blood flukes (digenea: aporocotylidae) of...

BLOOD FLUKES (DIGENEA: APOROCOTYLIDAE) OF ELOPOMORPHS: EMENDATION OF

PARACARDICOLOIDES, SUPPLEMENTAL OBSERVATIONS OF PARACARDICOLOIDES

YAMAGUTII, AND A NEW GENUS AND SPECIES FROM LADYFISH, ELOPS SAURUS,

(ELOPIFORMES: ELOPIDAE) IN THE GULF OF MEXICO

Stephen A. Bullard

Aquatic Parasitology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, College of Agriculture, Auburn University, 203 Swingle Hall,Auburn, Alabama 36849. Correspondence should be sent to: [email protected]

ABSTRACT. Monotypic Paracardicoloides Martin, 1974 is emended based on supplemental observations of 2 voucher specimens ofParacardicoloides yamagutiiMartin, 1974. Features of the anterior sucker, esophagus, and intestine as well as the male and female genitaliapreviously attributed to, or omitted from the diagnosis and description of, this fluke are resolved and further detailed herein. The holotype ofP.yamagutii, originally deposited in theAllanHancockParasitologyCollection, apparently has been lost, andQueenslandMuseumvoucherG222650 is designated as the neotype. Elopicola nolancribbi n. gen., n. sp. infects the ladyfish, Elops saurus, (Elopiformes: Elopidae) in thenorth-central Gulf of Mexico. The new genus resembles Paracardicoloides by having the combination of a bowl-shaped anterior sucker,inverse U-shaped intestine, short posterior ceca, post-cecal ovary, pre-ovarian ootype, compact uterus, and prominent excretory arms andvesicle. It differs from Paracardicoloides by lacking robust tegumental body spines and by having a single testis, post-testicular ovary, andprimarilypost-testicularuterus.Thepresent study is thefirst confirmedreportofanaporocotylid infectingamemberofElopiformesaswell asthe first report of an infection in an elopomorph outside of Australia and New Zealand or in marine waters.

Paracardicoloides yamagutii Martin, 1974 (Digenea: Aporocoty-

lidae) infects the blood vascular system of speckled longfin eel,

Anguilla reinhardtii Steindachner, 1867 (type host); the shortfin eel,

Anguilla australis Richardson, 1841; and the New Zealand longfin

eel, Anguilla dieffenbachiiGray, 1832 in rivers of Australia (Martin,

1974;NolanandCribb,2004) andNewZealand (Hine, 1978). It is the

only blood fluke known from the 24 families, 156 genera, and 856

species of Elopomorpha, the basal lineage (craniate subdivision)

including fisheswith an ‘‘eel-like’’ or ribbon-like larva (leptocephalus

type) (Greenwood et al., 1966; Forey et al., 1996; Inoue et al., 2004;

Nelson, 2006). The poor knowledge of fish blood fluke diversity

among basal fish lineages, e.g., Elopomorpha, is a barrier to

understanding the natural history of blood flukes (Schistosomatoi-

dea) as a whole but especially the evolutionary origins of fish blood

flukes.

Toward shedding light on the identities and morphological

features of blood flukes that infect these basal fishes, I herein (1)

emend the diagnosis of Paracardicoloides, (2) provide supplemental

observations of its type species, P. yamagutii, and (3) propose a

new genus and describe a new species of Aporocotylidae from

another elopomorph, the ladyfish, Elops saurus Linnaeus, 1766,

(Elopiformes: Elopidae) from the north-central Gulf of Mexico.

The present study is the first taxonomic description of an

aporocotylid infecting an elopomorph outside of Australia and

New Zealand or in marine waters (Martin, 1974; Hine, 1978;

Smith, 1997a, 1997b; Nolan and Cribb, 2004). Noteworthy is that

Prearo et al. (1995) reported ‘‘Sanguinicola sp.’’ from a European

eel, Anguilla anguilla (Linnaeus, 1758), but this record needs

confirmation because no anatomical feature of those specimens was

published and apparently no museum voucher material exists.

MATERIALS AND METHODS

Sixty ladyfish were captured by hook-and-line, seine, and cast net inMississippi Sound as well as off the Mississippi barrier islands Horn Island

(308140330 0N, 888460530 0W) and Ship Island (308120530 0N, 888560140 0W)from October 1999 through July 2013. All ladyfish were killed by spinalseverance in the field and kept on ice. Because of the lengthy duration ofthese dissections, infected individual ladyfish were first identified from thepool of captured ladyfish by searching for aporocotylid eggs lodged in thegill epithelium, which were examined as wet-mounts with the aid of acompound microscope at 3200–400 magnification. With the hope ofdetermining the specific site of infection for the adult fluke in the ladyfishthe heart, mesenteric vessels, branchial sinuses with associated vessels, andkidney were each separated into different beakers and immersed, thenrinsed, in an anticoagulant solution of ~5.0 gm NaCl and ~2.0 gm NaCl-citrate/L of distilled water. Each of the rinsed tissues were examinedseparately for the presence of blood flukes in a petri dish containing asmall amount of saline before the remaining contents of each beaker weretransferred to pilsner glasses, allowed to settle for 30 min, decanted, andexamined in a dish containing fresh saline. Observations of living flukeswere made with the aid of a compound microscope at 3100–1,000magnification. Living flukes intended as whole-mounts were killed withheat from an ethanol-burner flame under little or no coverslip pressureand transferred to a vial of 5% neutral buffered formalin ( n.b.f.) orshaken vigorously in saline for 30–45 sec before being fixed directly in theformalin solution. Whole mounts were stained in Van Cleave’s hematox-ylin with several additional drops of Ehrlich’s hematoxylin, made basic at70% ethanol with lithium carbonate and butyl-amine, dehydrated, clearedin clove oil, and mounted in Canada balsam. The specimens for scanningelectron microscopy (SEM) were dehydrated, immersed in hexamethyldi-silazane for 30 min, air-dried for 45 min, and sputter-coated with gold-palladium (Bullard and Jensen, 2008). Drawings were made with the aid ofa drawing tube and facilitated by differential interference contrast (DIC)optical components. Measurements are reported in micrometers (lm) andgiven as ranges with the sample size in parentheses. Type material isdeposited in the United States National Parasite Collection (USNPC),Beltsville, Maryland. The studied voucher material of Paracardicoloidesyamagutii comprises 2 slides, each with a single specimen, and wasborrowed from the Queensland Museum (Queensland, Australia) courtesyof Mal Bryant and Rob Adlard.

DESCRIPTIONS

Paracardicoloides Martin, 1974 emended(Figs. 1–5)

Diagnosis: Body of adult flat, oval, ventrally concave, lackingposterolateral body protuberance, anterior and posterior ends taperingapproximately equally, spined; tegumental body spines robust, spike-like,lacking recurved tip, distributing in a narrow field along ventrolateralbody margin, clearly not distributing in ventrolateral transverse rows, with

Received 11 September 2013; revised 15 January 2014; accepted 20January 2014.

DOI: 10.1645/13-391.1

305

J. Parasitol., 100(3), 2014, pp. 305–316

� American Society of Parasitologists 2014

FIGURES 1–3. Paracardicoloides yamagutii Martin, 1974 (Digenea: Aporocotylidae) (Queensland Museum voucher G222650) from blood vascularsystem of speckled longfin eel, Anguilla reinhardtii Steindachner, 1867 (Elopomorpha: Anguilliformes) in the Brisbane River, Australia. Scale values

306 THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 3, JUNE 2014

tips enveloped by tegument. Rose thorn-shaped spines absent. Anteriorsucker bowl-shaped, not comprising a spheroid anterior sucker with amedio-ventral mouth, demarcated from anterior body end by peduncle,aspinous in adult. Muscular pharynx lacking. Esophagus sinuous, ventralto anterior nerve commissure, extending posteriad ,1/2 body length,comprising morphologically distinctive anterior, middle, and posteriorregions; anterior and posterior portions of esophagus comprisingdistinctive swellings. Intestine inverse U-shaped, with long posterior cecaonly, lacking diverticula or secondary rami, extending sinuously posteriadbut lacking loops or coils, terminating in middle third of body. Testes 2 innumber, medial, flanking ovary anteriorly and posteriorly, deeply lobed.Vasa efferentia coalescing at posterior margin of anterior testis andanterior margin of posterior testis to form larger ducts that coalesce toform vas deferens; vas deferens extending anteriad in dextral half of bodybefore narrowing distally and connecting to cirrus sac. Auxiliary externalseminal vesicle absent. Cirrus sac sinistral, pre-ovarian; enclosing internalseminal vesicle and cirrus; cirrus everting dorsally in sinistral body half,post-cecal. Ovary single, medial, post-cecal, intertesticular, deeply lobed.Oviduct post-cecal; oviducal seminal receptacle present. Laurer’s canalpresent. Vitellarium follicular, an extensive network of narrow intercon-necting branching bands having granular vitelline material, extendinglaterad beyond ventrolateral nerve cords, occupying space from anteriornerve commissure posteriad to level of distal tips of posterior ceca. Ovo-vitelline duct longitudinal, connecting with ootype posteriorly. Ootypepre-ovarian. Uterus comprising short ascending and descending portions,pre-ovarian, intercecal; uterine eggs having ovoid body nearly as wide asuterus, lacking filaments. Metraterm pre-ovarian, medial to cirrus sac.Male and female reproductive tracts opening into common atrium andsharing a common pore; common genital pore dorsal, sinistral, post-cecal.Excretory vesicle prominent posteriorly, Y-shaped; excretory pore dorsal,subterminal. In blood vascular system of eels (Elopomorpha: Anguilli-formes).

Differential diagnosis: Body lacking posterolateral body protuberance;tegumental body spines robust, spike-like, lacking recurved tip, notdistributing in clearly-defined ventrolateral transverse rows, enveloped bytegument. Anterior sucker bowl-shaped, demarcated from anterior bodyend by peduncle, aspinous. Muscular pharynx lacking. Esophagus havinganterior and posterior swellings. Intestine inverse U-shaped. Testes 2 innumber, flanking ovary anteriorly and posteriorly, deeply lobed; vasaefferentia coalescing at posterior margin of anterior testis and anteriormargin of posterior testis to form larger ducts that coalesce to form vasdeferens; vas deferens extending anteriad in dextral half of body beforenarrowing distally and connecting to cirrus sac. Cirrus sac present. Ovarypost-cecal, inter-testicular, deeply lobed. Oviducal seminal receptaclepresent. Laurer’s canal present. Ovo-vitelline duct longitudinal, connect-ing with ootype posteriorly. Ootype pre-ovarian. Uterus pre-ovarian,intercecal; uterine eggs having ovoid body nearly as wide as uterus, lackingfilaments. Male and female reproductive tracts sharing common atriumand pore.

Type and only nominal species: Paracardicoloides yamagutii Martin,1974.

Specimens examined: Queensland Museum vouchers G222650 andG222651.

Remarks

Monotypic Paracardicoloides is most easily differentiated from all othernominal aporocotylid genera by a combination of morphological featuresassociated with the anterior sucker, tegumental body spines, intestine,Laurer’s canal, and gonads (Figs. 1–3). The anterior sucker of Para-cardicoloides is large, bowl-shaped, and centered on a large mouth (Fig. 1).Paracardicoloides has tegumental body spines that are spike-like, robust,lack a recurved tip, and are not distributed in ventrolateral rows (Fig. 2).Moreover, these spines are evidently sheathed in tegument, which is anunusual feature among the nominal aporocotylids known at the time ofwriting this manuscript. The intestine is inverse U-shaped, lacks anterior

aside each bar. (1) Body of adult showing anterior sucker (as), anterior esophageal swelling (aes), ventrolateral nerve commissure (vc), nerve cord (nc),esophagus (e), vitelline follicles (v), esophageal gland (eg), posterior esophageal swelling (pes), posterior ceca (pc), anterior testis (t1), and posterior testis(t2). Ventral view. (2) Lateral tegumental body spines. Ventral view. (3) Region of genitalia showing posterior ceca (pc), anterior (t1) and posterior testis(t2), vasa efferentia (ve), confluence of vasa efferentia (cve), internal seminal vesicle (isv), cirrus sac (cs), ovary (o), oviduct (ov), oviducal seminalreceptacle (osr), primary vitelline duct (vd), ootype (oo), location of Laurer’s canal (*), ascending uterus (au), descending uterus (du), uterine egg (e),metraterm (met), excretory duct (ed), excretory vesicle (ev), and excretory pore (ep). Ventral view.

FIGURES 4–5. Paracardicoloides yamagutii Martin, 1974 (Digenea: Aporocotylidae) from blood vascular system of speckled longfin eel, Anguillareinhardtii Steindachner, 1867 (Elopomorpha: Anguilliformes) in the Brisbane River, Australia. Scale values aside each bar. (4) Terminal genitalia(Queensland Museum voucher G222650) showing internal seminal vesicle (isv), cirrus sac (cs), and metraterm (met) proximal to common genital atriumand pore. Ventral view. (5) Proximal portion of female genitalia (Queensland Museum voucher G222651) showing medial portion of ovary (o), oviduct(ov), oviducal seminal receptacle (osr), Laurer’s canal (Lc; indicated by dashed line because the canal is dorsal to the vitelline duct), uterus (u), andvitelline duct (vd). Ventral view.

BULLARD—ELOPOMORPH BLOOD FLUKES 307

ceca, and extends to the middle third of the body (Fig. 1). I also confirmthat a Laurer’s canal is present (Figs. 3, 5) (not reported or described byMartin [1974], Smith [2002], or Nolan and Cribb [2004]) and that theovary is medial and resides between the 2 testes (Fig. 3). Considering thesefeatures, Paracardicoloides is most similar to monotypic AcipensericolaBullard, Jensen, Snyder, and Overstreet, 2008 by having a bowl-shapedanterior sucker, lateral tegumental body spines that lack a recurved tip, aninverse U-shaped intestine, an intertesticular ovary, and a Laurer’s canal.However, Paracardicoloides can be easily differentiated from Acipenser-icola by having 2 testes only, both of which are primarily post-cecal (Figs.1, 3), rather than having a testicular column comprised of severalintercecal testes plus a single testis posteriorly. Few aporocotylids have aLaurer’s canal, i.e., species of Chimaerohemecus van der Land, 1967,Hyperandrotrema Maillard and Ktari, 1978 (see Orelis-Ribeiro et al.,2013), and Acipensericola (see Bullard et al., 2008), but all of theaforementioned genera possess an inverse U-shaped intestine and do notreportedly mature in higher bony fishes of Euteleostei.

Paracardicoloides yamagutii Martin, 1974(Figs. 1–5)

Diagnosis of adult (measurements and illustrations based on 2 whole-mounted specimens): Body 1,360–1,400 (2) long, 300–480 (2) wide,approximately 3–53 longer than wide, with anterior and posterior endstapering approximately equally, with maximum width near midbody (Fig.1); dorsal and ventral surfaces of body lacking sensory cilia or sensorypores evident with light microscopy; tegumental body spines directinglaterally, slightly medially, or posteriorly, each spine 15 in maximumlength, each spine 3 in maximum width or approximately 53 longer thanwide, numbering approximately 281–287 (2) spines per side of body,distributing in a ventrolateral field (Fig. 2); ventrolateral field oftegumental spines approximately 15 wide, comprising a maximum of 3spines abreast but typically 1 or 2 spines abreast, not confluent posteriorly.Ventrolateral nerve cord 13–18 (2) in maximum width, becomingconfluent 28 (2) from posterior body end and extending anteriad untilbecoming less obvious; anterior nerve commissure 140–170 (2) or 10% ofbody length from anterior body end, 75–80 (2) across width of worm, 15–25 (2) in width, perpendicular to midline of body; secondary branches andventrolateral nerve cord and dorsolateral nerve cord indistinct (Fig. 1).

Anterior sucker 38–55 (2) in diameter or 11–16% of body width,strongly muscular, extending anteriad from anterior end of bodyapproximately 33–35 or 2–3% of body length or 64–87% of anteriorsucker diameter, with probable gland cells embedded in rim of sucker;mouth a large pore opening within anterior sucker and surrounded bymuscular rim of sucker (Fig. 1). Esophagus ventral to nerve commissure,475–515 (2) long or 34–38% of body length, including morphologicallydistinctive anterior, middle, and posterior portions; anterior portion ofesophagus comprising an anterior swelling, lacking discernible musclesurrounding lumen, appearing delicate in whole-mounted specimens, notextending laterally beyond nerve cord, between level of mouth andanterior nerve commissure, 93–95 (2) long or 18–20% of esophagus totallength, or approximately 7–9% of body length, 20–23 wide (2) orapproximately 4–8% of body width, 4.0–4.83 longer than wide, notmarkedly thick-walled; middle portion of esophagus sinuous, thin-walled,separated from anterior esophageal swelling by a marked constrictionanterior to level of nerve commissure, 280–325 (2) long or 59–63% ofesophagus total length or approximately 20–24% of body length, 45–60(2) wide, approximately 13–15% of body width, 4.7–7.23 longer thanwide, with wall approximately 1 thick; posterior esophageal swellingbulbous, thick-walled, separated from medial portion of esophagus by amarked constriction, immediately anterior to cecal bifurcation, 75–100 (2)long or 15–21% of esophagus total length or approximately 6–7% of bodylength, 40–45 (2) wide, approximately 9–13% of body width, 1.9–2.23longer than wide (Fig. 1). Esophageal gland evident in 1 specimen andprincipally surrounding posterior esophageal swelling, 130 long or 1.33posterior esophageal swelling length or 27% of esophagus total length, 138wide or 29% of body width. Cecal bifurcation immediately posterior toposterior esophageal swelling, 475–515 (2) or 34–38% of body length fromanterior body end; posterior ceca sinuous, arching posterolaterad fromcecal bifurcation and extending posteriad in parallel with lateral bodymargin, containing black or brown-colored contents in lumen; dextral andsinistral posterior ceca asymmetrical, dextral cecum longer in bothspecimens studied, 275–300 and 300–350 respectively, extending posteriadapproximately 300–312 or 22% of body length or 58–66% of esophagus

length, 30–50 in maximum width, ending approximately 600–700 (2) or43–51% of body length from posterior body end, not extending laterallybeyond ventrolateral nerve cord (Fig. 1).

Anterior testis 160–250 (2) long or 12–18% of body length, 140–240 (2)wide or 47–50% of maximum body width, 1–1.43 longer than wide,terminating 475 or 34% of body length from posterior body end (Figs. 1,3); posterior testis 190–255 (2) long or 14–18% of body length or 1–1.23anterior testis length, 145–255 (2) wide or 48–53% of maximum bodywidth, 1–1.33 longer than wide, terminating 200 or 14% of body lengthfrom posterior body end; testis lobes 30–38 (2) long, approximately 30wide or 1–1.33 longer than wide; vasa efferentia difficult to trace in fixedspecimens, an interconnecting meshwork of fine ducts entwiningthroughout testicular tissue, approximately 8–15 (2) wide, containingsperm in all specimens, coalescing in posterior portion of anterior testisand anterior portion of posterior testis to each form larger ducts(¼trunks); anterior trunk of vasa efferentia 38–50 long or 3–4% of bodylength, 25 (2) in maximum width (Figs. 1, 3); posterior trunk of vasaefferentia longer than anterior trunk, 80–100 (2) long or 1.6–2.633 longerthan anterior trunk of vasa efferentia or 6–7% of body length, 25–75 (2) inmaximum width; vas deferens forming from anterior and posterior trunksof vasa efferentia at level of ovary or between testes, extending directlyanteriad 195–225 (2) or 14–16% of body length in dextral half of body,18–38 (2) or 6–8% of body width in maximum width before archingmediad approximately 100 (2) and narrowing to approximately 10 (2),crossing midline and extending slightly posteriad and connecting withcirrus sac, containing sperm in all specimens. Cirrus sac 113–118 (2) long,33–43 (2) wide, 3.4–4.23 longer than wide, with wall 3 (2) thick, enclosingwell-delineated seminal vesicle, residing between anterior testis andsinistral nerve cord; internal seminal vesicle occupying much of theluminal space within cirrus sac, 60–80 (2) long or 4–6% of body length,30–43 (2) wide (Figs. 1, 3, 4).

Ovary residing between anterior and posterior testes, compressed, notextending laterad beyond nerve cords, 55–63 (2) long or 4–5% of bodylength, 163–250 (2) wide or 52–54% of maximum body width, 0.4–0.23longer than wide; post-ovarian space 445 long or 32% of body length(Figs. 1, 3, 5). Oviduct extending from anteroventral surface of ovary,extending anterosinistrad 75–83 (2) or 5–6% of body length before curvingdorsomediad and turning anteriad before meeting with oviducal seminalreceptacle, distal to oviducal seminal receptacle and connection ofLaurer’s canal continuing anteriad and joining with primary vitellinecollecting duct; oviducal seminal receptacle a thin-walled sac containingsperm in all specimens, 38–50 (2) long or 46–67% of oviduct total length,38–40 (2) wide, varying in length and width depending on amount ofsperm in duct (Figs. 1, 3, 5); Laurer’s canal comprising a swollen ductopening on dorsal surface and connecting to oviduct immediately distal tooviducal seminal receptacle, 38 (2) long, 13–18 (2) in maximum widthdepending on contents of canal. Primary vitelline collecting ductconnecting with oviduct anterior to oviducal seminal receptacle andLaurer’s canal and at level of cirrus sac, not evident except in posteriorportion of body before confluence with oviduct, originating in dextral halfof body along posterior margin of anterior testis, coursing betweenanterior testis and ovary, 28–30 (2) in maximum width, uniting withoviduct to form common duct (¼ovo-vitelline duct); ovo-vitelline duct anarrow duct extending anteriad 75–113 (2) or nearly entire length of cirrussac in sinistral half of body, 8–10 wide, connecting with ootype medially;ootype oblong, intercecal, at level of distal tips of ceca, 38–43 (2) long, 25–35 (2) wide or 1.2–1.53 longer than wide, medial or slightly sinistral,ventral to anterior testis and occupying space at level of anterior margin ofanterior testis, 670–730 (2) or 49–52% of body length from posterior bodyend; Mehlis’ gland indistinct in fixed material. Uterus abbreviate, curvingonce, lacking convolutions or coils, thick-walled, with ascending anddescending portions; ascending uterus straight, extending anteriad 100–200 (2) or 7–14% of body length before curving ventrally at midline andnear dextral posterior cecum and connecting with descending uterus, 20–23 (2) in maximum width; descending uterus straight, directing mediad,shorter than ascending uterus, extending posteriad 85–98 (2) or 6–7% ofbody length or 43–98% of ascending uterus length before meeting withmetraterm at midline or in sinistral half of body, 20–23 (2) in maximumwidth; uterine eggs 40 long, 23 wide or approximately 23 longer than wide,none containing seemingly well-formed miracidium, none having polarfilament-like structures or lateral extensions, with thin, membranous shellhaving brownish tint. Metraterm originating between distal tips ofposterior ceca, sinistral, a narrow duct when lacking eggs, straight,


extending posteriad from descending portion of uterus approximately 200or 14% of body length, 8 wide or 253 longer than wide, with muscularwall (Figs. 1, 3, 4). Common genital pore opening 510 or 36% of bodylength from posterior body end (Figs. 1, 3, 4).

Excretory system difficult to observe in fixed specimens; excretoryvesicle oblong, 100 long or 25 wide; excretory arms each approximately 80long, 25 wide; excretory pore ending 38 or 3% of body length from bodyend (Figs. 1, 3).

Taxonomic summary

Type host: Adults infecting Anguilla reinhardtii Steindachner, 1867(Elopomorpha: Anguilliformes), speckled longfin eel (Martin, 1974; Nolanand Cribb, 2004).

Other hosts: Adults infecting Anguilla australis Richardson, 1841,shortfin eel (Hine, 1978) and Anguilla dieffenbachii Gray, 1832, NewZealand longfin eel (Hine, 1978); sporocyst, rediae, and cercariae infectingPosticobia brazieri (Smith, 1882) (Hypsogastropoda: Hydrobiidae),freshwater snail (Hine, 1978; Nolan and Cribb, 2004).

Sites in host: ‘‘Blood vessels, dorsal aorta’’ (type specimens; Martin[1974]); dorsal aorta, atrium and ventricle of heart, gill, and blood vesselsof intestine, swim bladder, and kidney (Nolan and Cribb, 2004); gill (Hine,1978).

Type locality: Brisbane River and tributaries, Queensland, Australia(Martin, 1974; Nolan and Cribb, 2004).

Other localities: New Zealand rivers (Hine, 1978).Specimens examined: Queensland Museum vouchers G222650 and

G222651.

Remarks

Herein the diagnosis of Paracardicoloides is emended and supplementalobservations of P. yamagutii are provided such that several genericfeatures previously attributed to or omitted from the diagnosis anddescription of the genus and species, respectively, now require clarificationor correction. Comparing Martin’s (1974) description with Nolan andCribb’s (2004) voucher specimens should be done cautiously, however,because Martin’s (1974) specimens were small in body size (518–924 [mean¼ 704] long), had small gonads (testes 31–152 long; ovary 30–50 long), andreportedly lacked uterine eggs in 2 of 4 specimens studied; all of whichstrongly indicates that at least some of his specimens were juveniles oryoung adults and that the morphometric and anatomical details of Martin(1974) were based partly on immature specimens. Hence, such acomparison is likely one between juveniles or young adults (Martin’sspecimens) vs. older, larger adults (Nolan and Cribb’s specimens:G222650, G222651). Despite the size differences, all of these specimenswere sourced from the same locality and host species. Regardingterminology, I employ a few different terms to make the presentdescriptions consistent with post-1974 aporocotylid descriptions; Martin’s(1974) terms are in quotations preceding the term used herein inparentheses: ‘‘oral disc’’ (anterior sucker); ‘‘cerebral commissure’’ (anteriornerve commissure); ‘‘esophageal bulb’’ (posterior esophageal swelling),‘‘cirrus pouch’’ (cirrus sac); and ‘‘uterus with swollen, sausage-shapedportion’’ (metraterm).

Martin’s (1974) figure 1 is labeled as a ventral view but likely representsa dorsal view because the voucher specimens I studied clearly have asinistral metraterm and sinistral common genital pore. Although theanterior sucker in Martin’s (1974) figure 1 is illustrated ‘face-up’ and thenerve commissure is illustrated as coursing behind the esophagus, bothindicating an aporocotylid is in ventral view, the genitalia depicted in theillustration are in dorsal view. Smith’s (2002) diagnosis of Paracardico-loides is incorrect regarding ‘‘Male and female genital pores open. . . toright of ovary.’’ Nolan and Cribb’s (2004) figure 1 correctly illustrated thisfluke in ventral view, i.e., with anterior sucker facing ventrally, nervecommissure dorsal to esophagus, and genital pore sinistral and openingdorsolateral to the ovary.

Martin (1974) reported the presence of ‘‘spinelike projections on oraldisc (that) seem to be tegument only’’ (see Martin’s [1974] figure 3), but Icould not confirm the presence of this feature. The voucher specimensstudied herein lacked a spine-like feature on the dorsum of the anteriorsucker when viewed at 31,000 magnification using an oil immersionobjective, and the size of those features as illustrated by Martin (1974) andre-illustrated by Smith (2002) mandate that they would be detectable atthis magnification, if present. Figure 1 of Nolan and Cribb (2004) shows

that the sucker has spines. The fine structure and spination of the anteriorsucker of aporocotylids comprise valuable generic features (Bullard andOverstreet, 2003; Bullard et al., 2008), and the presence of such large,spine-like projections of the tegument certainly would make Para-cardicoloides unique among all other known aporocotylids. Acipensericolapetersoni has peg-like, straight spines lining the dorsal luminal surface ofthe anterior sucker about the mouth but not on the dorsal surface of thesucker itself (Bullard et al., 2008). Detailing the anterior sucker ofaporocotylids can be difficult, however, and some features are bestdemonstrated by studying living specimens (Bullard and Jensen, 2008).Perhaps these spine-like features are more apparent in live specimens orperhaps only in juveniles or young adults. Regarding spination of P.yamagutii, Martin (1974) reported that the tegumental body spines wereeach 6 long but I measured them as approximately 15 long (Fig. 2).

The esophagus of P. yamagutii has 3 morphologically-distinctiveregions which may also have distinct physiological functions. Althoughillustrated by Martin (1974) and Nolan and Cribb (2004), the presence ofthese distinctive regions has not been treated as a generic featurepreviously. Martin (1974) referred to the posterior esophageal swellingbut did not specifically provide details of the anterior esophageal swelling.He also reported that the esophagus was ‘‘glandular externally along mostof its length;’’ however, a well-formed esophageal gland was evident onlysurrounding the posterior esophageal swelling in the specimens studiedherein.

Martin (1974) reported that, ‘‘vasa efferentia unite anterior to anteriortestis to form a relatively short vas deferens which empties into anteriorend of cirrus pouch;’’ however, the voucher specimens studied hereinclearly show that the anterior and posterior trunks of the vasa efferentiaunite at level of the ovary and between the testes. No detail regarding theoviduct of Paracardicoloides was provided by Martin (1974) but thepresent study confirms its course and connection with the ovo-vitellineduct and the presence of a prominent oviducal seminal receptacle. Martin(1974) reported that the uterus was ‘‘slightly coiled;’’ however, hisillustration shows no coil and neither voucher specimen I studied had auterine coil. The uterus does, however, turn sharply between the distal tipsof the posterior ceca (Nolan and Cribb, 2004). Also significant is thatMartin (1974) reported that the uterine eggs in his specimens were ‘‘badlycollapsed during fixation,’’ which prevented him from detailing the eggs.Herein, I provide detail of those eggs and add this feature to the diagnosisof the genus.

Apparently without access to any extant type materials or newlycollected materials of P. yamagutii, Smith’s (2002) diagnosis of monotypicParacardicoloides was based wholly on the description published byMartin (1974). Smith (2002) listed ‘‘seminal receptacle absent’’ and‘‘Laurer’s canal absent;’’ however, P. yamagutii has a proximal portion ofthe oviduct that can be laterally expanded and hold sperm (¼oviducalseminal receptacle), and the present observations also confirm the presenceof a Laurer’s canal. Martin (1974) and Smith (2002) listed the uterushaving a swollen ‘‘sausage-shaped portion’’ in their generic diagnoses ofParacardicoloides. The distal portion of the uterus (¼metraterm) of P.yamagutii evidently can appear laterally-expanded when filled with eggsbut it can also appear as a narrow duct if devoid of eggs, as in G222650.Hence, the absence of the former character state should not discount afluke’s membership to Paracardicoloides (see Martin, 1974; Smith, 2002).

I could not locate any extant type material of P. yamagutii. Theholotype (No. 7113) or other potential deposited materials of P. yamagutiithat W. E. Martin reportedly deposited in the ‘‘Hancock ParasitologyCollection’’ (also known as the ‘‘Allan Hancock Parasitology Collection’’[AHPC], which is likely no longer a cohesive collection but formerly of theUniversity of Southern California), apparently have been lost. I contactedRalph Lichtenfels, Eric Hoberg, and Pat Pilitt (United States NationalParasite Collection), Scott Gardner (Harold W. Manter Laboratory ofParasitology), Agustın Jimenez-Ruiz (University of Southern IllinoisCarbondale), Eric Hochberg (Santa Barbara Museum of Natural History,University of California), Armand Kuris and Ryan Hechinger (Universityof Santa Barbara), and Todd Huspeni (University of Wisconsin StevensPoint); all presently serve or previously served as curators or collectionmanagers of North American parasitological collections or knew W. E.Martin personally and received portions of his collection before he died.Although generously giving of their time, and helpful in responding to myqueries concerning the holotype, together we failed to locate the holotypeor any original materials of P. yamagutii lodged by Martin. Moreover, the


FIGURES 6, 7. Elopicola nolancribbi n. gen., n. sp. (Digenea: Aporocotylidae) from the blood vascular system and viscera of ladyfish, Elops saurusLinnaeus, 1766, (Elopiformes: Elopidae) from the Northern Gulf of Mexico off Ship Island and adjacent waters. Scale values aside each bar. (6) Body ofadult (specimen #1 [holotype] USNPC No. 107974) showing location of muscular anterior sucker (as), pharynx (ph), ventrolateral nerve commissure (vc),esophagus (e), nerve cord (nc), vitelline follicles (v), posterior esophageal swelling (pes), posterior ceca (pc), testicular field (tf), vasa efferentia (ve),confluence of vasa efferentia (cve), cirrus sac (cs), internal seminal vesicle (isv), metraterm (met), uterus (u), ootype (oo), oviducal seminal receptacle (osr),oviduct (ov), ovary (o); ventral view. (7) Body of adult (specimen #2 [paratype] USNPC No. 107975) showing same features as in Fig. 1; dorsal view.


FIGURES 8–13. Elopicola nolancribbi n. gen., n. sp. (Digenea: Aporocotylidae) from the blood vascular system and viscera of ladyfish, Elops saurusLinnaeus, 1766, (Elopiformes: Elopidae) from the Northern Gulf of Mexico off Ship Island and adjacent waters. Scale values aside each bar. (8) Anteriorportion of gastrointestinal system of adult showing anterior sucker (as), muscular pharynx (ph), esophagus (e), esophageal gland (eg), which is nearlyindistinct in most specimens, and posterior esophageal swelling (pes) immediately anterior to cecal bifurcation. Ventral view. (9) Juvenile specimen(collected from a juvenile ladyfish, 296 mm in fork length, captured in Davis Bayou, Mississippi, on 29 July 2006) showing features as in Fig. 8 as well astegumental spines (ts), cecal bifurcation (cb), testicular anlage (ta), ovarian anlage (oa), and excretory vesicle (ev). Ventral view. (10) Genitalia of adultspecimen (specimen #1 [holotype] USNPC No. 107974) showing testis (t), vasa efferentia (ve), confluence of vasa efferentia (cve), cirrus sac (cs), internalseminal vesicle (isv), cirrus (c), common genital pore (cgp), oviducal seminal receptacle (osr), vitelline duct (v), ootype (oo), uterus (u), metraterm (mt).Ventral view. (11) Genitalia of adult showing features as in Figure 10. Dorsal view. (12) Genitalia of adult showing features as in Figure 11. Ventral view.(13) Everted cirrus (ec) showing internal seminal vesicle (isv), cirrus sac (cs), pore (p), base of cirrus (bs), and opening (op) of sperm tube (st). Lateralview.


present disposition of the AHPC is, unfortunately, indeterminate andlikely no longer extant as a parasitological collection.

Because the holotype as well as any other specimen(s) that may havebeen part of Martin’s original type series of P. yamagutii evidently arelost, I propose to designate the voucher specimen (Queensland MuseumCollection No. G222650) collected and prepared by M. J. Nolan anddetailed in Nolan and Cribb (2004) as the neotype of P. yamagutii. Thisspecimen is ideally suited for this role because it was collected from thetype host (A. reinhardtii), was collected from Warrill Creek, a tributaryof the type locality (Brisbane River), and is of high quality, i.e., well-stained and well-mounted, and demonstrates all of the key features of thegenus as diagnosed herein. The International Code of ZoologicalNomenclature defines ‘‘neotype’’ as, ‘‘The single specimen designatedas the name-bearing type of a nominal species or subspecies when there isa need to define the nominal taxon objectively and no name-bearing typeis believed to be extant.’’ Because stability and universality are notthreatened regarding P. yamagutii, a ruling by the Commission thatwould result in the designation of a neotype is not required. It should,however, also be noted that presently there is no uncertainty about theidentity of the species, as no other similar taxa have been named;however, as more species of Paracardicoloides are described, suchuncertainty may arise.

Elopicola n. gen.(Figs. 6–19)

Diagnosis: Body flat, oval, ventrally concave, lacking posterolateralbody protuberance, anterior and posterior ends tapering approximatelyequally; tegumental body spines of juvenile specimens minute, straight,delicate, barely discernable with light microscopy, lacking recurved tip,not distributing in ventrolateral transverse rows, enveloped bytegument. Rose thorn-shaped spines absent. Nervous system withventrolateral nerve cords and commissure; ventrolateral nerve cordpaired, contiguous anteriorly and posteriorly. Anterior sucker bowl-

shaped, not comprising a spheroid anterior sucker with a medio-ventralmouth, demarcated from anterior body end by peduncle, aspinous inadult. Muscular pharynx occupying space between anterior sucker andnerve commissure. Esophagus sinuous, ventral to anterior nervecommissure, extending sinuously posteriad approximately 1/3–1/2 ofbody length, comprising morphologically distinctive anterior, middle,and posterior regions; anterior and posterior portions of esophaguscomprising distinctive swellings. Intestine typically inverse U-shaped orwith abbreviated lobes of cecum directing anteriad, with long posteriorceca, lacking diverticula or secondary rami, extending sinuouslyposteriad but lacking loops or coils, terminating in middle third ofbody. Testis single, medial, intercecal, preovarian, deeply lobed; vasaefferentia coalescing in antero-sinistral region of testis to form vasdeferens; vas deferens extending posteriad before narrowing distallyand connecting to cirrus sac. Auxiliary external seminal vesicle absent.Cirrus sac sinistral, pre-ovarian, enclosing internal seminal vesicle andcirrus; cirrus everting dorsally near midline, post-cecal. Ovary single,medial, post-cecal, post-testicular, occupying posterior body extremity.Oviduct transverse, post-cecal; oviducal seminal receptacle present.Vitellarium an extensive network of narrow, interconnecting, branchingbands having granular vitelline material, extending laterad beyondventrolateral nerve cords, occupying space from anterior nervecommissure posteriad to level of distal tips of posterior ceca; primaryvitelline duct extending posteriad near dextral body margin or slightlyventral to testis, curving mediad before uniting with oviduct to formovo-vitelline duct. Ovo-vitelline duct short, transverse, connecting withootype laterally. Ootype pre-ovarian. Uterus comprising short ascend-ing and descending portions, pre-ovarian, post-cecal; uterine eggshaving tetrahedral body, with elongate polar filaments. Metraterm pre-ovarian, medial to cirrus sac. Male and female reproductive tractsopening into common atrium and sharing a common pore; commongenital pore dorsal, sinistral, post-cecal. Excretory vesicle prominent

FIGURES 14–19. Elopicola nolancribbi n. gen., n. sp. (Digenea: Aporocotylidae) from the blood vascular system and viscera of ladyfish, Elops saurusLinnaeus, 1766, (Elopiformes: Elopidae) from the Northern Gulf of Mexico off Ship Island and adjacent waters. Scale values aside each bar. Figs. 14–17,scanning electron micrographs; Figs. 18, 19, light microscopy. (14) Body of adult specimen. (15) Anterior sucker (as) and penduncle of sucker (p).Ventral view. (16) Probable sensory cilia (*) of dorsal body surface. (17) Higher magnification view of base of probable sensory cilium (detached)showing associated spoke- and hub-like features. (18) Developing egg in gill epithelium of ladyfish showing egg body (b) and elongate polar filaments(arrows). (19) Developing egg of E. nolancribbi (arrow) aside a putatively recently-deposited egg of Didymozoidae sp. (*).


posteriorly, Y-shaped; excretory pore dorsal, subterminal. Adultsinfecting members of Elopiformes.

Differential diagnosis: Body lacking posterolateral body protuberance.Anterior sucker bowl-shaped, demarcated from anterior body end bypeduncle, aspinous. Muscular pharynx occupying space between anteriorsucker and nerve commissure. Esophagus having anterior and posteriorswellings. Intestine inverse U-shaped or with abbreviated lobes of cecumdirecting anteriad. Testis single, pre-ovarian, deeply lobed; vasa efferentiacoalescing in antero-sinistral region of testis to form vas deferens; vasdeferens extending posteriad before narrowing distally and connecting tocirrus sac. Cirrus sac present. Ovary post-cecal, post-testicular, occupyingposterior body extremity. Oviducal seminal receptacle present. Ovo-vitelline duct short, transverse, connecting with ootype laterally. Ootypepre-ovarian. Uterus pre-ovarian, post-cecal; uterine eggs having tetrahe-dral body, with elongate polar filaments. Male and female reproductivetracts opening into common atrium and sharing a common pore.

Type and only known species: Elopicola nolancribbi n. sp.Etymology: ‘‘Elopicola’’ is for the host order, Elopiformes, and the

Latin ‘‘cola’’ for inhabitant or dwelling within.

Remarks

Elopicola, Paracardicoloides, and Acipensericola are the only nominalaporocotylid genera that have the combination of an ovoid body thatlacks a posterolateral protuberance, a bowl-shaped anterior sucker,inverse U-shaped ceca, tegumental body spines (when present, injuveniles of the new species) that lack distally recurved tips, a cirrussac, an ootype occupying the region between the gonads, a commongenital atrium and pore, and a prominent excretory vesicle and excretoryarms at level of the posterior nerve confluence. In addition, although notconfirmed in Elopicola, both Paracardicoloides and Acipensericola have aLaurer’s canal. The new genus is most similar to Paracardicoloides byhaving that combination of features plus (1) relatively short posteriorceca that terminate in the middle third of the body, (2) a cirrus sac that issinistral and pre-ovarian, (3) a metraterm that is highly muscular,sinistral, and primarily post-cecal, and (4) a common genital atrium andpore that is sinistral and post-cecal. Acipensericola differs from thesegenera by having (1) elongate posterior ceca that terminate near the levelof the excretory bladder, (2) a cirrus sac that is dextral and resides atlevel of the ovary, (3) a metraterm that is absent or weakly muscular,dextral, and intercecal, and (4) a common genital atrium and pore that isdextral and intercecal.

The new genus is most easily differentiated from Paracardicoloides byhaving a single testis (Figs. 6, 7) whereas Paracardicoloides has anteriorand posterior testes (Figs. 1, 3). The new genus further differs fromParacardicoloides by morphological characters associated with thepharynx, proximal male genitalia (vasa efferentia, vas deferens, cirrussac), female genitalia (oviduct, ovo-vitelline duct, and uterus), and uterineeggs. Regarding the pharynx, the new genus has a muscular pharnyxwhich is absent in Paracardicoloides (Figs. 6, 7, 10–13). Regarding the vasaefferentia and vas deferens, Elopicola has vasa efferentia that coalesce inthe antero-sinistral region of the testis to form a vas deferens that extendsdirectly posteriad in the sinistral half of the body (Figs. 10–12). InParacardicoloides, the vasa efferentia of each testis coalesce anterior andposterior to the ovary before uniting to form a vas deferens that runsanteriad in the dextral half of the body (Figs. 1, 3). In both genera, thecirrus sac receives the vas deferens anteriorly and is located near the distalend of the sinistral posterior cecum. Regarding the oviduct and ovo-vitelline duct, the new genus has an oviduct that is principally transverseand accommodates a large oviducal seminal receptacle. It also has a short,transverse ovo-vitelline duct that connects to the ootype laterally (Figs. 6,7, 10–12). Paracardicoloides has a longitudinal oviduct that accommodatesan oviducal seminal receptacle proximal to a long ovo-vitelline duct thatextends anteriad nearly the length of the cirrus sac and connects to theootype posteriorly (Figs. 1, 3, 5). Regarding the uterus, the new genus hasa post-cecal uterus that is lateral to the testis (Fig. 6, 7) whereasParacardicoloides has an intercecal, partly pre-testicular uterus (Fig. 1).Perhaps most significantly, Elopicola has eggs bearing tendril-likefilaments (Fig. 18, 19) whereas the uterine eggs of Paracardicoloides areovoid and lack filaments (Fig. 3).

I could not confirm the presence of a Laurer’s canal in Elopicola;however, given the morphological similarities shared among this taxonand its putative close relatives, P. yamagutii and A. petersoni, both of

which have a Laurer’s canal, the presence or absence of this key featurerequires further investigation.

Elopicola nolancribbi n. sp.(Figs. 6–19)

Diagnosis of adult (measurements and illustrations based on 8 whole-mounted specimens and 2 sputter-coated specimens): Body 848–1,482 (6)long, 270–390 (6) wide, approximately 3–43 longer than wide, withmaximum width near midbody (Figs. 6, 7); dorsum bearing probablesensory cilia and sensory pores (Figs. 16, 17); sensory cilium havingproximal base affixed to body surface (Fig. 16); proximal base bearingradial, sprocket-like features originating from center (Fig. 17). Ventrolat-eral nerve cord becoming confluent with paired cord 35–50 (2) or ~3% ofbody length from posterior body end (Figs. 6, 7); anterior nervecommissure 118–165 (3) or 10% of body length from anterior body end,63–75 (2) across width of worm, 8–25 (2) in maximum width,perpendicular to midline of body; secondary branches and dorsolateralnerve cord indistinct.

Anterior sucker 25–63 (5) in diameter or 8–24% of body width, stronglymuscular, extending anteriad from anterior end of body approximately15–38 (4) or ,2% of body length or 50–80% of anterior sucker diameter;mouth a large pore opening within anterior sucker and surrounded bymuscular rim of sucker (Figs. 6–8). Esophagus ventral to nervecommissure, 310–580 (6) long or 23–39% of body length, includingmorphologically distinctive anterior (muscular pharynx), middle, andposterior (posterior esophageal swelling) portions (Figs. 6–8); anteriorportion of esophagus comprising a muscular pharynx, not extendinglaterally beyond nerve cord, between level of mouth and anterior nervecommissure, 38–85 (5) long or 10–17% of esophagus total length orapproximately 4–6% of body length, 19–30 wide (5) or approximately 1–3% of body width, 1.4–2.83 longer than wide, markedly thick-walled andmuscular, with muscular wall of comparable thickness to muscular rim ofanterior sucker (Figs. 6–9); middle portion of esophagus sinuous, thick-walled, separated from muscular pharynx by a marked constriction, 275–500 (3) long or 87–91% of esophagus total length or approximately 25–34% of body length, 18–25 (3) wide, with wall 4–10 (3) thick or 22–40% ofmiddle portion of esophagus width; posterior esophageal swellingbulbous, thick-walled, separated from medial portion of esophagus by amarked constriction, immediately anterior to cecal bifurcation, 58–78 (3)long or 14–20% of esophagus total length or approximately 5–6% of bodylength, 23–55 (5) wide, approximately 6–17% of body width, 1.1–2.73longer than wide, with wall approximately 5–10 (3) thick or 9–22% ofposterior esophageal swelling width (Figs. 6–8). Esophageal glandtypically indistinct, narrowly-enveloping esophagus from level of pharynxto cecal bifurcation, lacking birefringent rodlet-like structures, concen-trating in an area surrounding posterior esophageal swelling 47 long or15% of esophagus length, 63 wide or 1.33 wider than long. Cecalbifurcation immediately posterior to posterior esophageal swelling, 300–700 (6) or 34–47% of body length from anterior body end; abbreviatedlobes of cecum directing anteriad discernable in 3 specimens, short, 10–75(6) long or 4–14% of esophagus length, 20–88 (6) wide or 6–7% of bodywidth, comprising a seemingly superficial outpocketing of the intestine;posterior ceca slightly sinuous, arching posterolaterad from cecalbifurcation and extending posteriad approximately in parallel with lateralbody margin, containing black or brown-colored contents in lumen;dextral and sinistral posterior ceca asymmetrical, 228–420 (6) and 201–400(6) respectively, extending posteriad approximately 215–410 (6) or 20–27% of body length or 22–103% of esophagus length, 13–44 (6) inmaximum width, ending approximately 325–500 (6) or 26–45% of bodylength from posterior body end, extending laterally beyond ventrolateralnerve cord, or not (Figs. 6, 7).

Testis midpoint at level of distal tips of posterior ceca, anterior halfoccupying intercecal space, posterior extension in dextral half of body,274–375 (5) long or 19–33% of body length, 150–193 (5) wide or 38–53%of maximum body width, 1.7–2.13 longer than wide, terminating 197–370(5) or 19–29% of body length from posterior body end; testis lobes 30–60(2) long, 28–45 (2) wide, 1.1–1.333 longer than wide; vasa efferentiadifficult to trace in fixed specimens, an interconnecting meshwork of fineducts entwining throughout testicular tissue, 5–13 (3) wide, containingsperm in all specimens; vas deferens extending a short distance posteriadbefore meeting cirrus sac and internal seminal vesicle, 24–80 (4) long or 2–7% of body length, containing sperm in all specimens (Figs. 6–9, 10–12).Cirrus sac 150–192 (2) long, 18–58 (2) wide, 2.6–3.43 longer than wide,


with wall 3 (2) thick, enclosing well-delineated internal seminal vesicle,residing between anterior testis and sinistral nerve cord; internal seminalvesicle occupying breadth and length of cirrus sac to varying degreesdepending on amount of sperm present in duct, directing posteriad andorienting parallel with long axis of body, 105–213 (6) long or 2.1–5.73 vasdeferens length, 47–68 (6) in maximum width (Figs. 6, 7, 10–13); cirruseverting dorsally near midline or slightly sinistral, with cylindricalproximal base 83 (1) long and 28 (1) wide plus a finger-like distal portion192 long and 57 wide, with sperm tube coursing near ventral surface,directing dextrad and extending from common genital pore to beyondlateral body margin, lacking spines (Fig. 13).

Ovary a loose aggregation of probable ova, immediately anterior toposterior nerve confluence, 100–125 (5) long or 7–10% of body length,100–160 (6) wide or 26–41% of maximum body width, 0.8–1.33 longerthan wide (Figs. 6, 7, 10–12); post-ovarian space 50–165 (4) long or 4–11%of body length. Oviduct S-shaped, extending anteriad from anteriormargin of ovary, 141–281 (5) long; oviducal seminal receptacle a thin-walled sac containing sperm in all specimens, 75–100 (2) long or 45–71%of oviduct length, 13–23 (2) wide, 3–83 longer than wide, varying in lengthand width depending on amount of sperm in duct (Figs. 6, 7, 10–12).Laurer’s canal not observed in specimens studied. Primary vitellinecollecting duct dextral, extending 115–325 (4) posteriad and coursingbetween testis and dextral body margin or dorsal to dextral margin oftestis, 7–25 (5) in maximum width, curving mediad at level of posteriormargin of testis, with distal portion coursing dorsal to and in parallel withoviducal seminal receptacle, uniting distally with oviducal seminalreceptacle immediately before forming ovo-vitelline duct (Figs. 6, 7, 10–12); ovo-vitelline duct a short duct formed by union of primary vitellineduct and distal portion of oviducal seminal receptacle, 13–33 (2) long or 9–20% of oviduct length, 8–10 (2) in maximum width, connecting withootype laterally (Figs. 10, 11); ootype difficult to delineate fromsurrounding tissue in most specimens, elongate (not spheroid), post-cecal,slightly posterior to or at level of posterior margin of testis, posterior tocirrus sac, 16–38 (3) long, 18 (3) wide, or 0.9–2.13 longer than wide, 175–230 (2) or 17–20% of body length from posterior body end; Mehlis’ glandindistinct in fixed material. Uterus extremely short, curving once;ascending uterus extending 50–75 (3) anterosinistrad from ootype, lessthan or equal to oviducal seminal receptacle length, 15–25 (3) in maximumwidth, with cuboidal cells lining lumen in some specimens, curving mediadbefore connecting with metraterm posterior to or at level of cirrus sac;descending uterus absent (Figs. 6, 7, 10–12). Metraterm between testis andcirrus sac, nearly medial, 65–115 (3) long or 4–11% of body length, 20–28(3) wide or 2.8–4.63 longer than wide, with muscular wall; metraterm eggshaving body 13–18 (3) long, each bearing 2 tendril-like filamentsmeasuring 8–38 (3) long or 44–253% of egg body length, with thinmembranous shell. Common genital pore 16–25 (2) in diameter, opening247–350 (5) or 20–29% of body length from posterior body end. Eggslodged in gill epithelium of ladyfish bearing elongate polar filaments withdistally recurved ends, enveloping ciliated miracidium (Figs. 18, 19).

Excretory system difficult to observe in fixed specimens; excretoryvesicle oblong, 34–64 (2) long or 3–7% of body length, 13–46 (2) wide;excretory arms each 63–90 (2) long, 8–29 (2) wide (Figs. 7, 11, 12).

Diagnosis of juvenile (based on 1 whole-mounted specimen): Body 970long, 160 wide or 63 longer than wide, bearing 80 embedded spines alongeach lateral body margin or totaling 160 spines (Fig. 9); tegumental bodyspines straight, lacking recurved distal tips, orienting at 458 angleposteriad (Fig. 9). Nervous system not evident.

Anterior sucker 35 in diameter, 30% of body width (Fig. 9). Esophagus305 long or 31% body length, 5 wide and having wall 3 thick near mouth,13 wide and having wall 5 thick near cecal bifurcation; anterior portion ofesophagus comprising a muscular pharynx, 53 long or 17% esophaguslength or approximately 5% of body length, 20 wide; posterior esophagealswelling resembling adult, 38 long or 12% of esophagus length, 18 wide.Cecal bifurcation 380 or 39% of body length from anterior end; posteriorceca 170 and 163 long or approximately 17% of body length orapproximately equal to esophagus length, 20 and 15 wide, ending 440 or45% of body length from posterior body end.

Testicular anlage 175 long or 18% of body length, 28 wide or 18% ofbody width, 6.33 longer than wide, terminating 280 or 29% of body lengthfrom posterior body end (Fig. 9). Probable cirrus anlage 30 long, 13 wideor 2.33 longer than wide, seemingly everted through a pore 350 or 36% ofbody length from posterior body end.

Ovarian anlage loosely formed in posterior body end, 45 or 5% of bodylength from posterior body end (Fig. 9).

Taxonomic summary

Type and only known host: Elops saurus Linnaeus, 1766 (Elopiformes:Elopidae), ladyfish.

Site in host: Indeterminate. Adults and juveniles in sediment derivedfrom rinsing macerated head, trunk, and body cavity; mature eggsembedded in gill epithelium, lodged beneath gill filament capping tissue.

Type locality: Ship Island (308120530 0N; 888560140 0W), Mississippi,Northern Gulf of Mexico. Other locality: Horn Island (308150040 0N;888420420 0W), Mississippi, Northern Gulf of Mexico.

Specimens deposited: Holotype USNPC No. 107974. Paratype USNPCNo. 107975.

Prevalence of infection: Thirty-one of 60 (52%) ladyfish off Mississippiwere infected with eggs or adults of Elopicola nolancribbi: 21 of 60 (35%)ladyfish were positive for eggs in gill but adults could not be collected; 10of 60 (17%) ladyfish were positive for eggs in gill as well as having adultsor the juvenile of Elopicola nolancribbi.

Etymology: The specific name ‘‘nolancribbi’’ is a conjoined patronymhonoring Matthew J. Nolan (Centre for Biodiscovery and MolecularDevelopment and Therapeutics, James Cook University) and Thomas H.Cribb (The University of Queensland) for their contributions to thetaxonomy of fish blood flukes in the southwest Pacific Ocean.

DISCUSSION

The taxonomic diversity of blood flukes among elopomorphs

has probably been underestimated and additional species

probably remain to be discovered and described in Elopidae (6

species of Elops), Megalopidae (2 species of Megalops),

Albulidae (3 species of Albula), Halosaridae (3 genera, 15

species), Notacanthidae (3 genera, 10 species), and especially

Anguilliformes (15 families, 141 genera, 791 species) (Nelson,

2006). The importance of examining additional elopomorphs for

the presence of blood fluke infections, discovering and describ-

ing new taxa, and assessing their morphological features is

underscored by the fact that the new species described herein is

clearly morphologically most similar to another elopomorph

blood fluke. This is highly suggestive that phylogenetically-

related fishes harbor phylogenetically-related aporocotylids, and

the results of the present study indicate to us that basal fish

lineages (sensu Nelson, 2006) such as Elopomorpha and

Acipenseriformes harbor morphologically similar fish blood

flukes that likely share a recent common ancestor. In specific,

Paracardicoloides yamagutii (see Martin, 1974; Hine, 1978;

Nolan and Cribb, 2004; present study), Acipensericola spp.

(blood flukes of North American sturgeons [Acipenser spp.] and

paddlefish [Polyodon spathula], Acipenseriformes; see Appy and

Dadswell, 1978; Bullard et al., 2008), and Elopicola nolancribbi

(present study) have a bowl-shaped anterior sucker, robust,

spike-like tegumental spines that lack recurved tips (if present),

an anterior esophageal swelling or muscular pharynx, U-shaped

intestine (intestine lacks anterior ceca), Laurer’s canal (needs

confirmation in E. nolancribbi), an inter-gonadal ootype, and a

common genital atrium and pore. The aporocotylids infecting

higher bony fishes (Euteleostei) differ from Paracardicoloides,

Acipensericola, and Elopicola most notably by lacking a bowl-

shaped anterior sucker, pharynx, and Laurer’s canal and by

typically having ventrolateral transverse rows of minute

tegumental spines each having a recurved tip (if present), an

X- or H-shaped intestine, separate genital pores, and a post-

gonadal ootype. Noteworthy also is that the anterior sucker of

Paracardicoloides, Acipensericola, and Elopicola, although


unique among aporocotylids of these genera, is not unlike that

observed in some turtle blood flukes (paraphyletic ‘‘Spirorchii-

dae’’ [see Snyder, 2004]). This type of sucker is markedly distinct

from the sucker that has been described in some aporocotylids

infecting members of Euteleostei, many of which have a spheroid

anterior sucker armed with concentric rows of minute spines

anterior to a pore-like, sub-terminal mouth located near the

sucker’s equator, e.g., species of Cardicola Short, 1953 and

Elaphrobates Bullard and Overstreet, 2003.

Rarely are blood fluke eggs in the gill of fishes drawn,

photographed, or described in the text of taxonomy papers

dealing with aporocotylids; however, morphological features of

the fully developed eggs of aporocotylids in the uterus,

metraterm, or definitive host’s gill epithelium may comprise

informative phylogenetic or generic features. For example,

Acipensericola and Paracardicoloides have large, ovoid uterine

eggs (Bullard and Overstreet, 2002, 2003; Bullard et al., 2008;

Bullard and Jensen, 2008) whereas the new species has an egg

that has elongate filaments in the fluke’s uterus as well as in the

fish’s gill epithelium. That the egg is well-formed in the uterus

seems to be a feature common to blood flukes of basal fishes, and

those of fishes of Euteleostei seemingly lack well-formed uterine

eggs.

Anguilla spp. are catadromous, infections of P. yamagutii are

known only from freshwater localities, and the molluscan

intermediate host for P. yamagutii is a freshwater snail (Martin,

1974; Nolan and Cribb, 2004). As such, the present study is the

first report of a blood fluke from an elopomorph captured in

high salinity marine waters. Similar to eels of Anguilla, juveniles

of Elops saurus are common in low salinity or freshwater areas.

They are commonly encountered off beaches, in the marshes,

and in rivers of the Gulf of Mexico where they can tolerate

freshwater and low salinity waters; however, as adults they are

abundant offshore and in high salinity waters of open sand bar

flats common around barrier islands. The juvenile specimen of

E. nolancribbi I describe herein was collected from a juvenile

ladyfish (296 mm in fork length) captured in Davis Bayou,

Mississippi, a few meters from shore, on 29 July 2006. The gill of

this infected fish harbored putative eggs of E. nolancribbi but no

adult specimen was collected from this infected ladyfish.

Discovery of these eggs in the gill indicates that the infected

fish harbored adult flukes at some point, either previously or at

necropsy, taking into account the possibility that I missed adults

during necropsy. Moreover, the presence of the juvenile

specimen indicates that this ladyfish was infected relatively

recently—both of which are suggestive that the life cycle of this

fluke, like that of P. yamagutii, occurs (i.e., cercariae infect the

definitive fish host) nearshore or in low salinity waters, perhaps

within a nearby river. Given that no aporocotylid life cycle has

been experimentally or molecularly demonstrated in the Gulf of

Mexico Basin, further searches and investigations on E.

nolancribbi in ladyfish and hydrobiids from this region could

be exciting and fruitful.

ACKNOWLEDGMENTS

I thank Michael Miller (Department of Biological Sciences, AuburnUniversity) for SEM assistance; Eric Hoberg and Pat Pilitt (USNPC,Beltsville, Maryland) for ensuring the safe deposition of our type materialsand for loaning type and voucher aporocotylids; Mal Bryant and RobAdlard (Queensland Museum, Queensland, Australia) for loaning the

voucher specimens of P. yamagutii; and, especially, Matt Nolan and TomCribb for contributing the invaluable voucher specimens of P. yamagutiito the helminthology community. This is a contribution of theSoutheastern Cooperative Fish Parasite and Disease Project (School ofFisheries, Aquaculture, and Aquatic Sciences, Auburn University) andwas supported in part by the National Science Foundation’s Division ofEnvironmental Biology with funds from NSF-DEB grants 1112729,1051106, and 1048523 to S.A.B.

LITERATURE CITED

APPY, R. G., AND M. J. DADSWELL. 1978. Parasites of Acipenserbrevirostrum LeSueur and Acipenser oxyrhynchus Mitchill (Osteich-thyes: Acipenseridae) in the Saint John River Estuary, N. B., with adescription of Caballeronema pseudoargumentosus sp. n. (Nematoda:Spirurida). Canadian Journal of Zoology 56: 1382–1391.

BULLARD, S. A., AND K. JENSEN. 2008. Blood flukes (Digenea: Apor-ocotylidae) of stingrays (Myliobatiformes: Dasyatidae): Orchispiriumheterovitellatum from Himantura imbricata in the Bay of Bengal andMyliobaticola richardheardi n. gen., n. sp. from Dasyatis sabina in theNorthern Gulf of Mexico. Journal of Parasitology 94: 1311–1321.

———, AND R. M. OVERSTREET. 2002. Potential pathological effects ofblood flukes (Digenea: Sanguinicolidae) on pen-reared marine fishes.Proceedings of the 53rd Gulf and Caribbean Fisheries Institute(2002): 10–25.

——— AND ———. 2003. Elaphrobates euzeti gen. and sp. n. (Digenea:Sanguinicolidae) from snappers (Lutjanidae) in the Gulf of Mexico.In Taxonomie, ecologie et evolution des metazoaires parasites.Taxonomy, ecology and evolution of metazoan parasites. (Livrehommage a Louis Euzet). Tome 1, C. Combes and J. Jourdane (eds.).PUP, Perpignan, France, p. 97–113.

———, S. D. SNYDER, K. JENSEN, AND R. M. OVERSTREET. 2008. Newgenus and species of Aporocotylidae (Digenea) from a loweractinopterygian, the American paddlefish, Polyodon spathula, (Aci-penseriformes: Polyodontidae) from the Mississippi Delta. Journal ofParasitology 94: 487–495.

FOREY, P. L., D. T. J. LITTLEWOOD, P. RITCHIE, AND A. MEYER. 1996.Interrelationships of elopomorph fishes. In Interrelationships offishes, M. L. J. Stiassny, R. Parenti, and G. D. Johnson (eds.).Academic Press, San Diego, California, p. 175–191.

GREENWOOD, P. H., D. E. ROSEN, S. H. WEITZMAN, G. S. MEYERS. 1966.Phyletic studies of teleostean fishes, with a provisional classificationof living forms. Bulletin of the American Museum of Natural History131: 339–456.

HINE, P. M. 1978. Distribution of some parasites of freshwater eels in NewZealand. New Zealand Journal of Marine and Freshwater Research12: 179–187.

INOUE, J. G., M. MIYA, K. TSUKAMOTO, AND M. NISHIDA. 2004. Mitogenicevidence for the monophyly of elopomorph fishes (Teleostei) and theevolutionary origin of the leptocephalus larva. Molecular Geneticsand Evolution 32: 274–286.

MARTIN, W. E. 1974. Paracardicoloides yamagutii gen. et sp. n. from anAustralian eel (Trematoda: Sanguinicolidae). Proceedings of theHelminthological Society of Washington 41: 22–25.

NELSON, J. S. 2006. Fishes of the world, 4th ed. John Wiley and Sons, Inc.,New York, New York, 601 p.

NOLAN, M. J., AND T. H. CRIBB. 2004. The life cycle of Paracardicoloidesyamagutii Martin, 1974 (Digenea: Sanguinicolidae). Folia Para-sitologica 51: 320–326.

PREARO, M., E. BOZZETTA, D. PATTONO, AND C. GHITTINO. 1995.Segnalazione di sanguincolosi in anguille d’allevamento. BollettinoSocieta Italiana di Patologia Ittica 7: 22–25.

ORELIS-RIBEIRO, R., C. F. RUIZ, S. S. CURRAN, AND S. A. BULLARD. 2013.Blood flukes (Digenea: Aporocotylidae) of epipelagic lamniforms:Redescription of Hyperandrotrema cetorhini from basking shark(Cetorhinus maximus) and description of a new congener fromshortfin mako shark (Isurus oxyrinchus) off Alabama. Journal ofParasitology 99: 835–846.

SMITH, J. W. 1997a. The blood flukes (Digenea: Sanguinicolidae andSpirorchidae) of cold-blooded vertebrates: Part 1. A review of thepublished literature since 1971, and bibliography. HelminthologicalAbstracts 66: 255–294.


———. 1997b. The blood flukes (Digenea: Sanguinicolidae and Spirorch-idae) of cold-blooded vertebrates: Part 2. Appendix I: Comprehensiveparasite-host list; Appendix II: Comprehensive host-parasite list.Helminthological Abstracts 66: 329–344.

———. 2002. Family Sanguinicolidae von Graff, 1907. In Keys to theTrematoda, Vol. 1, D. I. Gibson, A. Jones, and R. A. Bray (eds.).

CABI Publishing and The Natural History Museum, Wallingford,

U.K., p. 433–452.

SNYDER, S. D. 2004. Phylogeny and paraphyly among tetrapod blood

flukes (Digenea: Schistosomatidae and Spirorchiidae). International

Journal for Parasitology 34: 1385–1392.


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