Parasitol Res (2009) 105:1637–1642 DOI 10.1007/s00436-009-1604-9 ORIGINAL PAPER Abnormalities on cephalic hooklets of advanced third-stage larvae from Gnathostoma Owen, 1836 (Nematoda: Gnathostomidae) collected from Mexican rivulus Millerichthys robustus Costa, 1995 (Ciprinodontiformes: Rivulidae) in Tlacotalpan, Veracruz, Mexico Miguel Ángel Mosqueda-Cabrera & Angélica Ocampo-Jaimes Received: 7 January 2009 / Accepted: 14 August 2009 / Published online: 3 September 2009 # Springer-Verlag 2009 Abstract Morphological abnormalities were observed on the cephalic bulb hooklets of advanced third-stage larvae (AdvL3) of genus Gnathostoma. The larvae were obtained from the fish “Mexican rivulus” Millerichthys robustus collected from a seasonal pond near Tlacotalpan, Veracruz, Mexico. The abnormalities involved (1) extra rudimentary hooklets, located between the four rows and after the fourth row, (2) branched or lobulated hooklets, and (3) fragmented hooklets not uniformly disposed in rows. The alterations observed on the cephalic bulb hooklets do not represent intraspecific variations, and they may be considered as a potential tool for assessing the presence of pollutants or stressors located within the ecosystem. Introduction Because of their morphoanatomy, importance for human and animal health, and unique life cycle (which includes at least three hosts) gnathostomes are interesting. In this sense, the first intermediate host is a cyclopoid copepod, where an early third-stage larva (EaL3) develops. Commonly, the second intermediate host is a fish, where the advanced third-stage larva (AdvL3) develops. The definitive host is a carnivore mammal, where the larva completes its developM. Á. Mosqueda-Cabrera (*) : A. Ocampo-Jaimes Departamento El Hombre y su Ambiente, Universidad Autónoma Metropolitana-Xochimilco, Calzada del Hueso No. 1100, Col. Villa Quietud, Delegación Coyoacán, 04960 México, D. F., México e-mail: zitzitl@correo.xoc.uam.mx ment to the adult stage (Almeyda-Artigas et al. 2000; Miyazaki 1954 and 1960). In Mexico, the three species of Gnathostoma have been documented (i.e., G. binucleatum Almeyda-Artigas 1991, G. turgidum Lamothe-Argumedo et al. 1998, and G. lamothei Bertoni-Ruiz et al. 2005). The identification of AdvL3 of these species is important because of their possible participation as causative agents of the human disease called gnathostomiasis. According to Miyazaki (1960) some of the most useful criteria to differentiate the AdvL3 of the genus Gnathostoma are: (1) number of transverse rows of hooklets in the cephalic bulb and (2) quantity, shape, and size of the hooklets occurring in each row. Consequently, the study of the shape of the hooklets in the cephalic bulb has been utilized as a criterion for differentiating species. In this sense, Akahane et al. (1995) documented for the first time, abnormalities of the cephalic hooklets in larvae of the genus Gnathostoma. They observed a new kind of AdvL3 taking into account the shape of the hooklet which shows a ramified base (zigzag shape). On the other hand, two studies have documented morphological variations or abnormalities of the cephalic bulb hooklets of AdvL3 from G. spinigerum. The first, carried out by Rojekittikhun and Pubampen (1998), recorded that 15.5% of the larvae isolated from the liver of mice experimentally infected showed morphological variations or abnormalities. The most common variations were additional rudimentary hooklets after the fourth row and between the four rows of the cephalic bulb (10.8%). They concluded that the variations were due to intraspecific differences. The second study, by Rojekittikhun et al. (1998) about larvae isolated from the viscera of eels Fluta alba bought at a Bangkok marketplace, showed a higher percentage of 1638 Parasitol Res (2009) 105:1637–1642 morphological variations and abnormalities (25.4%). Such variations were considered as interspecific differences by these authors. Recently, Martínez-Salazar and LeónRègagnon (2005) have reported the intraspecific variability of AdvL3 G. binucleatum from freshwater fishes in Tres Palos Lagoon, Guerrero, Mexico. They documented hooklets additional to those of the regular rows of the cephalic bulb (8.6%). In the current study, we record the presence of AdvL3 from Gnathostoma in a naturally infected Mexican rivulus Millerichthys robustus as well as the abnormalities observed in the cephalic bulb hooklets. Fig. 1 Male M. robustus. Scale bar 10 mm Materials and methods Results Sampling was carried out near Tlacotalpan, Veracruz Mexico. The host M. robustus (Ciprinodontiformes: Rivulidae; Fig. 1) were collected from “El Tamarindo”, a drying seasonal pond (18°37′39.3″N, 95°38′53.0″W), using spoon-like nets. The host M. robustus is an annual fish (term applied to species living in seasonal freshwater biotopes) that inhabits the Papaloapan and Coatzacoalcos Rivers in Mexico (Costa 1995). This species is endemic to Mexico and according to the Mexican regulations (NOM-059-Ecol-2001), it is considered an “endangered species” (Diario Oficial de la Federación 2002). In order to harvest every larva, the fish were taken alive to the laboratory, where they were killed and digested with artificial gastric juice (7 g pepsin in 10 ml HCL/1,000 distilled water) during 4 h at 40°C. On the other hand, “guavinos” Gobiomorus dormitor (Pisces: Eloetridae) were collected from the Papaloapan River, near the town of Tlacotalpan (18°36′N, 95°39′W) using a net or a hook; once in the laboratory, a meticulous examination of the musculature was carried out. All larvae were fixed in hot 70% ethanol, cleared in Amann lactofenol for their study in temporary wet mounts on glass slides, and later stored in 70% ethanol. A light microscope was used for the study. All measurements are given in micrometers, unless otherwise stated, and they are presented as a range, followed by the mean±standard deviation in parentheses. Photomicrographs were obtained by using a Kodak Technical Pan black and white film. Specimens were deposited in the “Colección Helmintológica de la Universidad Autónoma Metropolitana Unidad Xochimilco (CHUAMX), D. F., México”; accession numbers are: CHUAMX-G017 for eight AdvL3 obtained from M. robustus from “El Tamarindo” (1995); CHUAMXG036 for four AdvL3 obtained from M. robustus from “El Tamarindo” (1998); and CHUAMX-G038 for 30 AdvL3 obtained from G. dormitor from Papaloapan River (1998). The hosts G. dormitor and M. robustus were identified according to Espinosa-Pérez et al. (1993) and Costa (1995), respectively. The infection parameters utilized are those proposed by Bush et al. (1997). Three collections were performed, one in October 1995 and two in February 1998. A total of 80M. robustus were collected at the studied pond in 1995, from which eight larvae were recovered and later examined. During 1998, 30 M. robustus collected from the same pond were examined and four larvae were obtained; additionally, 14 guavinos were examined from the Papaloapan River, five of them were infected and 13 AdvL3 were recovered. Comparison between measurements of larvae obtained from different hosts in 1995 and 1998 are shown in Table 1. In the larvae collected from M. robustus in 1995, two types of morphological variations and abnormalities of the cephalic hooklets were observed, that is, extra rudimentary hooklets below the fourth row (75%) or between the four rows of hooklets and lobulated or branched hooklets (25%; Fig. 2a). The measurements of the larvae collected in 1998 were not different from those of 1995, but the alterations of the cephalic bulb hooklets were more evident. All larvae showed fragmented hooklets not uniformly disposed in the rows, and without a point. This made it impossible to count the number of cephalic hooklets (Fig. 2b, c). The AdvL3 isolated from guavinos did not show cephalic hook alterations (Fig. 2d) and are similar to those described by Almeyda-Artigas et al. (1994) as G. binucleatum. The parasite abundance in M. robustus was 0.10 and 0.13 for collections of 1995 and 1998, respectively, and because of the method utilized for recovering the larvae, it was not possible to calculate other infection parameters. In the case of the larvae from G. dormitor, the prevalence was about 36%, the intensity was from 1 to 8, the mean intensity was 2.6, and the abundance was 0.93. Discussion The AdvL3 from every host species were slightly different in sizes. The largest were those isolated from G. dormitor; Parasitol Res (2009) 105:1637–1642 1639 Table 1 AdvL3 of Gnathostoma, naturally obtained from hosts collected in the Papaloapan River and seasonal ponds near the town of Tlacotalpan, Veracruz, Mexico Observation Gnathostoma sp.a Gnathostoma sp.a G. binucleatumb Total length Maximum width Esophagus length Esophagus width Cephalic bulb length Cephalic bulb width Location of cervical papillae Location of excretory pore Number of hooklet rows in the body I II III IV IV-I N 2,792.0–4,276.8 202.5–226.8 1,109.7–1,239.3 147.2–171.7 78.0–106.8 178.6–192.9 12–15 16–25 170–283 3,097.2–3,760.0 (3,356.3±445.3) 203.7–220.0 (211.1±18.6) ND ND 74.4–112.8 (86.1±12.8) 155.2–190 (171.7±9.1) 10–15 (12.4±1.4) 22–28 (25.2±2.4) 235–250 (240.5±20.2) 2,300.4–4,338.3 243.0–348.3 850.5–1,490.4 102.2–298.4 73.9–180.7 149.9–262.8 10–17 27–35 222–303 (3,799.6±433.7) (220.1±10.8) (1,170.4±54.9) (161.5±8.9) (85.8±10.12) (183.7±6.1) (13.3±1.3) (21.0±2.1) (250. 3±26.4) 36–40 (38±1.9) 39–45 (42.3±2.4) 39–46 (43.0±2.8) 48–52 (49.8±1.6) 10–15 (11.8±1.7) 8 4 (3,143.5±669.6) (317.7±26.3) (1,198.4±155.7) (178.5±43.0) (139.4±27.2) (220.1±26.4) (13.5±2.0) (30.0±2.3) (259.1±21.8) 36–44 (40.2±2.3) 35–47 (43.5±2.7) 39–51 (46.9±2.6) 44–54 (49.9±2.5) 4–16 (9.6±2.7) 13 ND not determined a Obtained from M. robustus collected from “El Tamarindo” (1995 and 1998, respectively) b Obtained from guavino G. dormitor collected from Papaloapan River (1998) which might be attributed to a larger muscular space available for the development of the larvae and/or to the longevity of the host. The number of hooklets in the rows of the cephalic bulb is similar to that reported for G. binucleatum and G. spinigerum (Table 2). The most important difference between G. binucleatum and G. spinigerum (absent in America according to AlmeydaFig. 2 Front view of cephalic bulbs of AdvL3 of Gnathostoma. a Larva from M. robustus collected in 1995, with abnormal hooklets. b–c Larva from M. robustus collected in 1998, with abnormal hooklets. d Larva of G. binucleatum from G. dormitor, with four rows of normal hooklets. Scale bar 50 μm Artigas et al. 2000) appears to be the average number of hooklets appearing in every row; G. spinigerum presents, on average, four hooklets more than G. binucleatum in all the rows, a peculiarity that was previously documented by Akahane et al. (1994). In the case of G. binucleatum, no more than 50 hooklets are observed in the second and third rows, as frequently occurring in G. spinigerum (Table 2). 1640 Parasitol Res (2009) 105:1637–1642 Table 2 Comparisons of number of hooklets in the four rows of the cephalic bulb of AdvL3 of Gnathostoma spp. Species Rows of cephalic bulb I G. spinigeruma G. binucleatumb G. turgidumc G. binucleatumd Gnathostoma sp.e 39–49 35–44 26–34 36–44 36–40 II (44.3) (38.7) (30.8) (40.2) (38.0) 42–54 38–47 29–38 35–47 39–45 III (47.3) (42.4) (34.0) (43.5) (42.3) 45–56 40–49 29–43 39–51 39–46 IV (49.6) (44.7) (36.7) (46.9) (43.0) a Miyazaki (1954), based on the measurements of 26 larvae from fish (Channa argus; Ophiocephalus argus) b Almeyda-Artigas (1991), 30 larvae from fish (Petenia splendida) c Mosqueda-Cabrera et al. (2009), 11 larvae from frog (R. zweifeli) d Current study, 13 larvae from G. dormitor e Current study, 8 larvae from M. robustus Consequently, the larvae isolated from the guavinos correspond to G. binucleatum (Almeyda-Artigas 1991). However, the comparison between the larvae from G. dormitor and from M. robustus shows the excretory pore of the latter, which is closer to the cephalic bulb (Table 1). This suggests that these larvae might correspond to a different species. The larvae from M. robustus do not correspond to G. turgidum, because the AdvL3 of this species are typically encysted in the musculature of the frog, Rana zweifeli (Mosqueda-Cabrera et al. 2009), have a lower number of hooklets in the cephalic bulb (Table 2), and are smaller 1,530–2,007.4 (1,670.2). A new species of gnathostome was recently described in Mexico, G. lamothei (BertoniRuiz et al. 2005); however, up to this date, the third stage of the larvae of this species has not been characterized, making the comparison impossible. Based on the morphology of the cephalic bulb hooklets of AdvL3, some authors have considered that such larvae may constitute a new species of the genus Gnathostoma (Setasuban et al. 1991; Akahane et al. 1995). Particularly in the case of AdvL3 of G. spinigerum naturally and experimentally obtained, morphological variations or abnormalities in the cephalic bulb were recorded (Rojekittikhun and Pubampen 1998; Rojekittikhun et al. 1998). These abnormalities were: presence of additional rudimentary hooklets before the first row and after the fourth one, as well as between the four rows of the cephalic bulb; presence of a fifth row of hooklets; as well as lobulated, branched, and fragmented hooklets. Taking into account such observations, they concluded that the AdvL3 experimentally obtained, were very similar to the new type of AdvL3 from eels of Nakhon Nayo, Thailand, described by Setasuban et al. (1991), and to those reported by Akahane et al. (1995). The AdvL3 collected from M. 45–58 43–52 33–42 44–54 48–52 IV-I (52.0) (48.2) (39.6) (49.9) (49.8) 7.7 9.5 8.8 9.6 12.0 robustus in 1995 show morphological alterations similar to those described by Rojekittikhun and Pubampen (1998), and Rojekittikhun et al. (1998; Fig. 2a). However, the AdvL3 isolated from M. robustus collected in 1998 show different morphological alterations: the hooklets are not disposed uniformly in rows and they do not have a clear shape (they show fragmented scales of different shapes and sizes, without the characteristic point of the hooklet; Fig. 2b, c). Contrary to Rojekittikhun et al. (1998), who obtained AdvL3 from naturally infected eels, we think that the abnormalities observed in our research are not due to interspecific variations but intraspecific. This fact is supported by the results shown by Rojekittikhun and Pubampen (1998), who recorded intraspecific variations on AdvL3 of G. spinigerum experimentally obtained. Therefore, the new type of larva reported by Akahane et al. (1995) is the result of intraspecific variations on the AdvL3 of G. spinigerum and not due to interspecific morphological variations as indicated by these authors. Furthermore, the intraspecific variability in Gnathostoma has been documented on the basis of the shape of the cephalic hooklets of AdvL3. In this sense, MartínezSalazar and León-Règagnon (2005) have recognized up to six morphotypes of G. binucleatum. These authors observed a minimum amount of variation among ITS2 sequences of three of their morphotypes. They conclude that the variation among morphotypes 1, 2, and 3 is the result of intraspecific variability of the species supported by morphology and DNA traits. Thus, the abnormalities of cephalic bulb hooklets from AdvL3 cannot be considered as valid characteristics for differentiating species. On the other hand, according to Möller (1987), there is a close relationship between parasitic diseases and pollution of aquatic environments; this relationship relies Parasitol Res (2009) 105:1637–1642 on the vulnerability of the free-living larval stages of the parasites and, therefore, the effect on the intermediate or definitive hosts. Free-living infective stages of parasites are directly exposed to toxins, and may be susceptible to pollution (Poulin 1992; Overstreet 1997; MacKenzie 1999; Pietrock and Marcogliese 2003). However, little is known about the direct effects of toxic agents on the freeliving stages of parasites, ectoparasites exposed to contaminants throughout their entire life, or the intermediate hosts (Poulin 1992). In this sense, Šebelová et al. (2002) have reported abnormalities in the attachment clamps of diplozoon Paradiplozoon homoion, P. ergensi, P. megan, and Diplozoon paradoxum (Monogenea: Diplozoidae) isolated from gills of fish exposed to water pollution in two river systems in Europe. The abnormalities observed in the clamps included a reduction in the number and size of clamps, asymmetry in their arrangement, and deformities. In this case, the authors concluded that pollutants have a direct impact on diplozoon morphology. Moreover, Rojekittikhun et al. (1998) state that abnormalities may be a consequence of a larger infection period on the natural life cycle. However, in our opinion the possible presence of toxins, or toxic agents in the pond might be the cause of the alterations of the AdvL3 found in M. robustus. Taking into account that in our case, the pond has no tributary source of water, and is located within an agricultural and livestock breeding zone (activities which involve an intensive use of plaguicides and other chemical products), the host–parasite relationship may be exposed to these pollutants. Such toxins may provoke the morphological alterations observed in the larvae. Initially, the toxic exposure may affect the free-living phases (egg and secondstage larvae), and the effects may continue in the EaL3 and AdvL3, during their development within the first intermediary host (Copepoda) and second intermediary host (Pisces), respectively. Finally, identifying the causes of the observed deformations of gnathostomes in M. robustus may provide important environmental information. 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