Physiological Alterations in Bradybaena Similaris

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Experimental Parasitology 139 (2014) 12–18

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Experimental Parasitology

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Physiological alterations in Bradybaena similaris (Stylommatophora: Bradybaenidae) induced by the entomopathogenic nematode Heterorhabditis indica (Rhabditida: Heterorhabditidae) strain LPP1 ⇑ Victor Menezes Tunholi a,b,c, , Caio Oliveira Monteiro a, Lidiane Cristina da Silva a, Claudia de Melo Dolinski d, Marcos Antônio José dos Santos e, Maria de Lurdes de Azevedo Rodrigues f, ⇑ Vânia Rita Elias Pinheiro Bittencourt f,1, Jairo Pinheiro b,1, Vinícius Menezes Tunholi-Alves a,b,

a Curso de Pós-Graduação em Ciências Veterinárias, Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Km 7, BR 465, Antiga estrada Rio-São Paulo, 23890-000 Seropédica, RJ, Brazil b Departamento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, Km 7, BR 465, Antiga estrada Rio-São Paulo, 23890-000 Seropédica, RJ, Brazil c Docente do curso de Medicina Veterinária da Faculdade de Castelo (FACASTELO), Av. Nicanor Marques s/n, Castelo, ES 29360-000, Brazil d Universidade Estadual do Norte Fluminense Darcy Ribeiro, Centro de Ciências e Tecnologias Agropecuárias, Campos dos Goytacazes, RJ, Brazil e Departamento de Biologia Animal, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, Km 7, BR 465, Antiga estrada Rio-São Paulo, 23890-000 Seropédica, RJ, Brazil f Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, RJ, Brazil

highlights graphical abstract

H. indica LPP1 induces mortality in B. similaris. Exposure by H. indica LPP1 induces proteolysis in B. similaris. H. indica LPP1 increase the activities of AST and ALT in B. similaris. Exposure by H. indica LPP1 changes the levels of nitrogen products in B. similaris.

article info abstract

Article history: Heterorhabditis is a nematode found in the soil that is used as an important biological control agent Received 3 June 2013 against various organisms. However, few studies have been performed of its use against snails and the Received in revised form 6 February 2014 present study is the ﬁrst to investigate the effect of experimental exposure of Bradybaena similaris to Het- Accepted 9 February 2014 erorhabditis indica LPP1. Two groups of 16 snails were formed: the control group (not exposed) and the Available online 22 February 2014 treatment, which was exposed for three weeks to infective juveniles (J3) of H. indica LPP1. The entire experiment was conducted in duplicate, using a total of 64 snails. After this period, the snails were dis- Keywords: sected to collect the hemolymph to evaluate the possible physiological alterations, namely total proteins, Entomopathogenic nematodes uric acid and hemolymph urea, as well as the activities of alanine aminotransferase (ALT) and aspartate Parasite–host interaction Biological control aminotransferase (AST) as a result of the infection. The terrariums were analyzed on alternate days

⇑ Corresponding authors at: Curso de Pós-Graduação em Ciências Veterinárias, Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Km 7, BR 465, Antiga estrada Rio-São Paulo, 23890-000 Seropédica, RJ, Brazil. Fax: +55 28 99946 7752. E-mail addresses: [email protected] (V.M. Tunholi), [email protected] (V.M. Tunholi-Alves). 1 Research Fellow, CNPq.

throughout the experiment to count the dead snails. Intense proteolysis was observed in the infected snails. An increase in the level of uric acid and reduction of the hemolymph urea content indicated that the infection by H. indica results in the inversion of the excretion pattern of the host snail. Variations in the aminotransferase activities were also observed, with the infected group presenting signiﬁcantly higher values (p < 0.05) than the control group for both ALT and AST. The exposure to H. indica LPP1 caused 55% mortality, with the highest rate observed in the ﬁrst week after exposure (30%). These results suggest that the use of H. indica LPP1 is a feasible alternative for the biological control of B. similaris. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction (Akhurst, 1995). The infection of the host occurs through natural openings as (mouth, anus and spiracles) or, in some cases, through Gastropod mollusks are organisms of great importance in hu- the cuticle. After entering the host’s hemocoel, the nematodes re- man and veterinary medicine because they are intermediate hosts lease their simbiotic bacteria, which are primarily responsible for of various parasites, such as trematodes and nematodes. Many of killing the host (Dowds and Peters, 2002). Recently, the release of these helminths afflict pets and farm animals as well as humans, EPNs in the environment by providing cadavers of infected insects causing economic losses to stock breeders and public health prob- has shown good results (Dolinski, 2006). In this method, after a few lems (Pinheiro et al., 2011; Tunholi-Alves et al., 2012). Because of days of application, the infective juveniles (J3 = IJs) begin to leave the intimate relationship observed between some parasite species the cadaver and seek new hosts in the soil, making the control of and host snails, the World Health Organization (WHO, 1983) rec- the target pest (Shapiro-ilan et al., 2006). Theses EPNs, in general, ommends the development of new methods to control host popu- have low specificity to the natural host, and under certain condi- lations as an effective strategy to eradicate parasitoses. tions can infect snail, resulting in significant pathological changes The Asian trampsnail Bradybaena similaris (Férussac, 1821) is a (Jaworska, 1993). land snail found throughout Brazil. It is classified as an agricultural There are few studies of the pathogenicity of EPNs in snails. One pest besides being the intermediate host of parasites such as of the few is described by Jaworska (1993), which describes the Angiostrongylus cantonensis (Chen, 1935) and Angiostrongylus cos- susceptibility of Deroceras agreste (Linnaeus, 1758) and Deroceras taricensis (Morera and Céspedes, 1971), which are etiological reticulatum (Müller, 1774) to infection by three EPN species. This agents of neural and abdominal angiostrongyliasis, both of which is not the earliest report, since Li et al. (1986) observed that certain are relevant public health problems (Caldeira et al., 2007), as well species of Steinernema and Heterorhabditis can infect, kill and de- as of the trematodes Eurytrema coelomaticum (Giard and Billet, velop in the semi-aquatic snail Oncomelania hupensis Gredler 1892), a parasite of the pancreatic ducts of ruminants (Pinheiro (1881). However, to our knowledge, no previous study has con- and Amato, 1995), and Postharmostomum gallinum (Witenberg, firmed the use of EPNs to control of B. similaris. Based on the fact 1923) (Amato and Bezerra, 1996), which afflicts the intestinal ce- that B. similaris inhabits an environment favorable to the presence cum of chickens. of EPNs, the aim of the present study was to evaluate for the first For many decades the control of snails has relied mainly on the time the possible physiological alterations and the mortality rate application of chemical molluscicides (Machado, 1982). But, this of B. similaris after exposure to infective juveniles (J3) of Heteror- control strategy is not sustainable because these compounds accu- habditis indica, isolate LPP1, during three weeks. mulate and contaminate the environment, compromising human and animal health (Henrioud, 2011). Additionally, studies have 2. Materials and methods demonstrated the biocidal effect of these substances on non-target plants and organisms. Finally, the high cost of their application 2.1. Source of the snails and nematodes limits their use in public control programs (Andrews et al., 1983). In this context, many experiments have been carried out to find The snails used in this study were obtained from a colony kept new control alternatives, such as the use of molluscicidal sub- in the Laboratório de Biologia de Moluscos do Museu Professor stances of plant origin (Lustrino et al., 2008; Mello-Silva et al., Maury Pinto de Oliveira of the Universidade Federal de Juiz de Fora 2010; Silva et al., 2012). Another alternative method to control (UFJF), located in the city of Juiz de Fora, Minas Gerais, Brazil. The agricultural pests is to use microorganisms that are pathogenic to nematodes of the species H. indica isolate LPP1 were donated by the host. In this respect, entomopathogenic nematodes (EPNs) of the Laboratório de Nematologia of Universidade Estadual Norte the genera Steinernema Chitwood and Chitwood (1937) and Het- Fluminense (UENF) and were maintained and multiplied in the erorhabditis Poinar (1976) have shown great promise and have Laboratório de Parasitologia of the Embrapa Dairy Cattle Research been successfully used in various countries to control insect pests Unit (Embrapa Gado de Leite) according to the methods proposed that present at least one development stage in the soil (Grewal by Lindegren et al. (1993) and Kaya and Stock (1997). et al., 2001; Dolinski, 2006). The infective juveniles of these nematodes are found in the soil. Upon infecting the host, they release 2.2. Exposure of the snails to the nematodes symbiont bacteria in the hemocele, which multiply and cause the insect’s death by septicemia between 24 and 72 h after infection Cadavers of the caterpillar Galleria mellonella (Linnaeus, 1958) (Grewal et al., 2001; Hazir et al., 2003). This nematode have a sim- were used as the source of nematodes to infect the snails, accord- ple life cycle that includes the egg, four juvenile stages (separated ing to the method proposed by Shapiro and Glazer (1996). To pre- by moults) and adult (Adams and Nguyen, 2002), in which, third pare the infected cadavers, six caterpillars were placed inside a stage juvenile represent the infecting form. The infective juveniles previously sterilized Petri dish (9 cm diameter) lined with two are the only free living (outside host), occurring naturally in the sheets of filter paper. Then the lining was moistened with 2 ml of soil where they infect and kill their insect host within 2 or 3 days, an aqueous suspension containing approximately 600 nematodes producing 2 or 3 generations in the host. As a result, infective juve- (100 EPNs/caterpillar) and the dish was sealed with plastic film niles are formed, emerging from host cadaver 1 or 2 weeks later (ParafilmÒ) and placed in a climate-controlled chamber (25 ± 1 °C). 14 V.M. Tunholi et al. / Experimental Parasitology 139 (2014) 12–18

After three days the caterpillars with signs of infection were 2,dinitrophenylhydrazine was added and the solution was kept at transferred to another previously sterilized Petri dish also lined 25 °C for 20 min. The reactions were interrupted by adding 5 mL with filter paper. The dishes were sealed with the same film and of 0.4 M NaOH. The readings were taken in a spectrophotometer kept in the chamber under the same conditions. After eight days, at 505 nm (Kaplan and Pesce, 1996) and the results were expressed the six infected caterpillars were buried in a terrarium as URF/ml. (12 Â 24 Â 14 cm) containing a 10-cm layer of autoclaved soil with the addition of 0.5 g of CaCO3. The terrarium was periodically 2.7. Determination of the concentrations of uric acid and urea moistened with dechlorinated water to establish favorable conditions for the maintenance of the snails and nematodes. To measure the uric acid level, 50 ll of hemolymph was mixed with 2 ml of the dye reagent (100 mmol/L of sodium phosphate 2.3. Maintenance of the snails and formation of the experimental buffer [pH 7.8] containing 4 mmol/L of dichlorophenol-sulfonate, groups 0.5 mmol/L of 4-aminoantipirina, 120U 6 uricase, 4.980U 6 ascor- bate oxidase, 1.080U 6 peroxidase). The mixture was homogenized Two experimental groups were formed: one control group and incubated at 37 °C for 5 min. The readings were taken in a (uninfected) and one infected group (infected). Each group con- spectrophotometer at 520 nm (Bishop et al., 1996) and the results tained 16 snails, reared in the laboratory from hatching, to be cer- were expressed as mg/dl. tain of their age and that the snails were free of infection by other The urea concentration was measured by adding 2 ml of a solu- parasites. The entire experiment was conducted in duplicate, using tion containing 60 mmol of sodium salicylate, 3.4 mmol of sodium a total of 64 snails, of which 32 snails constituted the control group nitroprusside and 1.35 mmol of disodium EDTA. Then 2 ll of ure- and 32 snails, the infected group. The terrariums were kept in a ase and 20 ll of hemolymph were added. This mixture was homog- room with controlled temperature of 25 °C throughout the enized and incubated at 37 °C for 5 min. The readings were taken experiment. in a spectrophotometer at 600 nm and the results were 202 ex- The snails were fed with fresh lettuce leaves (Lactuca sativa L.) pressed as mg/dl (Connerty et al., 1955). ad libitum. The terrariums were maintained every other day, when the lettuce leaves were replaced to prevent their fermentation. For 2.8. Histochemical analyses three weeks the terrarium were analyzed on alternate days to count the dead snails, by direct observation, with the dead snails Snails (three) from each group experimental were dissected and being immediately removed. The dead snails were then placed in transferred to Duboscq-Brasil fixative (Fernandes, 1949). Soft tis- White traps (White, 1927) for possible recovery of infective juve- sues were processed according to routine histological techniques niles and verification of completion of the cycle of H. indica LPP1 (Humason, 1979). Sections (5 lm) were stained using (hematoxy- in the snail. To avoid the influence of population density on the lin-eosin and Gomori trichrome) and observed under a Zeiss Axio- physiological patterns of B. similaris (Oliveira et al., 2008), the same plan light microscope. Images were captured by an MRc5 AxioCam number of specimens were removed from the control group as the digital camera and processed with the Axiovision software. dead snails in the treated group, to keep the number of snails in the two groups equal. 2.9. Statistical analyzes

2.4. Dissection and collection of the hemolymph The results obtained were expressed as mean ± standard deviation and the Tukey test was used to compare the means (P < 0.05) At the end of the three-week experimental period, the snails (InStat, GraphPad, v.4.00, Prism, GraphPad, v.3.02, Prism, Inc.). from the control and infected groups were dissected and the hemolymph was collected by cardiac puncture and maintained at À10 °C 3. Results until the biochemical analyses. All the samples were kept in an ice bath during dissection. The choice of the study period (three The exposure to H. indica LPP1 caused 55% mortality in B. simi- weeks) was based on Wilson et al. (1994) in function of the mortal- laris, with the highest rate observed in the ﬁrst week after exposure ity rate during the infection of D. reticulatum by Heterorhabditis sp. (30%) (Fig. 1). The levels of total proteins, uric acid and urea and activity of the 2.5. Determination of total proteins aminotransferases (ALT and AST) in the hemolymph of uninfected

This assay was performed according to the biuret technique (Weichselbaum, 1946). A mixture of 50 ll of hemolymph and 2.5 ml of the biuret reagent (0.114 M trisodium citrate, 0.21 M sodium carbonate and 0.01 M copper sulfate) was homogenized and left at room temperature for 5 min, after which the readings were taken in a spectrophotometer at 550 nm. The results were expressed as mg/dl.

2.6. Aminotransferases activities (AST and ALT)

To test for aminotransferases activities, 0.5 ml of substrate for EC2.6.1.2 L-alanine: 2 oxoglutarate aminotransferase (ALT) or EC2.6.1.1 L-aspartate: 2 oxoglutarate aminotransferase (AST) (solution containing 0.2 M L-alanine or 0.2 M L-aspartate; 0.002 M a- cetoglutarate and 0.1 M sodium phosphate buffer, pH 7.4) was incubated at 37 °C for 2 min. Then, 100 or 200 ll of hemolymph (for ALT and AST, respectively), were added, homogenized and Fig. 1. B. similaris mortality (%) after exposure by H. indica LPP1 during three weeks again incubated at 37 °C for 30 min. After this, 0.5 mL of 0.001 M (control = uninfected snails). V.M. Tunholi et al. / Experimental Parasitology 139 (2014) 12–18 15

Table 1 Variation in the total protein (mg/dl), uric acid and urea (mg/dl) contents in the hemolymph of B. similaris experimentally infected with H. indica LPP1 and uninfected (control).

Groups Total protein (g/dl) Uric acid (mg/dl) Urea (mg/dl) X ±SD X ±SD X ±SD Control 3.08 ± 0.22a 7.22 ± 0.59a 93.79 ± 0.46a Infected 1.87 ± 0.23b 12.26 ± 0.43b 79.34 ± 2.08b a,bMeans followed by different letters differ signiﬁcantly in line (a = 5%). X ± SD = mean ± standard deviation.

Table 2 Aminotransferases (ALT and AST) activity (UFR/ml) in the hemolymph of B. similaris experimentally infected with H. indica LPP1 and uninfected (control).

Groups ALT (UFR/ml) AST (UFR/ml) X ±SD X ±SD Control 41.27 ± 2.09a 54.05 ± 2.37a Infected 84.98 ± 4.23b 117.65 ± 0.88b a,bMeans followed by different letters differ significantly in line (a = 5%). X ± SD = mean ± standard deviation. and infected snails during exposure by H. indica LPP1 are shown in Tables 1 and 2. There were significant differences in all the param- eters observed between the infected and the control groups. In relation to the protein metabolism of the infected snails, a decrease of 39.28% in relation to control group (3.08 ± 0.22) was observed differ significantly (Fig. 2A). The infection of B. similaris by H. indica LPP1 caused alterations in the metabolism of nitrogen excretion products, inducing an increase in the concentrations of uric acid and a decrease in those of urea in the snails’ hemolymph. Significant difference in the levels of uric acid was observed in the infected group (12.26 ± 0.43), representing an increase of 69.80% in relation to the average value in the control group (7.22 ± 0.59) (Table 1). In contrast, a decrease in the contents of urea occurred in the infected snails (79.34 ± 2.08), differing significantly from the control group (93.79 ± 0.46) (Fig. 2B and C). There was also a significant increase in the activities of AST and ALT in the infected snails’ hemolymph (Fig. 3A and B). The activity of ALT increased 105.91% in exposed snails to nematode (84.98 ± 4.23) differing significantly from the control group (41.27 ± 2.09). A similar variation was observed in AST activity, with the infected group presenting an activity of (117.65 ± 0.88) Fig. 2. Relationship between the concentrations of protein total expressed in mg/dl indicating an increase of 117.66% in relation to the control group (A), uric acid expressed in mg/dl (B) and urea expressed in mg/dl (C) in the (54.05 ± 2.37) (Table 2). hemolymph of B. similaris infected by H. indica LPP1 and uninfected. (⁄) Means differ The histochemical results revealed the presence of larval stages significantly (mean ± SD) = (mean ± standard deviation). of H. indica LPP1 in the digestive gland of the infected snails, as well as the occurrence of granulomata with concentrations of haemocytes around the larvae of nematode (Fig. 4A–D). In relation to control snails, there were no observed larval stages of the parasite in D. reticulatum. The same observation was reported by Jaworska sections of digestive gland tissue, with its integrity and functioning (1993), studying the association of D. agrestes and D. reticulatum preserved (Fig. 4E and F). exposed to Steinernema carpocapsae (Weiser, 1955), Steinernema feltiae (Felipjev, 1934) and Heterorhabditis bacteriophora (Poinar, 4. Discussion 1975). In turn, Wilson et al. (1994), evaluating the pathogenicity of another isolate of S. feltiae on D. reticulatum, found that the snail Tan and Grewal (2001) demonstrated the pathogenic effect of was resistant to infection by the nematode, although the snail was Moraxella osloensis (gamma subdivision: Moraxellaceae), an aero- moderately susceptible when a symbiotic bacterium (Xenorhabdus bic gram-negative bacterium associated with the nematode Phas- bovienii) was inoculated directly in the hemocele. Therefore, the marhabditis hermaphrodita (Schneider, 1859), on the slug D. mortality of B. similaris exposed to H. indica LPP1 infective juveniles reticulatum, which is considered an agricultural pest (Wilson probably results from an analogous mechanism to those summa- et al., 1993). The authors attributed the mortality observed in the rized above, since the pathogenicity of this nematode mainly oc- host organisms to the production of endotoxins resulting from curs in association with the bacterium Photorhabdus luminescens the bacterial metabolism, validating the use of the complex (bacte- akhurstii, killing the host by septicemia (Forst and Nealson, 1996; rium nematode) as a potential alternative for biological control of Boemare, 2002; Forst and Clarke, 2002). 16 V.M. Tunholi et al. / Experimental Parasitology 139 (2014) 12–18

Fig. 3. Relationship between the AST (A) and ALT (B) expressed in URF per milliliter, in the hemolymph of B. similaris infected by H. indica LPP1 and uninfected. (⁄) Means differ signiﬁcantly (mean ± SD) = (mean ± standard deviation).

Fig. 4. Digestive gland of B. similaris (A–D) infected with H. indica (Hi) to show granulomata (Gr) with concentrations of haemocytes around the larvae of H. indica (Hi); (E and F) control B. similaris to show the digestive gland intact and the absence of granulomata. (A) and (B) Scale bar = 200 lm. (CÀF) Scale bar = 100 lm.

The mortality pattern observed during the experiment therefore reducing the number of infective forms. This condition appeared to be inﬂuenced by the degree of environmental contam- was observed by Kaya and Mitani (2000), who demonstrated a pro- ination, since the cadavers (dead snails) were regularly removed portional relationship between the concentration of Steinernema from the terrariums to recover the larval stages of the nematode, longicaudum/cm2 and the mortality percentage of D. reticulatum. V.M. Tunholi et al. / Experimental Parasitology 139 (2014) 12–18 17

Besides this, that pattern might also have resulted from the longev- carbohydrates are stored, proteins are recycled and nitrogen excre- ity of the Heterorhabditis infective juveniles in the soil, since this tion products are formed (Tunholi-Alves et al., 2012). In light of parameter appears to be strongly influenced by the time of expo- this, the increase in the activity of ALT and AST in B. similaris can sure to the nematodes in the environment and the strain used indicate tissue injuries caused by the migration of the H. indica (Shapiro-Ilan et al., 2006). LPP1 larvae within the host, contributing to the observed patho- To investigate the possible mechanisms associated with the genic effect, here confirmed through histopathological analysis. mortality of B. similaris during infection by H. indica LPP1, we ana- In recent years, histological studies have been carried out as lyzed some metabolic pathways of the host, since other studies tools to assess the degree of resistance and susceptibility of differ- have demonstrated that infection by nematodes (Tunholi-Alves ent strains of snails hosts the infection by helminth parasite (Souza et al., 2012) and exposure to molluscicides (Mello-Silva et al., et al., 1995; Tunholi-Alves et al., 2011). Borges et al. (1998) assess- 2011) induce an accumulation of toxic nitrogen products and an ing histopathological features associated with susceptibility and increase in the activities of aminotransferases, both of which ef- resistance of Biomphalaria spp. the infection by Schistosoma man- fects can contribute to snail mortality. soni, describe two mechanisms related resitance of snail after mira- The establishment of infection by helminths in snails provokes cidial penetration: one would implicate the immediate destruction severe physiological changes in the host organism, mainly due to of the miracidium leaving no traces in the tissues; the other involv- the competition for nutrients between the parasite and host. As a ing late reactions that seem to completely destroy invading spo- consequence, the host has to mobilize its polysaccharide reserves rocysts and leave histological changes. According to the authors, and non-glycidic substrates as alternatives to maintain its normal these reactions are characterized by a focal and diffuse prolifera- glycemia. Thus, activation of the protein catabolism is observed tion of amebocytes, accompanied by an expansion of the extracel- causing a significant decrease in this substrate in the digestive lular matrix, which sometimes simulated the process of fibrosis gland and hemolymph of infected organisms (Pinheiro et al., and calcification seen in vertebrate tissues. Our results indicate 2009; Tunholi et al., 2011). The reduction of the levels of total pro- that the process of encapsulation in B. similaris during experimen- teins in the hemolymph of B. similaris exposed to H. indica LPP1 tal infection by H. indica LPP1, initially involves the participation of suggests the snail uses the carbon structures obtained from deam- basophilic haemolymph cells, followed by formation of fibrous ination of glucogenic amino acids to synthesize glucose. Addition- nodular aggregates. Such reactions may contribute, in part, to the ally, the decline in the concentration of this substrate also can loss of function of the site studied (digestive gland), an organ sim- result from the absorption of amino acids by the nematode larvae, ilar to the liver, where carbohydrates are stored, proteins are recy- which use it to form energy reserves (Tunholi-Alves et al., 2012). cled and nitrogenous products of excretion are formed, That possibility was previously observed by Selvan et al. (1993), contributing to the loss of homeostasis and consequently death who found a protein reserve of 46% in Heterorhabditis obtained of snails. from the relationship established with the hosts. The present study is the first to report the effects of exposure of The increase in the level of uric acid observed in B. similaris ex- B. similaris to H. indica LPP1, focusing on the effect of this exposure posed to the EPNs can be explained by the intense process of on the snail’s metabolism. The results indicate this nematode has deamination of amino acids induced by the energy needs of the potential for use for biological control of B. similaris. However, snail and nematodes. At the same time, there was a reduction in additional investigations are necessary to better understand this the concentration of urea in the hemolymph, indicating that H. in- interface. Besides this, since B. similaris was susceptible to infection dica LPP1 induces na inversion of the excretion pattern of B. simi- by H. indica LPP1 and the few reports in the literature on the path- laris. A similar condition has been noted in other snail-nematode ogenicity of EPNs to gastropods indicate that nematodes of the Ste- associations (Tunholi-Alves et al., 2012). A study conducted with inernema genus are the most virulent, it is necessary to Galleria mellonella (L.) also indicated that infection by H. bacterio- conductfuture research into the effect of infection of B. similaris phora initially promotes a reduced concentration of organic nitro- by EPNs of this genus. gen compounds, probably related to the activity and growth of the symbiont bacteria (Shapiro et al., 2000). Those authors also Conflicts of interest pointed out that the increased concentration of those compounds found in the advanced phase of the infection provides favorable There are no conflicts of interest to be declared. conditions for reproduction of the nematode. These results thus help explain the changes of the levels of nitrogen compounds in the model studied, indicating an interesting adaptation between Acknowledgments H. indica LPP1 and B. similaris, over the long term being one of the causes of the host’s death. This study was supported in part by Conselho Nacional para o An increase in the activity of ALT and AST was observed in the Desenvolvimento Científico e Tecnológico (CNPq) and Fundação infected snails. The aminotransferases are a group of enzymes that Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de catalyze the transfer of amino groups from amino acids to a-ceto Janeiro (FAPERJ). acids or vice versa. Therefore, they are of fundamental importance to the metabolism of carbohydrates because they are related to References gluconeogenesis (Tunholi et al., 2011). The results presented here indicate that the increase in the activity of these enzymes is the re- Adams, B.J., Nguyen, K.B., 2002. Taxonomy and systematics. In: Gaugler, R. (Ed.), sult of greater energy demand, including mobilization of protein Entomopathogenic Nematology. CABI, New York, pp. 311–332. Akhurst, R.J., 1995. Bacterial symbionts of entomopathogenic nematodes the power sources, among them amino acids, in an attempt to reestablish behind the throne. In: Bedding, R., Akhurst, R., Kaya, H. (Eds.), Nematodes and the normal glycemia and energy state of organisms exposed to the Biological Control of Insect Pests. CSIRO Publications, East Melboune, infection, since this increase occurred at the same time as the Australia, pp. 127–135. Amato, S.B., Bezerra, J.C.B., 1996. 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