Acta Tropica 137 (2014) 95–98

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Detection of Paragonimus mexicanus (Trematoda) metacercariae in

crabs from Oaxaca, Mexico
Jaime Vargas-Arzola a , Aristeo Segura-Salvador a , Leobardo Reyes-Velasco a ,
Dylan L. Díaz-Chiguer b , Adrián Márquez-Navarro c , Gloria León-Avila c ,
Gabriela Ibañez-Cervantes d , Alejandro D. Camacho c , Rosa Ma. Sánchez-Manzano c ,
Benjamín Nogueda-Torres c,∗
a
Facultad de Ciencias Químicas, Universidad Autónoma “Benito Juárez” de Oaxaca, Oaxaca 68120, Mexico
b
Instituto de Seguridad y Servicios Sociales para los Trabajadores del Estado, ISSSTE, México DF 06720, Mexico
c
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México DF 11340, Mexico
d
Centro Nacional de la Transfusión Sanguínea, México DF 07360, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Metacercariae of Paragonimus mexicanus were collected in crabs Tehuana guerreroensis (Rathbun, 1933)
Received 4 December 2013 in the municipality of Putla, Oaxaca, Mexico. Metacercariae were found in 20.8% of the crabs collected,
Received in revised form 3 May 2014 with an average of 1.9 metacercarie per crab. Stained metacercariae showed the specific characteristics
Accepted 9 May 2014
of P. mexicanus by morphology and sequencing a fragment of the 28S ribosomal gene obtained by PCR.
Available online 16 May 2014
These findings reveal that T. guerreroensis is an intermediate host for P. mexicanus; this new report is
relevant considering the potential risk of transmission in the states of Oaxaca and Guerrero, Mexico.
Keywords:
© 2014 Elsevier B.V. All rights reserved.
Paragonimus mexicanus
Metacercariae
Crabs
Tehuana

1. Introduction Perú, Ecuador, Costa Rica, Panamá and Guatemala. In México,

Paragonimiasis is a food-borne parasitic disease of humans,
other mammals, molluscs and crustaceans; it is caused by flukes of
the genus Paragonimus spp. Human paragonimiasis is distributed
across three continents: South and Central America, East Asia and
West Africa. Typical symptoms of human paragonimiasis include
fever, cough, eosinophilia and hemoptysis, which together can be
misdiagnosed as tuberculosis. Praziquantel is the drug of choice for
paragonimiasis; however, other drugs have been used (Keiser and
Utzinger, 2010). Paragonimus spp. use snails as first intermediate
hosts and decapod crustaceans (crabs and freshwater shrimp) as
second intermediate hosts.
Infection occurs when man ingests either raw or uncooked
crustaceans infected with metacercariae or meat from paratenic
hosts. More than 30 species of the genus Paragonimus have been
described; of these, 10 species have been reported to infect
humans. Paragonimus mexicanus is located in México, Colombia,
∗ Corresponding author. Tel.: +52 5557296000x62399.
E-mail addresses: bnogueda@yahoo.com, bnogueda@hotmail.com
(B. Nogueda-Torres).
this parasite has been found in wild animals from Colima, Chia-
pas, Hidalgo, Michoacán, Nayarit, Puebla, San Luis Potosi, Tabasco,
Yucatán, Veracruz and State of México (Lamothe-Argumedo, 1985).
Several crustacean species of the family Pseudothelphusidae and
one of the family Trichodactylidae serve as second intermediate
hosts (Blair et al., 1999).
At least 13 genera and 48 species of the family Pheudothel-
phusidae have been described in México, as summarized elsewhere
(Rodríguez and Magalhães, 2005). P. mexicanus metacercariae
have been found in crustaceans such as Pseudothelphusa dilatata
(Lamothe-Argumedo et al., 1977), Ptychophallus tristani and P.
costaricensis (Brenes et al., 1980; Mongue et al., 1985), Odontothel-
phusa maxillipes and Raddaus tuberculatus (Lamothe-Argumedo,
1984), Pseudothelphusa americana belliana, P. nayaritae and P. ter-
restris (Lamothe-Argumedo, 1995), all of which serve as second
intermediate hosts. In many regions of Mexico, as in the state of
Oaxaca, people traditionally include wild crabs and other crus-
taceans as part of their diet. Improper cooking of these crabs
represents a potential risk for infection due to ingestion of Parag-
onimus spp. metacercariae. The aim of this study is to isolate and
evaluate the presence of P. mexicanus from crabs collected in the
state of Oaxaca, Mexico.

http://dx.doi.org/10.1016/j.actatropica.2014.05.004
0001-706X/© 2014 Elsevier B.V. All rights reserved.
96 J. Vargas-Arzola et al. / Acta Tropica 137 (2014) 95–98
Fig. 1. Map of Mexico country and localization of Putla Villa de Guerrero, state of Oaxaca, Mexico.
2. Materials and methods
2.1. Source of crabs
Crabs were collected at San Miguel Reyes, municipality of Putla
Villa de Guerrero, State of Oaxaca, Mexico (97◦ 55 45 West and,
17◦ 01 33 North at an altitude of 783 m.a.s.l.) (Fig. 1). A total of 120
crabs (Fig. 2) of various sizes were collected between February 16th
and March 13th 2011, in small freshwater streams. Water tempera-
ture at the time of capture was 21 ◦ C; pH was 7.0. Crabs were placed
in plastic containers and transported alive to the laboratory.
2.2. Isolation of metacercariae
In the laboratory, crabs were allowed to acclimatize for two days
and then weighed and measured with a caliper. Afterwards, crabs
were killed by thermal shock (2 ◦ C for 10 min) fixed in 5% formalin
and then placed in plastic containers. Crabs were then dissected:
they were cut in half from the cephalothorax in longitudinal sec-
tion in order to extract the gills, digestive system and ontocele.
This material was placed in Petri dishes with saline, the tissue was
finelly sliced with scalpel and carefully examined under a stereo
microscope in search of Paragonimus spp. When found, metacer-
cariae were rinsed in saline and preserved in 96% ethanol until used.
Individual metacercaria were observed under the microscope.
Fig. 2. Image of crab Tehuana guerreroensis collected in Oaxaca, Mexico.
2.3. DNA extractions from metacercariae
DNA was extracted from approximately 5 mg of metacercariae.
DNA was purified using the DNeasy Blood & Tissue Kit (Qiagen,
Valencia, CA, USA) and its concentration was measured using a
nanoDrop 2000 (Thermo Scientific Waltham MA, USA). DNA from
uninfected crabs maintained and bred in the laboratory was used
as negative control. All DNA samples were stored at −20 ◦ C until
use.
2.4. PCR assay
The DNA extracted was amplified in a 25-␮l reaction well
using 100 ng of DNA template, 0.8 ␮M each of forward and reverse
primers (final concentration), and Master Mix (Roche). Amplifi-
cation was run in a Tc-3000 (TECHNE)® . Cycling was performed
as follows: initial DNA denaturing (94 ◦ C, 5 min), followed by 30
cycles each of denaturing (92 ◦ C, 30 s), annealing (61 ◦ C, 30 seg),
extension (72 ◦ C, 1 min). The final extension was 72 ◦ C for 4 min.
The primers 28S-F 5 -GAGGGTGAAAGGCCCGTGGG-3 and 28S-R
5 -ACGCATGCACACACCTCRAGCCG-3 were designed in a conserva-
tive region bracketing a variable region of approximately 630 bp of
the 28 S rRNA. Amplicons obtained were analyzed in 1.5% agarose
gel stained with ethidium under UV light. Positive and negative
controls were always included.
2.5. Sequencing and BLAST
The PCR product was sequenced in both strands in the Unit
of Proteogenomics, UNAM, Juriquilla, Mexico. The sequences were
viewed in the Chromas program Lite and refined manually. The final
sequence was analyzed by BLAST in the NCBI server.
2.6. Identification of crabs
Adult crabs were sacrificed by thermal shock, fixed in 5% forma-
lin for 5 days and then preserved in glass jars with 70% ethanol.
Specimens were sexed and identified at the family and genus
levels based on external morphological features using a Nikon
SMZ1000 stereo microscope. Males were dissected and the gono-
pod was extracted to allow the specific identification by taxonomic
keys (Rodríguez and Smalley, 1696; Rodríguez, 1982). The updated
species name was taken from criteria of Alvarez and Villalobos
(1994).
 J. Vargas-Arzola et al. / Acta Tropica 137 (2014) 95–98 97

Table 1
Prevalence of Paragonimus metacercariae in crabs Tehuana guerreroensis in a com-
munity of Putla Villa de Guerrero, state of Oaxaca, Mexico.

No. of crabs Average of No. of

metacercariae/crab metacercariae
detected

Examined Infected

120 25 (20.8%) 1.9 48

3. Results

Of a total of 120 crabs examined, 25 specimens (20.83%) were

positive for Paragonimus spp. metacercariae (Table 1). A total of 48
metacercariae were found, averaging 1.9 metacercariae per host
(range: 1–6 metacercariae per host). Metacercariae were mainly
located in the digestive system, ontocele and gills.
Individual metacercariae were stained and the internal struc-
tures were observed. As seen in Fig. 3, the oral sucker,
excretory system, acetabulum and blind gut showed typical
morphological characteristics of the Paragonimus genus. The
metacercariae were oval (890 ± 195 × 418 ± 94 ␮m) covered with
a spinosed tegument; oral sucker is subapical (93 ± 17 ␮m) and
central acetabulum (diameter 66 ± 12 ␮m); pharynx rectangu-
lar (110 ± 12.3 × 123 ± 12.3 ␮m, base) and intestinal ceca ending
blindly and extended to the end of the body.
The identification of P. mexicanus was confirmed by sequencing
a fragment of the 28S ribosomal gene (Fig. 4).
In Figs. 5 and 6 we show the caparace and left gonopod that allow
the identification of crabs. We determined that crabs belong to
Tehuana guerreroensis (Rathbun, 1933) (Crustacea, Brachyura, Pseu-
dothelphusidae) (= Pseudothelphusa guerreroensis Rathbun, 1933).
Fig. 3. A metacercaria of Paragonimus mexicanus found in the crab Tehuana guer-
reroensis from Putla Villa de Guerrero, state of Oaxaca, Mexico. (1) Oral sucker, (2)
4. Discussion excretory system, (3) acetabulum, (4) blind gut. Scale bar represent 100 ␮m.

Foodborne trematode infections are still an emerging pub-
lic health issue. These are closely related to the proximity of
human settlements to freshwater bodies. Worldwide, an estimated
750 million people are at risk of infections with foodborne trema-
todes, and at least 292.8 million humans are specifically at risk of
infection with the lung fluke Paragonimus spp. (Keiser and Utzinger,
2005, 2009). This parasite can cause zoonosis in humans when
meat infested with it is consumed raw or undercooked. Although
these parasites occur in Southeast Asia and Western Pacific regions
due to aquaculture and traditional cooking practices, some of them
may emerge in other continents through international trade and
improved transportation and distribution systems (Dorny et al.,
2009). It has been proposed that “the rising demands on natural
resources increase the likelihood of encountering environments
and produce food products contaminated with parasites” (Slifko
et al., 2000). In addition, social and cultural changes that modify
the diet have led humans to explore and in many cases alter the
micro- and macro-environmental conditions resulting in the emer-
gence or re-emergence of parasitic zoonosis (Macpherson, 2005).
The implementation of common parasitological techniques sup-
ported by molecular methods such as PCR and DNA sequencing may
be useful in the identification of parasites from different sources,
including local fauna, involved in the transmission of parasites.
Identification of P. mexicanus infecting crabs T. guerreroensis
from the community of San Miguel Reyes, Municipality of Putla
Villa de Guerrero, in the State of Oaxaca, Mexico, lead us to con-
sider this crab species as a second intermediate host. The site of
Putla is close to the border with the state of Guerrero. Rodríguez and
Smalley (1696) and Alvarez and Villalobos (1994), report the locali-
ties of Copanatoyac and Malinaltepec, south of Tlapa, all in the state
of Guerrero, as reference localities for T. guerreroensis. Vegetational,
climatic and socioeconomic affinities in the distribution range of T.

Fig. 4. Alignment of the sequence corresponding to the 28 rRNA amplicon. The nucleotide sequence was analyzed in the NCBI web site. A 100% identity match (192/192)
was obtained between the sample sequence and Paragonimus mexicanus (Subject) 28S ribosomal RNA gene, partial sequence reported in GenBank (HM172619.1).
98 J. Vargas-Arzola et al. / Acta Tropica 137 (2014) 95–98
Fig. 5. Tehuana guerreroensis, frontal región and orbital región of caparace.
Fig. 6. Tehuana guerreroensis. Left gonopode.
guerreroensis allow us to consider this area of southwestern Mexico
as a potential focus of paragonimiasis.
The DNA sequence obtained has a 100% match with the sequence
reported for P. mexicanus. Additionally, it has 98% match with P.
pseudoheterotremus and P. heterotremus, both species distributed
in Asia; this distributional data corroborates our identification of P.
mexicanus. On other hand, the techniques used in this study (stain-
ing and molecular identification) demonstrate the presence of P.
mexicanus in Putla, western Oaxaca, this location also being a new
report. People living in this community are poor and are primar-
ily engaged in farming and traditional use of natural resources.
Although the prevalence of P. mexicanus was relatively low, it
should nonetheless be considered a potential risk zone for acquiring
paragonimiasis.
Components of local fauna and other environmental factors,
together with the diet of local inhabitants, favor the establishment
of Paragonimus spp. These conditions suggest that the study region
might be at risk of Paragonimus spp. outbreaks, and identifies this
parasite as a potential target in preventive public health campaigns.
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