SHORT COMMUNICATION

Effects of infection by larvae of Angiostrongyluscantonensis (Nematoda, Metastrongylidae) on the lipidmetabolism of the experimental intermediate hostBiomphalaria glabrata (Mollusca: Gastropoda)

Vinícius Menezes Tunholi-Alves &

Victor Menezes Tunholi & Patrícia Gôlo & Mariana Lima &

Juberlan Garcia & Arnaldo Maldonado Júnior &

Emerson Guedes Pontes &

Vânia Rita Elias Pinheiro Bittencourt & Jairo Pinheiro

Received: 9 October 2012 /Accepted: 18 January 2013 /Published online: 2 February 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Experimental infection of Biomphalaria glabrataby Angiostrongylus cantonensis induces significant changesin the concentrations of triacylglycerol and cholesterol in thehemolymph and of neutral lipids in the digestive gonad–gland (DGG) complex of the host snail. In this study, snailswere dissected after 1, 2, and 3 weeks of infection to collect

the hemolymph and DGG and to measure the levels ofcholesterol and triacylglycerol in the hemolymph and neu-tral lipid fractions in the tissues. The results show thatinfection by this nematode resulted in a significant decreasein the concentrations of both cholesterol and triacylglycerolin the hemolymph of B. glabrata during the parasite’s initialontogenic development period. This reduction indicates thepossible use of these molecules by both parasite and host notonly as energy substrates but also as structural factors re-quired during development of the parasite’s larval stages. Inparallel, changes in the neutral lipid profile in the DGG andlipase activity of the infected snails were observed, indicat-ing the importance of these molecules for successfulinfection.

Introduction

The success of nematodes in infecting the host depends notonly on behavioral and physiological adjustments but alsoon biochemical adaptations of the host. This situation hasrecently been demonstrated by Tunholi-Alves et al. (2011b,2012) in the Angiostrongylus cantonensis/Biomphalariaglabrata interface. According to the authors, experimentalinfection of A. cantonensis resulted in changes in the inter-mediate host’s reproductive biology and biochemical pro-file, characterizing co-adaptations that are vital to the host’ssurvival, which, in turn, allows the parasite to developcompletely (Grewal et al. 2003).

The pathophysiological effects of infection by larval stagesof helminths on the lipid metabolism of snails have been

Vânia Rita Elias Pinheiro Bittencourt, Arnaldo Maldonado Júnior andJairo Pinheiro are research fellows at CNPq.

V. M. Tunholi-Alves :V. M. Tunholi :M. Lima : J. Pinheiro (*)Departamento de Ciências Fisiológicas, Instituto de Biologia,Universidade Federal Rural do Rio de Janeiro (UFRuralRJ),Seropédica, Rio de Janeiro, Brazile-mail: jps@ufrrj.br

V. M. Tunholi-Alves :V. M. Tunholi : P. Gôlo :M. Lima :V. R. E. P. BittencourtCurso de Pós-Graduação em Ciências Veterinárias, Departamentode Parasitologia Animal, Instituto de Veterinária, UFRuralRJ,Seropédica, Brazil

J. Garcia :A. M. JúniorLaboratório de Biologia e Parasitologia de Mamíferos SilvestresReservatórios, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil,4365-Manguinhos,21040-900, Rio de Janeiro, Rio de Janeiro, Brazil

E. G. PontesDepartamento de Química, Instituto de Ciências Exatas,Universidade Federal Rural do Rio de Janeiro, BR 465, Km 7,23890-000, Seropédica, Rio de Janeiro, Brazil

V. R. E. P. BittencourtDepartamento de Parasitologia Animal, Instituto de Veterinária,Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro,Rio de Janeiro, Brazil

Parasitol Res (2013) 112:2111–2116DOI 10.1007/s00436-013-3308-4

investigated. Nevertheless, the little information that are avail-able are contradictory, indicating the lipid profile of snails ishighly variable. Fried et al. (1989) and Bandstra et al. (2006)observed a significant reduction in the levels of triacylglycerolin the digestive gland–gonad (DGG) complex of B. glabratainfected by Echinostoma caproni. In contrast, an increase inthe concentrations of the same neutral lipid fraction in theDGG of Helisoma trivolvis patently infected by Echinostomatrivolvis has been showed (Fried and Sherma 1990). Despitethese studies, data on changes in the metabolism of lipids insnails infected by nematodes have not yet been reported.

Angiostrongylus cantonensis is a nematode parasite ofthe lungs of wild rodents (Rattus rattus and Rattus norvegi-cus) and is classified as the main etiological agent of humaneosinophilic meningoencephalitis. Snails are the obligatoryintermediate hosts of this parasite, allowing the develop-ment of forms that can infect the definitive host (Stewartet al. 1985). Epidemiological studies have confirmed theoccurrence of this zoonosis in various regions of the world,including Brazil, highlighting for rapid dissemination of thisdisease (Wang et al. 2007).

Therefore, because of the importance of B. glabrata inthe biological cycle of A. cantonensis, the determination ofthe concentrations of neutral lipids in the DGG and their freefractions in the hemolymph is necessary to shed light on thisparasite–host relationship and to contribute to the develop-ment new protocols to control this snail, and consequently,human eosinophilic meningoencephalitis, as recommendedby the World Health Organization (WHO—World HealthOrganization 1983).

Material and methods

Maintenance of the snails and formation of groups

The snails were kept in aquariums containing 1,500 ml ofdechlorinated water, to which 0.5 g of CaCO3 was added, inthe Laboratório de Biologia e Parasitologia de MamíferosSilvestres Reservatórios, Instituto Oswaldo Cruz, Fiocruz,IOC. The water was replaced once a week. Six groups wereformed: three control groups (uninfected) and three infectedgroups (infected). Each aquarium contained ten snails,reared in the laboratory from hatching to be certain of theirage and that the snails were free of infection by other para-sites. The entire experiment was conducted in duplicate,using a total of 120 snails, 60 snails forming the controlgroups (uninfected) and 60 snails forming the infectedgroups. The aquariums were kept in a room with controlledtemperature of 25 °C throughout the experiment. The studywas performed for 3 weeks, a period that corresponds to theprepatent development of A. cantonensis in B. glabrata(Guilhon and Gaalon 1969; Tunholi-Alves et al. 2011b).

Parasites

Third-stage larvae (L3) of A. cantonensis, obtained fromspecimens of Achatina fulica collected in the municipal-ity of Olinda, Pernambuco (PE), Brazil in 2008, in thearea surrounding the home of a patient diagnosed witheosinophilic meningoencephalitis, were inoculated in R.norvegicus maintained in the Laboratório do Patologia ofInstituto Oswaldo Cruz (Fiocruz), according with protocolapproved by the Animal Use Ethics Committee, CEUAL-074/08. Identification of adult worms and larval stagesof A. cantonensis (PE isolate) was realized by polymer-ase chain reaction and morphological analysis (Thiengoet al. 2010). The first-stage larvae (L1) used in this studywere obtained from this experimental cycle.

Infection of the snails

The feces of parasitized R. norvergicus were collectedand used to obtain the L1 larvae by the Baermanntechnique, employed to separate and decant them(Willcox and Coura 1989). After processing the fecalsamples, specimens of B. glabrata (8–12 mm) at 90 daysof age on average were exposed individually to approx-imately 1, 200 L1 larvae on 24-hole plates. After 48 h,the snails from each group were individually examinedunder a stereomicroscope to detect larvae (L1 stage) inthe plates (Tunholi-Alves et al. 2011b). The absence oflarvae in the plates ensured the infection and susceptibil-ity of snails under laboratory conditions. Subsequently,the snails were removed from the plates and transferredto the aquariums for formation of the experimentalgroups. The snails were grouped according to their in-fection stage (1, 2, and 3 weeks post-exposure). In eachperiod analyzed, ten infected and ten uninfected snails(control) were dissected.

Dissection and collection of the hemolymph and tissues

Weekly, the hemolymph was collected by cardiac punctureof randomly chosen snails from each group (n=10), afterwhich, the specimens were dissected and the DGG wasseparated, weighed, and frozen at −80 °C. The hemolymphwas maintained in an ice bath during collection and storedat −10 °C.

Determination of the concentrations of triacylglyceroland cholesterol in the hemolymph

The contents of triacylglycerol (TAG) and cholesterol(CHO) were determined according to Trinder (1969)(Doles®), and their values were expressed as milligramsper deciliter.

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Thin-layer chromatography and image analysis

For analysis of different neutral lipids in snail tissues,thin-layer chromatography (TLC) was used as the stan-dard method due to its ability to assess several lipidsconcomitantly. Fifty milligrams of DGG tissue fromeach group was homogenized in 100 μL of lysis buffer(Tris 50 mM; EDTA 5 mM; Nonidet P-40 1 %; TLCK0.1 mM; PMSF 2 mM). The homogenates were centri-fuged at 2,520×g for 3 min. The total protein content inthe supernatant fractions was determined according tothe Lowry method with modifications (Markwell et al.1978), using bovine serum albumin as the standard.Samples containing 1,000 μg of total protein were sub-mitted to lipid extraction as described by Bligh andDyer (1959). The lipids extracted were analyzed byone-dimensional thin-layer chromatography (TLC)according to Tunholi-Alves et al. (2011a).

The DGG tissues of uninfected snails collected at the endof each week were polled. Thus, the mean result obtainedcould better express the variation observed in the infectedgroups during the 4 weeks of infection.

Preparation of the enzyme and determination of the lipaseactivity

The digestive glands were homogenized in Tris pH7.4 buff-er in a Potter–Elvehjem homogenizer with a Teflon pestle.The homogenate was centrifuged at 5,040×g for 10 min, andthe infranatant was analyzed. The protein concentrationswere measured using the modified Lowry method(Markwell et al. 1978), and then, the lipase activity wasdetermined by the method described by Choi et al. (2003).The absorbances of each sample were read in triplicate byELISA reader (Bio-Rad iMark™) at 405 nm, and the entireassay was performed twice (Fig. 1).

Statistical analysis

The results obtained in the tests to measure the concentrationsof CHO and TAG in the hemolymph were expressed asmean±standard deviation, and the Tukey’s test/ANOVAwas utilized for comparison of the means. A polynomialregression was estimated to analyze the relation betweenthe values obtained and the time of infection (P>0.05)(InStat, GraphPad, v.5.00, Prism). The values for TAGand CHO levels in the hemolymph of uninfected snailswere expressed as mean, represented by zero (0) week,since there was no significant difference among them atthe end of each week. The chromatographic results wereexpressed as mean±standard deviation in relation to thecontrol group (uninfected), also represented as zero (0)week of infection.

Results

After the prepatent period of A. cantonensis, B. glabrataspecimens were randomly selected and subjected to chemi-cal digestion process with pepsin-HCl to recover L3. Theresults showed infectivity rate above 95 %.

The infection in B. glabrata by A. cantonensis causedchanges in the hemolymph levels of CHO in the snails.There was a positive relation between the infection timeand CHO concentration in the hemolymph of the infectedspecimens (r2=0.84) (Fig. 2a). The lowest concentrations ofCHO in the hemolymph of infected snails were observed inthe first week (0.74±0.082) and second week after infection(0.72±0.069), representing decreases of 29.23 and 30.76 %,respectively, in relation to the control group average (1.05±0.09) (Table 1).

Changes in the concentrations of TAG in the hemolymphof the infected B. glabrata specimens were also observed.The smallest and largest values were obtained, respectively,in the first (0.87±0.098 mg/dl) and third (2.13±0.088 mg/dl)weeks after infection in relation to the control group (2.23±0.089 mg/dl). The polynomial regression test indicated asignificative positive relation between TAG level and timeof infection (r2=0.98) (Fig. 2).

After having detected these variations, we directed theanalyses to determine the possible effect of infection on theconcentrations of neutral lipids stored in the DGG of B.glabrata. The levels of CHO in the treated groups were lowerthan those in the control group throughout the experiment,except in the third week of infection. The CHO level in theDGG complex of the snails infected by A. cantonensis de-clined in comparison with the control group (0.024±0.0028),with the lowest values observed in the first (0.011±0.0014)

Fig. 1 Lipase activity in the digestive gland–gonad (DGG) complex ofBiomphalaria glabrata infected by Angiostrongylus cantonensis infunction of time of infection, expressed in weeks. Week0 (zero) rep-resents the average of the control group during the 3 weeks of infec-tion. Absorbance was read at 405 nm. Error bars represent standarddeviations. All assays were done in duplicate. mAbs, milli-absorbance

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and second (0.010±0.0007) weeks after infection, with re-spective declines of 54.16 and 58.33% (Fig. 3b). However, nosignificant variations were observed in the cholesterol esterlevels of infected snails, with decreased concentrations in thefirst week (0.009±0.0021) and increased in third week (0.011±0.007) after infection, not significantly different from thecontrol group (0.010±0.0007) (Fig. 3d).

The levels of TAG in the treated group were consistentlyhigher than those in the control group during the entire obser-vation period. The TAG levels in the DGG complex increasedsteadily from the first week after infection (0.067±0.0035),attaining increases of 48.88 % in relation to the control group(0.045±0.0055) (Fig. 3c). The same order of variation wasobserved in relation the fatty acid (FA) levels in the infectedgroup, with increases of 57.57 and 66.66 % after the first andsecond weeks of infection, respectively, in relation to thecontrol group (0.033±0.0005) (Fig. 3a).

To better understand the increase in the concentrations ofFA in the DGG of infected B. glabrata, we determined thelipase activity in this organ using the synthetic substrate 2,3-dimercapto-1-propanol tributyrate (DMPTB). Interestingly,an increase in the enzymatic activity of lipases in the DGG

of infected snails was observed in the first (0.149±0.02) andsecond (0.225±0.019) weeks of infection, increases of69.31 and 155.68 %, respectively, in relation to the controlgroup (0.088±0.017), coinciding with the increase in the FAconcentrations.

Discussion

In the present work, the infection by A. cantonensis resultedin a significant reduction in the hemolymph concentrationsof TAG in B. glabrata 1 and 2 weeks after infection, con-firming that this is the period of greatest competition fornutrients with the host. This reduction suggests that thelarvae are able to use this substrate as a source of energy.Tang and Wang (2011) demonstrated through enzyme teststhe presence of enzymes related to β-oxidation in larvalstages of A. cantonensis, confirming the capacity of thismetastrongylid to utilize lipids obtained from its host as anenergy substrate.

At the same time, the increase in concentrations of free FA inthe DGG of B. glabrata after infection is partly explained bythe mobilization of neutral lipids stored mainly in the formTAG. The same condition has been observed in snails infectedby the larval stages of trematodes, consisting of an importantmetabolic strategy of the host to minimize the deleteriouseffects of the infection (Humiczewska and Rajski 2005). Thatfact is better seen by the increase in the lipase activity in theDGG of the infected snails (first and second weeks), suggestingacceleration of the lipolytic process, necessary for formation ofFA, which once oxidized in the mitochondrial matrix generatesthe ATP required in the snail’s cell activities (Tripathi and Singh2002). Similar result was reported by Choubisa (1988) in theinterfaces Melanoides tuberculatus/Cercaria diglandulata andM. tuberculatus/Cercaria martini.

As observed for the FA fractions, infection by A. canto-nensis resulted in a sharp increase in the levels of TAG in the

Table 1 Levels of cholesterol and triacylglycerol in the hemolymph(mg/dl) of Biomphalaria glabrata infected by Angiostrongylus canto-nensis, in different post-infection periods, expressed in weeks. Week0(zero) represents the average of the control group during the 3 weeks ofanalysis since there were no significant differences in these weeks

Weeks Cholesterol (mg/dl) Triacylglycerol (mg/dl)

0 1.050±0.097a 2.23±0.089a

1 0.743±0.082b 0.98±0.066b

2 0.727±0.069b 0.87±0.098b

3 1.087±0.098a 2.13±0.088a

a, bMeans followed by different letters differ significantly, P<0.05,(mean±standard deviation)

Fig. 2 Levels of cholesterol and triacylglycerol in the hemolymph(mg/dl) of Biomphalaria glabrata infected by Angiostrongylus canto-nensis, in different post-infection periods, expressed in weeks. Week0(zero) represents the average of the control group during the 3 weeks of

analysis since there were no significant differences in these weeks.a, b=means followed by different letters differ significantly, P<0.05,(mean±standard deviation)

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DGG of B. glabrata during the entire experiment. Our resultsare in agreement with previous studies that indicated thecapacity of helminth parasites to excrete lipids (Fried andShapiro 1975). This possibility was also observed by Mulleret al. (2000), working with B. glabrata infected bySchistosoma mansoni, as well as by Tunholi-Alves et al.(2011a), studying the relation between B. glabrata andEchinostoma paraensei. Thus, the increase of this neutral lipidfraction (TAG) in the DGG of B. glabrata here showed can beexplained either through secretion of lipids by the parasite(Kwong 1989) or alteration of the metabolism of the host’sDGG as a consequence of the infection (Fried et al. 1998).

The decrease in the levels of cholesterol in the hemolymphobserved at the start of infection indicates the ability of A.cantonensis larvae to directly assimilate that substrate fromthe hemolymph, necessary to maintain the fluidity of itsmembranes, crucial to their establishment in the host(Yamoka et al. 1978). Moreover, the use of those moleculesby larvae of this helminth as precursors to synthesize new cellmembranes during their development in B. glabrata contrib-utes to the change evidenced here (Barrett et al. 1970). Thenormalization of the CHO levels in the hemolymph after thethird week of infection observed in this study indicates theparticipation of homeostatic mechanisms in B. glabrata, en-abling the host to synthesize new cholesterol endogenously

from precursors already identified, such as latosterol anddemosterol (Lustrino et al. 2010; Zhu et al. 1994).

The ontogenic development of A. cantonensis in B. glabratacauses an intense spoliation process, with consequent cell dis-organization in the host’s digestive gland because of migrationof the parasite’s larval stages (Tunholi-Alves et al. 2012). Inlight of these findings, the decline in the levels of CHO in theDGG in B. glabrata during the first weeks of infection can bepartly explained by the leakage this components through thecell membrane to the hemolymphatic circulatory system due tothe occurrence of lesions caused by the infection. The samesituation has been demonstrated by Tunholi-Alves et al.(2011a) in the B. glabrata/E. paraensei interface.

This study contains the first description of the pathophys-iological effect of infection by A. cantonensis on the metab-olism of lipids by B. glabrata. Our results indicate that boththe host and parasite are capable of using TAG as an energysubstrate and that the parasite can use CHO as a precursor tosynthesize new membrane components required during de-velopment, while the host can use it to preserve the plastic-ity of its tissues. Additionally, the greatest alterations in theconcentrations of the lipid fractions observed here occurredin the early stage of the infection, confirming that this is theperiod of greatest competition for nutrients and hence, themost important period for the success of this association.

Fig. 3 Concentration of neutral lipids in the digestive gland–gonad(DGG) complex of Biomphalaria glabrata infected by Angiostrongy-lus cantonensis in function of time of infection, expressed in weeks.Week0 (zero) represents the average of the control group during the

3 weeks of analysis. a level of fatty acids (FA), b level of cholesterol(CHO), c level of triacylglycerol (TAG), and d level of cholesterolester (CHOE). All values are expressed as mean±standard deviation. a,b=means followed by different letters differ significantly, P<0.05

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The results contribute to the efforts recommended by theWHO to control diseases transmitted by snails, by sheddingmore light on the metabolic profile of B. glabrata, to sup-port future studies focused on controlling this parasite andthus, human eosinophilic meningoencephalitis.

Acknowledgments This study was supported in part by ConselhoNacional para o Desenvolvimento Científico e Tecnológico (CNPq)and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estadodo Rio de Janeiro (FAPERJ).

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