Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2000073173
A KARYOLOGICAL STUDY OF THE SPIRURID NEMATODE MASTOPHORUS MURIS (NEMATODA: SPIROCERCIDAE) ŠPAKULOVÁ M.*, CASANOVA J.C.**, LAPLANA GUILLÉNN.**& KRÁL'OVÁ I.*
Summary : The karyotype of Mastophorus muris (Gmelin, 1 790) comprises four pairs of small autosomal chromosomes and two larger sex X chromosomes in females or one X chromosome in males (2n = 8 + XX/XO). All pairs of chromosomes possess rather uniform morphology without distinct primary or secondary constrictions. No heterochromatin bands were found by Cbanding analysis. The absolute chromosome length ranges from 4 . 0 2 to 2 . 2 4 µm. The mean total length of the haploid complement is 14.34 µm. The course of gametogenesis represents a typical pattern common in the order Spirurida. The recently available karyotypes of spirurid nematodes have been reviewed. KEY WORDS : Nematoda, Mastophorus muris, Spirurida, karyotype, chromosomes, Mus muscutus.
INTRODUCTION Mastophorus muris (Gmelin, 1979) is the cosmopolitan spirurid nematode parasitizing stomach of small rodents, often in associations with synantropic mice populations (Yamaguti, 1961, G e n o v 1 9 8 4 ) . It w a s originally described as Ascaris muris Gmelin, 1790 and was later combined with the genus Mastophorus by Chitwood (1938). This species was well characterized b y its morphology and biology (Skryabin & Sobolev, 1963). Since its systematic status changed several times, the present paper adopts the classification of the Nematoda published by Anderson et al. (19741983). T h e only information on the chromosome number of M. muris was published by Walton ( 1 9 2 4 e x Walton, 1974), as the part of the tabular review: 2n = 9-10, X = 1. As n o other information has b e e n available so far, the aim of this work is to describe the chromosome com plement of both sexes and visible events of gametoge nesis of M. muris, and to compare available karyological data on other nematodes of the order Spirurida.
* Parasitological Institute SAS, Hlinkova 3, 04001 Košice, Slovak Republic. ** Laboratori de Parasitologia, Facultat de Farmacia, Universitat de Barcelona, Avda Diagonal s/n, 08028 Barcelona, Spain. Correspondence: Marta Špakulová. Tel.: +421 95 6334455 - Fax: +421 95 6331414. E-mail
[email protected] Parasite, 2000, 7, 173-177
Résumé :
ÉTUDE DU CARYOTYPE DU NEMATODE MASTOPHORUS MURIS (NEMATODA: SPIROCERCIDAE)
Le caryotype de Mastophorus muris (Gmelin, 1790) comprend quatre paires de courts chromosomes autosaumaux et deux chromosomes X chez la femelle, ou un X chez le mâle (2n = 8 + XX/XO). Toutes le paires de chromosomes possèdent à peu près la même morphologie, sans constriction primaire ou secondaire. L'analyse par C-banding n'a pas permis de révéler la présence de bande d'hétérochromatine. La longueur totale du chromosome est comprise entre 4,02 et 2,24 µm. La plus grande longueur totale du chromosome haploïde est de 14,34 µm. Le cycle de la gamétogenèse représente un modèle commun dans l'ordre des Spirurida. Le caryotype d'autres spirurid nématodes a été revu. MOTS CLÉS : Nematoda, Mastophorus muris, Spirurida, caryotype, chromosomes, Mus musculus.
MATERIALS A N D M E T H O D S
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e m a t o d e s M. muris w e r e recovered from sto machs o f t w o m i c e Mus musculus L. collected in the farm Les Franqueses near Granollers (Spain) in July 15, 1998. Immediately after dissection, six males a n d nine females w e r e treated with colchi cine (O,5mg.ml ) in physiological saline for 30 min at 37° C. T h e n the w o r m s w e r e subjected to a hypotonic treatment (distilled water, 60 min), processed according to the air-drying m e t h o d and stained by conventional Giemsa and C-banding methods as described by Spa k u l o v á et al. ( 1 9 9 4 ) . Karyological analysis o f mitotic cells w a s carried out according to photographs o f 30 well-spread spermatogonial plates and 24 oogonial plates. Metric characteristics ( a b s o l u t e a n d relative lengths) w e r e calculated from the measuring o f the 10 best m e t a p h a s e spreads. -1
RESULTS
A
c h r o m o s o m a l c o m p l e m e n t consisted o f nine elements in males (Fig. 1A-C) and 10 in females (Fig. 2A, B ) . T h e s e x w a s determined by t w o X X c h r o m o s o m e s in female a n d o n e X c h r o m o s o m e in male (2n = 8 + XX/XO). T h e s e x c h r o m o s o m e X was the largest o n e out o f all and measured 4.02 ± 0.92 µm. T h e absolute lengths o f autosomal pairs w e r e 2.98 ±
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ŠPAKULOVÁ M.*, CASANOVA J.C.**, LAPLANA GUILLÉN N.** & KRÁL'OVÁ I.*
Fig. 1 - Chromosomes of Mastophorus muris male. A-C, three spermatogonial cells in a mitotic division. A, prometaphase, C-banding. B, C, metaphase. D, primary spermatocyte in the first meiotic metaphase. Complement comprising four bivalents and one univalent sex chromosome X. E, primary spermatocyte in the first meiotic anaphase. Univalent sex X chromosome located between two hapliod sets of autosome chromosomes. F, two secondary spermatocytes comprising haploid sets of four (n = 4, down) and five (n = 4 + X, upward) chromosomes. Sex chromosome X arrowed. B-F Giemsa staining. Scale bar 10 µm.
Fig. 2 - Chromosomes of Mastophorus muris female. A-B, two oogonial cells in mitotic prometaphase (A) and metaphase (B). C, oocyte in meiotic metaphase II. Chromosomes X arrowed. A-C Giemdsa staining. Scale bar 10 µm. 174
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A KARYOLOGICAL STUDY OF MASTOPHORUS
0.55 µm, 2.65 ± 0.45 pm, 2.45 ± 0.49 µm and 2.24 ± 0.43 µm. A gross c h r o m o s o m e morphology was better remarkable in the early metaphase cells (Figs 1A, B, 2A) then in the late metaphase cells (Figs 1C, 2 B ) . All pairs o f c h r o m o s o m e s possessed rather uniform morphology without distinct primary or secondary constrictions and were classified according to their length. C-banding ana lysis revealed n o heterochromatin bands (Fig. 2A). A n u m b e r o f spermatocytes (Fig. 1D-F) w e r e found in males being in various phases o f meiotic division, predominantly in the metaphases I and II. In females, o o c y t e s in the m e t a p h a s e II w e r e rarely o b s e r v e d containing five bar-form c h r o m o s o m e s (Fig. 2C) T h e subsequent stages of cleavage w e r e not observed.
DISCUSSION
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alton ( 1 9 5 9 ) s h o w e d the haploid c h r o m o some number o f M. muris to b e n = 5. Later, he specified the data m o r e accurately (2n = 9-10, X = 1) and published the figure o f t w o s e c o n dary spermatocytes with haploid sets o f four and five c h r o m o s o m e s (Walton, 1 9 7 4 ) . No information on the gross m o r p h o l o g y o f mitotic male and female c o m plements has b e e n available s o far. Present results correspond well with the a b o v e data o n the sex-determining m e c h a n i s m X X in females and X 0 in males. This fact has b e e n proved n o w b y the evaluation o f gametogonial mitotic divisions in both s e x e s . Dividing oogonial cells comprised 10 c h r o m o s o m e s while spermatogonial cells nine c h r o m o s o m e s . T h e h e t e r o c h r o m o s o m e X was the largest o f all and measured up to 4.02 pm. In our study, mitotic c h r o m o s o m e s neither s h o w e d clear primary constrictions in m e t a p h a s e nor the pericentromeric heterochromatin. T h e similar rod-shaped mitotic c h r o m o s o m e s were found in many other nema tode species belonging to the subclass Secernentea. T h e holokinetic c h r o m o s o m e structure w a s anticipated in the majority o f them (e.g. Walton, 1974; Triantaphyllou, 1983; Král, 1 9 9 4 ) . However, diffuse or polycentric c e n t r o m e r e w a s proved in only several n e m a tode species using ultrastructural, immunocytochemical and other special m e t h o d s studying k i n e t o c h o r e struc ture (for review s e e S p a k u l o v á et al, 1 9 9 5 ) . Within the order Spirurida, mitotic chromosomes showing n o clear secondary constrictions have been reported in several species: Brugia pahangi and B. malayi(Sakaguchi et al., 1983), Dirofilaria immitis (Sakaguchi et al., 1980), Gongylonema pulchrum (Valero et al., 1982), Physaloptera clausa (Mutafova, 1995), Onchocerca volvulus and O. gutturosa (Hirai et al., 1985, 1987; Procunier & Hirai, 1986) and Thelazia callipaeda (Sakaguchi & Kihara, 1984). However, this feature was interpreted in the different ways. S o m e authors did not c o m m e n t the chromosomal Parasite, 2000, 7, 173-177
MURIS
structure at all (Valero et. al., 1982; Hirai et al., 1985). In s o m e other cases, the monocentric character of chro m o s o m e s was supposed and the location of centromere was inferred from the formation of bivalents during meiotic prophase (Sakaguchi et al., 1980; Sakaguchi & Kihara, 1984; Procunier & Hirai, 1986). Finally, Goswami (1974), Podgornova et al. ( 1 9 7 9 ) and Sakaguchi et al. ( 1 9 8 3 ) discussed the possibility of the holokinetic cha racter of c h r o m o s o m e s in the members of the genera Camallanus, Setaria, Cystidicola and Brugia. According to the M. muris kayotype, the peculiarities of mitotic and meiotic chromosomes determined by a light microscopy enable us to anticipate their hypothetic holokinetic struc ture. Thus, the majority of mitotic chromosomes have a rod-shaped structure without a clear location of centro mere. During meiotic divisions, bivalents behave as telocentrics. Similar features were demonstrated also in a b o v e m e n t i o n e d spirurid s p e c i e s and s o m e other oxyurid nematodes (e.g. Mutafova & Vasilev, 1987; Spa kulovâ et al, 1994; Mutafova, 1995). However, further study is required for the decision on the character of attaching of mitotic c h r o m o s o m e s to the spindle fibers. Recently available karyological data on the spirurid n e m a t o d e species (Table I), s h o w e d that their karyo types are c o m p o s e d predominantly of 10 (sixteen spe cies) or 12 c h r o m o s o m e s (thirteen s p e c i e s ) . According to Triantaphyllou ( 1 9 8 3 ) , the s a m e numbers o f ele ments characterised also most species o f Strongylida and Oxyurida, w h e r e a s the diploid n u m b e r 2n = 12 and XX/XO mechanism of sex determination is the most widespread a m o n g all nematodes. There is a certain consistency of the type of the chro mosomal sex determination in spirurid species compri sing various genera. T h e heterogametic male configura tion of sex pair occurring in spirurids is either X 0 or X Y , and the former type was considered to b e evolutionary older (Triantaphyllou, 1983). For instance, males of phylogenetically older genera of the family Onchocercidae (including Litosomoides) have X O sex pair while more advanced genera have X Y (e.g. Brugia and Onchocerca, Table I). Phylogenetic relationships of these spirurid worms have b e e n assessed according to rRNA sequence comparison (Gill et al., 1988). Concerning higher taxonomic categories, both X 0 and X Y configurations has been found within the phylogenetically older families (Thelasiidae and Gongylonematidae) as well as within the younger ones (Onchocercidae) (see Table I). The elu cidation of the role of Y chromosome in the sex deter mination in nematodes has recently b e e n started by the identification of the T O Y locus specifically associated with the Y chromosome of Brugia malayi (Underwood & Bianco, 1999). Unfortunately, karyological data of the spi rurid nematodes are still rather scarce and insufficient for assessing of pathways of karyotype evolution within this systematic group or the phylogenetic relations in general.
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ŠPAKULOVÁ M.*, CASANOVA J.C.**, LAPLANA GUILLÉN N.** & KRÁL'OVÁ I.*
Superfamily, Family Camallanoidea, Camallanidae Gnathostomatoidea, Gnathostomatidae Physalopteroidea, Physalopteroidae Thelazioidea, Thelaziidae Spiruroidea, Gongylonematidae Spiniridae Spirocercidae Habronematoidea, Cystidicolidae
Acuarioidea, Acuriidae Filarioidea, Onchocercidae
Species Camallanus baylisi Camallanus lacustris Ancyracanthus cistidicola
Chi omosome No. + !>ex pair ?/
