International Journal for Parasitology 35 (2005) 1445–1451 www.elsevier.com/locate/ijpara
Synaptonemal complex karyotype of Eimeria tenella Emilio del Cachoa,*, Marc Pagesb, Margarita Gallegoa, Luis Monteagudoc, Caridad Sa´nchez-Acedoa a
Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Spain b HIPRA Laboratories, Gerona, Spain c Department of Anatomy, Embriology and Genetics, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Spain Received 8 April 2005; received in revised form 6 June 2005; accepted 16 June 2005
Abstract In most organisms, biological variability rests on the behaviour of the chromosomes in the meiotic context. Despite the importance of meiosis, very little is known about the meiotic behaviour of the Eimeria chromosomes. The aim of the present study is to describe the standard synaptonemal complex karyotype from Eimeria tenella oocyst spreads by electron microscopy. For that purpose, complete sets of pachytene synaptonemal complexes were obtained and the morphological pachytene karyotype was determined. The authors used a previously reported method that overcomes the difficulty of the extreme resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The chromosomes were selected under a light microscope and those selected were stained with phosphotungtic acid and studied by transmission electron microscopy. The authors confirmed 14 chromosomes, which were observed as synaptonemal complexes, and the karyotype was constructed by arranging synaptonemal complexes according to their relative lengths and kinetochore position. Components of the synaptonemal complex, lateral elements, central element, recombination nodules and kinetochore were observed. Measures of the kynetochore, width of the synaptonemal complex, diameter of the recombination nodule and length of the telomeres are given. Minimal and no significant differences were found between measures of chromosomes isolated from different Eimeria tenella strains. To the best of our knowledge, the present investigation for the first time identifies and describes the morphological characteristics of the synaptonemal complex of Eimeria tenella during the meiosis that occurs within the oocysts. In addition, the authors provide evidence of the presence of recombination nodules, suggesting that the recombination process may play an important role in the molecular evolution of this parasite. q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Eimeria tenella; Synaptonemal complex; Meiosis; Karyotype
1. Introduction The introduction of an air-dry method for the preparation of mammalian meiotic chromosomes (Evans et al., 1964) has allowed significant progress in our understanding of how meiotic chromosomes behave. Using this method, the synaptonemal complex (SC) has been studied and its structure and features extensively described. These studies
* Corresponding author. Address: Parasitologia y Enfermedades Parasitarias, Facultad de Veterinaria, Miguel Servet, 177, 50013-Zaragoza, Spain. Tel.: C34 976 76 15 56; fax: C34 976 76 16 12. E-mail address:
[email protected] (E. del Cacho).
have contributed to our knowledge of the biological variability that occurs as a consequence of the genetic recombination, which takes place during meiosis in animals and plants (Moses, 1977; Gillies, 1981; Kaelbling and Fechheimer, 1983). The SC, a proteinaceous structure that appears during the meiotic prophase, holds the homologous chromosomes together along their entire lengths, stabilises their pairing and provides for genetic recombination (Roeder, 1990). It is composed of two lateral elements, each of which connects a pair of replicated sister chromatids, together with a central element that joins the two. The structural components of the SC have remainded highly conserved throughout
0020-7519/$30.00 q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2005.06.009
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the evolution of protozoa, fungi, plants, insects and mammals (Westergaard and Wettstein, 1972), which is compelling evidence of the significant role they play in the meiotic process. Studies on Eimeria meiotic chromosomes are limited, since meiotic division occurs exclusively in oocysts within the first 9 h of sporulation (Canning and Anwar, 1968), as a consequence of which all the other stages contain a haploid number of chromosomes (Shirley and Harvey, 1996). Oocysts are extremely resistant to disruption by chemicals, enzymatic digestion, detergents or hypotonic solution. In addition, breakage of the walloocyst by sonication or vortexing with glass beads, because used for molecular extraction of DNA, does not permit visualization of meiotic figures, because these methods lead to the dispersion of the contents of the oocysts. The difficulty for the cytological observation of the Eimeria meiotic chromosomes has resulted in a scarcity of morphological data on Eimeria chromosomes. This lack of knowledge of the Eimeria chromosomes is in contrast to the importance of Eimeria tenella as a causative agent of cecal coccidiosis, a parasitic disease which in turn, results in significant economic loss to the poultry industry. Del Cacho et al. (2001) have reported a modified version of the air-dry technique, which permits visualisation of the SC by TEM when applied to eimerian oocysts in pachytene. Observation by TEM of SCs in whole-mount spreads is useful in the construction of the karyotype of species in which the length of the chromosomes is difficult to differentiate by light microscopy, as is the case with Eimeria spp. (Canning and Anwar, 1968; Del Cacho et al., 2001) The E. tenella karyotype has been controversial ever since Canning and Anwar (1968) used light microscopy to describe the formation of five chromosomes in the haploid form of E. tenella (10 chromosomes during meiosis in E. tenella). Subsequent, molecular genetic studies by Shirley et al. (1990) and Fernando and Pasternak (1991) failed to determine the exact number of chromosomes, due to the fact that these techniques were not sufficiently refined so as to be able to separate chromosomes of similar size from each other. More recently, and using different electrophoretic conditions, Shirley (1994) stated that the E. tenella karyotype consisted of 14 chromosomes ranging in size between 1 and more than 7 Mbp of DNA. To date, however, no further information has become available in the literature on the morphological identification of each individual E. tenella chromosome. Against this background, the aim of the present study was to describe the standard karyotype of E. tenella by TEM in whole-mount spread of the contents of the oocysts, with the ultimate objective of extending molecular genetic studies to morphological SC investigation.
2. Materials and methods 2.1. Animals Four-week old White Leghorn chickens were used. Chicken eggs were obtained from Doux Iberica, Zaragoza, Spain, and incubated in an automatically rotating incubator at 37 8C. The chickens were hatched and reared coccidia-free under routine laboratory conditions with free access to feed and water. No anticoccidial or antibiotic compounds were added to the food or water. All experiments were performed in accordance with the guidelines appoved by the Animal Ethics Committee of our institution. 2.2. Parasite Four E. tenella strains were used to infect the chickens: (i) the Houghton Laboratory strain, kindly provided by Dr R. Marshall; (ii) an E. tenella strain, obtained from Merck, Sharp and Dohme (Madrid, Spain); (iii) the Beltsville Laboratory strain, kindly provided by Dr H. Lillehoj; and (iv) an E. tenella strain, obtained from litter samples collected from broiler grower houses located in Zaragoza (Spain). 2.3. Experimental design A total of 40 chickens were divided into four groups, consisting of 10 birds each. All chickens in each group received a single dose of 10,000 sporulated oocysts (stored at 4 8C for less than 4 weeks) from one of the four strains mentioned above. Oocysts, isolated from the ceca at 7 days p.i., were allowed for initiation of sporulation under the experimental conditions reported by Raether et al. (1995). Five!106 oocysts of each strain were obtained at 4, 6 and 8 h after the start of the sporulation. These three time points were selected since in E. tenella meiotic events take place between 3 and 9 h after the start of the sporulation (Canning and Anwar, 1968). In addition, in previous reports on E. tenella, the authors had observed synaptonemal complexes at 4 h after the start of the sporulation (Del Cacho et al., 2001). 2.4. Transmission electron microscopy Sporulating oocysts obtained at 4, 6 and 8 h following the start of sporulation were treated according to the method described by Del Cacho et al. (2001) in order to permit isolation of chromosomes and their subsequent study by TEM. Briefly, oocysts were incubated in an ethanol:HCl solution for 15 min in order to partially digest and, thereby, lower the resistance of the oocyst-wall. After washing in PBS (pH 7.4), oocysts were re-suspended in PBS at a concentration of 2!105 oocysts/ml. Six to eight drops of the oocyst suspension were placed on a coat slide
E. del Cacho et al. / International Journal for Parasitology 35 (2005) 1445–1451
with colodion in amyl acetate. The slides were then dried at 37 8C, frozen at K20 8C for 30 min and subsequently thawed. The process of freezing and thawing was repeated three times to disrupt the oocyst-wall and permit the release and spread of intact meiotic chromosomes. Chromosomes were then fixed in paraformaldehide and stained with acetic orcein, in order to make a selection under the light microscope. Selected areas showing a high density of chromosomes were marked and then placed on a grid. They were subsequently stained for 3 min with phosphotungstic acid (PTA) following the method described by Switonski et al. (1987). Finally, chromosomes were observed by TEM, JEM 100C (Jeol). 2.5. Measurements Total length of SCs, length of both arms and telomeres, diameter of recombination nodules and width of the SCs were accurately measured on electron micrographs using the trace facility of an image analyser (IM50 Leica). The relative length of each SC was calculated as the total SC length divided by the sum of SC lengths. The centromere index was calculated as the length of the short arm!100, divided by total SC length. Measurements were calculated from 10 complete SC karyotypes for each of the four E. tenella strains used in the study. Images were processed with Adobe Photoshop 6.0 software on a Powerbook Macintosh G4.
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3. Results At 4-6 h after the start of sporulation, complete sets of 14 pachytene SCs were clearly identified in surface spreads of meiotic chromosomes from E. tenella oocysts (Fig. 1). The SC is selectively stained because of its protein nature and is visible along its entire length, being distinct from the surrounding unstained chromatin (Fig. 1). In a pair of homologous chromosomes the axes adhere to each other to become the lateral elements of the SC. Thus, SC, which had a constant width of 42 nm, were observed as two parallel electrondense lines, that is to say, the lateral elements (LE) (Figs. 1 and 2A). Twists of SC were commonly seen (Figs. 1 and 2B). SC formation was complete in all bivalents and along their total length. The central element (CE) was seen as a fine dense line between the LEs (Fig. 2A). Each LE ended in a densely stained thickening, the telomere (Fig. 1), whose length (32 nm) was constant in both short or long chromosomes. Recombination nodules with a diameter of 25 nm appeared as dense round structures, associated with the central region of the SC (Fig. 2A). Recombination nodules were more frequently observed in the short chromosomes. The centromere position along each SC was identified (Fig. 1). The centromeres were characterised by their kinetochores. The two homologous kynetochores of a bivalent were marked by short, densely stained prominences in the lateral elements
2.6. Statistical analyses Differences between means for multiple comparison purposes were assessed by Duncan’s New Multiple Range Test, with statistical significance inferred at P!0.05.
Fig. 1. Complete set of Eimeria tenella (Houghton strain) bivalents represented by 14 synaptonemal complexes from pachytene oocyst spread obtained 4 h after the start of sporulation. Kinetochores (arrows) are visible on each synaptonemal complex. Telomeres (arrowheads). Most of the bivalents contain twists in one arm.
Fig. 2. (A) Electron micrograph of a typical synaptonemal complex (Zaragoza strain), showing the lateral (arrowheads) and central (thin arrow) elements of the complex and including two recombination nodules (thick arrows). Pachytene oocyst spread obtained 4 h after the start of sporulation. (B) Electron micrograph of two tightly synapsed Eimeria tenella (Merck strain) homologous chromosomes containing the kinetochores (arrow). Note the presence of twists (arrowheads). Pachytene oocyst spread obtained 6 h after the start of sporulation.
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and were visible on each SC (Figs. 1 and 2B). Kynetochores were elongated in shape, having 62 nm in the longest diameter and 47 nm in the shortest one. Kynetochores were used to identify SC in surface spreads. Observation of kynetochores made it possible to determine the pachytene karyotype (Fig. 3). The criteria used for SC classification were relative length of the SC and centromere index (CI) (Table 1). The SCs were ranked by relative length, with No.1 having the largest relative length and No.14 the shortest. The average centromere indexes of the SC are given in Table 1 for each of the strains used. Bivalents Nos. 1, 2, 4, 5, 10 and 11 were sub-metacentric; Nos. 3, 6 and 12 were metacentric; Nos. 7 and 14 were acrocentric; and Nos. 8, 9 and 13 were near-acrocentric (Fig. 3). Analyses of variance between SCs revealed that there was no significant difference (PO0.05) in the length of a specific SC, neither between strains nor between spreads from the same strain (Table 1). Desynapsis of the paired homologous chromosomes was seen at 8 h. At that time, the SCs were partially dissolved in specific regions whereas, in others, persistent segments of SC held the homologs together (Fig. 4). That is to say, separation of the homologous chromosomes was incomplete, as the two homologs were in contact with each other at sites where persistent segments of SC were seen.
a constant and distinctive characteristic for each SC of E. tenella, despite variations in absolute length from oocyst to oocyst. The centromere index is distinctive for each SC and, together with relative length, serves to identify individual chromosomes. Shirley (2000) reported the existence of chromosome-size polymorphisms between E. tenella strains by means of Pulsed Field Gel Electrophoresis. However, our transmission electron microscopic study did not support his observation. An explanation for this finding may lie in the fact that, during meiosis, chromatin is highly spiralised when visualised under the microscope. As a consequence, length measurements on electron micrographs may be unable to detect slight molecular weight differences. The high degree of reproducibility of the relative SC lengths, which was observed among the 40 spreads measured, implies both biological stability and lack of physical distortion. The finding of this high degree of reproducibility seems to be in apparent contradiction to what might have been expected from a method that involves cell disruption, spreading by surface tension and flattening by air-drying. However, numerous reports have demonstrated the high reproducibility of results (Switonski et al., 1987; Westergaard and Wettstein, 1972). Indeed, the only morphological alteration observed by the authors in the chromosomes was twist. Twisting of the SC along the core of the bivalent may occur naturally, or it may be a consequence of the spreading method (Moses, 1977).
4. Discussion Using previously described methods (Del Cacho et al., 2001), the complete SC karyotype of E. tenella on surface spreads of oocysts was visualized. SCs were specifically recognised during E. tenella meiosis, which indicates that the E. tenella chromosomes condense in a similar manner to that of the metazoan chromosomes. The number of SCs observed has allowed us to identify 14 chromosomes in the E. tenella karyotype. Thus, we confirm the number of chromosomes reported by Shirley (1994), who defined the molecular karyotype of E. tenella by applying molecular genetic techniques. In addition, the present results offered very detailed information on the morphological characteristics of the E. tenella chromosomes during meiosis. The decisive factor for the observation of the SC was the use of phosphotungstic acid to stain the surface spreads of the oocyst content. The advantage of this over other staining methods is that it permitted the labelling of kinetochores as centromere-associated structures. In addition, by knowing the position of the kinetochore, the centromere index for each chromosome could be calculated. We found no significant differences between the relative length and the centromere index of a specific SC, neither between E. tenella strains nor between different spreads from the same strain. Relative length is
Fig. 3. Complete Eimeria tenella (Houghton strain) synaptonemal complex karyotype from a pachytene oocyst spread obtained 4–6 h after the start of sporulation. The synaptonemal complexes are arranged according to their relative lengths.
Table 1 Mean values of absolute and relative measurements for the synaptonemal complexes Chromosome#
1
2
Eimeria tenella Beltsville strain SC length 1.78 1.63 (mm) SD 0.04 0.06 12.6 11.6 Relative length (%) Centro17.3 36.8 mere index Eimeria tenella Zaragoza strain SC length 1.82 1.70 (mm) SD 0.06 0.06 Relative 12.3 11.5 length (%) Centro16.9 37.1 mere index
4
5
6
7
8
9
10
11
12
13
14
1.47
1.32
1.25
1.14
0.72
0.69
0.68
0.66
0.65
0.63
0.56
0.43
0.07 10.8
0.04 9.7
0.06 9.2
0.05 8.4
0.06 5.3
0.04 5.1
0.03 5
0.05 4.8
0.06 4.8
0.03 4.6
0.05 4.1
0.05 3.1
50
15.9
24.2
50
0
13.1
15.2
26.2
30
45.4
15.7
0
1.40
1.32
1.24
1.18
0.75
0.68
0.68
0.65
0.65
0.63
0.53
0.44
0.05 10.4
0.06 9.8
0.06 9.2
0.05 8.7
0.05 5.5
0.04 5
0.05 5
0.07 4.8
0.06 4.7
0.05 4.6
0.06 3.9
0.05 3.2
49.6
14.3
26.2
50
0
13.5
15.8
27.4
29.8
46.1
15.2
0
1.49
1.37
1.28
1.23
0.80
0.72
0.70
0.68
0.68
0.65
0.57
0.45
0.05 10.6
0.05 9.7
0.03 9.1
0.06 8.7
0.05 5.6
0.02 5.1
0.04 4.9
0.04 4.8
0.05 4.8
0.05 4.6
0.07 4
0.03 3.2
50
15.1
26
49.7
0
12.9
15.6
26.9
31.5
46.3
15.5
0
1.53
1.43
1.34
1.29
0.87
0.78
0.74
0.72
0.70
0.68
0.63
0.50
0.08 10.3
0.04 9.7
0.05 9
0.04 8.7
0.06 5.9
0.05 5.2
0.06 5
0.08 4.8
0.08 4.7
0.07 4.6
0.07 4.2
0.05 3.3
49.7
15.5
25.7
49.2
0
13.3
16.2
27.3
32.3
45.9
15.8
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Eimeria tenella Houghton strain SC length 1.75 1.56 (mm) SD 0.05 0.03 Relative 12.9 11.5 length (%) Centro18 35.8 mere index Eimeria tenella Merck strain SC length 1.73 1.58 (mm) SD 0.06 0.03 Relative 12.8 11.7 length (%) Centro17.5 37.2 mere index
3
0
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Fig. 4. Proteinaceus axes of the paired chromosomes separate (arrows), marking the end of the synapsis. In places the axes are held together by persistent distal segments (arrowheads) of SC. Oocyst spread (Beltsville strain) obtained 8 h after the start of sporulation.
shed light on the behaviour of the chromosomes during E. tenella meiosis. In addition to these findings, our results show both SCs during pachytene between 4 and 6 h following the start of sporulation, as well as separation of the axes of the SC (dipolotene stage) at 8 h after the start of sporulation. These findings establish the period of time when the chromosome rearrangement and segregation can be studied in E. tenella. As such, they provide an important basis to develop new lines of research aimed at relating structural modifications of the chromosomes and changes in their number with specific biological characterisrics of E. tenella, such as drug resistance, precocious development and pathogenicity.
Acknowledgements
The twists observed in the present study did not induce any variability in the length of the SC. In the present study, the size and morphological characteristics of the telomeres, as well as those of the components of the SC, are described for the first time in the chromosomes of E. tenella. SC provides the structural framework necessary for recombination events. However, the active recombination process is thought to be mediated, instead, by the recombination nodules. Consequently, in the present case, the most interesting finding was the observation of the recombination nodules, which are the structures of the SC involved in specific recombination events which causes alteration in the relative arrangement of nucleotide sequences in chromosomes (Roeder, 1990). This has the effect of increasing the genetic variability in organisms that reproduce sexually. Despite the importance of this process, we remain remarkably ignorant of the mechanisms through which recombination takes place during meiosis in the apicomplexan protozoa. As Canning and Anwar (1968), and Canning and Morgan (1975) did not find evidence of DNA synthesis before the meiosis takes place in E. tenella, they hypothesised preliminary duplication of chromosomes did not occur. On the basis of these reports, Raikov (1982) suggested that in E. tenella a simple type of chromosome reduction occurs, which is completed in one nuclear division (one-step meiosis). In such a meiosis, crossing-over is absent, because crossover occurs only at the stage of four-strand bivalents, which is absent in one-step meiosis. However, studies on the genetic linkage map of E. tenella have indicated a high meiotic crossover activity in this protozoan parasite (Shirley and Harvey, 2000). In this regard, the recombination nodules observed in the present study may be the site of a large multienzyme recombination machine that brings local regions of DNA together across the SC, as Rahn and Solari (1986) have suggested occurs in other organisms. On the basis of the present results, further morphological studies are needed to
We wish to thank Dr R. Marshall (Veterinary Laboratories Agency, Surrey, England), Dr H. Lillehoj (Agricultural Research Service, Beltsville, USA), Dr C. Montes (Merck Sharp and Dohme, Madrid, Spain) for providing oocysts. This work was supported by the Research Council of Aragon, Spain (218-184).
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