided into 4 clearly discernible sectors in the esophageal region, this arrangement difficult to determine posterior to esophageal region. Anteriormost row with smaller spines; spines increasing in size posteriorly in esophageal region before becoming finer, then smaller; sectors of spines in esophageal region becoming broadly interrupted, laterally, offset after rows 4 5. Spines on rows occurring singly, occasionally in doublets, triplets. Oral opening (mouth) dorsoventrally elongated, ovoid, bordered by 4 weakly developed, low, submedian labia (2 subdorsal, 2 subventral) bearing narrow (sclerotized?) sublabia and by 2 lateral, broad, pseudolabia, each with slightly narrowed midregion, dorsoventrally expanded inner margin; base of pseudolabia widening inside oral cavity. Oral cavity continuing as short prostom, followed by narrow, cuticularized 648

CHOUDHURY AND PÉREZ-PONCE DE LEÓN. S. OSORIOI N. SP. 649 FIGURES 1 4. Spinitectus osorioi (ex. Chirostoma estor). 1. Anterior portion of male. 2. Prespined anterior region of male (note 1 complete sector of spines in the first row). 3. Oral region of female showing cephalic papilla, amphid, pseudolabium (p), and submedian labia (s). 4. En face view of oral region of female showing lips and pseudolabia. Arrows point to papillae; asterisks indicate position of amphids. stoma, esophagus with anterior muscular region, longer posterior glandular region, intestine, short rectum leading to ventral slitlike anus. Anterior end with paired lateral amphids, 2 pairs of papillae, 1 on either side of amphid. Deirids single, narrow, finger-like, inconspicuous, bilateral, between spine rows 1 and 2. Excretory pore between spine rows 6 and 7, occasionally immediately above or closer to row 7. Males (measurements based on 10 specimens unless otherwise stated): Body 3,250 5,550 (4,370) long; maximum width 85 130 (85) in region of seminal vesicle. Spination beginning 52.5 80 (n 7) (70) from anterior end; 47 99 (78) rows of spines discerned, extending to posterior third of body, ending in region of vas deferens; distinct sectors of spines distinguished in anterior 11 19 (14) rows (esophageal region); first 2 rows of spines closer to each other, 10 17.5 (12.5) apart, distance between rows 2 and 3 15 25 (22.5); generally (in 8 of 10 worms) sectors becoming offset posterior to rows 3 8; anteriormost row bearing 28 40 (36) spines, 7 10 (9) per sector, 2.5 3.75 (3.75) long; spines increasing in size in esophageal region, maximum length 11.25 15 (12.5) in rows 11 14 (14). Caudal region coiled (1 1½ turns). Nerve ring 117 160 (n 7) (147.5) from anterior end. Stoma (vestibule) 72.5 105 (n 9) (87.5) long. Muscular esophagus 152.5 227.5 (n 9) (205) long, 15 20 (20) wide (maximum width); glandular esophagus 460 735 (n 8) (490) long, 30 50 (40) wide (maximum width), muscular: glandular esophagus 0.29 0.41 (0.41) (n 8); esophagus length:body length 0.14 0.19 (n 8) (0.15). Testes originating in midregion of body, extending anteriorly, looping 10 165 (n 8) (165) posterior to esophageal intestinal junction or 95 110 anterior to junction (in 2 specimens) before continuing posteriorly to form seminal vesicle occupying most of pseudocoelomic width, merging posteriorly with vas deferens. Area rugosa well developed, consisting of 2 longitudinal rows of conspicuous cuticular ridges or cleats in preanal region. Caudal region with 4 pairs of preanal papillae of which first and fourth pairs more ventrolateral with longer peduncles; first 2 pairs of papillae more closely situated; 6 pairs of postanal papillae, of which first 3 pairs more closely situated; sixth pair (likely phasmids) immediately posterior to fifth pair and more ventromedial. Spicules markedly unequal, longer (left) spicule slender, 480 520 (480) long, with conspicuous distal protuberance immediately anterior to tapered end, shaft length:blade length 0.84 0.92 (0.84), short (right) spicule broader than left spicule, gently arcuate, 120 160 (125) long with rounded distal end. Tail 138 225 (190) long, with narrow alae, tapering to dorsally rounded end with ventrally positioned spikelike mucron. Females (measurements based on 10 specimens): Body 6,100 9,750 (9,175) long; maximum width 105 185 (175) immediately anterior to vulva; spination beginning 67 95 (n 7) (75) from anterior end, extending over posterior third of body to within short distance of anus, first 2 rows of spines closer to each other, 13.7 17 (n 9) (17) apart, distance between rows 2 and 3, 22.5 27.5 (25); distance between rows increasing to maximum of 50 52.5 in region of posterior glandular

650 THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 3, JUNE 2001 FIGURES 5 8. Spinitectus osorioi. (ex. Chirostoma estor). 5. Spine rows 13 15 of male (at the level of posterior esophageal region). 6. Anterior third of body of female showing spines borne on narrow ridges. 7. Caudal region of male (note preanal area rugosa, spicule with distal protuberance). 8. Vulval region of female. esophagus or esophagus intestinal junction; sectors of spines posterior to row 4 or 5 offset; anteriormost row bearing 40 44 spines (10 11 [11] per sector), 3.75 5 (5) long; spines decreasing in number over first 4 rows, e.g., 11, 9, 9, 8, or 11, 9, 8, 6 spines per sector etc. (counted from sectors on same plane of view, different rows); spines increasing in size in esophageal region, maximum length 12.5 15 in row 10 13 (12). Stoma 100 112.5 long (n 9), muscular esophagus 202.5 262.5 (260) long, 22.5 25 (25) wide (maximum width); glandular esophagus 570 940 (805) long, 35 52.5 (45) wide (maximum width); muscular: glandular esophagus 0.28 0.35 (0.32), esophagus length:body length 0.12 0.16 (0.12). Reproductive system amphidelphic, each arm of reproductive system consisting of coiled and looped ovary, followed by looping oviduct, seminal receptacle, and uterus; anterior arm of reproductive system looping 25 670 (345) anterior to esophagus intestinal junction, loops of posterior ovary reaching 15 295 (295) anterior to anus (n 9) or 15 posterior to anus (n 1). Uteri voluminous, together occupying most of middle and posterior third of body, obscuring other internal organs. Muscular vagina posteriorly directed, at least 362.5 500 (437.5) long, with short distal chamber (ovijector) and longer posterior segment (vagina vera), junction with uterus difficult to determine precisely. Vulva bulging slightly; opening transverse, slitlike, with protruding lip. Eggs 35.5 40 long, 20 22.5 wide (30 eggs measured), without surface ornamentation or filaments; terminal uterine eggs containing coiled larva. Tail 106 152.5 long, tapering, with terminal mucron bearing pointed processes; phasmids ventrolateral, near posterior end of tail. Taxonomic summary Type host: Chirostoma attenuatum (Osteichthyes: Atherinidae). Other host: Chirostoma estor (Osteichthyes: Atherinidae). Site of infection: Intestine. Type locality: Lake Pátzcuaro, Michoacán, México, 19 32 19 42 N and 101 32 101 43 W. Deposition of specimens: Holotype (male) CNHE 4034, allotype (fe- FIGURES 9 13. Spinitectus osorioi. 9. Anterior region of male (ex. Chirostoma attenuatum). 10. Anterior region of female (ex. Chirostoma estor). 11. Anterior region of male with deirid. 12. Caudal end of female (ex. C. attenuatum) end showing mucron and phasmid. 13. Caudal end of female ex. C. attenuatum showing minor variation in mucron. Scale bars: Figures 9 12, 50 m; Figure 13, 25 m.

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CHOUDHURY AND PÉREZ-PONCE DE LEÓN. S. OSORIOI N. SP. 653 male) CNHE 4035. Paratypes: CNHE 4037 and CNHE 2261, USNPC 90643. Voucher specimens: CNHE 4036, HWML 15776. Etymology: The species is named in honor of David Osorio-Sarabia, Instituto de Biología, Laboratorio de Helmintología, UNAM, for his extraordinary humanity and for his dedication and contributions to parasitology. Remarks The decision to re-examine specimens deposited in the CNHE- UNAM as S. carolini was made after a perusal of the description of S. carolini in Osorio-Sarabia et al. (1986) in which the illustrations of the spicules indicated that this was likely a distinct species. Spinitectus osorioi can be readily distinguished from all its congeners in North and South America by the presence of the distal protuberance on the ventral side of the left spicule. It also possesses a simple curved right spicule that distinguishes it from S. gracilis and S. acipenseri that possess right spicules that are twisted like a rams horn with a lateral terminal heel and anteriorly directed vaginae, and from S. carolini and S. micracanthus that possess a ventral terminal barb on their right spicules. Furthermore, the excretory pore in S. osorioi is situated between spinerows 6 and 7, in contrast to that in other North American and middle- American species of Spinitectus. It is situated between rows 4 and 5 in S. gracilis and in S. acipenseri, between rows 8 and 9 in S. carolini, and between rows 7 and 8 (in males) or between rows 8 and 9 (in females) in S. micracanthus. Spinitectus osorioi can be readily distinguished from the only other freshwater species of Spinitectus described from México, S. mexicanus, which possesses a unique pattern of spination (see Caspeta-Mandujano et al., 2000) although both Mexican species possess spines that are arranged in sectors. Seven species of Spinitectus have been described from the freshwaters of the neotropics. Moravec (1998) considers 5 of these valid. However, Moravec s taxonomic remark that S. pachyuri is undoubtedly conspecific with S. rodolphiheringi is an apparent misprint. Moravec (1998) considers Spinitectus sternopygi Petter, 1984 conspecific with Spinitectus rodolphiheringi Vaz and Pereira, 1934, not S. pachyuri, which is a markedly different species. Three of the 7 nominal South American species, viz. S. pachyuri Petter, 1987, Spinitectus asperus Travassos, Artigas and Pereira, 1928, and Spinitectus jamundensis Thatcher and Padilha, 1977, possess spination that is markedly different from S. osorioi. Spinitectus pachyuri Petter, 1984 also possesses fewer (12) and much longer anterior spines and males of the species have 6 pairs of precloacal papillae, Spinitectus multipapillatus Petter, 1987 possesses 7 8 pairs of precloacal papillae, and Spinitectus yorkei Travassos, Artigas, and Pereira, 1928 reportedly possesses 15 pairs of precloacal papillae. Spinitectus rodolphiheringi, S. sternopygi, and S. yorkei have a posteriorly situated vulva. The position of the excretory pore also differs in the neotropical species in being situated as follows; at the base of row 4 in S. sternopygi and S. rodolphiheringi, at the base of or at the level of row 6 in S. pachyuri, between rows 13 and 14 in S. jamundensis, between rows 4 and 5 in Spinitectus multipapillata, These characteristics readily distinguish S. osorioi from its neotropical congeners. All former records of S. carolini from México refer to S. osorioi. This includes USNPC 82163 (S. carolini from C. attenuatum, Lake Pátzcuaro, México). DISCUSSION Spinitectus osorioi is the first species of its genus to be described from freshwater atherinids. Its hosts, C. estor and C. attenuatum, are endemic to the neovolcanic lakes of the Mesa Central region; C. attenuatum inhabits lakes Pátzcuaro and Zirahuén (as a nominal subspecies), whereas C. estor is found in lakes Pátzcuaro, Zirahuén (apparently as a subspecies) and in Lake Chapala (Pérez et al., 1993). Spinitectus osorioi has thus far been found only in Lakes Pátzcuaro and Zirahuén (Espinosa-Huerta et al., 1996) and only in these 2 atherinid hosts. Its absence in the other atherinids such as C. grandocule in Lake Pátzcuaro is possibly due to differences in their ecology (see Pérez Ponce de León et al., 2000). This pattern of specificity for certain Chirostoma spp., accompanied by a high level of endemism, makes it difficult to disentangle host and geographical specificty. No other species of fish in Lake Pátzcuaro is host to any Spinitectus species (Pérez-Ponce de León et al., 1996, 2000), and no species of Spinitectus has been reported from other Mesa Central lakes or from the Lerma-Santiago drainage (Pérez-Ponce de León et al., 1996). This precludes hypotheses of speciation by host shifts from extant hosts in the area. The historical biogeography of Chirostoma spp. may provide a clue to the origins of S. osorioi. The genus Chirostoma comprises a possibly diphyletic group of freshwater atherinids endemic to the lakes and drainages of the Mesa Central, and many species are endemic to the high altitude lakes, e.g., Pátzcuaro, Zirahuén, Zacapu, etc., of the neovolcanic axis of the region (Barbour, 1973a). The origin of the freshwater atherinids is thought to be linked to the colonization of the Lerma-Santiago River basin by descendants of marine or brackish water ancestors and subsequent speciation after the Mesa Central lakes became isolated following orogenic events and accompanying vulcanism (Barbour, 1973b; Barbour and Chernoff, 1984). Phylogenetic analyses (Eschelle and Eschelle, 1984) suggest that Chirostoma is closely related to Menidia, species of which inhabit drainages of northwest México and southeast Texas. This indicates a historical pre-pleistocene northern connection with atherinids of the Rio Grande (Rio Bravo) drainages (Eschelle and Eschelle, 1984), in turn suggesting an original Gulf-of- Mexico derivation. Intriguingly enough, the geological history of the Mesa Central (Miller and Smith, 1986) indicates that the Lerma-Santiago system has always been a Pacific drainage and that the present-day isolated lakes in the Mesa Central, home to the Chirostoma spp., were historically part of this drainage. The fact that Chirostoma spp. are endemic to these water bodies raises the possibility of a historical Pacific connection. Furthermore, Eschelle and Eschelle s (1984) analysis did not include atherinids such as Melaniris spp. that inhabit the Pacific drainages of central México. Given this information about the historical biogeography of the freshwater atherinids and the geological history of the Mesa Central, the present distribution of S. osorioi and its host associations allow us to develop 2 hypotheses about the origin and distribution of this nematode. First, S. osorioi evolved from a marine ancestral Spinitectus species that was carried into the freshwaters of México by its ancestral atherinid host. The fauna of marine atherinids from the Pacific or Gulf of Mexico coast and North America is poorly known, but the above hypothesis predicts that a sister species of S. osorioi will be found in marine atherinids of the area in question. This hypothesis can be extended to make the same prediction of close relationship between S. osorioi and species of Spinitectus in other freshwater atherinids such as from Me- FIGURES 14 16. Spinitectus osorioi. 14. Vagina (worm ex. Chirostoma attenuatum) showing terminal chamber with egg. 15. Male caudal region (worm ex. C. attenuatum) in side view, showing spicules and caudal papillae. 16. Male caudal region in ventral view showing papillae (note: sixth pair of papillae not visible due to the flexed distal end of the tail, approximate end of tail in stippled outline). Scale bars, 50 m.

654 THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 3, JUNE 2001 laniris balsanus and Melaniris crystallina, if and when found in these hosts. Second, S. osorioi originated in the freshwaters of the Lerma Santiago River system by host switching from other freshwater hosts. This hypothesis will be corroborated if the sister species of S. osorioi is found only in more distantly related freshwater fish species and if that sister species pair in turn nests within a wholly freshwater clade of Spinitectus. The hypothesis will be falsified if S. osorioi is more closely related to a marine Spinitectus sp. (see above). Both hypotheses are testable but clearly require a phylogenetic analysis of Spinitectus spp. and sampling of marine and brackish water atherinids in the Gulf of Mexico and in the Pacific coastal areas of México. In addition, the parasite fauna of other freshwater atherinids in neighboring Pacific drainages of México, e.g., Melaniris spp., and from the Gulf of Mexico drainages, e.g., Menidia spp., Xenatherina lisa, require more thorough investigation. It is hoped that such parascript studies (see Brooks and McLennan, 1993) will provide insights into the historical biogeography and coevolutionary history of S. osorioi and its endemic freshwater atherinid hosts, as well as other host parasite systems, in this unique and fascinating juxtaposition of endemic, nearctic, and neotropical faunas that is México. ACKNOWLEDGMENTS We thank the following persons for their help during this study: Sara Fuentes, Instituto de Biología, UNAM, for operating the SEM unit and for assistance in SEM photomicrography, Luis García-Prieto for loan of specimens, Patrick Nelson, Department of Zoology, University of Manitoba, for help in sampling in Manitoba, Canada, and Fernando Marques for help in sampling in Algonquin Park, Ontario. We also thank Luis García-Prieto, Berenit Mendoza-Garfias, and Virginia León Règagnon for help with field work in México. This work was supported by a grant from Consejo Nacional de Ciencia y Tecnologia (CONACyT) no. 27996N and Program PAPIIT-UNAM no. IN-219198 to G.P.P.D.L. A.C. acknowledges an NSERC postdoctoral fellowship at the University of Toronto and an NSERC grant 7696 to Dan Brooks, at whose laboratory A.C. held the fellowship. A.C. also thanks Rebecca Cole, USGS National Wildlife Health Center, Madison, Wisconsin, for continued support for fish parasite studies. 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Helminth community structure of Chirostoma attenuatum (Osteichthyes: Atherinidae) in two Mexican lakes. Southwestern Naturalist 41: 288 292. GARDUÑO-LUGO, M., I. SOFER-CHICUREL, AND P. ADAME DE PAASCH. 1987. Prevalencia de helmintos gastrointestinales en las lisas Agonostomus monticola de rio Bobos de Tlapacoyan, Veracruz. Veterinaria (Mexico) 18: 139 141. HOFFMAN, G. L. 1999. Parasites of North American freshwater fishes, 2nd ed. Cornell University Press, Ithaca, New York, 539 p. MILLER, R. R., AND M. L. SMITH. 1986. Origin and geography of the fishes of Central Mexico. In Zoogeography of North American freshwater fishes, C. H. Hocutt and E. O. Wiley (eds.). John Wiley & Sons, New York, New York, p. 491 517 MORAVEC, F. 1994. Parasitic nematodes of freshwater fishes of Europe. Kluwer Academic Publishers, Dordrecht, The Netherlands, 473 p.. 1998. Nematodes of freshwater fishes of the neotropical region. Academia, Prague, The Czech Republic, 464 p. MUELLER, J. F., AND H. J. 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