Lymnaea schirazensis, an overlooked snail distorting fascioliasis data: genotype, phenotype, ecology, worldwide spread, susceptibility, applicability

Abstract

Background: Lymnaeid snails transmit medical and veterinary important trematodiases, mainly fascioliasis. Vector specificity of fasciolid parasites defines disease distribution and characteristics. Different lymnaeid species appear linked to different transmission and epidemiological patterns. Pronounced susceptibility differences to absolute resistance have been described among lymnaeid populations. When assessing disease characteristics in different endemic areas, unexpected results were obtained in studies on lymnaeid susceptibility to Fasciola. We undertook studies to understand this disease transmission heterogeneity.

Methodology/principal findings: A ten-year study in Iran, Egypt, Spain, the Dominican Republic, Mexico, Venezuela, Ecuador and Peru, demonstrated that such heterogeneity is not due to susceptibility differences, but to a hitherto overlooked cryptic species, Lymnaea schirazensis, confused with the main vector Galba truncatula and/or other Galba/Fossaria vectors. Nuclear rDNA and mtDNA sequences and phylogenetic reconstruction highlighted an old evolutionary divergence from other Galba/Fossaria species, and a low intraspecific variability suggesting a recent spread from one geographical source. Morphometry, anatomy and egg cluster analyses allowed for phenotypic differentiation. Selfing, egg laying, and habitat characteristics indicated a migration capacity by passive transport. Studies showed that it is not a vector species (n = 8572 field collected, 20 populations): snail finding and penetration by F. hepatica miracidium occur but never lead to cercarial production (n = 338 experimentally infected).

Conclusions/significance: This species has been distorting fasciolid specificity/susceptibility and fascioliasis geographical distribution data. Hence, a large body of literature on G. truncatula should be revised. Its existence has henceforth to be considered in research. Genetic data on livestock, archeology and history along the 10,000-year post-domestication period explain its wide spread from the Neolithic Fertile Crescent. It is an efficient biomarker for the follow-up of livestock movements, a crucial aspect in fascioliasis emergence. It offers an outstanding laboratory model for genetic studies on susceptibility/resistance in F. hepatica/lymnaeid interaction, a field of applied research with disease control perspectives.

Figure 1. Maps of the Old and New Worlds showing localities where Lymnaea schirazensis was collected: A) Old World: 1 = Taleb Abad river, Bandar Anzali, Gilan province, Iran; 2 = Medicine Faculty, Rasht, Gilan province, Iran; 3 = El Kazza, Hosh Esa district, Behera governorate, Egypt; 4 = Tiba, Delengate district, Behera governorate, Egypt; 5 = Boulin El Aly, Kafr El Dawar district, Behera governorate, Egypt; 6 = Albufera of Valencia, Valencia province, Spain; 7 = Nules-Moncofar, Castellon province, Spain; B) New World: 8 = Constanza, Departamento de La Vega, the Dominican Republic; 9 = Río Grande, Constanza, Departamento de La Vega, the Dominican Republic; 10 = Los Molinos, subcuenca Nexapa, Atlixco, Puebla, Mexico; 11 = Escuela A. Obrego, La Trinidad Tepango, Atlixco, Puebla, Mexico; 12 = Xalpatlaco, Atlixco, Puebla, Mexico; 13 = Jiutepec, Morelos, Huauchinango, Mexico; 14 = Laguna de Fe y Alegria, El Valle, Estado de Merida, Venezuela; 15 = Hotel Valle Grande, El Valle, Estado de Merida, Venezuela; 16 = Guarandauco, Chillogallo, Ecuador; 17 = La Buena Esperanza, Cayambe, Ecuador; 18 = Machachi, Santo Domingo, Ecuador; 19 = Baños del Inca, Cajamarca, Peru; 20 = Rio Lurin, Lima, Peru.

For a higher resolution situation of each locality, see respective coordenates in text.

Figure 2. Nucleotide differences in a total of 64 variable positions found in the complete 18S rDNA sequence of the lymnaeid species compared and their location in the secondary structure.

Helix, Position and Difference number = numbers to be read in vertical. Position = numbers refer to positions obtained in the alignment made with MEGA 4.0. Identical = .; Indel = −. Shaded area corresponds to Helix E10-1 of the variable area V2 where differences in the 18S rRNA gene of Lymnaeidae are concentrated . GenBank Accession Nos. = Z73980−Z73985 ; Z83831 ; AM4122222 ; FN182190 ; FN598151- FN598152 ; L. schirazensis from present paper. Sequence correspondences: * L. cubensis, L. viatrix, L. humilis and L. cousini without definitive genus ascription; 18S identical in L. viatrix and L. neotropica ; 18S identical in L. cousini and L. meridensis .

Figure 3. Variable positions showed by the mtDNA 16S sequence fragment in a 433-bp-long alignment including the two haplotypes of Lymnaea schirazensis and other Galba/Fossaria species.

Numbers (to be read in vertical) refer to positions obtained in the alignment made with MEGA 4.0. Identical = . ; Indel = − . Haplotype codes only provisional due to incomplete sequences of the gene. L. cubensis 16S-HB (FN182204), L. humilis 16S-HA (FN182195) and L. humilis 16S-HB (FN182196) ; F. bulimoides (AF485657) and F. obrussa (AF485658) ; * the sequence ascribed to the stagnicoline C. elodes (EU038305) concerns in fact a Galba/Fossaria species-see analysis in .

Figure 4. COX1 amino acid sequence differences detected in the alignment of the haplotypes of the lymnaeid species studied, together with species of the Galba/Fossaria group and other proximal lymnaeid species available in GenBank.

Only cox1 sequence fragments of a lenght similar to that of sequences obtained in present paper are included. Variable positions = Numbers (to be read in vertical) refer to positions obtained in the alignment made with MEGA 4.0. − = position not sequenced; ? = undetermined amino acid. Haplotype codes only provisional due to incomplete sequences of the gene. * Sequences somewhat shorter and including a few undetermined amino acids; ** sequences somewhat shorter although presumably identical to haplotype cox1a of the same species.

Figure 5. Phylogenetic trees of lymnaeid species studied based on maximum-likelihood (ML) estimates: A) data set of ITS-1 and ITS-2, with the planorbid B. pfeifferi as outgroup (−Ln = 10016.27013); B) same data set of ITS-1 and ITS-2 with B. pfeifferi as outgroup, after adding Radix species (−Ln = 10078.46520).

Scale bar indicates the number of substitutions per sequence position. Support for nodes a/b/c: a: bootstrap with NJ reconstruction using PAUP with ML distance and 1000 replicates; b: bootstrap with ML reconstruction using PAUP with 1000 heuristic replicates; c: Bayesian posterior probability with ML model using MrBayes. See Table S1 for systematic-taxonomic notes.

Figure 6. Phylogenetic trees of lymnaeid species studied based on maximum-likelihood (ML) estimates: A) data set of 18S, ITS-1 and ITS-2, with B. alexandrina as outgroup (−Ln = 13171.38533); B) data set of 16S and cox1, with B. alexandrina as outgroup (−Ln = 5282.96177).

Scale bar indicates the number of substitutions per sequence position. Support for nodes a/b/c: a: bootstrap with NJ reconstruction using PAUP with ML distance and 1000 replicates; b: bootstrap with ML reconstruction using PAUP with 1000 heuristic replicates; c: Bayesian posterior probability with ML model using MrBayes. See Table S1 for systematic-taxonomic notes.

Figure 7. Shells of Lymnaea schirazensis in ventral, dorsal and from-below views, showing intraspecific variability: A,B) specimen (7.20 mm high) from Tiba, Delengate district, Behera governorate, Egypt; C) specimen (7.00 mm) from Albufera of Valencia, Valencia province, Spain; D–F) specimen (7.10 mm) from Albufera of Valencia, Valencia province, Spain; G,H) specimen (7.80 mm) from Nules-Moncofar, Castellon province, Spain; I,J) specimen (7.84 mm) from Laguna de Fe y Alegria, El Valle, Estado de Merida, Venezuela. Scale bars: A−E,G−J = 4 mm; F = 2 mm.

Figure 8. External aspect of Lymnaea schirazensis: A–E) living specimens showing (i) large, round, black eyes, (ii) long, slender tentacles and (iii) dark shell (A = lighted from down; B–D = lighted from above; F = epi− and infralighted simultaneously); F–I) dark brown to blackish mantle roof of specimens from Spain (F, G) and Mexico (H, I) showing small unpigmented white-greyish round spots, including several tiny circles (artificially remarked in white with computer effects in I) at the beginning of the border of the pulmonary region (I = yellow rectangle in H).

Figure 9. Aspects of soft part anatomy of Lymnaea schirazensis: A, B) renal tube and ureter in renal region extending between pericardium and mantle collar; C) carrefour in detail, with arrows indicating ducts to albumen gland (ag), spermiduct (spd) and nidamental gland (ng); D) oviducal crown turned to show detail of the region of oviduct pouch; E, F) reproductive system in two ventral views; G) female complex in dorsal view; H) prostate section showing absence of internal folds. Scale bars: A = 1.5 mm; B = 1.2 mm; C = 0.5 mm; D = 0.9 mm; E = 0.8 mm; F = 1 mm; G = 0.6 mm; H = 250 µm (drawings R. Rojas; plate configuration S. Mas-Coma).

Figure 10. Gradual evolution of egg cluster lays in experimentally raised Lymnaea schirazensis and Galba truncatula: A–O) L. schirazensis: note trend to kidney shape in early lays (E–K) and final trend to banana-shape in late trends (L–O); only in very early lays, when shape is still round-oval (A, B) and may sometimes become elongate (C, D), it can be confused with clusters of G. truncatula. P–Z) G. truncatula: note general trend to round-oval shape (P–R, V–X) and occasional variation to elongate shape (S–U, Y, Z).

Materials of L. schirazensis from strains originally collected in Xalpatlaco, Mexico (A, B, F, I, J), Jiutepec, Mexico (C), Albufera of Valencia, Spain (D, E, G, L, O), Tiba, Egypt (H), Escuela Obrego, Mexico (K). Materials of G. truncatula from strains originally collected in Qued Tiout, Marrakesh, Marocco (P–T, Y, Z) and Albufera of Valencia, Spain (U–X). Scale bar = 2 mm.

Figure 11. Environments of localities where Lymnaea schirazensis was collected: A) Garden of the Medicine Faculty, Rasht, Gilan province, Iran; B) Taleb Abad river, Bandar Anzali, Iran; C) El Kazza, Behera governorate, Egypt; D) Nules-Moncofar, Castellon province, Spain; E) Albufera of Valencia, Valencia province, Spain; F) Constanza, Departamento de La Vega, the Dominican Republic; G) Río Grande, Constanza, Departamento de La Vega, the Dominican Republic; H) Xalpatlaco, Atlixco, Mexico; I) Escuela A. Obrego, La Trinidad Tepango, Atlixco, Puebla, Mexico; J) Laguna de Fe y Alegría, El Valle, Merida, Venezuela; K) Hotel Valle Grande, El Valle, Merida, Venezuela; L) Guarandauco, Chillogallo, Ecuador; M) Machachi, Santo Domingo, Ecuador; N) Baños del Inca, Cajamarca, Peru.

Figure 12. Comparison showing interpopulational differences of mean characteristics of life span and selfing reproduction capacity of isolatelly-maintained Lymnaea schirazensis specimens of four populations from different geographical origins, experimentally followed up from the day of their individual isolation immediately after hatching.

Populations studied: Albufera of Valencia, Valencia, Spain; Tiba, Delengate district, Egypt; Escuela A. Obrego, La Trinidad Tepango, Atlixco, Puebla, Mexico; and Jiutepec, Morelos, Huauchinango, Mexico. n = 10 snail specimens followed per population (for details on intrapopulational variability ranges during the laying period, see Table 6 and Table 7); tlc = mean total laying capacity (number of clusters/life span in days); lrsp = mean laying rate in sexually active period (number of clusters/laying period in days); tnld = mean total non-laying days within laying period.

Figure 13. Galba truncatula: A–G) Shells in ventral and dorsal views of specimens from Albufera of Valencia in Spain (A,B: 9.33 mm high), Sachsen in Germany (C: 6.20 mm), Nules in Spain (D,E: 6.00 mm), and Qued Tiout in Morocco (F,G: 8.29 mm). H) Eyes and tentacles in living specimen. I) Comparison of living L. schirazensis (left) and G. truncatula (right) showing differences in (i) eyes, (ii) tentacles and (iii) mantle roof colour through the shell (by infralighting). J,K) Mantle roof in specimens from Nules, Spain (J) and Iran (K). L) Part of reproductive system in ventral view. M,N) Male terminal organs in specimens from Nules, Spain (M) and Iran (N).

Scale bars: A−G = 3 mm; H = 1.2 mm; I = 1.5 mm; J,K = 2 mm; L = 2.5 mm; M,N = 1 mm.

Figure 14. Old World spread of Lymnaea schirazensis combined haplotype ITS-2 H1-ITS-1 HA-16S HA-cox1 Ha during the livestock postdomestication 10,000-year period from the “Fertile Crescent” region of origin in the Near East, including expansion route into the New World from southern Spain during the early period of colonization about 500–400 years ago (green lines and red dots).

For historical and archeological data supporting this recent spread, see text. Green dot of Shiraz, Iran = type locality of L. schirazensis; black lines = spreading routes into Europe and Africa to cover the geographical distribution suggested by the presumed species and variety synonyms (see text) and “resistant” Galba truncatula populations described in France ; brown lines = spreading routes into Asia according to the geographical distribution noted by Kruglov .

Figure 15. New World spread of the different combined haplotypes of Lymnaea schirazensis from remote Old World source(s) in Europe (only or mainly southern Spain) during the early period of colonization about 500–400 years ago (green lines and red dots), illustrating the dispersal role played by Hispaniola island.

Combined haplotype ITS-2 H2-ITS-1 HB: (i) origin whether in Mexico itself after introduction from Hispaniola (green broken line) or from an unknown European source (yellow ? broken line), and (ii) subsequent introduction into Ecuador and Peru by a sea route from Mexico. Introduction of ITS-2 H1 in northern Peru whether by a maritime route through southern Central America (Panama isthmus) or by a terrestrial route from Venezuela (alternative dark broken lines). For historical and archeological data supporting this recent spread, and further details on mtDNA gene isolated haplotypes (16S HB and cox1 Hb, c and d), see text.