Behavioral changes caused by Austrodiplostomum spp. in Hoplias malabaricus from the São Francisco River, Brazil Lincoln L. Corrêa, Geza T. R. Souza, Ricardo M. Takemoto, Paulo S. Ceccarelli & Edson A. Adriano Parasitology Research Founded as Zeitschrift für Parasitenkunde ISSN 0932-0113 Parasitol Res DOI 10.1007/s00436-013-3679-6
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Author's personal copy Parasitol Res DOI 10.1007/s00436-013-3679-6
ORIGINAL PAPER
Behavioral changes caused by Austrodiplostomum spp. in Hoplias malabaricus from the São Francisco River, Brazil Lincoln L. Corrêa & Geza T. R. Souza & Ricardo M. Takemoto & Paulo S. Ceccarelli & Edson A. Adriano
Received: 31 May 2013 / Accepted: 4 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Traira (Hoplias malabaricus) is a neotropical fish that is widely distributed in freshwater environments in South America. In the present study, we documented the occurrence of metacercariae of Austrodiplostomum spp. (Diplostomidae) in the eyes and cranial cavity of H. malabaricus and described parasite-induced behavioral changes in the host. The fish were collected from the upper São Francisco River, in the Serra da Canastra mountain range, Minas Gerais, transported alive to
L. L. Corrêa (*) : E. A. Adriano Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6109, CEP 13083-970 Campinas, SP, Brazil e-mail:
[email protected] E. A. Adriano e-mail:
[email protected] G. T. R. Souza : R. M. Takemoto Laboratório de Ictioparasitologia, Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura-Nupélia, Universidade Estadual de Maringá, Bloco G-90, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil G. T. R. Souza e-mail:
[email protected] R. M. Takemoto e-mail:
[email protected] P. S. Ceccarelli Centro Nacional de Pesquisa e Conservação de Peixes Continentais (CEPTA), Instituto Chico Mendes de Conservação da Biodiversidade (ICMbio), Rod. SP 201, Km 6.5, Caixa Postal 64, CEP 13630-970 Pirassununga, SP, Brazil e-mail:
[email protected] E. A. Adriano Departamento de Ciências Biológicas, Universidade Federal de São Paulo (UNIFESP), Rua Professor Artur Riedel, 275, Jardim Eldorado, CEP 09972-270 Diadema, SP, Brazil
the laboratory, observed for 2 weeks, and subsequently examined for parasites. Of the 35 fish examined, 28 (80 %) had free metacercariae in the vitreous humor (mean intensity=95.4; mean abundance = 76.3), and 24 (68.57 %) had free metacercariae in the cranial cavity, mainly concentrated below the floor of the brain, at the height of the ophthalmic lobe (mean intensity=12.91; mean abundance=8.85). Specimens of H. malabaricus with a high intensity of infection in the brain displayed changes in swimming behavior.
Introduction Hoplias malabaricus (Bloch, 1794) is a characiform fish in the Erythrinidae family, popularly known in Brazil as traira. Larval fish feed on plankton, juveniles feed on insects, and adults over 15 cm are piscivorous (Loureiro and Hahn 1996; Martins et al. 2009). H. malabaricus is benthic and sedentary and uses an ambush strategy to capture prey during both the day and night (Oliveira 1994; Loureiro and Hahn 1996; Hahn et al. 1997; Shibatta et al. 2002). H. malabaricus is infected by a variety of helminthes, including digeneans in the family Diplostomatidae. Diplostomatids utilize a three host life cycle where adults typically parasitize a wide range of piscivorous birds, freshwater snails act as first intermediate hosts, and freshwater fishes (occasionally amphibians) act as second intermediate hosts. The cercariae released from host snails infect the fishes and develop into metacercariae, which are found encysted and encapsulated in tissue, or free in the skin, eyes, musculature, and central nervous system. These metacercariae pass into the definitive host when it eats an infected fish (William and Charlie 1989; Eiras 1994; Karvonen et al. 2006; Santos et al. 2012). At high densities, metacercariae can cause exophthalmos, retinal detachment,
Author's personal copy Parasitol Res
lenticular opacities, blindness (Silva-Souza 1998), and cranial distortion with disruption of brain tissue, which ultimately results in reduced host survival (Bauer et al. 1964; Heckmann 1992; Sandland and Goater 2001). Metacercariae of the diplostomid Austrodiplostomum spp. infect the eyes and more rarely the brain of various species of South American fish (Zica et al. 2011). Diplostomids found outside of the lens tend to be host specific, and the ones in the lens tend to be generalists. However, these metacercariae cannot be identified solely based on morphology, and species identification requires the use of molecular markers (Locke et al. 2010). As such, in the present study, the forms found infecting the eyes and brain of H. malabaricus were considered to be a complex of cryptic species, named Austrodiplostomum spp. The central nervous system (CNS) is key for the coordination of host behavior, and parasites that occupy the CNS are ideally located to manipulate behavior either through damage or more subtle manipulation (Lafferty and Shaw 2013). Manipulation of the behavior of hosts caused by parasites has been reported in numerous host taxa (e.g., Shaw et al. 2009; Yanoviak et al. 2008; Berdoy et al. 2000), including trematodes (Poulin 2010; Lafferty and Shaw 2013). Most of these cases involve only subtle changes in one aspect of the host behavior or appearance, while some are more significant, as in the classical case of the trematode Dicrocoelium dendriticum. This parasite causes infected ants to climb to the tip of grass blades to patiently wait for a grazing sheep. Leucochloridium spp. alters the size, shape, and coloration of the tentacles of its snail intermediate host and causes them to pulsate violently in response to light, attracting the attention of birds to which the parasite must be transmitted (Poulin 2010). Parasite-induced changes in host behavior may be caused by a variety of mechanisms, the majority of which remain poorly understood (Lafferty and Shaw 2013). For example, if parasites extract energy from their hosts in the form of nutrition, hosts may become more sluggish or display diminished physical performance. Alternatively, the host may compensate for the parasitic infection by becoming more active and increasing its foraging rates. Both changes may increase the likelihood that the infected host is consumed by a predator (Lafferty and Shaw 2013; Poulin 2010). Another form of manipulation has been demonstrated in which some parasites appear to rely heavily on neuropharmacological methods to alter host behavior (Poulin 2010; Lafferty and Shaw 2013; Adamo 2013). Changes in the monoamine production have been observed in several host species infected with larval parasites. Altered indoleamine 5HT metabolism underlies changes in host behavior in fish infected by larval helminths (Lafferty and Shaw 2013; Adamo 2013). Specimens of Gasterosteus aculeatus infected with larval tapeworm Schistocephalus solidus displayed
elevated 5-HT activity in their brainstems and leading to exhibit altered behaviors, including increased surfacing and impaired escape responses, which should make infected fish more susceptible to predation by birds, the final hosts for S. solidus (Barber et al. 2000; Overli et al. 2001). The present study described the prevalence and intensity of Austrodiplostomum spp., diplostomid trematodes found infecting the eyes and brain of H. malabaricus in the São Francisco River Basin, as well as reported on the behavioral changes observed in host fish.
Material and methods Specimens of H. malabaricus (n =35) were collected in February 2010 from the Inhuma Lake, in the municipality of Puín, MG, near the Serra da Canastra mountain range (20°10′ 53″S and 45°50′32″W), in the São Francisco River Basin. The fish were collected with gillnets with different meshes (30 and 35 mm) and transported to the laboratory. For 2 weeks, all the fish were kept in a 2,000-l tank with running, filtered water; fed with small fishes; and observed for 30 min each morning. Fish with erratic swimming patterns were photographed. At the end of 2 weeks, the fish were euthanized by cervical cord transection, weighed, and measured (total length). All organs were examined with the aid of a stereomicroscope. The recovered metacercariae were compressed between a slide and cover slip, fixed in AFA (70 % ethanol, 93 ml; formalin, 5 ml; acetic acid, 2 ml), and preserved in 70 % ethanol. Subsequently, the helminths were either stained with carmine or diaphanized in beechwood creosote, mounted on permanent slides using Canada balsam, and identified in accordance with Gibson et al. (2002). Prevalence, mean intensity, and abundance were calculated in accordance with Bush et al. (1997). To identify the metacercariae, a Sony DSC-W5 digital camera was used coupled to a Stemi SV11 stereomicroscope. Morphometric analysis was performed using a computerized imaging system (QWin Lite 3.2, Leica). The Mann–Whitney U test was used to analyze differences in intensity of infection in different host tissues. The Duncan multiple mean comparison test was used to test if there was a difference in means of intensity of the larvae of Austrodiplostomum spp. between fish with altered swimming behavior and those with normal behavior. We calculated the relative condition (Kn) using the standard length (Ls) and total weight (Wt) of each host to estimate coefficients a and b for the length–weight relationship: Wt=a Ltb . The values of a and b were used to estimate expected weight (We) using the equation: We=a.Ltb . Then, the relative condition factor (Kn), which corresponds to the quotient between the weight observed and expected weight for a given length (Kn=Wt/We) (Le Cren 1951), was calculated for each host.
Author's personal copy Parasitol Res Table 1 Data of infection by metacercariae of Austrodiplostomum spp. in the eyes and cranial cavity of H. malabaricus taken from the São Francisco River Basin Infection site
Infected Ax fish
x and s
P (%) MI
MA
RE
25
1-138 39.17±38.91 71.42
54.84 39.17
LE
28
1-117 37.17±34.09 80.00
46.46 37.17
RE and LE
28
1-138 38.17±36.33 80.00
95.42 76.34
CC
24
2-42
12.91
RE and CC
24
1-138 24.01±32.25 68.57 111.33 76.34
LE and CC
24
(RE and LE) CC 24
13.27±9.78
68.57
1-117 23.01±28.93 68.57
8.85
67.12 46.02
1-138 28.40±33.30 68.57 124.25 85.20
Ax variation amplitude, x arithmetic mean, s standard deviation, MI mean intensity, MA mean amplitude, RE right eye, LE left eye, CC cranial cavity
The Mann–Whitney U test was used to analyze differences between the relative condition of infected and uninfected fish specimens, and Spearman's correlation coefficient was used to evaluate the relationship between condition and intensity of infection (Zar 1996). Statistical analysis was performed using the SAS (SAS Inc. 1996) statistical software program. Fish capture was authorized by IBAMA/ICMBio (Proc. no. 27447-2/2010-2011). The capture procedures and euthanasia of the fish were approved by the UNICAMP-CEUA (Proc. no. 2090-1) ethics commission. Representative specimens were deposited in the CEPTA/ICMBio Ichthyology Laboratory.
Results Thirty-five juvenile to adult fish specimens with length varying from 11 to 16 cm (mean=14.12 cm, standard deviation=1.08 cm) and weight varying from 11.40 to 32.30 g (mean=22.86 g, standard deviation=4.59) were used in this study. A total of 28 (80 %) of the fish had metacercariae of Austrodiplostomum spp. in the eyes (mean intensity [MI] of 95.4 and mean abundance [MA] of 76.3).
Fig. 1 Light photomicrographs of H. malabaricus eye infected with metacercariae of Austrodiplostomum spp. a Eye with free metacercariae in the vitreous humor (arrow). b Austrodiplostomum sp. (Trematoda: Diplostomidae) collected from the eyes. Scale bar =500 μm
Twenty-four (68.57 %) also had larvae in the cranial cavity (MI of 12.91 and MA of 8.85) (Table 1). In the eyes, the larvae were free in the vitreous humor, with no increase in the opacity of the infected eyes (Fig. 1). In the skull, the metacercariae were also free and mainly observed below the brain, on the floor, at the height of the ophthalmic lobes, and close to the optic nerve. The ophthalmic lobes, particularly the veins that irrigate this region, were dilated and clearly visible in infected fish. This alteration was not observed in noninfected fish or fish with infections only in the eyes. The Mann–Whitney U test showed that the infection intensity was not significantly different between the right and left eyes (Z(U )=3.51; n (A +B)=35; p =0.0002), but infection intensity did differ significantly between the eyes and the skull (Z(U )=0.041; n (A +B )=35; p
