Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 99(2): 131-136, March 2004 131 Phenotypic Plasticity in Adult Worms of Schistosoma mansoni (Trematoda:Schistosomatidae) Evidenced by Brightfield and Confocal Laser Scanning Microscopies Renata Heisler Neves, Michele Costa-Silva, Elaine Machado Martinez, Thiago B Branquinho*, Regina Maria Figueiredo de Oliveira, Henrique Leonel Lenzi*/++, Delir Corrêa Gomes**/++, José Roberto Machado-Silva/+/++ Disciplina de Parasitologia, Departamento de Patologia e Laboratórios, Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rua Prof. Manoel de Abreu 444, 5º andar, 20551-170 Rio de Janeiro, RJ, Brasil *Departamento de Patologia **Laboratório de Helmintos Parasitos de Vertebrados, Departamento de Helmintologia, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil A comparative morphometric study was performed to identify host-induced morphological alterations in Schis- tosoma mansoni adult worms. A wild parasite population was obtained from a naturally infected rodent (Nectomys squamipes) and then recovered from laboratory infected C3H/He mice. Furthermore, allopatric worm populations maintained for long-term under laboratory conditions in Swiss Webster mice were passed on to N. squamipes. Suckers and genital system (testicular lobes, uterine egg, and egg spine) were analyzed by a digital system for image analysis. Confocal laser scanning microscopy (CLSM) showed details of the genital system (testicular lobes, vi- telline glands, and ovary) and the tegument just below the ventral sucker. Significant morphological changes (p < 0.05) were detected in male worms in all experimental conditions, with no significant variability as assessed by CLSM. Significant changes (p < 0.05) were evident in females from the wild population related to their ovaries and vitelline glands, whereas allopatric females presented differences only in this last character. We conclude that S. mansoni worms present the phenotypic plasticity induced by modifications in the parasite’s microenvironment, mainly during the first passage under laboratory conditions. Key words: Schistosoma mansoni - phenotypic plasticity - adult worms - Nectomys squamipes - C3H/He mice - confocal laser scanning microscopy Vertebrate animals live in different environments where somiasis (Rodrigues-Silva et al. 1992, Maldonado Jr. et al. they are exposed to several parasites. The outcome of 1994, Ribeiro et al. 1998). Although this parasitism can this interaction is that both individuals have developed provoke severe damages to humans, rodents are permis- strategies for adaptation, aiming to reach an adjusted re- sive hosts with life-long infections (Machado-Silva et al. lationship (Hart 1992). In order to be established within 1997), which do not affect their life span (Rodrigues-Silva the host, a successful parasite must possess the ability to et al. 1992) or reproductive capacity (D’Andrea et al. 2000). tolerate the host immune responses (Zelmer 1998). N. squamipes develops peculiar granulomas consisting The trematode Schistosoma mansoni is a parasite mainly of large macrophages, many of them full of schis- worm naturally present in some rodents from Africa tosome pigment, characterizing an exudative-macrophage (Duplantier & Sène 2000) and Neotropical region (Combes granuloma type, usually smaller than the equivalent granu- 1990, Théron et al. 1992, Rey 1993, Alarcón de Noya et al. loma type in mouse, and they never acquire Symmer’s 1997, D’Andrea et al. 2000, 2002). The water-rat Nectomys fibrosis pattern (Silva & Andrade 1989, Costa-Silva et al. squamipes (Rodentia: Sigmondontinae) acts as a possible 2002). Such granulomas have been also observed in the reservoir of schistosomiasis mansoni in Brazil (Rey 1993, rat Calomys callosus (Lenzi et al. 1995). D’Andrea et al. 2002) and can also be used as an alterna- Parasitic flatworms that have been maintained in dif- tive animal model in basic biological studies on schisto- ferent hosts rather than in natural ones have undergone strong morphological changes (phenotypic plasticity) (Mouhaid et al. 1997). S. mansoni adult male worms were bigger in Rattus rattus than in R. norvegicus (Jourdane & Imbert-Establet 1980) as well as in N. squamipes than in Swiss Webster (SW) mice. Moreover, they presented a Financial support: Universidade do Estado do Rio de Janeiro shorter distance between suckers and the number of their and Fundação Oswaldo Cruz testicular lobes was enhanced, when an isolate of S. This work is part of a MSc Thesis in Morphology, Instituto de Biologia, Uerj, RJ, Brasil mansoni from N. squamipes has been developed in albino +Corresponding author. Fax: +55-21-2587.6148. E-mail: mice (Machado-Silva et al. 1994). In the present study we [email protected] demostrate the occurrence of phenotypic plasticity in S. ++Fellowships CNPq mansoni adult worms induced in two host changes: N. Received 23 July 2003 squamipes to C3H/He mice and albino mice to N. Accepted 21 January 2004 squamipes. 132 Phenotypic Plasticity of S. mansoni • Renata Heisler Neves et al. MATERIALS AND METHODS RGB) and transferred to a computer containing software Animals - SW and C3H/He mice were supplied by the for image analysis (Image Pro Plus - Media Cybernetics, Animal Facility Center from the Oswaldo Cruz Founda- US). The following morphological characters were ana- tion, Fiocruz, Rio de Janeiro, Brazil, and the laboratory- lyzed: oral and ventral sucker areas, distance between them reared N. squamipes (Ns) by the Laboratory of Biology (both sexes); number, area, major and minor diameter, and and Control of Schistosomiasis Mansoni, Department of perimeter of the testicular lobes; area, major and minor Tropical Medicine, Oswaldo Cruz Institute, Rio de Janeiro diameter, perimeter of the egg and its spine (Neves et al. (D’Andrea et al. 1996). The animals were kept in polypro- 1998). All measurements are in micrometers, unless other- pylene cages (40 cm x 33 cm) with stainless steel screened wise stated. Five specimens from each population (SN, covers. All animals received a well-fed diet for mice SR, BE/Ns, and BE/Sw) were also studied by confocal (Nuvilab CR1, Colombo, Paraná, Brasil) and water ad libi- laser scanning microscopy (CLSM) (LSM-410, Zeiss), tum. Sacrifice of the animals followed standard ethical pro- analyzing the following morphometric characteristics: cedures for laboratory animals (GV-SOLAS 1985). thickness of the tegument below the ventral sucker (for both sexes); length and width of the first, third, and last Experimental design normal testicular lobes; length, width, and area of the vi- Wild S. mansoni population - Twelve N. squamipes telline glands, and length and width of the ovary. On what were captured in a transmission area of schistosomiasis refers to the body length, values were those previously mansoni in the municipality of Sumidouro, state of Rio de reported by Machado-Silva et al. (1995): 9.5 ± 1.3 mm for Janeiro, Brazil (22°02’46"S; 42°41’21"W). They were killed the wild strain and 6.9 ± 1.55 mm for laboratory strain. under laboratory conditions and perfused for adult worm Statistical analysis - The data were submitted to the recovering (Machado-Silva et al. 1994) (Ibama license 061/ Student’s t-test (brightfield microscopy) and Mann- 2003 – CGFAU/LIC). This parasite population was termed Whitney test (confocal microscopy), and statistical sig- as SN. nificance was assessed at p ≤ 0.05. The statistical pro- Sympatric laboratory-reared Biomphalaria glabrata gram SPSS 9.0 for Windows was used to facilitate calcula- snails, measuring 5 mm in shell diameter, were exposed to tions. ten hatched miracidia collected from the rodent’s feces. The snails were kept in glass vials with dechlorinated tap RESULTS water and fed with fresh lettuce (Lacttuca sativa). Wild strain - Biometric evaluations of male worms from Seven-day-old C3H/He mice were exposed individu- the SN population showed higher values than those ob- ally to cercariae (n 50) shed by the infected snails. After served in SR, except in relation to the number of testicular eight weeks post infection, the mice were killed by cervi- lobes. Most differences were considered to be significant cal dislocation and adult worms were recovered by por- (p < 0.05), except for the major and minor diameters of the tal-hepatic perfusion, establishing another isolate desig- testicular lobes (Table I). Testicular lobes in atypical posi- nated as SR. tion (supernumerary) were present in adult worms from Laboratory S. mansoni population - Laboratory-reared both populations. Two specimens (SN population) pre- N. squamipes and SW mice (five to six week old) were sented only one supernumerary lobe, while six worms had infected with four different laboratory worm strains that one, three or six lobes (SR population). The distance be- have been maintained for more than five years under labo- tween the last testicular lobe and the nearest supernu- ratory conditions in B. glabrata derived from the respec- merary one in the SN population varied from 254.66 µm to tive geographical areas: state of Pará (01°28’03"S; 517.24 µm, while in the SR population the occurrence of 48°29’18"W), state of Ceará (03°45’47"S; 38°31’23"W), higher number of supernumerary testicular lobes was fol- state of Rio Grande do Norte (05°45’54"S; 35°12’04"W), lowed by longer distance between them. For instance, in and state of Pernambuco (08°45’48"S; 34°54’47"W). The case with three and six testicular lobes the distances were parasites isolated from N. squamipes and SW were desig- 976.63 µm and 1251.80 µm, respectively. nated as BE/Ns, CE/Ns, CM/Ns, and CMO/Ns or BE/Sw, All female characteristics of the SR population were big- CE/Sw, CM/Sw, and CMO/Sw, respectively. All popula- ger than those from the SN population (p < 0.05) (Table I). tions were routinely maintained in SW mice and B. glabrata Both male and female worms from the SR population snail host at the Department of Malacology, IOC-Fiocruz, examined by CLSM presented all measurements bigger Rio de Janeiro, Brazil.