Parasitol Res (2008) 103:1105–1110 DOI 10.1007/s00436-008-1101-6
ORIGINAL PAPER
Comparative karyological analysis of three members of Allocreadiidae (Digenea): taxonomic and phylogenetic implications Romualda Petkevičiūtė & Gražina Stanevičiūtė
Received: 14 March 2008 / Accepted: 12 June 2008 / Published online: 9 July 2008 # Springer-Verlag 2008
Abstract Chromosome number and morphology were studied in three allocreadiid species from mitotic metaphases of intramolluscan stages using karyometric analysis. Bunodera luciopercae has a diploid number 2n=14, with two large pairs of metacentrics, one pair of submeta-subtelocentrics, and four pairs of acrocentrics. Karyotype of Allocreadium isoporum, 2n=14, with two large pairs of metacentrics and five pairs of acrocentrics was recognized to be closely related to that of B. lucioperca. The most conspicuous interspecific difference occurs in the centromere position of the chromosomes of the pair 3. The possible reasons of the discrepancies between data reported therein and the karyological information previously published on these two species are discussed. Crepidostomum sp. has 2n=12, with one pair of large metacentrics and five pairs of acrocentrics and is more distantly related karyologically to the other two members of the family. Evolutionary considerations based on comparison of the chromosomes of Allocreadiidae and other studied families of the suborder Xiphidiata suggest that allocreadiids are karyotypically distinct. The placement of Allocreadiidae and Opecoelidae in the superfamily Allocreadioidea is questioned.
Introduction Members of the family Allocreadiidae Looss, 1900 are among the most common and widely distributed parasites of the intestines of freshwater teleosts. In Europe, the family includes the genera Allocreadium looss, 1900, Bunodera Railliet, 1896, R. Petkevičiūtė (*) : G. Stanevičiūtė Institute of Ecology, Vilnius University, Akademijos 2, Vilnius LT-08412, Lithuania e-mail:
[email protected]
and Crepidostomum Braun, 1900 (Niewiadomska and Valtonen 2007). Known allocreadiid cercariae are of the ophthalmoxiphidiocercaria type (with distinct eye-spots and stylet) and overwhelmingly use sphaeriid bivalves as the first intermediate hosts (e.g., Wiśniewski 1958; Cannon 1971; Caira 1989). Much disagreement regarding taxonomy, systematics, and phylogeny of allocreadiids exists (see Caira 1989; Caira and Bogea 2005; Cribb 2005). Overall, body plasticity makes the identification of systematically useful morphological traits of allocreadiids, as well as of many other digeneans, a difficult task and leads to the search for new characteristics. Karyological data define one of the basic genetic characteristics of eukaryotic organisms and enable the phylogenetic relationships to be determined, especially in taxons, which contain species that show poor morphological differentiation. While the first karyotypes of trematodes had been described in the beginning of twentieth century (see review of Britt 1947), the subsequent rate of karyological descriptions has been low, and data on chromosome sets of only six allocreadiid species are currently known (Table 1). However, even this sparse available information is controversial. In his early study, Britt (1947) revealed seven meiotic chromosomes in the cells of testis of mature Bunodera luciopercae. Later observations on meiotic divisions of B. luciopercae from lake Opeongo, Canada showed six chromosomes in haploid complements (Cannon 1971). Next examination of larval B. luciopercae from Sphaerium rectidans in Northwestern Chukotka, Russia revealed the diploid number 2n= 26 (Barshene and Orlovskaya 1990). The purpose of the present study was to provide karyological information on three allocreadiid species, B. luciopercae (Müller 1776), Allocreadium isoporum (Looss 1894), and Crepidostomum sp. commonly occurring in Palearctic. Comparison of karyotypes of these species, in the context of wider study, might throw light on their relationships as well as on
1106 Table 1 Available information on chromosome numbers and morphology in Allocreadiidae
m metacentric, sm submetacentric, st subtelocentric, a acrocentric chromosomes
Parasitol Res (2008) 103:1105–1110 Species
No and morphology of chromosomes
References
Allocreadium isoporum A. fasciatusi A. handiai Bunodera luciopercae
N=8 3n ¼ 21 ¼ 3m þ 12sm þ 6st 2n ¼ 14 ¼ 12sm þ 2st n=7 n=6 2n ¼ 26 ¼ 4sm=m þ 2st þ 20a 3n=23 n=8
Britt 1947 Ramanjaneyulu and Madhavi 1984 Ramanjaneyulu and Madhavi 1984 Britt 1947 Cannon 1971 Barshene and Orlovskaya 1990 Cannon 1971 Britt 1947
B. sacculata Crepidostomum serpentinum
the systematic position of Allocreadiidae in suborder Xiphidiata which include digeneans with penetrating stylet in the cercariae. In addition, we wanted to clear discrepancies in existing karyological data.
Materials and methods The material for karyological analysis was obtained from spontaneously infected sphaeriid bivalves. Individuals of Pisidium amnicum infected with B. luciopercae were collected in Nava Lake and in water reservoir of the dammed up River Nemunas near Kaunas in Lithuania. Sphaerium rivicola infected with A. isoporum were collected in water reservoir of the dammed up River Nemunas near Kaunas in Lithuania and in River Teterev in Ukraine. Sphaerium corneum infected with Crepidostomum sp. were collected in River Belka in Ukraine. Larval allocreadiids were identified using morphological features of the cercariae and rediae. Identifications followed descriptions by Wiśniewski (1958) and Caira (1989). Before dissection, clams were treated in 0.01% colchicines in well water for 3–4 h at room temperature. Chromosome plates were prepared from material (presumably germinal cells and cercarial embryos) removed from infected clams. The tissues containing parthenites were treated in distilled water for hypotony for 30–40 min. The saturated tissues were transferred into fresh-made fixative (ethanol/acetic acid=3:1). Fixed material was stored at −20°C. Slide preparations were made using an air-drying technique (Petkevičiūtė and Stanevičiūtė 1999). Preparations were left to dry overnight. Preparations were then treated with 1 N HCl for 10–15 min, rinsed three times in distilled water, and stained for 30 min using freshly prepared 4% Giemsa solution in phosphate buffer (pH 6.8). After staining, slides were washed with tap water and dried. The chromosome preparations were examined with Olympus BX51 microscope with an oil immersion objective and digital camera (3 megapixels). The best chromosome spreads were photographed, and karyotypes were constructed by arranging the chromosomes in order of decreasing size. For each chromosome pair, the mean absolute and relative length and mean centromeric
index with their standard deviations (SD) were calculated. The relative length was expressed as 100×absolute chromosome length (in micrometer) divided by the total length of the haploid complement. Centromeric index was calculated by dividing 100×the length of the short arm by the total chromosome length. The chromosome classification system follows Levan et al. (1964). Data were analyzed by the Student’s t test. Results were considered significant when P
