First Record of Transversotrema Witenberg, 1944 (Digenea)...

J. Parasitol., 101(6), 2015, pp. 717–725

� American Society of Parasitologists 2015

First Record of Transversotrema Witenberg, 1944 (Digenea) from the Americas, with

Comments on the Taxonomy of Transversotrema patialense (Soparkar, 1924) Crusz and

Sathananthan, 1960, and an Updated List of Its Hosts and Geographic Distribution

Matthew R. Womble, Stephanie J. Cox-Gardiner, Thomas H. Cribb*, and Stephen A. Bullard

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

ABSTRACT: Specimens of Transversotrema patialense (sensu lato)

(Soparkar, 1924) Crusz and Sathananthan, 1960 (Digenea:

Transversotrematidae) infected the skin (epidermal spaces beneath scales

near pectoral fins) of 4 of 126 (prevalence 3%; mean intensity 1.8)

zebrafish (Danio rerio (Hamilton, 1822) [Cypriniformes: Cyprinidae])

purchased in 2009 and cultured by a California (USA) fish supplier. These

fish were sold as ‘‘laboratory-reared’’ and ‘‘specific pathogen free,’’

purportedly raised in a recirculating aquaculture system that included

zebrafish only. We herein describe the morphological features of this

transversotrematid using light and scanning electron microscopy, provide

a comprehensive list of hosts (snails and fishes) and geographic locality

records for specimens reported as T. patialense, which is perhaps a species

complex, and provide a brief historical synopsis of the taxonomic and life

history research that has been conducted on this fluke. No species of

Transversotrema previously had been reported from the Americas;

however, this discovery is not surprising given that: (1) a suitable

intermediate host (red-rimmed melania, Melanoides tuberculata (Muller,

1774) [Cerithioidea: Thiaridae]) has been established in California and

elsewhere in North America, (2) the zebrafish is a susceptible definitive

host, and (3) T. patialense reportedly matures on a broad ecological and

phylogenetic spectrum of freshwater fishes. To our knowledge, this is the

northern-most geographic locality record for a species of this genus. We

suspect this case study represents an example of a parasite that may now

be established in North America by the fortuitous co-occurrence of a

susceptible, exotic snail host (the red-rimmed melania) and a susceptible,

widely distributed, exotic fish host (the zebrafish).

Transversotrema Witenberg, 1944 (Digenea: Transversotrematidae)

comprises digeneans that infect the epidermal surfaces beneath and

between overlapping cycloid and ctenoid scales of freshwater and marine

bony fishes (Actinopterygii). Published host and geographic locality

records for species of Transversotrema that infect freshwater fishes are

more abundant in the literature than they are for marine species, but those

reports total many fewer parasite species. The genus presently includes 25

accepted species (Cribb et al., 2014), with all but 6 nominal species

infecting marine bony fishes of the southwest Pacific Ocean (Cribb et al.,

1992, 2014; Hunter and Cribb, 2012; Hunter et al., 2012). Trans-

versotrematids are morphologically unique in having adults with an

extremely dorsoventrally flattened body that is markedly broader than

long (transversely elongate), lacking a demonstrable oral sucker, and

possessing a diminutive ventral sucker; features that are presumably

adaptive for life beneath and between the scales of bony fishes.

Transversotrematids are unique also in having a cercarial body that

closely resembles that of the adult (similar to some azygiids [Womble et

al., 2015]) and that directly attaches to the external body surface of the

definitive host as well as having well-developed genitalia prior to infecting

the fish host. No life cycle for any member of the genus is known to

include a second intermediate host.

Transversotrema patialense (sensu lato) (Soparkar, 1924) Crusz and

Sathananthan, 1960, which likely comprises a species complex (Cribb,

1988), is the most wide-ranging freshwater species of the genus (Table I). It

undergoes asexual reproduction in a snail (i.e., 3 species of Melanoides

Olivier, 1804 [Cerithioidea: Thiaridae] and Sermyla riquettii (Grateloup,

1840), with most records sourcing from red-rimmed melania, Melanoides

tuberculata (Muller, 1774)) and matures in 43 species of freshwater and

euryhaline bony fishes assigned to 33 genera, 16 families, and 8 orders that

range in the Philippines, India, Australia, Japan, Sri Lanka, Thailand,

Malyasia, the Solomon Islands, Zaire, and Israel (Table I). Available

evidence suggests that T. patialense exhibits high specificity to the snail

host and low specificity to the fish host. However, no morphological and

molecular study has compared specimens and/or sequences of ‘‘T.

patialense’’ from across the diversity of these fish hosts and geographic

localities. In addition, and relevant to the identity of T. patialense,

apparently no type material exists.

The taxonomy of T. patialense and its congeners is interesting because

their unique life cycles initially caused confusion and intrigue among the

first investigators who studied them. Prior to the first description of an

adult specimen of a species of Transversotrema, i.e., Transversotrema haasi

Witenberg, 1944, early reports focused on morphological descriptions of

cercariae shed from red-rimmed melania. These specimens were assigned

to the collective genus group name Cercaria (e.g., ‘‘Cercaria patialensis’’

[see Soparkar, 1924]), and most researchers took note of the unusually

well-developed genitalia of the cercaria (Soparkar, 1924; Miller, 1926;

Witenberg, 1944; Olivier, 1947; Anantaraman, 1948). Witenberg (1944)

first described the adult of a species of Transversotrema. Realizing that his

specimens were adults by the presence of uterine eggs and well-developed

genitalia, but apparently not realizing their morphological similarity to C.

patialensis of Soparkar (1924), he assigned the new species to Trans-

versotrema and diagnosed the Transversotrematinae Witenberg, 1944,

which he assigned to the Mesometridae Poche, 1926, based on the

presence of a cyclocoel and ventral sucker as well as the unique

arrangement of the excretory system. Velasquez (1958, 1961) conducted

morphological and life history studies with T. patialense (as Trans-

versotrema laruei Velasquez, 1958) but maintained that fish were infected

by unencysted metacercariae, not adults. Olivier (1947), Anantaraman

(1948), Yamaguti (1953), and Velasquez (1958) acknowledged that

specimens from snails (C. patialense and Cercaria koliensis) and from

fishes (T. haasi, T. laruei) were likely congeners, but Crusz and colleagues

were the first to specifically state that ‘‘Cercaria patialensis’’ was a

progenetic larval form of a species of Transversotrema (see Crusz and

Sathananthan, 1960) and that its life cycle involved a snail and a fish only

(Crusz et al., 1964). Subsequently, the collective genus group name

‘‘Cercaria,’’ as applied to transversotrematid cercariae, ceased to be used.

As an aside and also regarding nomenclature, Witenberg (1944) did not

formally diagnose Transversotrema nor specify a type species. Articles

11.5.2., 13.1., 13.3., and 13.4. of the International Code for Zoological

Nomenclature (5th ed.) indicate that the genus group name Trans-

versotrema, therefore, was not made available by Witenberg (1944).

Yamaguti (1953) diagnosed Transversotrema, fixed the type species, and

could be regarded as the taxonomic authority for Transversotrema.

However, doing so could create confusion where none exists now;

* School of Biological Sciences, The University of Queensland, Brisbane,Queensland, 4072 Australia.

DOI: 10.1645/15-799

717

TABLE I. Wild hosts for Transversotrema patialense (Soparkar, 1924) Crusz and Sathananthan, (1960).

Fish hosts Locality Reference

Elopiformes

Megalopidae

Megalops cyprinoides Malabon, Philippines Velasquez (1961; as Transversotrema laruei)*

Clupeiformes

Clupeidae

Anodontostoma chacunda Malabon, Philippines Velasquez (1961; as T. laruei)*

Cypriniformes

Cyprinidae

Amblypharyngodon mola North India Pande and Shukla (1972; as Transversotrema

soparkari)*

Barbodes binotatus (as Puntius binotatus) Penang, Malaysia Leong (1988)

Barbus sp. (as ‘‘Barbus puntius’’) Trivandrum, India Mohandas (1973; as Transversotrema chackai)*

Cirrhinus reba North India Pande and Shukla (1972; as T. soparkari)*

Discherodontus ashmeadi Chiang Mai, Thailand Wongsawad et al. (2004)

Esomus danricus Waltair (Andhra Pradesh), India Hanumantha Rao and Ganapati (1967)

ns Madhavi and Jhansilakshmibai (1994)†

Danio albolineatus (as Brachydanio albolineatus) Penang Island, Malaysia Betterton (1979)

Danio rerio (as Brachydanio rerio) ns Whitfield et al. (1975)†; Anderson et al. (1977)†; Mills

(1979)†; Mills et al. (1979)†

Danio rerio‡ California, USA Present study

Gibelion catla (as Catla catla) Samalkot, India Hanumantha Rao and Ganapati (1967)†

Puntius chola North India Pande and Shukla (1972; as T. soparkari)*

Puntius sophore North India Pande and Shukla (1972; as T. soparkari)*

Rasbora argyrotaenia Chiang Mai, Thailand Wongsawad et al. (2004)

Rasbora sumatrana Penang Island, Malaysia Betterton (1979)

Penang, Malaysia Leong (1988)

Systomus orphoides Chiang Mai, Thailand Wongsawad et al. (2004)

Systomus sp. (as ‘‘Systomus stoliezkae’’) Chiang Mai, Thailand Wongsawad et al. (2004)

Cyprinodontiformes

Aplochelidae

Aplocheilus panchax (as Panchax panchax) Waltair, India Hanumantha Rao and Ganapati (1967); Murty and

Hanumantha Rao (1968)

Penang Island, Malaysia Betterton (1979)

Aplocheilus panchax Penang, Malaysia Leong (1988)

Visakhpatnam, India Vasantha and Hanumantha Rao (1989)

ns Madhavi and Jhansilakshmibai (1994)†

Poecilidae

Gambusia affinis‡ Jerusalem, Israel Ben-Ami et al. (2005)†

Gambusia sp. (as ‘‘Gambusia striatus’’) Wongsawad et al. (2004)

Poecilia latipinna (as Mollienesia latipinna) Malabon, Philippines Velasquez (1961; as T. laruei)‡

Mugiliformes

Mugilidae

Liza macrolepis Visakhapatnam, India Rekharani and Madhavi (1985)

Liza subviridis Queensland, Australia Cribb et al. (1992)

Moolgarda cunnesius (as Valamugil cunnesius) Visakhapatnam, India Rekharani and Madhavi (1985)

Moolgarda seheli (as Valamugil seheli) Queensland, Australia Cribb et al. (1992)

Mugil cephalus Visakhapatnam, India Rekharani and Madhavi (1985)

Mugil sp. Malabon, Philippines Velasquez (1961; as T. laruei)*

Beloniformes

Hemiramphidae

Rhynchorhamphus georgii (as Hemiramphus

georgii)

Malabon, Philippines Velasquez (1961; as T. laruei)*

Synbranchiformes

Mastacembelidae

Macrognathus siamensis Chiang Mai, Thailand Wongsawad et al. (2004)

Perciformes

Latidae

718 THE JOURNAL OF PARASITOLOGY, VOL. 101, NO. 6, DECEMBER 2015

TABLE I. Continued.

Fish hosts Locality Reference

Lates calcarifer Malabon, Philippines Velasquez (1958, 1961; as T. laruei)*

Nandidae

Nandus nandus North India Pande and Shukla (1972; as T. soparkari)*

Lucknow, India Agrawal and Singh (1981; as Transversotrema

chauhani)*

Tetrapontidae

Terapon argenteus Malabon, Philippines Velasquez (1961; as T. laruei)*

Cichlidae

Oreochromis mossambicus (as Tilapia

mossambica)

Malabon, Philippines Velasquez (1961; as T. laruei)*†

Batalagoda, Sri Lanka Crusz et al. (1964)

Oreochromis mossambicus‡ Ishigakijima Island, Japan Maneepitaksanti and Nagasawa (2012)

Coptodon zillii (as Tilapia zillii) Jerusalem, Israel Ben-Ami et al. (2005)†

Scatophagidae

Scatophagus argus Malabon, Philippines Velasquez (1961; as T. laruei)*

Helostomatidae

Helostoma temminckii (as Helostoma temincki) Nakhonsithammarat, Thailand Lerssutthichawal (2008; as Transversotrema partialense)

Osphronemidae

Betta pugnax Penang Island, Malaysia Betterton (1979)

Pseudosphromenus cupanus (as Macropodus

cupanus)

Batalagoda, Sri Lanka Crusz and Sathananthan (1960); Crusz et al. (1964)

Trivandrum, India Mohandas (1973; as T. chackai)*

Pseudosphromenus dayi (as Macropodus cupanus

var. dayi)

Trivandrum, India Mohandas (1973; as T. chackai)*

Trichopodus pectoralis (as Trichogaster

pectoralis)

Nakhonsithammarat, Thailand Lerssutthichawal (2008; as T. partialense)

Trichopodus trichopterus (as Trichogaster

trichopterus)


Penang , Malaysia Leong (1988)

Trichopsis vittata (as Trichopsis vittatus) Chiang Mai, Thailand Wongsawad et al. (2004)

Channidae

Channa gachua Chiang Mai, Thailand Wongsawad et al. (2004)

Channa punctata (as Ophicephalus punctatus) Batalagoda, Sri Lanka Crusz et al. (1964)

Channa punctata (as Channa punctatus) North India Pande and Shukla (1972; as T. soparkari)*

Snail Hosts

Melanoides anomala Zaire, Africa Brien (1954)

‘‘Melanoides scabra’’ Kerala, India Nadakal et al. (1969; as Cercaria chackai)*

Melanoides tuberculata§jj Punjab, India Soparkar (1924; as Cercaria patialensis)

Madras, India Anantaraman (1948; as C. patialensis)

Peradeniya, Sri Lanka Crusz (1956; as C. patialensis)

Colombo, Sri Lanka Crusz and Sathananthan (1960)

Batalagoda, Sri Lanka Crusz and Sathananthan (1960); Crusz et al. (1964)

Waltair, India Hanumantha Rao and Ganapati (1967)

Kerala, India Nadakal et al. (1969; as C. chackai)*

Lucknow, India Pandey (1971; as Cercaria soparkari)*


ns Mills et al. (1979)†

Maharashtra, India Deoray (1988)

Penang, Malaysia Leong (1988)

Thailand Krailas et al. (2014; as T. laruei)*

Melanoides tuberculata (as ‘‘Melanoides terebra

(?)’’)

Guadalcanal, Solomon Islands Olivier (1947; as Cercaria koliensis)*

Melanoides tuberculata (as Thiara tuberculata) Visakhparnam, India Vasantha and Hanumantha Rao (1989)

ns Madhavi and Jhansilakshmibai (1994)

Sermyla riquettii (as Thiara riquettii) Malabon, Philippines Velasquez (1961; as T. laruei)*

* Reported as a synonym of T. patialense; see Cribb et al. (1992).† Experimental infection only.‡ Introduced species in reported locality.§ Type host.jj Frequently reported as ‘‘Melanoides tuberculatus.’’ns¼Not specified.

SHORT COMMUNICATIONS 719

moreover, Witenberg and Yamaguti clearly were not in disagreement

about the concept of the genus nor who proposed it.

Cribb et al. (1992) regarded Transversotrema koliensis, T. laruei,

Transversotrema chackai, and Transversotrema soparkari as junior

subjective synonyms of T. patialense based on their similar body shape,

arrangement of gonads, and distribution of vitelline follicles. They

emphasized that absolute measurements unreliably differentiate Trans-

versotrema spp. because fluke body size can depend on fish host body size

(Cribb, 1988). Cribb and colleagues also noted that records for these

species were collectively sourced from a wide geographic area and from a

wide diversity of fish hosts. As such, they indicated the provisional nature

of these synonymies pending studies of newly collected material. Hunter et

al. (2010), Hunter and Cribb (2012), and Cribb et al. (2014) described

several new species of Transversotrema from marine teleosts based on

morphological evidence in the light of ITS2 sequence differences.

We herein describe the morphological features of several transverso-

trematid specimens that infected several experimental zebrafish, Danio

rerio (Hamilton, 1822) (Cypriniformes: Cyprinidae) purchased from a fish

dealer in California.

Several hundred allegedly laboratory-reared zebrafish were purchased

and shipped to our laboratory from a California-based fish supply

company. These fish were intended for use as experimental subjects in

pathobiology trials employing immersion challenge with the freshwater

bacterium Flavobacterium columnare, the etiological agent of ‘‘columnaris

disease’’ (Bullard et al., 2011). At challenge, fish were refractive to showing

signs of disease and exhibited unusually low mortality. Subsequently, 126

zebrafish from this batch were euthanized and necropsied with the aid of a

dissecting microscope, whereupon several trematodes were discovered

infecting the space beneath the scales (4 of 126 [3%] zebrafish infected by

12 digenean specimens [mean intensity ¼ 1.8]). These specimens were

removed alive from the fish using fine forceps, heat-killed with freshwater

heated to 60 C, immediately fixed in and held in 10% neutral buffered

formalin for 48 hr, placed overnight in distilled water, stained overnight in

Van Cleave’s hematoxylin with several additional drops of Ehrlich’s

hematoxylin, made basic in 70% ethanol with lithium carbonate and

butyl-amine, dehydrated, cleared in clove oil, and permanently mounted

on glass slides using Canada balsam. Illustrations of stained, whole-

mounted specimens were made with the aid of a Leica DM-2500

microscope (Leica Microsystems, Buffalo Grove, Illinois) equipped with

differential interference contrast optical components and a drawing tube.

Measurements are herein reported in micrometers (lm), followed by their

mean and number of specimens measured in parentheses. After all

specimens were measured and studied using light microscopy, 4 specimens

intended for scanning electron microscopy (SEM) were demounted by

soaking slides in xylene overnight, rinsed in 100% EtOH, critical point

dried in liquid CO2, mounted on standard aluminum SEM pin stubs with

double-sided carbon tape, sputter-coated with gold palladium (19.32g/

cm3; 25 mA), and viewed with a Zeiss EVO 50VP scanning electron

microscope (Carl Zeiss, Munich, Germany). Whole-mounted voucher

specimens are deposited in the U.S. National Museum, Smithsonian

Institution, Washington D.C. Fish scientific names, taxonomic authori-

ties, and dates for fish taxa follow Eschmeyer (electronically accessed 13

May 2015) and Eschmeyer and Fong (electronically accessed 5 May 2015).

Higher-level fish classification and nomenclature follows Nelson (2006).

Results from light and scanning electron microscopy of 12 specimens of

T. patialense (USNM Coll. Nos. 1283177, 1283178) follow (Figs. 1–15).

Body transversely elongate, 400–530 (461; 12) long at level of midbody or

41–52% (47%; 12) of maximum body width, 245–390 (297; 11) long

sinistrally at level of vitelline margin, 255–390 (313; 12) long dextrally at

level of vitelline margin, 820–1,060 (967; 12) in maximum body width;

with eyespots (Fig. 1) and ventral sucker (Figs. 1–5), demonstrable oral

sucker lacking (Fig. 6); anterior body margin relatively straight, having

delineated velum (flap) along its entire breadth anteriorly (Fig. 3);

posterior body margin markedly more curved, slightly indented medially

(where cercarial tail was attached) (Figs. 1–3). Eyespots paired, equal in

size, immediately anterior to or at level of ventral sucker, 15–33 (23; 12) in

diameter, spaced approximately 133–195 (158; 12) or 14–20% (16%; 12)

of body width apart, occupying space 100–160 (132; 12) or 24–32% (28%;

12) from anterior body end (Fig. 1). Tegumental spines distributing on

ventral and dorsal body surfaces (Figs. 8–15); ventral tegumental body

spines approximately 4 wide and protruding from tegument approximate-

ly 4–6 anteriorly (Figs. 8, 9), becoming smaller toward posterior body

margin (Figs. 10, 11); dorsal tegumental body spines approximately 7–8

long, 7–8 wide, seemingly sheathed in tegument (Figs. 12, 13), becoming

diminutive posteriorly (Fig. 14), absent from posterolateral body margin

(Fig. 15). Ventral sucker medial, nearly circular in outline, thin, lacking

robust musculature, 108–128 (117; 12) long or 21–31% (25%; 12) of body

length, 115–133 (121; 12) wide or 12–29% (14%; 12) of body width,

having anterior margin 113–183 (146; 12) or 26–39% (31%; 12) of body

length from anterior body extremity and posterior margin 153–235 (197;

12) or 38–47% (42%; 12) of body length from posterior body extremity,

with inner ventral surface accommodating spines sheathed in tegument

(Figs. 4, 5). Mouth medial, flanked by aspinous zone immediately

surrounding opening (Fig. 6), occupying space immediately anterior to

ventral sucker, 30–53 (41; 12) long, 8–38 (17; 12) wide, or 1.3–6 times

longer than wide, approximately 83–125 (101; 12) or 20–24% (22%; 12) of

body length from anterior body extremity (Figs. 1, 6). Pharynx 63–88 (72;

12) long or 51–78% (62%; 12) of ventral sucker diameter and 13–19%

(16%; 12) of body length, 65–90 (77; 12) wide or 7–9% (8%; 12) of body

width, occupying space 70–123 (93; 12) or 17–23% (20%; 12) of body

length from anterior end (Fig. 6). Esophagus 66–105 (88; 11) long or 14–

23% (19%; 11) of body length, extending 23–48 (35; 11) posteriad from

pharynx. Intestine bifurcating immediately anterior to genital cavity 163–

225 (195; 10) or 39–47% (42%; 10) from anterior body end and 210–300

(254; 10) or 50–61% (55%; 10) from posterior body end, having sinistral

and dextral branches reflecting slightly anterolaterad, with each branch of

intestine having a distinctive thin-walled anterior portion and a thick-

walled, glandular posterior portion; sinistral anterior portion of intestine

165–218 (194; 11) long or 39–48% (42%; 11) of body length and 18–22%

(20%; 11) body width; dextral anterior portion of intestine 160–213 (186;

10) long or 33–47% (41%; 10) of body length and 16–24% (20%; 10) of

body width; posterior portion of intestine highly glandular and thick-

walled, having myriad papilla-like outward projections, forming cyclocoel,

approximately mirroring curvature of body (including posteromedial

indentation), 996–1,138 (1,067; 10) long (curved total length), terminating

63–115 (91; 12) or 15–23% (20%; 12) of body length from posterior end,

enclosing space 350–430 (378; 12) wide or 35–44% (39%; 12) of body

width.

Testes 2, approximately equal in size, intercecal, occupying space

immediately postero-lateral to level of ventral sucker, approximately 46–

61% (54%; 11) of body length from anterior body end, spaced 75–120 (91;

11) or 7–12% (9%; 11) of body width apart, oriented with long axis

transverse relative to long body axis (breadth greater than length); sinistral

testis 115–165 (138; 11) long, 68–120 (92; 11) wide; dextral testis 115–150

(132; 11) long, 60–105 (83; 11) wide; post-testicular space 100–140 (123;

11) or 21–30% (26%; 11) of body length (Fig. 1). Vasa efferentia and vas

deferens not observed. Seminal vesicle originating between testes at level

of ventral sucker, 425–683 (547; 10) long, extending 100–150 (128; 11)

within cyclocoel and 325–555 (417; 10) external to cyclocoel.

Ovary sinistral, at level immediately posterior to level of pharynx

between sinistral testis and sinistral branch of esophagus, 58–110 (73; 12)

in maximum length, 48–105 (71; 12) in maximum breadth, or 0.67–1.08

times longer than wide, occupying space 128–208 (166; 12) or 30–44%

(36%; 12) of body length from anterior body end and 175–225 (205; 12) or

37–51% (45%; 12) of body length from posterior body end (Fig. 1).

Oviduct sinistral, intercecal, immediately posterior to sinistral branch of

esophagus, a short duct emanating from the dextral aspect of ovary,

extending 40–63 (47; 9) mediad before curving posterolaterad. Vitellarium

occupies space between cyclocoel and body margin, extends medially to

near level of the eyespots, comprising large follicles with spheroid, ova-like

structures and putative vitelline material. Vitelline reservoir 60–110 (87;

10) in breadth, 50–120 (83; 10) in width, occupying space approximately


FIGURES 1–7. (1) Transversotrema patialense (sensu lato) from epidermal space beneath scales of zebrafish,Danio rerio (Hamilton, 1822) (Cypriniformes:Cyprinidae) examined atAuburnUniversity (Auburn,Alabama) andpurchased fromaCaliforniafishdealer.Body, ventral view, anteriorbodyflap (f), eyespot(e), ventral sucker (vs), mouth (m), pharnyx (ph), esophagus (es), thin-walled anterior portion of intestine (ai), cyclocoel (cy), dextral testis (dt), sinistral testis(st), seminal vesicle (sv), ovary (o), vitelline follicles (vf), ootype (oo), Laurer’s canal (lc), Laurer’s canal pore (lcp), vitelline reservoir (vr), uterus (ut), commongenital pore (cgp), and excretory pore (ep). Ventral viewof voucher (USNMColl.No. 1283177). Bar¼100 lm. (2–7) Scanning electronmicrographs. (2) Body,ventral view. Bar¼200 lm. (3) Body, dorsal view. Bar¼200 lm. (4) Ventral sucker (vs) and position of mouth (m), ventral view. Bar¼50 lm. (5) Spines ofventral sucker, ventral view. Bar¼10 lm. (6)Mouth, ventral view. Bar¼20 lm. (7) Excretory pore and adjacent dorsal body spines, dorsal view. Bar¼20 lm.


FIGURES 8–15. Spines of Transversotrema patialense (sensu lato) from epidermal space beneath scale of zebrafish, Danio rerio (Hamilton, 1822)(Cypriniformes: Cyprinidae) examined at Auburn University (Auburn, Alabama) and purchased from a California fish dealer. Scanning electronmicrographs. (8–11) Ventral surface. Bar ¼ 10 lm. (8) Spines in anterior portion of body at level of mouth. (9) Spines at level of ventral sucker. (10)Spines posterior to level of ventral sucker. (11) Spines of posterior body margin. (12–15) Dorsum. Bar¼ 5 lm. (12) Spines in anterior portion of body atlevel of mouth. (13) Spines at level of ventral sucker. (14) Spines posterior to level of ventral sucker. (15) Spines of posterior body margin.


173–260 (216; 12) or 40–52% (47%; 12) of body length from anterior body

end. Ootype post-ovarian, intercecal, coursing posterolaterad between

ovary and sinistral testis, 60–93 (73; 10) long, 13–20 (15; 10) wide, curving

ventrally at level of cyclocoel before joining with Lauer’s canal. Laurer’s

canal sinistral, occupying space between ventral sucker and cyclocoel,

opening dorsally, comprising proximal and distal portion; proximal

portion of Laurer’s canal a glandular, oblong, sac-like structure

containing sperm, 123–210 (166; 7) long, 25–30 (28; 7) wide; distal portion

of Laurer’s canal a narrow tube extending diagonally posterolaterad,

dorsal to proximal portion of Laurer’s canal, extending dorsal to anterior

margin of sinistral testis, 40–95 (75; 7) long; Laurer’s canal pore opening

130–163 (140; 6) or 26–35% (31%; 6) of body length from posterior body

end. Uterus extending laterad across breadth of genital cavity 275–375

(327; 12) or 30–37% (34%; 12) of body width, crossing ventral to

cyclocoel in dextral half of body before curving anteriad and extending

225–318 (269; 11) or 54–65% (59%; 11) of body length to meet with

common genital pore; common genital pore associated with prominent

genital sucker (Fig. 1). Uterine eggs (not illustrated) present in 2 of 12

specimens, 0–3 in number, approximately 110–143 (131; 3) long, 38–48

(44; 3) wide, or 2.9–3.1 (3; 3) times longer than wide.

Excretory system difficult to see in fixed specimens; excretory pore

massive in relation to body, medial, subterminal, opening at level between

cyclocoel and posterior body margin (Fig. 7).

These specimens were identified as Transversotrema patialense by

comparing our specimens with published accounts of cercariae and adults

of T. patialense by Soparkar (1924) and Crusz et al. (1964) and the

taxonomic key provided by Cribb et al. (1992). Our specimens generally fit

the descriptions provided by the former 2 references and unambiguously

keyed to T. patialense by the latter reference. Specifically, our specimens

have vitelline follicles that extend medially to near the level of the

eyespots, a body width:length ratio equal to ,3, no intercecal vitelline

follicles, a ventral sucker that is much larger in diameter than the pharynx,

and uterine eggs that are .100 lm in length (Cribb et al., 1992). No

feature we describe in the studied specimens eliminates the possibility that

these specimens comprise T. patialense; however, to our knowledge the

holotype and other type materials are no longer extant or, at least, not

available for study. As a result, and in light of the recent molecular

taxonomic results of Hunter and Cribb (2012) and Cribb et al. (2014) that

indicate the existence of cryptic species with morphologically similar

adults among Transversotrema spp., we hesitate to definitively identify our

specimens as conspecific with the material of Soparkar (1924). The

zebrafish is a widely reported host for specimens identified as T. patialense

(Table I).

In addition to the specimens of T. patialense that we document herein,

the zebrafish we examined also were infected with gill-encysted

heterophyid metacercariae and a single gyrodactylid. Material from these

infections was insufficient to report in detail, but will be detailed later if

additional specimens become available.

To our knowledge, this is the first confirmed report of a species of

Transversotrema from North America, and no record of this genus, nor

any transversotrematid, exists from Central or South America (Table I).

The red-rimmed melania is a widely reported first intermediate host for T.

patialense as well as an invasive, wide-ranging species in North America

(Facon et al., 2003). We suspect this case study may represent an example

of an exotic parasite species that is established in North America by the

fortuitous co-occurrence of a susceptible, introduced intermediate host

(red-rimmed melania) and a susceptible, widely distributed, non-native

experimental fish host (zebrafish). Dechtiar and Christie (1988) conducted

a survey (from 1961–1971) of the parasites of Lake Ontario fishes,

including 1,965 individual hosts examined representing 56 fishes and 212

parasite species. They reported specimens of ‘‘Prototransversotrema sp.’’

from beneath the scales of common shiners, Notropis conutus (Mitchill,

1817) (Cypriniformes: Cyprinidae), captured in the Credit River, Ontario,

Canada. They noted that this was the first record of the parasite from

North America and indicated that it likely comprised a new species

awaiting further study. This taxon was not treated in a subsequent

publication, and we were unsuccessful in locating the voucher material at

the time of the writing of the present paper. This record indicates that

other transversotrematid infections might remain undocumented in the

Americas or the record may be dubious. Dechtiar and Christie (1988)

likened those specimens to Prototransversotrema steeri Angel, 1969, which

has been reported from a variety of euryhaline perciforms in Australia

(Angel, 1969; Cribb, 1988; Cribb et al., 1992), including several hosts that

are widely distributed and occur in Canada and the northwestern Atlantic

Ocean (i.e., striped mullet, Mugil cephalus Linnaeus, 1758 [Perciformes:

Mugilidae], western mosquitofish, Gambusia affinis [Baird and Girard,

1853] [Cyprinidontiformes: Poeciliidae], and bluefish, Pomatomus saltatrix

(Linnaeus, 1766) [Perciformes: Pomatomidae]). Perhaps no additional

transversotrematid hosts have been discovered in this region because few

workers inspect the space beneath the scales of fishes for trematode

infections. One of us (SAB) has examined mullets (M. cephalus and white

mullet, Mugil curema Valenciennes, 1836) in the Gulf of Mexico for

ectoparasite infections but without observing a transversotrematid

infection.

Transversotrema patialense (sensu lato) has been introduced on

ornamental or aquaculture fishes in Israel (Ben-Ami et al., 2005) and

Japan (Maneepitaksanti and Nagasawa, 2012). Ben-Ami et al. (2005)

documented infections in an aquarium containing several fish species and

indicated alarm at the finding of this non-native fluke in Israel. The finding

led them to examine host specificity of T. patialense in a native fish species

(redbelly tilapia, Coptodon zillii [as Tilapia zillii] [Gervais, 1848]

[Perciformes: Cichlidae]) and 2 non-native fishes [western mosquitofish

and common carp, Cyprinus carpio Linnaeus, 1758 (Cypriniformes:

Cyprinidae)]. Common carp were not susceptible, but redbelly tilapia

and western mosquitofish had experimental prevalences of infection of

38% and 77%, respectively. In redbelly tilapia, most flukes were observed

beneath the scales at the level of the pectoral fins, similar to the position of

the specimens we report herein. Maneepitaksanti and Nagasawa (2012)

reported T. patialense from Mozambique tilapia, Oreochromis mossambi-

cus (Peters, 1852) (Perciformes: Cichlidae), from an irrigation canal on

Ishigaki-jima Island, Western Pacific Ocean. This comprised the first

record of the parasite from Japan and, at that time, comprised the

northern-most record of the parasite in the eastern Pacific Ocean region.

In the present study, that these zebrafish were infected with the

transversotrematid is clear evidence that they were cultured in a system

that shared water with infected snails (probably a species of Melanoides).

Although intuitive to parasitologists, the use of parasite infections to

confirm the status of so-called ‘‘laboratory reared’’ or ‘‘specific pathogen

free’’ fish stocks for experimental studies is not frequently touted as a

useful tool. Doing so is indeed helpful and offers assurances that the fish

being studied have not been exposed to the myriad opportunistic

microbial pathogens in aquatic systems prior to a disease challenge. In

the case of the present study, we necropsied the infected zebrafish because

(1) they were resistant to challenge with a bacterial pathogen (F.

columnare) and (2) we were curious to see if they harbored symbionts

that would indicate they had already been exposed. That these zebrafish

were cultured in a flow-through system supplied with water that carried

transversotrematid cercariae is somewhat disconcerting: other freshwater

fishes in that aquatic system may also be infected. Examinations of

freshwater fishes in North America where large populations of red-rimmed

melania are established could reveal wild populations of transversotre-

matids infecting native freshwater fishes. Examinations of red-rimmed

melania from California should test for the presence of transversotrematid

cercariae. Conversely, the presence of transversotrematid infections in

local fishes may indicate the presence of invasive melania snails.We thank Cova Arias (School of Fisheries, Aquaculture, and Aquatic

Sciences, Auburn University) for allowing us to conduct parasitologicalwork related to the bacterial pathogen challenge. This is a contribution ofthe Center for Aquatic Surveillance and Health (CASH; formerlySoutheastern Cooperative Fish Parasite and Disease Project) and wassupported in part by the National Science Foundation’s Division ofEnvironmental Biology with funds from NSF-DEB grant numbers1112729, 1051106, and 1048523 to SAB.


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