ORIGINAL PAPER. Cristiane L. G. F. Mendonça & Omar S. Carvalho & Ester M. Mota & Henrique L. Lenzi

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1 Prsitol Res (2008) 102: DOI /s y ORIGINAL PAPER Development of Angiostrongylus costricensis Morer nd Céspedes 1971 (Nemtod: Angiostrongylide) lrve in the intermedite host Srsinul mrgint (Semper 1885) (Mollusc: Soleolifer) Cristine L. G. F. Mendonç & Omr S. Crvlho & Ester M. Mot & Henrique L. Lenzi Received: 8 October 2007 / Accepted: 29 November 2007 / Published online: 22 Februry 2008 # Springer-Verlg 2007 Abstrct In life cycle of Angiostrongylus costricensis, veronicellide mollusks prticipte s the invertebrte host while rodents s the min vertebrte host. The current work shows sequentil lrvl development of A. costricensis in Srsinul mrgint, individully killed nd digested from dy 1 to 43, post infection. Some lrve, recovered from sedimenttion, were submitted to selective stining fter prffin embedded or inclusion in JB-4 to study inner structures. As control, four slugs were used, two killed t the beginning of infection nd the others t the end of the experiment. At dy 2 post infection, lrve were motionless nd thick, presenting initil retention of grnules. At dy 4, L 2 were detected, persisting until 43 dys post infection. Lrve L 2 displyed lrge mount of grnules rich in lipids nd crbohydrtes through its overll body, with more ccumultion t the medil third corresponding to the esophgus intestine trnsition site. Lipid grnules, the min energetic source, were locted t the bsl nd picl regions of intestinl cells. Both L 1 nd L 3 presented bilterl le, which is lso common in other nemtodes. Trnsition forms between L 2 to L 3 molts were lso observed. C. L. G. F. Mendonç : O. S. Crvlho (*) Lbortory of Intestinl Helminthisis, Centro de Pesquis René Rchou/Fiocruz, Avenid Augusto de Lim 1715, CEP Belo Horizonte, Mins Geris, Brzil e-mil: E. M. Mot : H. L. Lenzi Deprtment of Pthology, Instituto Oswldo Cruz/Fiocruz, Av Brsil 4365, CEP Rio de Jneiro, Rio de Jneiro, Brzil Introduction The Metstrongilide Angiostrongylus costricensis Morer nd Céspedes 1971, order Strongylid nd fmily Angiostrongylide, is heteroxenic prsite. Throughout its life cycle, dult worms re locted into rodent mesenteric rteries, requiring veronicellide mollusks s intermedite hosts. The first description on its evolutionry cycle ws performed by Morer (1973) nd further revisions hve dded extensive dt to the cycle both in vertebrtes (Mot nd Lenzi 1995, 2005) nd invertebrtes (Mendonç et l. 1999, 2003). Studies hve demonstrted tht the nemtode lrvl development in mollusks my be influenced by environmentl fctors such s humidity, temperture, estivtion, intern fctors such s infection intensity, nd specific chrcteristics of the host like ge, size, nd susceptibility (Cheng nd Alict 1965; Gerichter 1948; Guilhon nd Glon 1969; Hlvorsen nd Skorping 1982; Hori et l.1985; Ishii 1984; Kutz et l.1999; Rchford 1976; Rose 1957; Smson nd Holmen 1984; Solomon et l. 1996; Yousif nd Lmmler 1975; Wllce nd Rosen 1969; Kutz et l.2001; Lv et l.2006; Jenkins 2006). The current work detiled sequentil morphologicl spects of A. costricensis lrvl development in the intermedite host, chrcterizing the min chemicl composition of the lrve body, the cells responsible for lipid secretion nd the sequentil structurl modifictions in the molting process from L 1 to L 3 stges. Mterils nd methods A totl of 185 slugs, weighting round 1.0 g, were infected with 5,000 L 1 during 24 h. Five slugs were killed dily, for

2 862 Prsitol Res (2008) 102: ech period of time, s follows: from the first to 30th dy nd t 33, 34, 35, 36, 40, 41 nd 43 dys post infection. They were individully digested (HCl + pepsin) nd the sediment ws recovered in Petri dishes nd exmined with inverted microscope for L 1, L 2, nd L 3 identifiction, ccording to Morer (1973). Purified lrve were stined with Sudn Blck nd Oil Red (Perse 1968) to verify the presence of lipids. For negtive control, lrve were previously treted with xylol nd lcohol for grese dissolution, followed by nother Sudn stining. Serch for glycoprotein ws performed with periodic cid Schiff (PAS) stining nd fluorescence ssy using Glycine mx (soyben) nd Lens culinris (Lentil)-fluorescein isothiocynte (FITC) lbeled. Amylse digestion ws used s control of PAS rection to glycogen. The L 1,L 2 nd L 3 were lso fixed in Crson s formlin (Crson et l. 1973) nd wshed nd embedded in hydrophilic resin JB-4 (Junqueir et l. 1979); this procedure excluded the lcoholic dehydrtion s requirement for resin infiltrtion. Semi-thin sections obtined with ultrcut microtome (Leic) were stined with Lee s methylene blue-bsic fuchsine (Bennett et l. 1976). Stined sections were nlyzed by bright-field microscopy nd entire lrve were studied by lser scnning confocl microscopy (LSM-410, Zeiss), pplying reflected mode to fluorescein lbeled mteril, while unstined lrve were seen under Nomrski mode. Results At dy 2 post infection, L 1 were thick nd motionless with initil expression of lipid grnules. At dy 4, L 2 were detected, persisting until dy 43 post infection. Throughout the molting process, lrvl thickness vried from thin with few grnules, when young, to thick with gret mount of grnules, fter mturtion (Figs. 1,b nd 2). Together with mture L 2, young L 3 were found with residul cuticle (Fig. 1c) t dys 11, 15, 18, 20 nd 28 post infection. Concomitnt mture L 3 nd L 2 nd young L 3 were observed t dys 11, 14, 15, 16, 18, 21, 22, 26, 28 nd 43 post infection. Lrve L 2 displyed gret mount of lipid grnules throughout the whole body, with more density in the medil third, corresponding to the esophgus intestine trnsition site (Fig. 2 c). Grnules vried in size nd initilly were more homogeneously distributed nd, throughout molting from L 2 to L 3, they grdully moved to the subnucler region of the intestinl epithelium (Fig. 3,b), disppering in mture L 3 (Fig. 3c). Lipid nture of grnules ws confirmed by previous tretment with xylol nd lcohol, turning Sudn stining completely negtive. Lrve L 2 were PAS positive, indicting the presence of neutrl glycoproteins which did not vnish fter mylse digestion, precluding the glycogen presence. Lrve L 1 nd L 3 were not stined by Sudn nd PAS. G. mx-fitc nd L. culinris-fitc lectins, with ffinity for N-cetyl-D-glctosmine, α-d-mnosyl nd α-d-glycosyl, respectively, developed grnules strongly stined nd locted predominntly in the medil third of L 2 (Fig. 2d). The density of crbohydrte grnules ws lower when compred with the lipid ones, minly t the ends of lrvl body. Lectins lso developed, besides lrge grnules, diffuse nd thin grnulr pttern, more evident in the medil nd proximl thirds of the lrvl body. Lrve L 1 nd L 3 presented typicl bilterl le (Fig. 3c,d). 14 µm 24 µm b c 27 µm Fig. 1 Lrve of Angiostrongylus costricensis in different stges of development. Externl spect of L 1 (DIC, LSCM, Br=14 μm). b L 2 undergoing L 3 with lipids lining up close to the cuticle. The inset detils the lipid grnules disply (DIC, LSCM, Br=24 μm). c Young L 3 with residul L 2 cuticle (DIC, LSCM, Br=27 μm). DIC=Differentil interference contrst, LSCM=lser scnning confocl microscopy

3 Prsitol Res (2008) 102: Fig. 2 Lrve L 2 with mny lipid grnules, diminishing in the nterior end, seen in color code, from tomogrphy sections (LSCM). b Lrve L 2 with Oil- Red-stined lipid grnules (LSCM, reflection mode). c Molting lrve L 2 showing residul cuticle nd lipid grnules in peripherl loction (LSCM). d Lrve L 2 with Glycine mx- FITC-positive dense grnules t the medil third, with shortge t the extremities, minly in the nterior end (Glicine mx-fitc, LSCM). FITC=Fluorescein isothiocynte, LSCM=lser scnning confocl microscopy 21 µm b 23 µm c 12 m d 20 µm Discussion The present work focused on morphologicl nd biochemicl chrcteristics of A. costricensis lrve in the invertebrte host Srsinul mrgint, using diverse methodologicl nlysis. It is demonstrted tht the second molt (L 2 L 3 ) occurred from dy 4 to 43 post infection, nd the min energetic source of L 2 consisted of lipids nd crbohydrtes, which were predominntly locted in the medil third of the lrve, corresponding to the esophgus intestine trnsition site nd primordil genitl system. The first description of A. costricensis life cycle in the intermedite host, reported by Morer (1973), referred the rpid chnges tht occurred in L 1, which becme thicker due to the ccumultion of numerous grnules. Such trnsformtions were more evident t dy 3 nd 4, when the first molt (L 1 L 2 ) took plce. Lrve L 2 grew bigger from dy 4 to 10, presenting grdul increse in the mount of grnules, mking the visuliztion of inner orgns very difficult. The second molt (L 2 L 3 ) ws observed from dy 11 to 14. Between 16 nd 19 dys, lrve reched mturity, the moment t which they becme infective to the vertebrte host. In the current study, the second molting period (L 2 L 3 ) ws longer nd extended up to dy 43. This event could be relted to the low temperture during the cold wether (My, June, nd July) in Brzil, nturl fctor ble to dely lrvl development in its intermedite host. Indeed, Greff-Teixeir et l. (1991) observed tht sesonl trnsmission of A. costricensis in the south of Brzil (lte spring nd erly winter) might be due to ecologicl fctors ssocited with the prsite host interction. At low tempertures, lrve evolution in the mollusk my be delyed nd in the presence of het nd humidity they keep evolving, which coincides with their highest ctivity nd the mollusks reproduction, enhncing chnces to rech mn. A similr pttern ws lso observed in Angiostrongylus cntonensis (Yousif nd Lmmler 1975; Ishii 1984; Hori et l. 1985; Lv et l. 2006), Angiostrongylus vsorum (Guilhon nd Glon 1969), Umingmstrongylus pllikuukensis (Kutz et l. 1999; Kutz et l. 2001), Protostrongylus spp (Smson nd Holmen 1984), Elphostrongylus rngiferi (Skorping 1984; Schjetlein nd Skorping 1995), Cystoculus ocretus (Gerichter 1948), Muellerius cpillris (Gerichter 1948; Rose 1957) nd Prelphostrongylus odocoilei (Jenkins 2006) for which strong ssocition between temperture, lrvl development nd infection rtes in invertebrte hosts ws shown. Other fctors hve lso been mentioned s possible interference gents in

4 864 Prsitol Res (2008) 102: b c 8 µm d 8 µm 8 µm 8 µm Fig. 3 Trnsversl section of L 2 showing lipid drops in intestinl epithelil cells, which re locted t supr nd subnucler positions in the cytoplsm (rrow; JB-4 resin, MBBF). b Molting L 2 L 3, with decrese in lipid vcuoles, displyed in line position tending to the bsl region of intestinl epithelil cells (rrow; JB-4 resin, MBBF). c Trnsversl section of L3: intestinl lumen nd epithelium; cells locted between intestinl epithelium nd cuticle, musculr lyer nd prominent bilterl le (rrow; JB-4 resin, MBBF). d Trnsversl section of L 1 t the esophgus level showing bilterl le (semi-thin section, JB-4 resin, MFB, Br=8 μm). MBBF=Lee s methylene bluebsic fuchsine lrvl cycles of nemtodes in their snil hosts such s ge (Cbret 1987; Cheng nd Alict 1965; Wllce nd Rosen 1969), prsitic lod (Gerichter 1948), susceptibility (Gerichter 1948; Hlvorsen nd Skorping 1982), lck of nutrients - estivtion (Solomon et l. 1996), nd the host constitution (Rchford 1976). Therefore, we lso believe tht temperture my be the min environmentl fctor tht directly influences lrvl development of Metstrongilide, which constitutes importnt dt in epidemiology nd cycle mintennce in lbortory. The min energetic source of prsite nemtodes is glycogen, but during free-living life cycle lipid is the min one. Such reserves my be kept in hypoderm, into musculr cells nd epithelil cells of intestine nd reproductive orgns. During the lrvl period, lipid is kept in intestine nd during encpsulting phse, in intestine lume (Rey 2001). Other studies hve shown tht the stored grnules in intestinl cells used s energetic source consisted of glycogen nd ft. Herein, Sudn Blck nd Oil red stining substntited the predominnt lipid composition of the grnules during L 2 phse (Fig. 2,b), which dditionlly contin lso crbohydrte nd/or glycoprotein, without evidence of glycogen. The crbohydrte content ws prtilly constituted of N-cetyl-D-glctosmine nd α- mnosyl nd α-d-glycosyl (Fig. 2d). Therefore, lipid nd crbohydrte (glycolipid, glycoproteins ) were the min energetic source, not glycogen, implicted in the following lrvl development phse (L 3 ). Anlysis of the semi-thin sections (JB-4) of L 2 demonstrted tht lipid grnules were secreted by intestinl cells, which re locted t supr nd subnucler positions in the cytoplsm (Fig. 3). During the trnsition of L 2 L 3, lipid moved preferentilly to the subnucler region of such cells (Fig. 3b), which probbly, lso synthesizes the glicídios. The metbolic process which is triggered in the consumption of lrge mount of synthesized lipid nd glycoprotein during L 2 stge is still unknown. Some nemtode use cetyl CoA, derived from β-oxidtion of ftty cid, in order to mke use of it during the Krebs cycle (Roberts nd Jnovy 2000). Probbly, s in Ancylostom tubeforme Dubini 1843 nd Hemonchus contortus Rud 1803 (Onwuliri 1985), lrve L 1 nd L 2 of A. costricensis re nerobic, while infective L 3 re erobic nd ble to perform β- oxidtion of gret quntity of lipid synthesized by L 2. This is the first report in the literture tht emphsizes some internl morphologicl nd biochemicl chrcteristics of L 2.

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