Biological, biochemical and histological features of Bradybaena similaris (Gastropoda: Pulmonata) infected by Heterorabditis indica (Rhabditida: Heterorhabditidae) strain LPP1

Experimental Parasitology 179 (2017) 28e35

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Biological, biochemical and histological features of Bradybaena similaris (Gastropoda: Pulmonata) infected by Heterorabditis indica (Rhabditida: Heterorhabditidae) strain LPP1 Victor Menezes Tunholi a, *, Vinícius Menezes Tunholi-Alves b, g, Caio Oliveira Monteiro c,
udia de Melo Dolinski e, Rosane Nora Castro f, Lidiane Cristina da Silva d, Cla ^nia Rita Elias Pinheiro Bittencourt g, Jairo Pinheiro da Silva b, g, Va Isabella Vilhena Freire Martins a
ria, Centro de Ci^
rias, Universidade Federal do Espírito Santo e UFES, Alegre, ES, Brazil Departamento de Medicina Veterina encias Agra
gicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, Serop
Departamento de Ci^ encias Fisiolo edica, RJ, Brazil c ~o em Ci^
s e Avenida Esperança, s/n, Campus Samambaia, Goia ^nia GO, 74.690
s-graduança Programa de Po encia Animal da Universidade Federal de Goia 900, Brazil d ~o em Ci^
s-graduaça
gicas, Comportamento e Biologia Animal da Universidade Federal de Juiz de Fora, Rua Jos
Programa de Po encias Biolo e Lourenço Kelmer,
rio, Bairro Sa ~o Pedro, Juiz de Fora MG, 36036-900, Brazil s/n - Campus Universita e
rias, Campos dos Goytacazes, RJ, Brazil Universidade Estadual do Norte Fluminense Darcy Ribeiro, Centro de Ci^ encias e Tecnologias Agropecua f Departamento de Química, Instituto de Ci^ encias Exatas, Universidade Federal Rural do Rio de Janeiro, BR 465, Km 7, 23890-000, Serop
edica RJ, Brazil g
ria, Universidade Federal Rural do Rio de Janeiro, Serop
Departamento de Parasitologia Animal, Instituto de Veterina edica, RJ, Brazil a

b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


H. indica LPP1 induce inversion in the oxidative metabolism of B. similaris.
Exposure by H. indica LPP1 changes the levels of glucose and glycogen in B. similaris.
H. indica LPP1 induces parasitic castration in B. similaris.
H. indica LPP1 increase the activity of LDH in B. similaris.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 October 2016 Received in revised form 25 May 2017 Accepted 23 June 2017 Available online 24 June 2017

This study investigated the possible biological, biochemical and histological changes in Bradybaena similaris (Gastropoda: Pulmonata) infected by Heterorhabditis indica (Rhabditida: Heterorhabditidae), strain LPP1. Two groups of 16 snails were formed: the control group (unexposed) and the treated group, which was exposed for three weeks to infective juveniles (J3) of H. indica LPP1. The experiment was conducted in duplicate, using a total of 64 snails. After the exposure period, the snails were dissected to collect the hemolymph and tissues, for evaluation of the physiological changes caused by the infection. The number of eggs laid/snail and the viability of these eggs were also assessed as indicators of the reproductive activity of B. similaris. Intense glycogenolysis was accompanied by a significant reduction (p < 0.05) in the glucose content of the hemolymph of the exposed snails, indicating that infection by H. indica induces breakdown of the host's glycemic homeostasis. Significant variations (p < 0.05) in the

Keywords: Entomopathogenic nematodes Parasite-host relationship Biological control

* Corresponding author. E-mail addresses: [email protected] [email protected] (V.M. Tunholi-Alves). http://dx.doi.org/10.1016/j.exppara.2017.06.004 0014-4894/© 2017 Published by Elsevier Inc.

(V.M.

Tunholi),

vinicius_

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lactate dehydrogenase activity occurred together with changes in the concentration of pyruvic and lactic acid in the hemolymph of the infected B. similaris snails, corroborating the transition from aerobic to anaerobic metabolism in the hosts. These metabolic alterations reflect the parasitic castration process in this interface. The results suggest that the use of H. indica LPP1 is a potential alternative for biological control of B. similaris. © 2017 Published by Elsevier Inc.

1. Introduction
russac, 1821) is a The land snail Bradybaena similaris (Fe gastropod with great medical and veterinary importance, for acting as the intermediate host of trematodes and nematodes that affect the health of animals and humans (Alves et al., 2014; Tunholi-Alves et al., 2014). Among the relevant helminths, Eurytrema coelomaticum (Giard et Billet, 1892) Looss, 1907, a parasite of the pancreatic ducts of ruminants, stands out. It is endemic in various regions of the world, including South America, Asia and Europe (Bassani et al., 2006). The species is the etiological agent of bovine eurytrematosis, parasitic disease characterized by cachexia and anemia, resulting in significant economic losses from reduced production of meat and milk (Ilha et al., 2005; Quevedo et al., 2013). Additionally, epidemiological studies have demonstrated the participation of this gastropod as intermediate host of Angiostrongylus cantonensis (Chen, 1935), the main agent causing human eosinophilic meningoencephalitis. In recent decades that disease has spread to many regions of the world, and is currently classified as an emerging parasitosis (Wang et al., 2007; Morassutti et al., 2014). Due to the proven importance of this gastropod as a link in the transmission chain of these and other helminthiases, the World Health Organization (WHO, 1983) has urged control of the population of these organisms as one of the effective ways to eradicate these diseases. The control of snails that act as intermediate hosts of parasites has traditionally been based on the use of chemical molluscicides (Machado, 1982). However, application of these compounds is not sustainable, showing low selectivity to the target organism and high ecotoxicity, posing a hazard to human and animal health (Henrioud, 2011). In reaction to this problem, experiments have been conducted to find new control alternatives, such as the use of plant-based molluscicidal substances and application of pathogenic microorganisms. The use of molluscicides of plant origin is one of the most promising methods to control snails (Mello-Silva et al., 2010; Silva et al., 2012). In this respect, Lustrino et al. (2008) were the first to demonstrate the negative physiological effect of Allamanda cathartica I. (Apocynacea) on B. similaris. They observed a significant decline in the amount of galactogen stored in the albumen gland and of glycogen stored in the cephalopedious mass and digestive gland of the infected snails. Likewise, Mello-Silva et al. (2010), studying the molluscicidal action of the latex of Euphorbia splendes var. hislopii on Biomphalaria glabrata (Say, 1818), an intermediate host of Schistosoma mansoni (Sambon, 1822), observed alterations in the host's carbohydrate metabolism. According to the authors, the depletion of polysaccharide reserves resulted in relevant modifications in the snail's reproductive biology, specifically a reduction of the oviposition rate and egg viability. Biological control agents such as fungi and pathogenic nematodes have also been indicated as promising alternatives to control snail populations (Jaworska, 1993; Rocha et al., 2009; Baron et al., 2013). In a recent study, Duarte et al. (2015) assessed under laboratory conditions the vulnerability of egg masses of B. glabrata to infection by Metarhizium anisopliae. The viability of the host's eggs

and maturation of the egg masses diminished significantly after exposure to conidia and hyphal bodies of the fungus, suggesting the possibility of its use in programs for biological control of B. glabrata. Tunholi et al. (2014) for the first time described the pathogenicity of Heterorhabditis indica LPP1 in B. similaris. According to them, exposure to the nematode induced severe histopathological alterations in the host, resulting in a mortality rate of 55%. Heterorhabditis and Steinernema are genus of free-living nematode found in Brazilian soils widely, in which establish a symbiosis relationship with the bacteria of the genus Photorhabdus and Xenorhabdus, respectively (Burnel and Stock, 2000), being therefore used in insect control programs that act as agricultural pests (Hazir et al., 2003). The infection of nematode-infecting juveniles (J3) in their natural hosts occurs through natural openings as (mouth, anus and spiracles) or, in some cases, through the cuticle. After entering the host's hemocoel, the nematodes release their simbiotic bacteria, which are primarily responsible for killing the host by septcemia (Dowds and Peters, 2002). In addition to the ability to infect a wide variety of arthropods (Dolinski et al., 2012; Monteiro and Prata, 2013), H. indica may accidentally infect gastropod snails, causing these hosts significants pathological changes (Jaworska, 1993). Li et al. (1986)has documented under laboratory conditions the susceptibility of Oncomelania hupensis, a semiaquatic snail that acts as an intermediate host of Schistosoma japonicum, to certain species of Sterneinema and Heterorhabditis. According to these authors, these nematodes had the ability to infect, develop and kill the host snail causing severe histopathological changes in the snail. Despite these studies, no information about the carbohydrate metabolism and reproductive parameters of B. similaris infected by H. indica LPP1 has yet been reported in the literature. Therefore, to better understand the B. similaris/H. indica LPP1 relationship, the aim of this study was to evaluate the concentrations of glycogen in the digestive gland (DG) and cephalopedious mass (CPM), as well as the amounts of glucose and enzymatic activity of D- and L-lactate dehydrogenase (EC 1.1.1.27 and EC 1.1.1.28) (LDH) in the hemolymph of snails exposed to infective juveniles (J3) of H. indica, strain LPP1, three weeks after exposure. In parallel, we measured the levels of galactogen stored in the albumen gland and performed histopathological analysis to shed more light on the reproductive alterations observed in this interface. We also applied HPLC to determine the levels of pyruvic and lactic acid in the hemolymph, to obtain conclusive information regarding the host's metabolic state. 2. Materials and methods 2.1. Source of the snails and nematodes The snails used in this study were obtained from a colony kept in
rio de Biologia de Moluscos do Museu Professor Maury the Laborato Pinto de Oliveira of the Universidade Federal de Juiz de Fora (UFJF), located in the city of Juiz de Fora, Minas Gerais, Brazil. The nematodes of the species H. indica isolate LPP1 were donated by the
rio de Nematologia of Universidade Estadual Norte Laborato

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Fluminense (UENF) and were maintained and multiplied in the
rio de Parasitologia of the Embrapa Dairy Cattle Research Laborato Unit (Embrapa Gado de Leite) according to the methods proposed by Lindegren et al. (1993) and Kaya and Stock (1997).

2.2. Exposure of the snails to the nematodes Cadavers of the caterpillar Galleria mellonella (Linnaeus, 1958) were used as the source of nematodes to infect the snails, according to the method proposed by Shapiro and Glazer (1996). To prepare the infected cadavers, six caterpillars were placed inside a previously sterilized Petri dish (9 cm diameter) lined with two sheets of filter paper. Then the lining was moistened with 2 ml of an aqueous suspension containing approximately 600 nematodes (100 EPNs/ caterpillar) and the dish was sealed with plastic film (Parafilm®) and placed in a climate-controlled chamber (25 ± 1  C). After three days the caterpillars with signs of infection were transferred to another previously sterilized Petri dish also lined with filter paper. The dishes were sealed with the same film and kept in the chamber under the same conditions. After eight days, the six infected caterpillars were buried in a terrarium (12  24  14 cm) containing a 10 cm layer of autoclaved soil with the addition of 0.5 g of CaCO3. The terrarium was periodically moistened with dechlorinated water to establish favorable conditions for the maintenance of the snails and nematodes.

2.3. Maintenance of the snails and formation of the experimental groups Two experimental groups were formed: one control group (uninfected) and one infected group (infected). Each group contained 16 snails, reared in the laboratory from hatching, to be certain of their age and that the snails were free of infection by other parasites. The entire experiment was conducted in duplicate, using a total of 64 snails, of which 32 snails constituted the control group and 32 snails, the infected group. The terrariums were kept in a room with controlled temperature of 25  C throughout the experiment. The snails were fed with fresh lettuce leaves (Lactuca sativa L.) ad libitum. The terrariums were maintained every other day, when the lettuce leaves were replaced to prevent their fermentation. For three weeks the terrarium were analyzed on alternate days to count the dead snails, by direct observation, with the dead snails being immediately removed. The dead snails were then placed in White traps (White, 1927) for possible recovery of infective juveniles and verification of completion of the cycle of H. indica LPP1 in the snail. To avoid the influence of population density on the physiological patterns of B. similaris (Oliveira et al., 2008), the same number of specimens were removed from the control group as the dead snails in the treated group, to keep the number of snails in the two groups equal.

2.5. Determination of glucose concentration and LDH activity For the determination of glucose, a 10 ml of sample was added to 1 mL of color reagent (0.05 M phosphate buffer solution, pH 7.45 ± 0.1; 0.03 mM aminoantipyrine and 15 mM of sodium phydroxybenzoate; 12 kU of glucose oxidase, and 0.8 kU peroxidase per liter). The product formed by oxidation of 4-aminoantipyrine was determined by spectrophotometry with maximum absorption at 510 nm, using a standard solution of glucose at a concentration of 100 mg/dL (Mello-Silva et al., 2010). The readings were expressed in mg/dL. For the determination of LDH activity, mixtures were prepared of 1 ml of solution containing substrate (0.1 M lactate solution, 0.005 M o-phenanthroline in 0.2 M Tris, pH 8.8), a drop of 0.012 M ferric ammonium sulfate and 25 ml of sample, and the mixture were incubated at 37  C for 2 min. Next a drop of solution was added containing 15.82 mM of nicotinamide adenine dinucleotide (NAD) and 3.73 mM of phenazine metasulphate and the mixture was incubated at 37  C for 5 min. The final reaction was stabilized by adding 1 ml of 0.5 M hydrochloric acid. After homogenization, the readings were taken in a spectrophotometer at 505 nm and the results were expressed in UI. 2.6. Determination of the glycogen and galactogen concentration The glycogen contents of the DGG and cephalopedal mass were determined according to the method 3.5 DNS (Sumner, 1924; Pinheiro and Gomes, 1994) and expressed as mg glucose/g tissue, wet weight. 2.7. Analysis of the reproductive biology of Bradybaena similaris exposed to Heterorhabditis indica LPP1 On alternate days, until the end of the three weeks of exposure the number of eggs laid was recorded by direct observation. After counting the eggs numbers, these were placed in new free terrariums without snails. Subsequently, the eggs were observed periodically for count the snails hatching. The egg viability, expressed as a percentage, is the number of snails hatched divided by the number of eggs laid in each experimental group, multiplied by 100. 2.8. Chemicals Standards of pyruvic and lactic acids were purchased from SigmaeAldrich (Steinheim, Germany) in the highest purity grade available. Acetonitrile, sodium dihydrogen phosphate and phosphoric acid were of analytical purity or for chromatographic use. Ultrapure water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Stock standard solutions were dissolved in mobile phase, phosphate buffer adjusted to pH 2.2 with phosphoric acid, and stored at 4  C as described in study of Tunholi et al. (2013). 2.9. HPLC analysis

2.4. Dissection and collection of the hemolymph At the end of the three-week experimental period, the snails from the control and infected groups were dissected and the hemolymph was collected by cardiac puncture and maintained at 10  C until the biochemical analyses. All the samples were kept in an ice bath during dissection as described by Tunholi et al. (2014). The choice of the study period (three weeks) was based on Wilson et al. (1994) in function of the mortality rate during the infection of D. reticulatum by Heterorhabditis sp.

All HPLC experiments were carried out in a Shimadzu LC-20AT system equipped with photodiode array detector (PDA; SPDM20A, Shimadzu, Japan) coupled to an LCSolution ChemStation data-processing station. Separations were carried out with reversed phase column C18 (150  4.5 mmI.D., 5 mm, Allure® Organic Acids, Restek) in isocratic conditions. The mobile phase consisted of 1% acetonitrile in 20 mol L1 NaH2PO4 aqueous solution, adjusted to pH 2.2 with H3PO4. The temperature was set at 36  C and the flow rate was 0.8 mL/min. The chromatograms were monitored at 210 nm and the injection volume was 20 ml. The

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identification of organic acids present in the samples was based on a comparison of UV spectra and retention times with those of the pure standard solutions. Quantification was performed on the basis of linear calibration plots of peak area against concentration. Calibration lines were constructed based on five concentration levels of standard solutions. The calibration graphs for pyruvic and lactic acids were linear (r ¼ 0.99) in all cases. All experiments were performed in triplicate (Tunholi et al., 2013). The hemolymph was vortexed and centrifuged for 10 min at 2520g. The supernatant was separated and undissolved particles were removed by filtration using 45 lm membrane filters. Aliquots of 20 ml were used for the chromatographic analysis (Tunholi et al., 2013). 2.10. Histochemical analyses Snails (three) from each group experimental were dissected and transferred to Duboscq-Brasil fixative (Fernandes, 1949). Soft tissues were processed according to routine histological techniques (Humason, 1979). Sections (5 mm) were stained using (hematoxylin-eosin and Gomori trichrome) and observed under a Zeiss Axioplan light microscope. Images were captured by an MRc5 AxioCam digital camera and processed with the Axiovision software. 2.11. Statistical analyzes The results obtained were expressed as mean ± standard deviation and the Tukey test was used to compare the means (P < 0.05) (InStat, GraphPad, v.4.00, Prism, GraphPad, v.3.02, Prism, Inc.). 3. Results The exposure to H. indica LPP1 resulted 50% mortality in B. similaris, with the highest rate observed in the first week after exposure (35%) (Fig. 1). These results are in accordance with according Tunholi et al. (2014) that reported mortality rate of 55% in B. similaris infected by LPP1 H. indicates. Changes in the glycemia of the infected snails occurred, characterized by a significant decrease in the glucose content in the hemolymph of snails exposed to the nematode (21.79 ± 3.24 mg/dL) compared to the control group (44.29 ± 4.94 mg/dL) (Fig. 2A). Alteration in the LDH activity was also observed. In this respect, the infection induced a sharp increase in the LDH activity in relation to the uninfected snails (2.09 ± 0.5UI) (Fig. 3A), a rise of about 105%, indicating recruitment of anaerobic fermentation pathways. For the purpose of better understanding the alterations in glycemia of infected B. similaris, the concentrations of glycogen stored

Fig. 1. Bradybaena similaris mortality (%) after exposure by H. indica LPP1 during three weeks (control ¼ uninfected snails).

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in the host's digestive gland and cephalopedious mass were analyzed. Regarding the glycogen stored in the digestive gland, the exposure to H. indica LPP1 infective juveniles caused severe depletion of this polysaccharide, with the lowest values being obtained three weeks after exposure (19.75 ± 2.24 mg/g), differing significantly from the average of the control group (44.81 ± 1.58 mg/g) (Fig. 2B). The same variation tendency was observed for the levels of glycogen stored in the cephalopedious mass of the infected snails, indicating induction of glycogenolysis (Fig. 2C). Additionally, the infection caused a significant decline in the content of pyruvic acid, of 60% in relation to the control group (1047.60 ± 34.42 mM) (Fig. 3B). At the same time, the levels of lactic acid increased in the infected snails (72.93 ± 3.46 mM) compared to the uninfected ones (34.84 ± 4.38 mM) (Fig. 3C), indicating that the infection promotes a transition from aerobic to anaerobic metabolism in the host. Variations in the reproductive performance of B. similaris infected by H. indica LPP1 were also observed. The infection reduced the number of eggs laid/snail (11.97 ± 5.19) in comparison with the control group (31.47 ± 5.65) (Fig. 4B). The infection also significantly reduced the hatching rate three weeks after exposure (7.84 ± 3.69), when the rate was 65.5%, compared to 98.41% in the uninfected snails (Fig. 4C). The content of galactogen decreased in the snails experimentally exposed to the nematode (0.93 ± 0.07 mg/g) (Fig. 4A), suggesting that the parasitic castration mechanism involving the B. similaris/H. indica LPP1 interface results from an indirect process, i.e., metabolic. Lastly, the histopathological results revealed the presence of an intense cellular disorganization process, in different tissues of the host, as a consequence of the establishment of an inflammatory response in the infected snails, thus impairing the integrity and functioning of the affected organs. In relation to the control snails, the absence of larval stages of the nematode was demonstrated in sections of the digestive gland, with maintenance of homeostasis in these organisms (Fig. 5AeD). 4. Discussion Over the past several decades, many authors have studied the relationship between larval stages of helminths and their intermediate hosts, in order to characterize the metabolic pathways in these hosts and to detect possible interferences during the parasites' development. According to those studies, the physiological changes in host snails in response to infection result from a series of biochemical, immunological and biological events, which assure not only the host's survival but also the complete development of the parasite. These findings can be used to support future studies focused on validating measures to control snails using biological control agents, since this relation also involves the penetration, colonization and multiplication of the control agent inside the host snail. Tunholi et al. (2014), evaluating the exposure of B. similaris to H. indica LPP1, suggested that the pathogenic effect of the nematode's larval stage results from, among other factors, in the acceleration of protein catabolism, promoting a process of deamination of amino acids and accumulation of nitrogen compounds like urea and uric acid. These products are highly toxic and over the long term compromise the host's homeostasis, explaining the high mortality rate recorded in the first week of infection. These physiological changes result from the direct action established by both, nematode larval stages and by Photorhabdus luminescens akhurstii, an aerobic gram-negative bacterium associated with the nematode, within the host snail, which compete directly for nutrients. After infection, H. indica, carrier of bacteria Photorhabdus luminesces,

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Fig. 2. Relation between glucose concentration, expressed in mg/dL, in the hemolymph (A), and glycogen content, expressed in mg of glucose/g tissue, wet weight, in the digestive gland (B) and in the cephalopedal mass (C) of Bradybaena similaris infected by Heterorabditis indica LPP1 e uninfected (control) after three weeks. (***) Means differ significantly (mean ± SD) ¼ (mean ± standard deviation). P < 0.001.

Fig. 3. Relation between lactate desidrogenase activity, expressed in UI (A), and the levels of pyruvic acid (B) and lactic acid (C) expressed in mM in the hemolymph of Bradybaena similaris infected by Heterorabditis indica LPP1 e uninfected (control) after three weeks. (*) Means differ significantly (mean ± SD) ¼ (mean ± standard deviation). P < 0.001.

eliminates in the hemolymph of the host, in case B. similaris, bacterial endosymbionts. Because of bacterial metabolism, there is the production of numerous enzymes and endotoxins which cause septicemia, favoring the occurrence of pathophysiological changes that contribute to the death of the host. In the same line of investigation, Lima et al. (2012) and Mello-Silva et al. (2007) confirmed

that the molluscicidal effect of the latex of Euphorbia spp. in B. glabrata results from a series of physiological and reproductive alterations, respectively. With the aim of complementing the data previously published by Tunholi et al. (2014), here we report changes in the carbohydrate metabolism and in the reproductive profile of B. similaris exposed to H. indica LPP1, to shed more light on

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Fig. 4. Relation between the and galactogen content, expressed in mg of galactose/g tissue, wet weight, in the albumen gland (A), as well as the number of eggs laid/snail (B) and the viability rate of the eggs (%) (C) in Bradybaena similaris infected by Heterorabditis indica LPP1 and uninfected (control) after three weeks. The results of the concentration of galactogen were expressed as (mean ± standard deviation). Since the data related to number of eggs laid/snail and the hatchability of embryos were expressed as (mean ± standard error) (***) Means differ significantly. P < 0.001.

Fig. 5. Digestive gland (A), mantle (B) and pericardic sac (C) of Bradybaena similaris infected by Heterorhabditis indica LPP1 showing granulomata ( ) with concentrations of haemocytes around the larvae of H. indica; (D) control B. similaris to show the digestive gland intact and the absence of granulomata.

this interface and to supports the potential of using formulations based on this nematode for integrated control of bovine eurytrematosis and human eosinophilic meningoencephalitis. The results obtained in the present study indicate that infection by the nematode resulted in a significant reduction in the

concentrations of glycogen, both in the digestive gland and the cephalopedious mass, and of glucose in the hemolymph of B. similaris. This probably was caused by the ability of P. luminescens to assimilate a wide variety of carbon sources, such as glucose, directly from the host's hemolymph, necessary for maintaining

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their intense metabolic processes during development, contributing in part to the establishment of this metabolic scenario, i.e negative energy balance, in snails exposed to nematodes (Gil et al., 2002; Jeffke et al., 2000). In response to the resulting hypoglycemia, the snail activates alternative metabolic pathways, such as glycogenolysis, allowing normalization of the circulating glucose by mobilizing polyosidic chains. This response evidences the existence of precisely regulated homeostatic pathways between tissue and hemolymph. However, the depletion of these reserves observed here was not able to reestablish the basal levels of this sugar, characterizing breakdown of glycemic homeostasis. That condition has also been observed in other snail/trematode and snail/nematode infection models (Mello-Silva et al., 2010; Pinheiro et al., 2009; Brockelman et al., 1976). Tunholi-Alves et al. (2014) and Tunholi et al. (2013) claim that under basal conditions gastropods have predominantly aerobic oxidative metabolism toward obtaining energy. However, when snails are subjected to physiological stress, such as exposure to molluscicides, various researchers have reported overlap of enzymatic centers related to fermentative pathways, such as LDH (Singh et al., 1999; Bakry et al., 2012). This enzyme catalyzes an important step in the cell metabolism, reducing pyruvate to lactate, thus increasing the concentration of the latter carboxylic acid (lactic acid) in the medium. As a consequence, the pH of the hemolymph declines, inducing a series of physiological changes. Therefore, the increase in the activity of LDH in the hemolymph of B. similaris infected by H. indica LPP1 helps to explain the pathogenic effect of this nematode. This reduction in the hemolymph pH increases the transfer of calcium carbonate (CaCO3) from the shell to the hemolymph, fundamental in the step for formation of bicarbonate, a buffering agent that helps normalize the altered pH (Tunholi et al., 2011). However, the activation of this pathway in parallel promoted decalcification and weakening of the shell, making the infected snails become more susceptible to predators, an additional factor for their biological control (Tunholi et al., 2011). To better understand the effect of infection on induction of anaerobic metabolism in B. similaris, the concentrations of two carboxylic acids (pyruvic and lactic) in the hemolymph were measured. The increase in the activity of LDH was accompanied by a decrease in the levels of pyruvic acid and an increase in the concentrations of lactic acid, corroborating the transition from aerobic to anaerobic metabolism in the host. Similar results have been reported by other authors, studying different snail/helminth interfaces (Bezerra et al., 1997; Tunholi et al., 2013). In this metabolic scenario, the energy yield related to the production of ATP is significantly lower compared to that of the aerobic pathways, explaining the damages caused by the infection. The changes in the carbohydrate metabolism were accompanied by variations in the reproductive performance of B. similaris. The infection by H. indica LPP1 resulted in a significant decline in the oviposition rate as well as the egg hatching rate, suggesting impairment of embryo development. According to Baudoin (1975), the reduction of reproductive activity observed in infected snails is called “parasitic castration” and can result from two mechanisms: (i) a direct process, related to the tissue damages in organs and structures attached to the host's reproductive system induced by the parasite, or (ii) an indirect process, associated with depletion of energy reserves, especially polysaccharides. In gastropods, these energy reserves consist of glycogen, present in different tissues, and galactogen, stored exclusively in the albumen gland, a structure attached to the feminine reproductive system, involved in the production of vitellum, essential for embryo development (Faro et al., 2013). For the purpose of elucidating the possible mechanisms involved in the parasitic castration process of the interface studied here, we carried out biochemical measurements to

determine the concentration to galactogen. Interestingly, the reduction in the reproductive parameters analyzed (number of eggs laid/snail and egg hatching rate) was accompanied by depletion of the galactogen reserves stored in the albumen gland of B. similaris. This finding suggests that the parasitic castration demonstrated in B. similaris infected by H. indica LPP1 probably results from a secondary mechanism, in function of the depletion of energy of the host snail. An analogous condition was observed by Tunholi-Alves et al. (2011), studying the relationship of B. glabrata and A. cantonensis, in which suggested that in infection models involving nematodes, the parasitic castration mainly results from metabolic interferences related to the profile of carbohydrates. Finally, besides the physiological and reproductive alterations, histopathological changes also have been demonstrated. Several authors have used histopathological tools to assess the susceptibility of snails to different helminth species (Harris and Cheng, 1975; Tunholi-Alves et al., 2014, 2015). According to these authors, the infection induces severe cellular disorganization characterized by proliferation of amebocytes and expansion of the extracellular matrix, triggering a process of fibrosis and metastatic calcification in the affected tissue. Our results indicate that the infection by H. indica LPP1 culminated in sharp histopathological alterations in B. similaris, such as production of a hemocytic infiltrate followed by formation of fibrous nodular aggregates. These reactions possibly impair the function of the organ (gonad-digestive gland complex), contributing to the loss of homeostasis and thus causing death of the snails. This study reports the effects of exposure of B. similaris to infective juveniles of H. indica LPP1. The results indicate the infection induced significant alterations in the carbohydrate metabolism of the host, characterized by intense glycogenolysis and lowering of the glycemic levels. Additionally, the increase in activity of LDH in the hemolymph was accompanied by a decrease in the concentrations of pyruvic acid and an increase in the concentrations of lactic acid, corroborating the transition from aerobic to anaerobic metabolism in the infected snails. The parasitic castration phenomenon was also observed, classified as an indirect and partial process, and probably explained by the drainage of the galactogen stored in the albumen gland of B. similaris infected by H. indica LPP1. In closing, the histopathological results reveal that the presence of larval stages of the nematode inducing accentuated process of cell disorganization in the host, thus impairing the organ's functional capability and maintenance of homeostasis. These findings indicate the potential use of this nematode in programs for biological control of B. similaris. Acknowledgments This study was supported in part by Conselho Nacional para o
gico (CNPq), Fundaç~ Desenvolvimento Científico e Tecnolo ao Carlos  Pesquisa do Estado do Rio de Janeiro Chagas Filho de Amparo a ~o de Amparo  (FAPERJ) and Fundaça a Pesquisa do Estado do Espírito Santo (FAPES). References ^lo, P., Bittencourt, V.R.E.P., Alves, H.R., Tunholi-Alves, V.M., Tunholi, V.M., Go
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