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 nematodeHeterorhabditis indica (Rhabditida: Heterorhabditidae) strain LPP1

http://dx.doi.org/10.1016/j.exppara.2014.02.0050014-4894/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding authors at: Curso de Pós-Graduação em Ciências Veterinárias, Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Fededo 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: victortunholi@yahoo.com.br (V.M. Tunholi), vinnytunholi@yahoo.com.br (V.M. Tunholi-Alves).1 Research Fellow, CNPq.

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, Brazilb 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, Brazilc Docente do curso de Medicina Veterinária da Faculdade de Castelo (FACASTELO), Av. Nicanor Marques s/n, Castelo, ES 29360-000, Brazild Universidade Estadual do Norte Fluminense Darcy Ribeiro, Centro de Ciências e Tecnologias Agropecuárias, Campos dos Goytacazes, RJ, Brazile 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, Brazilf Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, RJ, Brazil

h i g h l i g h t s

� 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.

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

a r t i c l e i n f o

Article history:Received 3 June 2013Received in revised form 6 February 2014Accepted 9 February 2014Available online 22 February 2014

Keywords:Entomopathogenic nematodesParasite–host interactionBiological control

a b s t r a c t

Heterorhabditis is a nematode found in the soil that is used as an important biological control agentagainst various organisms. However, few studies have been performed of its use against snails and thepresent study is the first to investigate the effect of experimental exposure of Bradybaena similaris to Het-erorhabditis indica LPP1. Two groups of 16 snails were formed: the control group (not exposed) and thetreatment, which was exposed for three weeks to infective juveniles (J3) of H. indica LPP1. The entireexperiment was conducted in duplicate, using a total of 64 snails. After this period, the snails were dis-sected to collect the hemolymph to evaluate the possible physiological alterations, namely total proteins,uric acid and hemolymph urea, as well as the activities of alanine aminotransferase (ALT) and aspartateaminotransferase (AST) as a result of the infection. The terrariums were analyzed on alternate days

ral Rural

V.M. Tunholi et al. / Experimental Parasitology 139 (2014) 12–18 13

throughout the experiment to count the dead snails. Intense proteolysis was observed in the infectedsnails. An increase in the level of uric acid and reduction of the hemolymph urea content indicated thatthe infection by H. indica results in the inversion of the excretion pattern of the host snail. Variations inthe aminotransferase activities were also observed, with the infected group presenting significantlyhigher values (p < 0.05) than the control group for both ALT and AST. The exposure to H. indica LPP1caused 55% mortality, with the highest rate observed in the first week after exposure (30%). These resultssuggest that the use of H. indica LPP1 is a feasible alternative for the biological control of B. similaris.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Gastropod mollusks are organisms of great importance in hu-man and veterinary medicine because they are intermediate hostsof various parasites, such as trematodes and nematodes. Many ofthese helminths afflict pets and farm animals as well as humans,causing economic losses to stock breeders and public health prob-lems (Pinheiro et al., 2011; Tunholi-Alves et al., 2012). Because ofthe intimate relationship observed between some parasite speciesand host snails, the World Health Organization (WHO, 1983) rec-ommends the development of new methods to control host popu-lations as an effective strategy to eradicate parasitoses.

The Asian trampsnail Bradybaena similaris (Férussac, 1821) is aland snail found throughout Brazil. It is classified as an agriculturalpest besides being the intermediate host of parasites such asAngiostrongylus cantonensis (Chen, 1935) and Angiostrongylus cos-taricensis (Morera and Céspedes, 1971), which are etiologicalagents of neural and abdominal angiostrongyliasis, both of whichare relevant public health problems (Caldeira et al., 2007), as wellas of the trematodes Eurytrema coelomaticum (Giard and Billet,1892), a parasite of the pancreatic ducts of ruminants (Pinheiroand Amato, 1995), and Postharmostomum gallinum (Witenberg,1923) (Amato and Bezerra, 1996), which afflicts the intestinal ce-cum of chickens.

For many decades the control of snails has relied mainly on theapplication of chemical molluscicides (Machado, 1982). But, thiscontrol strategy is not sustainable because these compounds accu-mulate and contaminate the environment, compromising humanand animal health (Henrioud, 2011). Additionally, studies havedemonstrated the biocidal effect of these substances on non-targetplants and organisms. Finally, the high cost of their applicationlimits their use in public control programs (Andrews et al., 1983).

In this context, many experiments have been carried out to findnew control alternatives, such as the use of molluscicidal sub-stances of plant origin (Lustrino et al., 2008; Mello-Silva et al.,2010; Silva et al., 2012). Another alternative method to controlagricultural pests is to use microorganisms that are pathogenic tothe host. In this respect, entomopathogenic nematodes (EPNs) ofthe genera Steinernema Chitwood and Chitwood (1937) and Het-erorhabditis Poinar (1976) have shown great promise and havebeen successfully used in various countries to control insect peststhat present at least one development stage in the soil (Grewalet al., 2001; Dolinski, 2006). The infective juveniles of these nema-todes are found in the soil. Upon infecting the host, they releasesymbiont bacteria in the hemocele, which multiply and cause theinsect’s death by septicemia between 24 and 72 h after infection(Grewal et al., 2001; Hazir et al., 2003). This nematode have a sim-ple life cycle that includes the egg, four juvenile stages (separatedby moults) and adult (Adams and Nguyen, 2002), in which, thirdstage juvenile represent the infecting form. The infective juvenilesare the only free living (outside host), occurring naturally in thesoil where they infect and kill their insect host within 2 or 3 days,producing 2 or 3 generations in the host. As a result, infective juve-niles are formed, emerging from host cadaver 1 or 2 weeks later

(Akhurst, 1995). The infection of the host occurs through naturalopenings as (mouth, anus and spiracles) or, in some cases, throughthe cuticle. After entering the host’s hemocoel, the nematodes re-lease their simbiotic bacteria, which are primarily responsible forkilling the host (Dowds and Peters, 2002). Recently, the release ofEPNs in the environment by providing cadavers of infected insectshas shown good results (Dolinski, 2006). In this method, after a fewdays of application, the infective juveniles (J3 = IJs) begin to leavethe cadaver and seek new hosts in the soil, making the control ofthe target pest (Shapiro-ilan et al., 2006). Theses EPNs, in general,have low specificity to the natural host, and under certain condi-tions can infect snail, resulting in significant pathological changes(Jaworska, 1993).

There are few studies of the pathogenicity of EPNs in snails. Oneof the few is described by Jaworska (1993), which describes thesusceptibility of Deroceras agreste (Linnaeus, 1758) and Derocerasreticulatum (Müller, 1774) to infection by three EPN species. Thisis not the earliest report, since Li et al. (1986) observed that certainspecies of Steinernema and Heterorhabditis can infect, kill and de-velop in the semi-aquatic snail Oncomelania hupensis Gredler(1881). However, to our knowledge, no previous study has con-firmed the use of EPNs to control of B. similaris. Based on the factthat B. similaris inhabits an environment favorable to the presenceof EPNs, the aim of the present study was to evaluate for the firsttime the possible physiological alterations and the mortality rateof B. similaris after exposure to infective juveniles (J3) of Heteror-habditis indica, isolate LPP1, during three weeks.

2. Materials and methods

2.1. Source of the snails and nematodes

The snails used in this study were obtained from a colony keptin the Laboratório de Biologia de Moluscos do Museu ProfessorMaury Pinto de Oliveira of the Universidade Federal de Juiz de Fora(UFJF), located in the city of Juiz de Fora, Minas Gerais, Brazil. Thenematodes of the species H. indica isolate LPP1 were donated bythe Laboratório de Nematologia of Universidade Estadual NorteFluminense (UENF) and were maintained and multiplied in theLaboratório de Parasitologia of the Embrapa Dairy Cattle ResearchUnit (Embrapa Gado de Leite) according to the methods proposedby 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, accord-ing to the method proposed by Shapiro and Glazer (1996). To pre-pare the infected cadavers, six caterpillars were placed inside apreviously sterilized Petri dish (9 cm diameter) lined with twosheets of filter paper. Then the lining was moistened with 2 ml ofan 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).

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

14 V.M. Tunholi et al. / Experimental Parasitology 139 (2014) 12–18

After three days the caterpillars with signs of infection weretransferred to another previously sterilized Petri dish also linedwith filter paper. The dishes were sealed with the same film andkept 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 withthe addition of 0.5 g of CaCO3. The terrarium was periodicallymoistened with dechlorinated water to establish favorable condi-tions for the maintenance of the snails and nematodes.

2.3. Maintenance of the snails and formation of the experimentalgroups

Two experimental groups were formed: one control group(uninfected) and one infected group (infected). Each group con-tained 16 snails, reared in the laboratory from hatching, to be cer-tain of their age and that the snails were free of infection by otherparasites. The entire experiment was conducted in duplicate, usinga total of 64 snails, of which 32 snails constituted the control groupand 32 snails, the infected group. The terrariums were kept in aroom with controlled temperature of 25 �C throughout theexperiment.

The snails were fed with fresh lettuce leaves (Lactuca sativa L.)ad libitum. The terrariums were maintained every other day, whenthe lettuce leaves were replaced to prevent their fermentation. Forthree weeks the terrarium were analyzed on alternate days tocount the dead snails, by direct observation, with the dead snailsbeing immediately removed. The dead snails were then placed inWhite traps (White, 1927) for possible recovery of infective juve-niles and verification of completion of the cycle of H. indica LPP1in the snail. To avoid the influence of population density on thephysiological patterns of B. similaris (Oliveira et al., 2008), the samenumber of specimens were removed from the control group as thedead snails in the treated group, to keep the number of snails in thetwo groups equal.

2.4. Dissection and collection of the hemolymph

At the end of the three-week experimental period, the snailsfrom the control and infected groups were dissected and the hemo-lymph was collected by cardiac puncture and maintained at �10 �Cuntil the biochemical analyses. All the samples were kept in an icebath during dissection. The choice of the study period (threeweeks) was based on Wilson et al. (1994) in function of the mortal-ity rate during the infection of D. reticulatum by Heterorhabditis sp.

2.5. Determination of total proteins

This assay was performed according to the biuret technique(Weichselbaum, 1946). A mixture of 50 ll of hemolymph and2.5 ml of the biuret reagent (0.114 M trisodium citrate, 0.21 M so-dium carbonate and 0.01 M copper sulfate) was homogenized andleft at room temperature for 5 min, after which the readings weretaken in a spectrophotometer at 550 nm. The results were ex-pressed as mg/dl.

2.6. Aminotransferases activities (AST and ALT)

To test for aminotransferases activities, 0.5 ml of substrate forEC2.6.1.2 L-alanine: 2 oxoglutarate aminotransferase (ALT) orEC2.6.1.1 L-aspartate: 2 oxoglutarate aminotransferase (AST) (solu-tion 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) wasincubated at 37 �C for 2 min. Then, 100 or 200 ll of hemolymph(for ALT and AST, respectively), were added, homogenized andagain incubated at 37 �C for 30 min. After this, 0.5 mL of 0.001 M

2,dinitrophenylhydrazine was added and the solution was kept at25 �C for 20 min. The reactions were interrupted by adding 5 mLof 0.4 M NaOH. The readings were taken in a spectrophotometerat 505 nm (Kaplan and Pesce, 1996) and the results were expressedas URF/ml.

2.7. Determination of the concentrations of uric acid and urea

To measure the uric acid level, 50 ll of hemolymph was mixedwith 2 ml of the dye reagent (100 mmol/L of sodium phosphatebuffer [pH 7.8] containing 4 mmol/L of dichlorophenol-sulfonate,0.5 mmol/L of 4-aminoantipirina, 120U 6 uricase, 4.980U6 ascor-bate oxidase, 1.080U 6 peroxidase). The mixture was homogenizedand incubated at 37 �C for 5 min. The readings were taken in aspectrophotometer at 520 nm (Bishop et al., 1996) and the resultswere expressed as mg/dl.

The urea concentration was measured by adding 2 ml of a solu-tion containing 60 mmol of sodium salicylate, 3.4 mmol of sodiumnitroprusside and 1.35 mmol of disodium EDTA. Then 2 ll of ure-ase and 20 ll of hemolymph were added. This mixture was homog-enized and incubated at 37 �C for 5 min. The readings were takenin a spectrophotometer at 600 nm and the results were 202 ex-pressed as mg/dl (Connerty et al., 1955).

2.8. Histochemical analyses

Snails (three) from each group experimental were dissected andtransferred to Duboscq-Brasil fixative (Fernandes, 1949). Soft tis-sues were processed according to routine histological techniques(Humason, 1979). Sections (5 lm) were stained using (hematoxy-lin-eosin and Gomori trichrome) and observed under a Zeiss Axio-plan light microscope. Images were captured by an MRc5 AxioCamdigital camera and processed with the Axiovision software.

2.9. Statistical analyzes

The results obtained were expressed as mean ± standard devia-tion 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 caused 55% mortality in B. simi-laris, with the highest rate observed in the first week after exposure(30%) (Fig. 1).

The levels of total proteins, uric acid and urea and activity of theaminotransferases (ALT and AST) in the hemolymph of uninfected

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

Groups Total protein (g/dl)X ± SD

Uric acid (mg/dl)X ± SD

Urea (mg/dl)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 2Aminotransferases (ALT and AST) activity (UFR/ml) in the hemolymph of B. similarisexperimentally infected with H. indica LPP1 and uninfected (control).

Groups ALT (UFR/ml)X ± SD

AST (UFR/ml)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.

Fig. 2. Relationship between the concentrations of protein total expressed in mg/dl(A), uric acid expressed in mg/dl (B) and urea expressed in mg/dl (C) in thehemolymph of B. similaris infected by H. indica LPP1 and uninfected. (⁄) Means differsignificantly (mean ± SD) = (mean ± standard deviation).

V.M. Tunholi et al. / Experimental Parasitology 139 (2014) 12–18 15

and infected snails during exposure by H. indica LPP1 are shown inTables 1 and 2. There were significant differences in all the param-eters observed between the infected and the control groups. Inrelation to the protein metabolism of the infected snails, a decreaseof 39.28% in relation to control group (3.08 ± 0.22) was observeddiffer significantly (Fig. 2A).

The infection of B. similaris by H. indica LPP1 caused alterationsin the metabolism of nitrogen excretion products, inducing an in-crease in the concentrations of uric acid and a decrease in thoseof urea in the snails’ hemolymph. Significant difference in the lev-els of uric acid was observed in the infected group (12.26 ± 0.43),representing an increase of 69.80% in relation to the average valuein the control group (7.22 ± 0.59) (Table 1). In contrast, a decreasein 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 andALT in the infected snails’ hemolymph (Fig. 3A and B). The activityof 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)indicating an increase of 117.66% in relation to the control group(54.05 ± 2.37) (Table 2).

The histochemical results revealed the presence of larval stagesof H. indica LPP1 in the digestive gland of the infected snails, as wellas the occurrence of granulomata with concentrations of haemo-cytes around the larvae of nematode (Fig. 4A–D). In relation to con-trol snails, there were no observed larval stages of the parasite insections of digestive gland tissue, with its integrity and functioningpreserved (Fig. 4E and F).

4. Discussion

Tan and Grewal (2001) demonstrated the pathogenic effect ofMoraxella osloensis (gamma subdivision: Moraxellaceae), an aero-bic gram-negative bacterium associated with the nematode Phas-marhabditis hermaphrodita (Schneider, 1859), on the slug D.reticulatum, which is considered an agricultural pest (Wilsonet al., 1993). The authors attributed the mortality observed in thehost organisms to the production of endotoxins resulting fromthe bacterial metabolism, validating the use of the complex (bacte-rium nematode) as a potential alternative for biological control of

D. reticulatum. The same observation was reported by Jaworska(1993), studying the association of D. agrestes and D. reticulatumexposed to Steinernema carpocapsae (Weiser, 1955), Steinernemafeltiae (Felipjev, 1934) and Heterorhabditis bacteriophora (Poinar,1975). In turn, Wilson et al. (1994), evaluating the pathogenicityof another isolate of S. feltiae on D. reticulatum, found that the snailwas resistant to infection by the nematode, although the snail wasmoderately susceptible when a symbiotic bacterium (Xenorhabdusbovienii) was inoculated directly in the hemocele. Therefore, themortality of B. similaris exposed to H. indica LPP1 infective juvenilesprobably results from an analogous mechanism to those summa-rized above, since the pathogenicity of this nematode mainly oc-curs in association with the bacterium Photorhabdus luminescensakhurstii, killing the host by septicemia (Forst and Nealson, 1996;Boemare, 2002; Forst and Clarke, 2002).

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. (⁄) Meansdiffer 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 andF) control B. similaris to show the digestive gland intact and the absence of granulomata. (A) and (B) Scale bar = 200 lm. (C�F) Scale bar = 100 lm.

16 V.M. Tunholi et al. / Experimental Parasitology 139 (2014) 12–18

The mortality pattern observed during the experimentappeared to be influenced by the degree of environmental contam-ination, since the cadavers (dead snails) were regularly removedfrom the terrariums to recover the larval stages of the nematode,

therefore reducing the number of infective forms. This conditionwas observed by Kaya and Mitani (2000), who demonstrated a pro-portional relationship between the concentration of Steinernemalongicaudum/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-ity of the Heterorhabditis infective juveniles in the soil, since thisparameter appears to be strongly influenced by the time of expo-sure to the nematodes in the environment and the strain used(Shapiro-Ilan et al., 2006).

To investigate the possible mechanisms associated with themortality of B. similaris during infection by H. indica LPP1, we ana-lyzed some metabolic pathways of the host, since other studieshave demonstrated that infection by nematodes (Tunholi-Alveset al., 2012) and exposure to molluscicides (Mello-Silva et al.,2011) induce an accumulation of toxic nitrogen products and anincrease in the activities of aminotransferases, both of which ef-fects can contribute to snail mortality.

The establishment of infection by helminths in snails provokessevere physiological changes in the host organism, mainly due tothe competition for nutrients between the parasite and host. As aconsequence, the host has to mobilize its polysaccharide reservesand non-glycidic substrates as alternatives to maintain its normalglycemia. Thus, activation of the protein catabolism is observedcausing a significant decrease in this substrate in the digestivegland and hemolymph of infected organisms (Pinheiro et al.,2009; Tunholi et al., 2011). The reduction of the levels of total pro-teins in the hemolymph of B. similaris exposed to H. indica LPP1suggests the snail uses the carbon structures obtained from deam-ination of glucogenic amino acids to synthesize glucose. Addition-ally, the decline in the concentration of this substrate also canresult from the absorption of amino acids by the nematode larvae,which use it to form energy reserves (Tunholi-Alves et al., 2012).That possibility was previously observed by Selvan et al. (1993),who found a protein reserve of 46% in Heterorhabditis obtainedfrom the relationship established with the hosts.

The increase in the level of uric acid observed in B. similaris ex-posed to the EPNs can be explained by the intense process ofdeamination of amino acids induced by the energy needs of thesnail and nematodes. At the same time, there was a reduction inthe concentration of urea in the hemolymph, indicating that H. in-dica LPP1 induces na inversion of the excretion pattern of B. simi-laris. A similar condition has been noted in other snail-nematodeassociations (Tunholi-Alves et al., 2012). A study conducted withGalleria mellonella (L.) also indicated that infection by H. bacterio-phora initially promotes a reduced concentration of organic nitro-gen compounds, probably related to the activity and growth ofthe symbiont bacteria (Shapiro et al., 2000). Those authors alsopointed out that the increased concentration of those compoundsfound in the advanced phase of the infection provides favorableconditions for reproduction of the nematode. These results thushelp explain the changes of the levels of nitrogen compounds inthe model studied, indicating an interesting adaptation betweenH. indica LPP1 and B. similaris, over the long term being one ofthe causes of the host’s death.

An increase in the activity of ALT and AST was observed in theinfected snails. The aminotransferases are a group of enzymes thatcatalyze the transfer of amino groups from amino acids to a-cetoacids or vice versa. Therefore, they are of fundamental importanceto the metabolism of carbohydrates because they are related togluconeogenesis (Tunholi et al., 2011). The results presented hereindicate that the increase in the activity of these enzymes is the re-sult of greater energy demand, including mobilization of proteinsources, among them amino acids, in an attempt to reestablishthe normal glycemia and energy state of organisms exposed toinfection, since this increase occurred at the same time as thereduction of the concentration of total proteins in the hemolymph(Pinheiro et al., 2001).

Studies have also validated that these enzymes can serve asexcellent biomarkers of cell injury in gastropods, especially in thedigestive gland, an organ similar to the liver of vertebrates, where

carbohydrates are stored, proteins are recycled and nitrogen excre-tion products are formed (Tunholi-Alves et al., 2012). In light ofthis, the increase in the activity of ALT and AST in B. similaris canindicate tissue injuries caused by the migration of the H. indicaLPP1 larvae within the host, contributing to the observed patho-genic effect, here confirmed through histopathological analysis.

In recent years, histological studies have been carried out astools to assess the degree of resistance and susceptibility of differ-ent strains of snails hosts the infection by helminth parasite (Souzaet al., 1995; Tunholi-Alves et al., 2011). Borges et al. (1998) assess-ing histopathological features associated with susceptibility andresistance of Biomphalaria spp. the infection by Schistosoma man-soni, describe two mechanisms related resitance of snail after mira-cidial penetration: one would implicate the immediate destructionof the miracidium leaving no traces in the tissues; the other involv-ing late reactions that seem to completely destroy invading spo-rocysts and leave histological changes. According to the authors,these reactions are characterized by a focal and diffuse prolifera-tion of amebocytes, accompanied by an expansion of the extracel-lular matrix, which sometimes simulated the process of fibrosisand calcification seen in vertebrate tissues. Our results indicatethat the process of encapsulation in B. similaris during experimen-tal infection by H. indica LPP1, initially involves the participation ofbasophilic haemolymph cells, followed by formation of fibrousnodular aggregates. Such reactions may contribute, in part, to theloss of function of the site studied (digestive gland), an organ sim-ilar to the liver, where carbohydrates are stored, proteins are recy-cled and nitrogenous products of excretion are formed,contributing to the loss of homeostasis and consequently deathof snails.

The present study is the first to report the effects of exposure ofB. similaris to H. indica LPP1, focusing on the effect of this exposureon the snail’s metabolism. The results indicate this nematode haspotential for use for biological control of B. similaris. However,additional investigations are necessary to better understand thisinterface. Besides this, since B. similaris was susceptible to infectionby H. indica LPP1 and the few reports in the literature on the path-ogenicity of EPNs to gastropods indicate that nematodes of the Ste-inernema genus are the most virulent, it is necessary toconductfuture research into the effect of infection of B. similarisby EPNs of this genus.

Conflicts of interest

There are no conflicts of interest to be declared.

Acknowledgments

This study was supported in part by Conselho Nacional para oDesenvolvimento Científico e Tecnológico (CNPq) and FundaçãoCarlos Chagas Filho de Amparo à Pesquisa do Estado do Rio deJaneiro (FAPERJ).

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