ZEBRAFISH

Volume 00, Number 00, 2021

ª Mary Ann Liebert, Inc.
DOI: 10.1089/zeb.2020.1950

First Record of Clinostomum sp. (Digenea: Clinostomidae)

in Danio rerio (Actinopterygii: Cyprinidae) and the Implication
of Using Zebrafish from Pet Stores on Research

Tony Silveira,1 Mateus T. Kütter,1 Camila M.G. Martins,1 Luis Fernando Marins,1
Robert T. Boyle,1 Vinicius F. Campos,2 and Mariana H. Remião2
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Abstract

Many scientific studies still use zebrafish from pet stores as animal models, even cutting-edge researches.
However, these animals differ genotypically and phenotypically between them. The importance of the use of
standardized models is widely recognized. Besides that, another consequence of using zebrafish from unknown
origins is the acquisition of parasitized animals. This study aimed to relate the infection by Clinostomum sp. in
zebrafish. Animals sold as ‘‘high standard’’ were acquired from a commercial company. Swimming alterations
and superficial yellow dots were observed in five zebrafish with clinical signs, which were isolated, euthanized,
and necropsied. Muscular yellow cysts with metacercaria associated with lesions were observed. The muscular
cysts were responsible for the superficial yellow dots as well as the swimming alterations. The prevalence was
2.5%, and the mean infection intensity was 7 digeneans/host. The cysts measured a mean of 1251.43 lm
long · 784.28 lm wide. Metacercariae measured a mean of 4847 lm long · 1353 lm wide. This first report
about infection by Clinostomum sp. in zebrafish is globally relevant since the host and the parasite genus
currently overlap worldwide. Furthermore, this study sheds light on the importance of the specific pathogen-free
commercial creations or laboratory-reared zebrafish for research.

Keywords: laboratory animal, metacercaria, parasite, pet store, yellow grub

Introduction One of the most severe consequences of using zebrafish

from an unknown origin in research is acquiring parasitized

S ince the seminal popularization of Danio rerio

(Hamilton, 1822) (zebrafish) as an experimental model,1
the use of zebrafish in scientific research is growing rapidly
around the world.2,3 Many studies, even cutting-edge inves-
tigations, still use zebrafish from pet stores and commercial
fishkeeping providers for experimentation.
Many zebrafish strains, such as AB, Tübingen, the Uni-
versity of Toronto Mississauga strain, and the transgenic F0104
strain of Federal University of Rio Grande (FURG), were es-
tablished from the breeding of zebrafish obtained from local pet
stores.4–6 However, zebrafish purchased from typical pet stores
differ genotypically and phenotypically,5 which contributes to
the lack of replicability. An alternative is to buy the zebrafish
from pet stores, breed them, and establish a population for
experiments.4 This procedure would help achieve a standard-
ized population with an adequate health history.
animals. The indiscriminate inclusion of healthy and sick
animals in the same experimental protocol leads to the mis-
interpretation of results and invalidation of the method,
making experimental reproducibility impossible.7–10 Various
infectious diseases have been recorded in zebrafish.11 Several
of these pathogens interfere negatively in zebrafish breeding,
survival, welfare, and management.12 Furthermore, some of
them have zoonotic potential,13–15 but few of these infectious
agents are digenean.
Clinostomum Leidy, 1856, is a genus of digenean parasites
distributed across all continents, except the Antarctic.16 Their
life cycles are indirect and involve at least three hosts: one
definitive host and two intermediate hosts. Several piscivo-
rous birds have been described as definitive hosts of Clin-
ostomum spp.16 Also, gastropods are obligatorily required as
the first intermediate host.17–20 Furthermore, many fish, frog,

1
Instituto de Ciências Biológicas, Universidade Federal do Rio Grande—FURG, Rio Grande, Brazil.
2
Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas—UFPEL, Capão do Leão, Brazil.

1
 2 SILVEIRA ET AL.
newt, and salamander species are included in the cycle as
second intermediate host.16,18–24 There is also the possibility
of the occurrence of paratenic hosts in the ecology of Clin-
ostomum spp. In this case, the parasite’s juvenile stage does
not develop in the host after the infection, the host serving
only as a reservoir and transport carrier.25,26 The definitive
host acquires the infection by ingesting the infected second
intermediate or paratenic hosts.
Although the increment in the use of zebrafish2,3 and the
fact that digeneans are the largest group of monozoic
platyhelminths and possibly the largest group of internal
metazoan parasites,26 the records of new infections by di-
geneans in zebrafish are scarce. Parasites can threaten the
animals, researchers, methods, and results of the zebrafish
research or the scientific method as a whole. Hence, the
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knowledge of a new parasitic agent able to infect zebrafish
has a capital relevance. In light of this, this study aimed to
relate, for the first time in Danio rerio, the infection by
metacercariae of Clinostomum sp. using zebrafish as a
second intermediate host and point the risks of using zeb-
rafish from pet stores on research.
Materials and Methods
A batch of 200 six-month-old wild-type zebrafish (males
and females) was purchased from a Brazilian commercial
breeding company. These animals were received at Aquatic
Bioterium of FURG and would be used in ecotoxicological
tests (approved by the Ethics Committee of the FURG,
Brazil, under the code Pq015/2018). These zebrafish were
sold as ‘‘high-standard animals.’’ Before transfer to the
recirculating housing system (Altamar Sistemas Aquáticos,
Brazil), the fish were quarantined in static aquaria, and some
swimming alterations and superficial yellow dots were no-
ticed in some animals. No initial mortality was observed.
The Aquatic Bioterium has recirculating housing systems
for zebrafish. The recirculating systems have ultraviolet
treatment (minimum dose 60,000 mWsec/cm2) for water
disinfection. The water of zebrafish systems comes from local
urban treatment system and undergoes dechlorination and
filtration before coming into contact with animals. Due to it,
diseases caused by metazoans are not common. During the
quarantine (14 days), the water conditions are maintained at
27 – 1C, pH 7.2 – 0.5, dissolved oxygen content of *7 mg/L,
and photoperiod of 12 h-light/12-h dark. Water parameters
were daily measured, and zebrafish were fed twice a day with
Ovo-vit flakes (Tropical, Germany).
Five zebrafish with the suspected affections were isolated
and euthanized with an overdose of buffered tricaine (tricaine
methanesulfonate, MS-222; Sigma; pH 7.2; 0.4 mg/mL). Later,
the animals were necropsied to investigate the cause of the
clinical signs. Two female and three males (total length =
34.3 – 2.3 mm and weight = 408.2 – 38.6 mg; mean – standard
deviation) were necropsied. The eyes, gills, heart, intestine,
liver, spleen, kidney, gonads, and swim bladder were exam-
ined. Coelomic cavity and body wall, as well as musculature
cuts, were also analyzed. It was not possible to investigate the
entire batch due to the necessity of returning the live animals to
the company. Thus, only the suspect animals were investigated.
The sampled material was distributed among Petri dishes,
washed with 0.9% physiological saline in a 150 lm mesh
sieve, and observed under a stereomicroscope. Helminths
were collected, counted, and processed according to Amato
et al.,27 fixed in alcohol-formaldehyde-acid solution (93 parts
of 70% ethanol, 5 parts of commercial stock formalin 37%–
40%, and 3 parts of glacial acetic acid; Vetec, Rio de Janeiro,
Brazil), stained with carmine (Dinâmica, Indaiatuba, Brazil),
cleared with beechwood creosote (Vetec), and mounted be-
tween a slide and a coverslip. Parasites were identified to
genus level based upon their morphological characteristics
according to Bray et al.28 and Travassos et al.29 Para-
sitological indices, prevalence, mean infection intensity, and
variation of infection intensity, were calculated according to
Bush et al.30 The morphometric values in micrometers (lm)
are presented as a range of measures (minimum and maxi-
mum values) followed by the average in parentheses.
Results
The main clinical signs observed in the animals were tor-
tuous swimming (without the normal wave motion of the
body, as if the movements of the tail were limited and im-
paired), yellow lumps and swellings visible on the body
surface, and lateral curvature of the spine (Fig. 1A–D).
Muscular yellow cysts with metacercaria were found in all
zebrafish with these clinical signs. The metacercariae were
identified as Clinostomum sp. The prevalence of infection
was at least 2.5%. The cysts presented oval or subcircular
format and yellowish color, with measurement of 1080–1900
(1251.43) long · 580–1200 (784.28) wide (based on 10 in-
tegrated cysts). The muscular cysts were responsible for the
superficial yellow dots as well as the other signs observed in
the fish.
The mean infection intensity was 7 encysted metacercariae
per host, and the variation of infection intensity was 1–16.
The lower infection intensity was observed in an animal,
which presented only one yellow dot with an associated cyst
on the left side of the dorsal musculature of the cranial third
of the body at the immediately caudal level end of the
operculum (Fig. 1A, C). The higher infection intensity was
observed in a zebrafish with 16 cysts: 12 at the right side of
the lateral caudal and dorsal musculature, with some super-
ficially distributed and others deeper; other 2 in the left side of
the body wall musculature at the middle level; and 2 at the
ventral region, close to the caudal peduncle (Fig. 1B, D).
Some of the largest cysts were opened during the necropsy,
and some metacercariae were still alive, showing vigorous
movements. Metacercariae of Clinostomum sp. from D. rerio
(Clinostomum sp. Dre) sampled in necropsy presented mor-
phological characteristics typical for the genus (Fig. 2A–C).
The following description is based on six specimens: mor-
phometric measures are shown in Table 1 as well as the data
of other authors that described Clinostomum in the Americas.
Metacercariae bodies are stout, linguiform, flat, covered by
thick cuticular spines, and convex dorsally and concave
ventrally. Their greatest width is at the level of the genital
complex and posses an evident oral collar surrounding the
subterminal oral sucker. The oral sucker is smaller than the
strongly muscular ventral sucker. Intestinal ceca are lateral to
the ventral sucker and genital complex and extend from the
terminus of the esophagus to the posterior end of the body.
Testes are located on the posterior half of the body and at the
middle third of the body, slightly triangular, with digitations,
facing each other with a concave face. The anterior testis is
 CLINOSTOMUM SP. IN ZEBRAFISH 3
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FIG. 1. Danio rerio (zebrafish) infected by encysted metacercaria of Clinostomum sp. (A) View in toto of the parasitized
adult female. (B) View in toto of the parasitized adult male after skin dissection during the necropsy. (C) View of the cranial
half of the parasitized specimen evidencing the yellow cyst. (D) Lateral view of the parasitized specimen evidencing the
cysts in the caudal musculature. Dashed circle (C) and black arrows (D) indicate the cyst containing metacercaria of
Clinostomum sp., also known as yellow grub or yellow spot. Scale bars: 5 mm (A, B); 3 mm (C, D). Color images are
available online.

located left of the cirrus sac. The cirrus sac is oval, opens into
a genital pore, is connected to a metraterm, and extends from
intertesticular space to the middle right margin of the anterior
testis. The genital pore is ventral, located at the level of the
right external margin of the anterior testis (slightly to the right
of the median plane). The ovary is oval and underdeveloped,
located dextrally in intertesticular space. Ootype is tangled at
intertesticular level, encircling the left margin of the anterior
testis and connecting to the posterior half of the uterus. Ex-
cretory ducts connect at the posterior end, and the excretory
pore is terminal.

FIG. 2. Metacercaria of Clinostomum sp. from Danio re-
rio (zebrafish). (A) Whole mount, view in toto. (B) Anterior
end of the body evidencing the oral sucker, oral collar,
distance between suckers, and partial view of ventral sucker.
(C) Middle third of the body evidencing the genital com-
plex. Ventral views. Scale bars: 500 lm (A); 300 lm
(B, C). Color images are available online.
Discussion
Clinostomum spp. is popularly known as yellow grubs or
yellow spot disease.42–46 This characteristic color of the cysts
and the metacercariae explains the clinical findings in this study.
The disease caused by Clinostomum spp. is widely noticed in
fishes, both in the natural environment and in commercial
creations. However, even the trematodes being present in Indian
territory, from which zebrafish is native,47 this infection by a
Clinostomum species has never been reported to D. rerio until
nowadays. Knowing a new infectious agent in zebrafish is very
important to adequate health monitoring in facilities, besides
having a capital relevance due to the current worldwide distri-
bution of zebrafish as a scientific research model.
Scientific experiments require animals in the best health
condition,3,12 which is not possible if parasitized. The pres-
ence of cysts of Clinostomum spp. lead to a loss of vigor and
appetite; retard growth; weight loss; behavioral alteration;
inflammatory response with vascular congestion, varying
from very mild inflammatory infiltrate to a severe acute le-
sion potentially fatal; eosinophil, lymphocyte, and granulo-
cyte proliferation; decrease in the content of hemoglobin and
erythrocyte count; adipocyte destruction; muscle fiber de-
formity; a decrease of innate immunity barriers; compression
malfunction; and may lead to death, mainly in the excystation
process.36,48–52 These local and systemic immunologic,
physiological, and metabolic alterations in fishes infected by
Clinostomum spp. result in a significant limitation to the use
of sick zebrafish in an experimental protocol.
In another way, the treatment of the infected fish is
not recommended since many antihelminthics may be toxic
to zebrafish53 and can skew the research. Furthermore, the
 Table 1. Morphometric Data of Clinostomum sp. Metacercariae from Zebrafish (Danio rerio) Compared
to Other Metacercariae of Clinostomum spp. Described in Fishes from New World by Different Authors
Parasite Clinostomum sp. Dre C. marginatum Clinostomum sp. C. heluans
Hosts Danio rerio Rhamdia quelen Poecilia reticulata Cichla temensis
Locality Brazil Brazil Brazil Brazil
Reference Present study Vianna et al.31 Pinto et al.32 Vicente et al.33
OCW 677–737 (701) — — —
BL 4495–5250 (4847) 3050–7380 (4920) 3610–5553 (4224) 2000–2200
BW 1250–1438 (1353) 780–1470 (1050) 1031–1616 (1215) 640–740
BL/BW 3.17–3.84 (3.59) — — —
OSL 155–220 (188) 130–222 (191) 170–239 (213) 120–150
OSW 215–265 (241) 193–285 (255) 232–273 (251) 120–140
OSW/BW 0.15–0.20 (0.18) — — —
VSL 639–764 (685) 460–750 (603) 602–746 (685) 200–420
VSW 580–668 (629) 460–730 (603) 602–756 (684) 230–290
VSW/OSW 2.32–3.11 (2.63) — — —
VSW/BW 0.45–0.50 (0.47) — — —
DBS 542–662 (600) — 515–860 (659) —
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ATL 210–270 (233) 180–550 (345) 137–226 (142) 84–120

ATW 141–190 (167) 135–550 (311) 137–206 (159) 56
ATW/ATL 0.61–0.90 (0.72) — — —
PTL 166–294 (241) 180–510 (362) 116–311 (172) 84–120
PTW 130–220 (175) 148–440 (307) 178–259 (217) 56
PTW/PTL 0.49–1.33 (0.80) — — —
DBT 187–240 (218) — — —
OL 50–100 (77) 83–118 (143) 89–147 (110) 50–110
OW 42–55 (50) 55–220 (118) 68–120 (85) 90–150
OW/OL 0.47–1.06 (0.70) — — —
CSL 250–340 (306) 127–530 (334) 150–205 (183) —
CSW 80–126 (108) 82–290 (143) 89–164 (121) —
CSL/BL 0.05–0.07 (0.06) — — —
Parasite C. marginatum C. detruncatum Clinostomum sp. Clinostomum sp.
Hosts Pterygoplichthys pardalis Synbranchus marmoratus Characidium rachovii Crenicichla vittata
Aphanotorulus emarginatus
Locality Brazil Brazil Argentina Argentina
Reference Souza et al.34 Acosta et al.35 Montes et al.36 Montes et al.36
OCW — 1117–1371 (1263) 1028–1355 (1200) 774–994 (913)
BL 3820 – 354 8022–9271 (8436) 5603–8163 (6714) 3614–5215 (4398)
BW 1506 – 160 3220–3695 (3411) 1483–1824 (1647) 1037–1617 (1377)
BL/BW – 2.26–2.88 (2.48) 3.78–4.47 (4.08) 2.69–3.74 (3.13)
OSL 237 – 10 315–406 (383) 203–326 (286) 232–310 (266)
OSW 344 – 22 422–441 (433) 246–413 (327) 236–327 (295)
OSW/BW — — 0.17–0.23 (0.20) 0.20–0.23 (0.21)
VSL 695 – 31 1021–1073 (1042) 751–908 (832) 692–890 (784)
VSW 906 – 31 1050–1149 (1096) 748–894 (816) 654–921 (821)
VSW/OSW — 2.42–2.73 (2.53) 2.11–3.04 (2.54) 2.55–3.9 (2.94)
VSW/BW — 0.30–0.36 (0.32) 0.44–0.55 (0.50) 0.51–0.59 (0.56)
DBS 578 – 85 763–997 (893) — —
ATL 271 – 5 359–655 (498) 154–308 (244) 178–275 (227)
ATW 475 – 68 890–1266 (1113) 242–349 (317) 468–619 (546)
ATW/ATL — 1.73–3.39 (2.35) 0.98–1.55 (1.25) 2.2–2.95 (2.49)
PTL 254 – 38 528–708 (601) 150–335 (231) 237–341 (289)
PTW 462 – 30 802–1318 (1020) 238–443 (369) 493–646 (581)
PTW/PTL — 1.25–2.44 (1.72) 0.89–2.63 (1.78) 1.63–2.46 (2.03)
DBT 578 – 85 383–608 (461) 238–295 (270) 199–297 (243)
OL — 157–206 (186) 107–162 (138) 140–185 (166)
OW — 103–156 (131) 81–109 (90) 129–171 (150)
OW/OL — 0.55–0.85 (0.71) 0.54–0.76 (0.64) 0.74–1.08 (0.91)
CSL — 604–685 (649) 254–373 (302) 355–400 (377)
CSW — 181–226 (200) 157–205 (184) 118–131 (126)
CSL/BL — 0.07–0.08 (0.08) 0.04–0.06 (0.04) 0.09–0.13 (0.11)
Parasite Clinostomum sp. C. marginatum C. tataxumui C. caffarae
Hosts Gymnogeophagus balzanii Dormitator maculatus Dormitator maculatus Astyanax aeneus
Locality Argentina Mexico Mexico Mexico
Reference Montes et al.36 Sereno-Uribe et al.37 Sereno-Uribe et al.37 Sereno-Uribe et al.38
OCW 901–1117 (1036) 555–670 (594) 480–690 (541) 563–836 (675)
(continued)
4
 Table 1. (Continued)
Parasite Clinostomum sp. C. marginatum C. tataxumui C. caffarae
BL 3667–4821 (4384) 2770–4100 (3300) 2390–3950 (2940) 3692–6609 (5207)
BW 1329–1927 (1556) 600–850 (730) 340–840 (593) 840–1417 (1101)
BL/BW 2.5–3.25 (2.84) 3.5–6.21 (4.5) 3.98–7.9 (5.1) 4.16–4.66 (4.72)
OSL 162–265 (216) 160–220 (183) 140–210 (160) 213–328 (264)
OSW 208–299 (262) 170–220 (196) 150–210 (174) 220–347 (276)
OSW/BW 0.19–0.22 (0.21) 0.25–0.30 (0.26) 0.25–0.47 (0.31) —
VSL 608–707 (649) 430–620 (496) 350–560 (420) 607–937 (752)
VSW 748–1038 (886) 450–620 (530) 320–570 (431) 591–89.00 (735)
VSW/OSW 2.5–4.19 (3.35) 2.4–3.1 (2.7) 2.00–2.71 (2.4) —
VSW/BW 0.54–0.59 (0.56) 0.66–0.84 (0.72) 0.51–1.19 (0.76) —
DBS — 380–700 (539) 350–610 (468) 407–687 (563)
ATL 234–255 (245) 180–370 (224) 120–250 (171) 205–402 (319)
ATW 591–786 (658) 200–350 (247) 140–275 (200) 180–399 (315)
ATW/ATL 2.32–2.55 (2.43) 0.8–1.4 (1.12) 0.93–1.02 (1.24) —
PTL 302–562 (432) 170–270 (214) 115–230 (172) 174–526 (318)
PTW 627–1103 (785) 250–400 (280) 180–320 (230) 243–535 (393)
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PTW/PTL 1.26–2.08 (1.67) 0.60–0.96 (0.74) 1.03–1.06 (1.3) —

DBT 208–237 (223) 110–170 (137) 90–210 (136) 205–310 (244)
OL 166 80–120 (98) 72–130 (96) 144–258 (219)
OW 85 60–90 (73) 42–111 (70) 80–199 (151)
OW/OL 0.51 0.54–0.88 (0.75) 0.54–1.11 (0.73) 0.56–0.93 (0.69)
CSL 343–461 (402) 180–220 (200) 137–225 (179) 257–463 (375)
CSW 111–120 (116) 90–120 (106) 65–125 (91) 113–202 (168)
CSL/BL 0.09–0.11 (0.10) 0.05–0.07 (0.06) 0.05–0.07 (0.06) 0.07–0.08 (0.07)
Parasite C. arquus C. cichlidorum C. marginatum
Hosts Profundulus punctatus Cichlidae Menidia beryllina
Profundulus balsanus
Pseudoxiphophorus jonesii
Locality Mexico Mexico United States of America
Costa Rica
38
Reference Sereno-Uribe et al. Sereno-Uribe et al.38 Rosser et al.39
OCW 581–776 (646) 563–773 (655) —
BL 3200–4516 (3783) 3251–4221 (3631) 3128–5407 (3968)
BW 676–1074 (818) 841–1110 (962) 904–1303 (1068)
BL/BW 3.90–5.31 (4.67) 3.53–3.91 (3.77) —
OSL 189–291 (222) 200–254 (220) 192–281 (222)
OSW 198–317 (227) 190–228 (207) 191–241 (215)
OSW/BW — — —
VSL 452–604 (508) 451–549 (505) 468–666 (560)
VSW 479–676 (539) 483–608 (548) 485–606 (542)
VSW/OSW — — —
VSW/BW — — —
DBS 508–732 (604) 454–698 (579) —
ATL 180–309 (226) 179–330 (237) 130–242 (190)
ATW 174–277 (229) 225–325 (277) 213–361 (278)
ATW/ATL — — —
PTL 148–258 (206) 191–275 (230) 153–230 (189)
PTW 211–378 (271) 258–404 (336) 158–306 (239)
PTW/PTL — — —
DBT 180–246 (214) 121–223.00 (163) —
OL 113–175 (137) 92–139.00 (115.88) 121–171 (143)
OW 74–113 (90) 70–121 (96) 169–275 (233)
OW/OL 0.42–0.87 (0.68) 0.68–1.13 (0.84) —
CSL 162–276 (220) 197–248 (221) —
CSW 100–144 (125) 102–142 (125) —
CSL/BL 0.05–0.07 (0.06) 0.06–0.06 (0.06) —
Parasite C. album C. marginatum
Hosts Pimephales promelas Perciformes
Locality United States of America Canada
United States of America
Reference Rosser et al.40 Caffara et al.41
OCW — 502–874 (721)
BL 4081–4938 (4509) 4215–6346 (5402)
BW 737–874 (805) 979–1532 (1329)
(continued)
5
 6 SILVEIRA ET AL.

Table 1. (Continued)
Parasite C. album C. marginatum
BL/BW — 3.05–4.40 (4.03)
OSL 227–267 (247) 238–599 (312)
OSW 268–297 (283) 194–586 (290)
OSW/BW — 0.82–1.05 (0.92)
VSL 593–666 (629) 510–757 (669)
VSW 548–673 (610) 571–804 (708)
VSW/OSW — 0.987–1.118 (1.060)
VSW/BW — 0.47–0.62 (0.53)
DBS — 959–1.49 (1.24)
ATL 270–294 (282) 231–413 (307)
ATW 333–334 (333) 264–498 (389)
ATW/ATL — 0.68–1.72 (1.29)
PTL 273–364 (318) 233–457 (327)
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PTW 287–301 (294) 295–492 (405)

PTW/PTL — 0.89–1.52 (1.25)
DBT — 196–393 (320)
OL 144–148 (146) 85–142 (115)
OW 125–132 (128) 62–97 (80)
OW/OL — 0.43–0.97 (0.71)
CSL — 172–343 (256)
CSW — 119–153 (137)
CSL/BL — 0.03–0.06 (0.05)
Data are expressed in micrometers (lm) as minimum and maximum values followed by the mean between parentheses, except for C.
heluans described by Vicente et al.33 and C. marginatum described by Souza et al.,34 measures of which are represented as minimum and
maximum values and mean – standard deviation, respectively.
OCW, oral collar width; BL, body length; BW, body width; OSL, oral sucker length; OSW, oral sucker width; VSL, ventral sucker
length; VSW, ventral sucker width; DBS, distance between suckers; ATL, anterior testis length; ATW, anterior testis width; PTL, posterior
testis length; PTW, posterior testis width; DBT, distance between testis; OL, ovary length; OW, ovary width; CSL, cirrus sac length; CSW,
cirrus sac width.

use of inappropriate animals leads to a violation of the re-
duction and refinement principles of the three Rs guiding
principles of laboratory animal science (Replacement, Re-
duction, and Refinement). The use of animals with higher
standards represents a refinement and leads to improved
experimental procedures. The use of animals directly from
pet stores, besides not contributing to the refinement, leads
to the need for a higher number of animals because some of
these may not be used or considered in the interpretation of
results.8
The infected zebrafish reported in this study took the place
of the second intermediate host in the life cycle of Clin-
ostomum spp. For this, the infection occurred through the
penetration of the skin or the mucous of fish by the cercariae,
which developed in snails (the first intermediate host) and
swam in freshwater by wagging their forked tails.20 Snails
could be used in zebrafish facilities to maintain water quality,
eating algae and detritus accumulated in the tanks.54 For this,
due to the high infectious risk, it is advisable not keep snails
and zebrafish together in research facilities. If this is neces-
sary, a reasonable sanitary control of both snails and fish is
essential to prevent emergent diseases as clinostomosis and
centrocestosis.55 Alternatively, it is still possible that zebra-
fish acts as a paratenic host. For this, the second intermediate
host must suffer cannibalism. Cannibalism is a common sit-
uation that causes concern in facilities around the world and
must be avoided.56 Thus, a noninfected zebrafish eats the cyst
with metacercaria and acquires the parasitiasis. Although
infecting this new host, the metacercaria will not change the
stage of development.
Clinostomum is the most diverse clinostomid genus with
21 valid species.37,38,40,45,57,58 In this study, it was impos-
sible to ascribe the Clinostomum sp. Dre to any known
species. Currently, the main morphological characters used
to differentiate species are body size; ceca shape; position of
the genital complex along the longitudinal axis; position of
the ovary in relationship to the cirrus sac, cirrus sac size, and
position in relationship to the anterior testis; distance be-
tween testes; post-testicular space; and presence of spines
on the tegument.37,38,59 Due to the observation of unique
characteristics in Clinostomum sp. Dre, we do not rule out
the possibility that it represents a new species. This pos-
sibility should be investigated in future studies using
molecular approaches.
In fact, Clinostomum systematics has been revised recently
using integrative taxonomy techniques.32,35–38,41,57,59,60 For
example, C. complanatum, the most reported species of the
genus, was considered a synonym of C. marginatum for
decades,31,61 before both being validated as independent
species.60 Recently, it is consensus that C. complanatum is
restricted to the Old World and C. marginatum to the New
World.37,41,57 Furthermore, several lineages and species of
Clinostomum have been described in North and Middle
Americas.37,38,40,45 In South America, new lineages have
been described in Argentina and Brazil, suggesting that the
biodiversity of this region may be underestimated.32,36
Future studies may reveal a higher diversity of Clin-
ostomum, especially for species with testes in the middle
third of the body,32 like the specimens reported in this
study.
 CLINOSTOMUM SP. IN ZEBRAFISH
Because of the recent elucidation of C. complanatum
distribution, many previous records have been made for
Brazil.35,62 This species has even been described in the same
area of this report; however, C. complanatum was considered
a synonym of C. marginatum.31,63 Probably all records of
C. complanatum from Brazil are actually C. marginatum or
species not yet described. Nevertheless, several records of
C. marginatum in fishes have been presented in Brazil.34,35,64
The parasites of this study have several morphometric charac-
teristics similar to C. marginatum, mainly that described by
Vianna et al.,31 and Caffara et al.41 (Table 1). However, Clin-
ostomum sp. Dre does not have the genital pore opening ante-
riorly to the anterior testis at midline and has the distance
between suckers smaller than that described by Caffara et al.41
Furthermore, Clinostomum sp. Dre presents more deeply lobated
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testes, diverticulated ceca, and higher post-testicular space.
The other species reported from Brazil are C. heluans and
C. detruncatum.35,65,66 Clinostomum sp. Dre have almost all
measures higher than that observed in C. heluans and smaller
than in C. detruncatum (Table 1). Also, while Clinostomum
sp. Dre has a genital complex in the middle third of the body,
C. heluans and C. detruncatum have in the posterior third of
the body. Our specimens also have dimensions comparable to
those presented by Clinostomum sp. described by Pinto
et al.32 and with species that occur outside of Brazil, mainly
with the metacercariae of C. caffarae38 and with the Argen-
tine lineages.36 However, Clinostomum sp. Dre have a
smaller testis and ovary; wider and deeply diverticulate ceca;
and more anterior genital complex, with a larger post-
testicular space. The cirrus sac is comparable in measures
with C. caffarae and C. arquus, but with disposition similar to
that of C. tataxumui38 and C. marginatum described by
Rosser et al.39 (Table 1; Fig. 2).
The plasticity of the life cycle of Clinostomum spp. can be
perceived in the parasitological indices variation among its
hosts. The prevalence, the mean infection intensity, and the
variation of infection intensity may vary from 2.45% to
60.8%, from 1 to 39 parasites/host, and from 0 to 1–585
parasites/host, respectively, in the same study area depending
on the host.19 The prevalence in zebrafish was similar to the
found in Squalius cephalus,46 Hoplias malabaricus, and
Loricaria sp.19 The infection intensity in zebrafish was close
to that found in Capoeta gracilis,67 Cichlasoma para-
naense,22 and Parauchenipterus galeatus.19 The variation of
infection intensity in zebrafish was similar to that found in
Hoplosternum littorale19 and Myxocyprinus asiaticus.42
Although until now only C. complanatum has been recorded
as zoonotic agent, all genus specimens are potentially zoonotic
and generate concerns about public health for over 80
years.35,46,68,69 In facilities and laboratories, the occupational
risk is low because the transmission depends on eating raw
infected fish. Even so, the risk of infection exists. In the re-
search environment, the researchers frequently are exposed to
small in natura biological material that may keep in contact
with the mouth of the researcher if he is not careful (e.g., in
necropsy, sample collection, and removal of dead animal re-
mains from the tanks). Hence, the staff who handle zebrafish
should be aware of this risk to personnel safety.70 If the acci-
dental infection occurs, humans act as definitive hosts.26 After
the ingestion, the metacercaria is freed in the stomach and
migrates toward the esophagus or the oral cavity. Usually, the
parasitism causes acute pharyngitis or laryngitis.24,26
7
Encysted metacercariae are common in farmed ornamental
fishes and fishes commercialized in pet stores.71–76 In the re-
search ambit, infections by metacercariae are pointed as im-
portant in zebrafish facilities, but the identification of the
etiologic agent sometimes is not possible.11 In the wild, zeb-
rafish are naturally parasitized by metacercariae of digenea
from Strigeida, Heterophyidae, Haplorchis sp., Acanthosto-
mum sp., and Centrocestus formosanus in Indian rivers.77 An
unidentified metacercaria has already been recorded infecting
zebrafish commercialized in a pet store in Brazil.71 The first
record in the Americas of the digenean Transversotrema pa-
tialense was in purchased experimental zebrafish in the United
States of America.78 Finally, parasitic gill infections by me-
tacercariae of C. formosanus in apparently healthy zebrafish
were recorded in Italy,55,79 Thailand,80 and Mexico.81
In light of this, due to the importance of the health moni-
toring and traceability of the zebrafish used in science, Italy
and the European Union require that all zebrafish used in
experimental procedures came from authorized establish-
ments.55 Another essential fact is that as Clinostomum sp.
Dre, Haplorchis sp., and C. formosanus are digenetic trem-
atodes that use zebrafish as an intermediate host and have
zoonotic potential. Thus, it is crucial that all researchers who
handle zebrafish know about these parasites and their cycles.
Compared to the European, the current Brazilian legislation
about laboratory fishes is not so strict regarding the origin of
the animals. The fish from this study, for example, were sold as
‘‘high-standard animals.’’ Still, they were sent without any
health certificate or veterinarian monitoring until the arrival in
the facility. This gap in Brazilian legislation makes this type of
health problem not unusual or isolated. As previously said, a
metacercaria had already been reported in zebrafish purchased
from pet stores in Brazil.71 After returning the animals to the
supplier company, no zebrafish kept in the facility showed new
signs of clinostomiasis, indicating that the source of infection
was not in our aquatic bioterium, but in the company plant.
This first report about infection by Clinostomum sp. in
zebrafish purchased from pet stores and commercial companies
to use directly as scientific experimental models is globally
relevant since both hosts and parasites of genus Clinostomum
currently overlap worldwide distribution. Furthermore, this
report sheds light on the importance of the specific pathogen-
free commercial creations or laboratory-reared zebrafish and
the difference between the standard of animals for ornamental
fishkeeping and use in scientific experiments. Thus, the general
recommendation of acquiring zebrafish from a pet store to
breeding it and using only their descendants in experiments is
justified.4 Although the Aquatic Bioterium of FURG does not
yet adopt this practice for all zebrafish strains, the authors un-
derstand its importance since the first transgenic fish in Brazil
(zebrafish of F0104 strain) was created and maintained under
these conditions.6 Therefore, it is urgent that the researchers of
‘‘zebrafish community’’ follow the footsteps of Louis Pasteur,
which replaced purchased animals by those raised in laboratory
at the end of the 19th century, toward the more refined use of
animals for scientific purposes.
Data Availability Statement
Data supporting the conclusions of this article are included
in the report. The complete data sets used during this study
are available from the corresponding author.
 8 SILVEIRA ET AL.
Ethical Statement
All procedures in animals were previously approved by the
Ethics Committee of the Federal University of Rio Grande,
Brazil, under the code Pq015/2018.
Acknowledgments
The authors gratefully acknowledge the contribution of the
Conselho Nacional de Desenvolvimento Cientı́fico e Tec-
nológico (CNPq) and Companhia Riograndense de Sanea-
mento (CORSAN). The authors are grateful to Dr. Martin M.
Montes; Dr. Gerardo Pérez-Ponce de León; Dr. Renato Za-
carias Silva; and Dr. Rogério Tubino Vianna for the help in
parasite identification. The authors also are grateful to the
anonymous reviewers who improved this study.
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Disclosure Statement
No competing financial interests exist.
Funding Information
L.F.M. and V.F.C. are research fellows from Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico
(CNPq, Proc. no. 309634/2018-0 and 313674/2019-1, re-
spectively). The project that originated the data published
here was partially financed by Companhia Riograndense de
Saneamento (CORSAN) through the technical cooperation
term no. 006/16—DTEC.
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Address correspondence to:
Mariana H. Remião, PhD
Centro de Desenvolvimento Tecnológico
Universidade Federal de Pelotas—UFPEL
Av. Eliseu Maciel, s/n Campus Capão do Leão
Prédio 20
Capão do Leão 96160-000
E-mail: mariana.remiao@ufpel.edu.br