Experimental Parasitology 144 (2014) 52–56

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Experimental Parasitology

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Research Brief

Changes in the lipid profile of Bradybaena similaris (Férussac, 1821)(Gastropoda, Xanthonychidae) during the development of Eurytremacoelomaticum (Giard and Billet, 1892) (Digenea, Dicrocoeliidae)

http://dx.doi.org/10.1016/j.exppara.2014.06.0100014-4894/� 2014 Published by Elsevier Inc.

⇑ Corresponding author at: Curso de Pós-Graduação em Ciências Veterinárias,Departamento de Parasitologia Animal, Instituto de Veterinária, UniversidadeFederal Rural do Rio de Janeiro, Km7, BR 465, Antiga estrada Rio-São Paulo,23890-000 Seropédica, RJ, Brazil.

E-mail address: victortunholi@yahoo.com.br (V.M. Tunholi).1 Research fellow CNPq.

Hélio Alves a, Vinícius Menezes Tunholi-Alves a,b, Victor Menezes Tunholi a,b,⇑, Patrícia Gôlo b,Vânia Rita Elias Pinheiro Bittencourt b,c,1, Jairo Pinheiro a,b,1

a Departamento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, Km7, BR 465, Antiga estrada Rio-São Paulo, 23890-000 Seropédica,RJ, Brazilb Curso de Pós-Graduação em Ciências Veterinárias, Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Km7, BR 465,Antiga estrada Rio-São Paulo, 23890-000 Seropédica, RJ, Brazilc 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

� Eurytrema coelomaticum changes inlipid contents in Bradybaena similaris.� Triacylglycerol decreases in the DGG

complex of B. similaris infected by E.coelomaticum.� Cholesterol decreases in the DGG

complex of B. similaris infected by E.coelomaticum.� TAG decreases and CHO increases in

the hemolymph of the infected 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 29 April 2014Received in revised form 7 June 2014Accepted 12 June 2014Available online 21 June 2014

Keywords:LipidsHost–parasite relationshipBradybaena similarisEurytrema coelomaticum

a b s t r a c t

The effect of experimental exposure of Bradybaena similaris by Eurytrema coelomaticum on the cholesteroland triglycerides total levels circulating in the hemolymph and the neutral lipids in the digestivegland–gonad (DGG) complex of the host snail were studied. In this study, snails were dissected afterone, two, and three months of infection to collect the hemolymph and DGG to measure the cholesteroland triacylglycerol levels in the hemolymph and neutral lipid fractions in the tissues. The results forthe hemolymph showed that the infection by this trematode resulted in a significant decrease in the con-centrations of triacylglycerol during all periods analyzed, as well as, increase in the levels of cholesterolobserved after the second and third months of study. This decrease suggests the possible use of triacyl-glycerol by both parasite and host as alternative substrate in maintaining their energy metabolisms. Inparallel, the increase in the contents of cholesterol results from a severe cellular disorganization in snail,especially DGG, as a consequence of the intense plastic processes showed by parasite. Additionally,changes in the neutral lipid profile in the DGG of the infected snails were observed, indicating theimportance of these molecules for successful infection.

� 2014 Published by Elsevier Inc.

1. Introduction

The digenetic trematode Eurytrema coelomaticum (Giard etBillet, 1892) Looss, 1907 is a parasite of the pancreatic ducts ofruminants that is endemic in several regions of the world,

H. Alves et al. / Experimental Parasitology 144 (2014) 52–56 53

including countries in South America, Europe and Asia (Bassaniet al., 2006). It is an etiological agent of bovine eurytrematosis, aparasitic disease of great interest in veterinary medicine becauseit results in significant economic losses from reduced productionof milk and meat (Bossaert et al., 1989; Ilha et al., 2005). Recently,Quevedo et al. (2013) described clinical and pathological findingsattributed to massive infection by this parasite in cattle in southernBrazil, mainly characterized by clinical features of cachexia andanemia, as well as histopathological changes consistent withchronic interstitial pancreatitis. In its life cycle, the terrestrial snailspecies Bradybaena similaris (Férussac, 1821) acts as the primaryintermediate host, so it plays an important role in the ontogeneticdevelopment of this helminth. After infection, the miracidial stagesof the trematode develop two generations of sporocysts in the coe-lomic cavity of the host snail, and the cercariae form within theselarvae, through an intense process of asexual multiplication. Thedaughter sporocysts containing cercariae emerge from theintermediate host and remaining in the environment until beingeaten by a grasshopper (Conocephalus sp.), which is the secondintermediate host and in which the metacercariae develop(Brandolini and Amato, 2001).

Studies on the E. coelomaticum/B. similaris interface havedemonstrated significant changes in the metabolism of carbohy-drates, inorganic compounds and reproductive aspects during thepre-patent development of the trematode (Paschoal and Amato,1996; Pinheiro and Amato, 1994, 1995; Brandolini and Amato,2001; Pinheiro et al., 2001). The results of these studies indicatethat infection results in a decrease in the content of glucose inthe hemolymph and polysaccharide reserves stored in the diges-tive gland and cephalopedal mass of B. similaris. Moreover, a signif-icant reduction in the number of eggs laid, as well as their viability,was also demonstrated (Pinheiro and Amato, 1995), indicating theexistence of parasitic castration, probably resulting from an indi-rect mechanism, causing metabolic depletion (Baudoin, 1975).Despite these studies, changes in the lipid metabolism B. similarisexperimentally infected by E. coelomaticum have not yet been pub-lished, limiting the knowledge about this relationship.

Due to the biological importance of these molecules, acting notonly as energetic substrate, but also as precursors in the synthesisof membranes observed during development and multiplication ofparasites in the host snail, authors have investigated the patho-physiological effects of infection by larval stages of the helminthson the lipid metabolism of the host snail (Tunholi-Alves et al.,2013; Bandstra et al., 2006). Nevertheless, the scanty informationavailable is contradictory, indicating the lipid profile of snails ishighly variable (Fried et al., 1989; Fried and Sherma, 1990).

Thus, in order to better understand the E. coelomaticum/B. similaris, the present study evaluated the influence of infectionon the profile of neutral lipids present in the DGG host snail, overthree months of infection, a period that corresponds to the pre-patent development of the trematode. Additionally, the contentof the hemolymphatic cholesterol and triacylglycerol were alsodetermined, allowing a better understanding of possible changes.

2. Materials and methods

2.1. Source and maintenance of the snails and formation of groups

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 Juiz de Fora City, Minas Gerais, Brazil. Thesnails were fed with fresh lettuce leaves (Lactuca sativa L.) ad libi-tum. The terrariums were maintained every other day, when thelettuce leaves were replaced to prevent their fermentation.

Six groups were formed: three control groups (uninfected) andthree infected groups (infected). Each terrarium contained tensnails, reared in the laboratory from hatching to be certain of theirage and that the snails were free of infection by other parasites.The entire experiment was conducted in duplicate, using a totalof 120 snails, 60 snails forming the control groups (uninfected)and 60 snails forming the infected groups. The terrariums werekept in a room with controlled temperature of 25 �C throughoutthe experiment. The study was performed for 3 months, a periodthat corresponds to the prepatent development of E. coelomaticumin B. similaris (Pinheiro et al., 2012).

2.2. Collection of E. coelomaticum, isolation of eggs, and experimentalinfection

The adult worms were collected from the pancreases of natu-rally infected cattle slaughtered at an abattoir (Frigorífico Irapuru,Barra Mansa, RJ, Brazil). The adult worms were left in Petri disheswith Lockes saline solution (Humason 1979) overnight at roomtemperature (25 �C). After that, the eggs were sedimented, andthe adult worms were discarded. The eggs were washed threetimes in Locke’s saline solution and stored at 4 �C until theirutilization.

The eggs were selected according to criteria of Tang (1950) andsmeared on pieces of cabbage leaves placed in Petri dishes linedwith filter paper moistened with tap water. Teen eggs/snail wasplace on these leaves and left overnight. The snails were addedover the lettuce leaves. The Petri dishes were closed, and the snailswere maintained in contact with the eggs overnight. After this,they were transferred to terrariums and maintained as describedabove.

2.3. Determination of the concentrations of triacylglycerol andcholesterol in the hemolymph

To determine the TAG concentration, 10 ll of hemolymph wasmixed with 1 ml of a solution containing 50 mmol/L of pipera-zine-N,N’-bis (2-ethanesulfonic acid) (PIPES) [pH 7.4], 1.25 mmolof 4-chlorophenol, 4.600 U of lipase, 40 U of glycerol kinase,125 U of glycerol-3-phosphate oxidase, 60 U of peroxidase,0.024 mmol of ATP and 0.018 mmol of 4-aminoantipyrine. Themixture was homogenized and incubated at 37 �C for 10 min.The absorbances were read with a spectrophotometer at 510 nm(Kaplan and Pesce, 1996). The results were expressed as mg/dl.

To measure the CHO level, 10 ll of hemolymph was mixed with1 ml of the color reagent (buffer of 35 mmol/L of PIPES [pH 7.0]containing 0.5 mmol/L of sodium cholate, 0.5 mmol/L of 4-amino-antipyrine, cholesterol esterase P P300 U, cholesterol oxi-dase P 204 U and peroxidase P 828 U). The resulting solutionwas then homogenized and incubated at 37 �C for 10 min. In thiscase, the absorbances were read with the spectrophotometer at500 nm (Kaplan and Pesce, 1996) and the results were expressedas mg/dl.

2.4. Monodimensional thin-layer chromatography and image analysis

For analysis of different neutral lipids in snail tissues, TLC wasused as standard methodology, due its ability to assess several lip-ids concomitantly. Moreover, this methodology has been used inother studies related to neutral lipid changes in DGG of snails, asdiscussed here (Bandstra et al., 2006; Fried et al., 1998, 2001).

Fifty milligrams of DGG tissue from each group was homoge-nized in 100 lL of lysis buffer (TRIS 50 mM; EDTA 5 mM; NonidetP-40 1%; TLCK 0.1 mM; PMSF 2 mM). The homogenates were cen-trifuged at 2520g for 3 min. The protein content in the supernatantfractions was determined according to Lowry with modification

54 H. Alves et al. / Experimental Parasitology 144 (2014) 52–56

(Markwell et al., 1978), using bovine serum albumin as thestandard. Samples containing 300 lg of total protein were submit-ted to lipid extraction as described by Bligh and Dyer (1959). Thelipids extracted were analyzed by one-dimensional-thin-layerchromatography (TLC) for neutral lipids using hexane, ethyl etherand acetic acid (60:40:1 by volume) as eluent (Kawooya andLaw, 1988). The neutral lipids used as standard curves were CHO,cholesterol ester (CHOE), TAG and fatty acids (FA). Both the stan-dards (20 lg of each) and sample extracts were spotted onto silicaplates. After that, each plate was developed until the eluent frontreached one centimeter from the edge of the plate. The lipids inthe plate were charred by dipping the plate in 10% cupric sulphate(w/v) and 8% phosphoric acid (v/v). After being dried, each platewas heated at 150 �C for 10 min (Ruiz and Ochoa, 1997). Theimages were analyzed by the Image Quant software.

The DGG tissues of uninfected snails collected at the end of eachmonth were polled. Thus, the mean result obtained could betterexpress the variation observed in the infected groups during the3 months of infection.

2.5. Statistical analysis

The results obtained in the tests to measure the concentrationsof CHO and TAG in the hemolymph were expressed as mean ± stan-dard deviation, and the Tukey’s test/ANOVA was utilized for com-parison of the means (P > 0.05) (InStat, GraphPad, v.5.00, Prism).The values for TAG and CHO levels in the hemolymph of uninfectedsnails were expressed as mean, represented by zero (0) month,since there was no significant difference among them at the endof each week. The chromatographic results were expressed asmean ± standard deviation in relation to the control group(uninfected), also represented as zero (0) month of infection.

3. Results

The infection in B. similaris by E. coelomaticum caused changesin the hemolymph levels of CHO in the snails. The highest concen-trations of CHO in the hemolymph of infected snails were observedin the second (57.98 ± 2.98) and third month after infection(106.15 ± 19.59), representing increases of 352.96 and 729.29%,respectively, in relation to the control group average(12.79 ± 2.03) (Fig. 1A).

Changes in the concentrations of TAG in the hemolymph of theinfected B. similaris specimens were also observed. The smallestand largest values were obtained, respectively, in the first(16.26 ± 5.55 mg/dl) and second (72.16 ± 7.3 mg/dl) months after

Fig. 1. Levels of cholesterol (CHO) (A) and triacylglycerol (TAG) (B) in the hemolymph (minfection periods, expressed in months. Month 0 (zero) represents the average of the contin these months. ⁄ = means different significantly from of the control group, P < 0.05, (m

infection, differing significantly in relation to the control group(126.40 ± 10.51 mg/dl) (Fig. 1B).

After having detected these variations, we directed the analysesto determine the possible effect of infection on the concentrationsof neutral lipids stored in the DGG of B. similaris. The levels of CHOin the treated groups were lower than those in the control groupthroughout the experiment. The CHO level in the DGG complexof the snails infected by E. coelomaticum declined in comparisonwith the control group (14.96 ± 1.13), with the lowest valuesobserved in the second (2.21 ± 0.52) and third (1.93 ± 0.22) monthsafter infection, with respective declines of 85.22 and 87.09%(Fig. 2C). Same pattern of variation was determined for the choles-terol ester levels (CHOE), with decreased concentrations beingshowed in the second (1.21 ± 0.39) and third month (1.20 ± 0.45)after infection, significantly different from the control group(17.58 ± 0.65) (Fig. 2D).

Infection by E. coelomaticum also resulted in significant changesin the TAG concentrations in the DGG of B. similaris. Duringthroughout the experiment (1, 2 and 3 months), the contents ofthis substrate were not detected in infected snails, indicating thattheir concentrations were lower compared to those used to plotthe standard curve (Fig. 2A). In relation the (FA) levels, theinfection by this trematode resulted in a significant decrease of thisfraction, with the lowest values observed in the second(9.49 ± 1.62) and third (2.93 ± 0.11) months after infection, differ-ent from the control group (18.25 ± 1.63) (Fig. 2B).

4. Discussion

According to Becker (1980), when invading the host snail,trematodes induce a series of metabolic alterations, caused bythe competition for nutrients, which are drained by the larvae tosupport their development and multiplication. This depletes thesnail’s energy reserves, mainly of polysaccharides (Tunholi et al.,2013; Pinheiro et al., 2009), forcing the snail to use other sub-strates, such as proteins (Tunholi-Alves et al., 2012; Tunholiet al., 2011) and lipids (Tunholi-Alves et al., 2013) to support notonly its own vital functions, but also to assure the complete devel-opment of the parasite. In the present study, the infection byE. coelomaticum resulted in a significant reduction in TAG levelsin the hemolymph of B. similaris, suggesting the use of this sub-strate as a source of energy by the developing larvae, principallyin the second month of infection. Franco-Acuña et al. (2011)showed that this period corresponds to the greatest asexualreproduction of the parasite, resulting in the formation ofdaughter sporocysts, which later form the numerous cercariae.

g/dl) of Bradybaena similaris infected by Eurytrema coelomaticum, in different post-rol group during the 3 months of analysis since there were no significant differencesean ± standard deviation).

Fig. 2. Concentration of neutral lipids in the digestive gland–gonad (DGG) complex of Bradybaena similaris infected by Eurytrema coelomaticum in function of time of infection,expressed in months. Month 0 (zero) represents the average of the control group during the 3 weeks of analysis. (A) level of triacylglycerol (TAG), (B) level of fatty acids (FA),(C) level of cholesterol (CHO) and (D) level of cholesterol ester (CHOE). All values are expressed as mean ± standard deviation. ⁄ = Means different significantly from of thecontrol group, P < 0.05. ND = Values of TAG not detected.

H. Alves et al. / Experimental Parasitology 144 (2014) 52–56 55

As a consequence of this process, besides the high energy demand,the developing larvae also require large quantities of molecules toserve as building blocks of new membranes, mainly recruitinglipids for this purpose. Similar results were presented byTunholiI-Alves et al. (2011), studying the infection of Biomphalariaglabrata (Say, 1818) by Echinostoma paraensei (Lie and Basch,1967). The authors noted a reduction in TAG concentration in thehemolymph of B. glabrata as a result of the process of multiplica-tion of the parasite, indicating the importance of this moleculeduring its development. These results are in agreement with thosepublished by Erasmus (1972), who identified the presence ofenzymes in trematodes related to the process of lipid metabolism,especially non-specific lipases and esterases, confirming the use ofthe TAG as an energy source by the parasite.

The infection by E. coelomaticum also resulted in failure todetect TAG reserves, as well as a significant reduction of the levelsof FA in the DGG of the infected snails. These alterations occurredmainly in the final stage of the parasite’s development, indictingthis is the period of greatest competition for nutrients, and thusgreatest energy demand, both by the parasite and host. Thus,TAG is initially hydrolyzed into free FA, which is then oxidizedwithin the mitochondrial matrix (Tripathi and Singh, 2002), gener-ating the energy necessary to meet the host’s metabolic needs andminimizing the deleterious effects of the infection (Humiczewskaand Rajski 2005). A similar condition was reported by Choubisa(1988) in the interfaces Melanoides tuberculata (Müller, 1774)/Cercaria diglandulata and M. tuberculata/Cercaria cartini. In thatstudy the authors mention that the reduction of the FA stored inthe DGG of infected snails occurred in parallel with the increase

in the activity of lipases in the host’s tissues, confirming that undersuch conditions of metabolic stress, the activation of the lipolyticprocess occurs as an alternative biochemical way to maintain thehost organism’s energy.

Variations in the concentrations of CHO in the DGG and theirhemolymphatic fraction in B. similaris experimentally infected byE. coelomaticum were also observed. Results previously publishedby Paschoal and Amato (1996), studying the B. similaris/E. coelomatucum interface, demonstrate a series of cellular altera-tions in the host’s tissues, with accentuated impairment of theovotestis and digestive gland, indicating that in this interactionthe migratory behavior of the larvae associated with their intensemultiplication results in tissue spoliation, compromising not onlythe structure, but also the functions of the affected organs(Tunholi et al., 2013). In light of these findings, the reduction ofthe concentrations of CHO in the DGG of B. similaris, especially inthe final development period of E. coelomaticum, can be partlyexplained by the leakage of these molecules through the cell mem-brane to the hemolymph, due to the infection, particularly in theDGG of the snail, which is the main site for development andreproduction of the parasite, and where neutral lipids are stored.This pattern occurred at the same time as the significant increasein the levels of CHO in the hemolymph of the infected snails,indicating that when both fractions (DGG and hemolymph) areanalyzed together, the CHO molecules can be used as markers ofcell lesion in the DGG of snails.

This study contains the first description of the pathophysiolog-ical effect of infection by E. coelomaticum on the lipid metabolismof B. similaris. Our results indicate that both the host and parasite

56 H. Alves et al. / Experimental Parasitology 144 (2014) 52–56

are capable of using TAG as an alternative energy substrate,thereby ensuring the survival of the snail and the full developmentof the helminth. In parallel, the establishment of E. coelomaticum inits intermediate host results from severe cellular disorganization inthe snail, especially the DGG, as a consequence of the intense plas-tic processes observed throughout its development. Additionally,the greatest alterations in the concentrations of the lipid fractionsobserved here occurred in the final stage of the infection, confirm-ing that this is the period of greatest competition for nutrients andhence the most important period for the success of this association.This information can be used in developing measures to control thehost snails, to avoid metabolic strategies that allow their prolongedsurvival and preventing this trematode from completing develop-ment and infecting in its definitive host.

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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