| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Mycology, Moscow State University, Leninskye Gory, 119992 Moscow, Russia
Philippe Callac 1
INRA, MYCSA (Mycologie et sécurité des aliments), BP 81, 33883 Villenave d’Ornon cedex, France
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
In Agaricus bisporus all cytological studies performed until now concerned the pseudohomothallic and bisporic var. bisporus. In the past 12 y two tetrasporic varieties have been described, the heterothallic var. burnettii and the homothallic var. eurotetrasporus. Our aim was to compare the behavior of the nuclei in the vegetative and reproductive cells of the three varieties with light microscopy (Feulgen and DAPI staining) and transmission electron microscopy. Most of the vegetative cells contained 3–5 nuclei in the three varieties. Nuclear migrations through the septum were detected. In the basidia relative locations of nuclei and vacuoles, meiotic spindle alignments, relative content of nuclear DNA and synaptonemal complexes were measured or observed. From the observation of numerous asynchronous second division of meiosis within basidia of var. bisporus and var. burnettii a new hypothesis emerges to explain the nonrandom distribution of the four meiotic products in the two spores of the bisporic basidia. Karyogamy and meiosis similarly occurred in the three varieties. In the case of A. bisporus var. eurotetrasporus this implies that the reproductive mode is sexual and therefore homothallic in the strict sense. The three different types of life cycles are described.
Key words: Agaricus bisporus var. burnettii, Agaricus bisporus var. eurotetrasporus, cytology, homothallism, meiosis, nucleus
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) with multiple alleles (Imbernon et al 1995) at the MAT locus on chromosome I (Xu et al 1993). In this species three different varieties differ in their life cycles, spore sizes and average spore number per basidium (Callac et al 2003). Most of the wild populations and all the traditional cultivars belong to the variety bisporus, which has a predominantly pseudohomothallic (= "secondary homothallic") life cycle (Raper et al 1972). Most basidiospores are produced via an intramictic process by bisporic basidia and receive two nonsister postmeiotic nuclei carrying different mating type alleles, so they give rise to heterokaryotic fertile mycelia (n + n) and tend to maintain the parental genotype in the offspring (Pelham 1967, Summerbell et al 1989, Kerrigan et al 1993).
Callac et al (1993) described specimens of an isolated population of A. bisporus var. burnettii Kerrigan et Callac in the Sonoran Desert of California; the life cycle of these specimens is preponderantly heterothallic. Most basidiospores are produced via a heteromictic process by tetrasporic basidia and give rise to self-sterile homokaryotic mycelia (n). Plasmogamy between two sexually compatible homokaryons restores a fertile heterokaryon.
Callac et al (1998, 2003) described rare specimens of A. bisporus var. eurotetrasporus Callac et Guinberteau found in Europe, in which most of the basidia are tetrasporic, as they are in var. burnettii, but the life cycle is putatively homothallic (= "primary homothallic"). In this life cycle the fruiting body is haploid and produces homokaryotic spores that give rise to fertile homokaryotic mycelia producing similar sporophores. However in the strict sense homothallism is a condition in which sexual reproduction can occur through a homomictic process (Hawksworth et al 1995). Until karyogamy and meiosis are demonstrated in var. eurotetrasporus we cannot be sure it truly has a primary homothallic life cycle. Without meiosis an amictic process would occur.
Agaricus bisporus var. bisporus exhibits limited heterothallism in conjunction with a low percentage of tetrasporic basidia (1.3% on average), and A. bisporus var. burnettii exhibits limited pseudohomothallism in conjunction with a low percentage of bisporic basidia (Kerrigan et al 1994, Callac et al 1996). The life cycle of both varieties is therefore amphithallic (Lange 1952) (i.e. both heterothallic and pseudohomothallic life cycles operate). Amphithallism is not rare because of the approximately 500 species of holobasidiomycetes with lamellae only 9% are considered amphithallic (Lamoure 1989). This percentage probably is underestimated because it was based mainly on the observation of a high proportion of bisporic basidia, therefore among the amphithallic species those that do not exhibit such a proportion generally are not considered.
Although the three varieties are characterized by ITS polymorphisms (but on small samples; see Challen et al 2003, Callac et al 2003) their phylogenetic relationships are unresolved. In the two tetrasporic varieties the tetrasporic trait results from the presence of a dominant Bsn-t allele at the BSN locus, which determines the basidial spore number per basidium, and which is linked to MAT on Chromosome I (Imbernon et al 1996). In var. eurotetrasporus no determinant for the haploid fruiting ability is known, but recent analyses strongly suggest that such a presumed determinant, if one exists, would not be linked to MAT (Couture et al 2004).
In the case of A. bisporus all cytological studies preceded the discoveries of the tetrasporic varieties and therefore concerned only var. bisporus. Our aim is to compare the behavior of the nuclei in the vegetative and reproductive cells of the three varieties and more particularly to determine whether meiosis and sexual homothallism occur in var. eurotetrasporus. The comparative study of the behavior of the nuclei is an important step before further studies on the three varieties in view of better understanding the spore volume variations, the variation of the number of spores per basidia and the nonrandom distribution of the four meiotic products in the spores of the bisporic basidia.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). Bs 423 (ATCC: MYA-2386; INRA collection) was isolated by tissue culture from a French specimen of the homothallic var. eurotetrasporus (Callac et al 2003). Cultivation and fructification.— – Mycelia for light microscopy were grown on 2% malt extract agar, on cellophane, at 25 C for 5–7 d according to the growth rate of each strain. Fruiting tests were performed in plastic trays containing pasteurized compost that had been inoculated with mycelia grown on wheat grain. Fruiting was conducted at 15–17 C in a controlled environment. Fruit bodies were harvested well after veil rupture.
Light microscopy with Feulgen staining.— –
Sections of gill tissue 10–15 µm thick were stained and squash preparations were made for observations on meiosis and basidiospore formation. Mycelia and gill sections were fixed in modified Carnoy’s fluid (Evans 1959) for 10 min, then washed in 96% ethanol (2–3 min) and stored in 70% ethanol. Before hydrolysis the material was washed in distilled water and placed in 1N HCl at room temperature 1–2 min. Then it was hydrolyzed in 1N HCl at 60 C for 10 min and placed in cold 1N HCl to stop hydrolysis. Preparations were stained in Schiff’s reactive 3–3.5 h (sections) or 3.5–4 h (mycelium) at room temperature. The material was washed in three series of sulfurous water (5 min in each) and in distilled water (3–5 min). The sections and the mycelia were mounted in glycerol solution (pH 8.2). An Olympus BX41 microscope was used (100x magnification). Photos were shot with an Olympus C4040 zoom digital camera.
Light microscopy with DAPI staining.— –
The standard DAPI (4',6-Diamidino-2-phenylindole) technique (Ota et al 1998) was modified for the object. After Carnoy’s fixation the lamellae were dehydrated through an ethanol gradient, acetone and isobutanol and embedded into Paraplast Plus media (SIGMA). Sections of 10–15 µm were cut by MSE-London microtome then gently squashed to 4 µm and stained with DAPI solution (pH 6.9) after Paraplast removal. The sections and mycelia were mounted in glycerol solution (pH 8.2). An Axioplan microscope (100x magnification) was used for fluorescent microscopy. The emission wavelengths of the fluorescence filters was 390–400 nm. Photos were shot with a Photometric SenSys KAF0401 G2 digital camera and analyzed with PMIS and ScionImage software.
Transmission electron microscopy (TEM).— –
Pieces of gill tissue were fixed 4 h in 4% KMnO4 solution or 2 h in solution of 5% glutaraldehyde (Merck) buffered at pH 7.2 with 0.1 M sodium phosphate, 5% DMSO and 1 M sucrose at room temperature with one change of the solution after 1 h. Material was rinsed in the buffer and postfixed with 1% OsO4 for 1 h. It was washed in the buffer, dehydrated through an ethanol gradient and embedded into EPON (Ferak). Sections were cut with an LKB-8800 ultratome with glass knives, stained with aqueous uranyl acetate 30 min and poststained with lead citrate solution (Reynolds 1963). Sections were examined with a Jeol (JEM-100B) transmission microscope.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). It was noted that the wild strains JB 3 and Bs 423 were more similar to each other; 57% (±0,3) of their intercalary vegetative cells contained three or four nuclei, whereas 70% (±0,4) of vegetative cells of the cultivar U1 contained three or four nuclei.
|
), or number of nuclei for 10 µm hyphal length, were significantly higher in the apical zones of hyphae (<50 µm from apex) than in the subapical zone (next 50 µm hyphal section); t-values for U1, JB 3 and Bs 423 were respectively 2.71, 3.17 and 2.93 (t-test at the P < 0.05 level with 83, 67 and 34 df). Moreover in the apical zone the NCR of Bs 423 was significantly higher than the NCRs of U1 and JB 3 (t-values were respectively 2.09 and 2.50; t-test with 59 and 47 df; P < 0.05). In the subapical zone the NCR of Bs 423 was significantly higher than only the NCR of JB 3 (t = 2.31, 54 df, P < 0.05). In previous NCR comparisons between homokaryons and heterokaryons of var. bisporus the NCR of the homokaryons was higher than the NCR of the heterokaryons but only in the subapical zone (unpublished data).
|
), and recovers spherical shape afterward.
Nuclear behavior and basidium morphogenesis (Feulgen staining).— –
The majority of subhymenial cells contained two nuclei, and we have rarely observed one or three. Similar nuclear behavior was observed during the basidiogenesis for the three strains. So we divided the process into five stages: (i) two nuclei placed along the longitudinal axis of the basidium (before karyogamy); (ii) one nucleus (after karyogamy); (iii) two nuclei aligned at right angles to the longitudinal axis of the basidium (after the first division of meiosis); (iv) four nuclei (after the second division of meiosis); (v) nuclei in spores. The sizes of the nuclei at these stages were recorded (TABLE II).
|
). Probasidia were usually 4 µm wide and their two nuclei were situated at the center of the cell, on the longitudinal axis. Two vacuoles, one apical and one basal, were observed to occupy almost all the rest of the space within the basidium, but sometimes 2–3 vacuoles were found at the apex. The premeiotic nucleus, resulting from the fusion and therefore presumably diploid, enlarges to 4.5–5.5 µm diam. The basidium becomes clavate at this point. Basidia of JB 3 enlarge after nuclear fusion and becomes higher than the other cells of the hymenium, but in U1 and Bs 423 all the basidia remain tightly packed. The apical vacuole generally disappears and the diploid nucleus moves to the apex of basidium to undergo meiosis. After the first division occurs in the apex, we observed two nuclei located close to the cell walls, opposite each other. The size of these nuclei was similar to the size of the two initial haploid nuclei.
|
). Their frequencies were similar for the three strains: types a and d were rare (1–2%), while frequencies of types b, c, e and f were almost equal. Among the six types of spindle alignments, three (a, d, f) were observed for the first time, while b, c and e correspond respectively to the Evans types a, b and d2 (Evans 1959). In conclusion karyogamy and meiotic spindle alignments apparently occurred similarly in the three varieties.
|
DNA content of basidia and asynchronous second divisions of meiosis (DAPI staining).— –
At different stages of basidiogenesis (FIGS. 4–7) nuclear DNA content was measured in haploid and diploid nuclei. Although the modified DAPI technique and the sensitivity of microscope did not let us quantify exactly the DNA content, the method distinguished the diploid nuclei in A. bisporus even for nuclei having less than 1 µm diam. DNA fluorescence intensity in diploid nuclei was on average 1.22 (in Bs 423), 1.38 (in JB 3) and 1.24 (in U1) times higher than in haploid ones. In other respects we often observed asynchronous second divisions of meiosis (FIGS. 8–9) for U1 and JB 3 but not for Bs 423. In bisporic basidia the two nuclei of the early second division migrated in first into the sterigmata.
|
|
). Probasidia contained two nuclei at the center of the cell; both apical and basal vacuoles were observed. Karyogamy occurred, but nuclear fusion was not observed. The nucleolus diameter in diploid nucleus is larger than in haploid one (1.2 and 0.6–0.7 µm respectively). The fusion nucleus migrated to the apex of the basidium and underwent the first meiotic division. At that moment the basidium enlarged and became higher than the other hymenial cells. The pachytene nucleus contained synaptonemal complexes, or SC (FIGS. 11–12). SCs usually were straight but some looked loop-shaped, with one end inserted on the inner nuclear membrane. The widths of the SCs of U1, JB 3 and Bs 423 were respectively about 0.20 µm, 0.16 µm and 0.13 µm.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) who observed mainly three with Feulgen staining (as we did) and those of (Hou and Elliott 1978) who primarily observed mainly 5–8 with the HCl-Giemsa technique. The number of nuclei per cell in the homokaryotic strain Bs 423 does not sensibly differ from those of the heterokaryotic strains. Such an absence of difference between homokaryons and heterokaryons was reported in A. bisporus (Hou and Elliott 1978) and in A. arvensis (Sonnenberg and Fritsche 1989). In the three strains NCR is greater in the apical zone than it is the subapical zone probably because the nuclear divisions are more frequent in the apex. Of note, Bs 423 has a greater NCR value in the apical zone than the two other strains, indicating that its hyphae extension is not so intensive as its nuclear division; this could reflect the slower growth rate of the Bs 423 homokaryon. NCR is correlated with the growth rate of the mycelium, which generally is higher for heterokaryons than for homokaryons (Kamzolkina 1996).
The phenomenon of nuclear migration through the septal pore in mycelium is surprising because the nuclei have to pass through numerous septa in the hyphae. The dolipore apparatus includes parenthosomes with small perforations about 0.04–0.06 µm diam (Stepanova and Vasilyev 1994). Chang (1978) and (Raper 1972) reported that nuclear migration could occur in monosporous vegetative hyphae of Volvariella volvacea and in haploid homokaryotic mycelia of Schizophyllum commune. A study of enzymatic degradation of septa in hyphal wall preparations from the monokaryotic and dikaryotic mycelia of S. commune showed that the dolipore apparatus is not a permanent structure in basidiomycetes (Korhonen 1983). The dissolution of septa results in the removal of the dolipore swellings and parenthosomes during nuclear migration (Chang 1978, Korhonen 1983). The migrations we observed might occur in this way.
Our main objective was to detect whether karyogamy and meiosis occurs in the homothallic var. eurotetrasporus. With Feulgen staining karyogamy and meiosis were indubitably observed in numerous basidia of the strain of this variety as in the strains of the two other varieties. Karyogamy and meiosis also were observed with DAPI staining and the DNA fluorescence intensity, as expected, was greater in the diploid nuclei than in the haploid ones. Our TEM data on basidiogenesis globally are similar to those obtained with light microscopy, and synaptonemal complexes were observed in all three strains.
We conclude that, in the basidia of var. eurotetrasporus, karyogamy occurs between two presumably identical nuclei and meiosis occurs through a homomictic process. Therefore var. eurotetrasporus has a sexual reproductive mode that corresponds to the homothallic life cycle in the strict sense. In this variety cytological approach was necessary to demonstrate this process because allelic segregation cannot be detected among homokaryotic and fertile homogeneous offspring (Callac et al 2003). However allelic segregations and crossovers were observed among the homokaryotic offspring of the U1-2 x Bs 423 intervarietal hybrid (Couture 2004). This suggested that the genetic background of the meiosis was intact in the Bs 423 strain of var. eurotetrasporus or that the U1-2 pathways were dominant; with the present data we now can consider that the background of the meiosis is not only intact but also functional in the homothallic variety.
Until now only a spatial explanation had been proposed to explain the migration of nonsister postmeiotic nuclei in the spores of the bisporic basidia. For the nuclear behaviors and the meiotic spindle alignments, we have not observed any major spatial difference between the three varieties. This suggests that the process of the nonrandom distribution of the spores would be the same at least for the bisporic basidia of the two amphithallic varieties. However, in comparison with other basidiomycetes, nuclear behavior in A. bisporus is characterized by these four items:
investigated some Agaricales species and reported that the nuclei migrated from the apex to the center of the basidium after the first meiotic division and returned to the apex before the second meiotic division. The four postmeiotic nuclei migrate again to the center of the cell and form a stable group existing until nuclear migration to the spores occurs. The same phenomenon was observed by (Ross and Margalith 1987) in Coprinus bilanatus and by (Kamada and Tanabe 1995) in C. cinereus. In A. bisporus, in contrast, such migration was not observed neither by us nor by Evans (1959). , Stepanova and Vasilyev 1994, Kuhner 1977, Evans 1959). In bisporic basidia these four characteristics provide conditions in which the migration of the two nuclei of the early second division can migrate, in first, into the two sterigmata, as was observed by us. Although the migration, in second, of the two nuclei of the late second division into the two sterigmata was not observed directly, we suspect that this occurred because, in a general manner, we did not observe degenerating nuclei in basidia of A. bisporus. Further studies using technique where temporal events could be explored will be necessary to confirm definitively such a process, which represents a temporal alternative explanation of the nonrandom distribution of the four meiotic products in the two spores.
We observed that basidia enlarged much more in var. burnettii than in the other varieties after nuclear fusion. We cannot explain this phenomenon, but it is in agreement with our previous description of the tetrasporic varieties: Basidia are 28–35 µm long in var. burnettii and 18–27 µm long in var. eurotetrasporus (Callac et al 1993, 2003).
The three sexual reproductive modes known in the basidiomycetes (heteromixis, intramixis and homomixis) exist in A. bisporus. The corresponding life cycles (heterothallism, pseudohomothallism, homothallism) are represented (FIG. 13) by the nuclear behavior observed in the present study. It must not be forgotten that these life cycles may not individually characterize a strain (e.g. a strain of var. bisporus is preponderantly pseudohomothallic and partially heterothallic according to the type of considered spores). In certain cases the type of spore (heterokaryotic or homokaryotic) is not characteristic of a life cycle because certain homokaryotic spores can participate in a homothallic life cycle as well as in a heterothallic one.
|
) or can be performed easily in vitro (but only with a limited number of partners). We do not exclude that the mating type allele of var. eurotetrasporus could be characteristic, but we have no evidence for this. In absence of major biological or genetic divergence from the other varieties, the apparently intact and functional background of the meiosis reinforce the interest of using strains of var. eurotetrasporus for further genomic investigations in A. bisporus.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Corresponding author. E-mail: callac{at}bordeaux.inra.fr
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
———, Imbernon M, Kerrigan RW, Olivier JM. 1996. The two life cycles of Agaricus bisporus. In: Royse DJ, ed. Mushroom biology and mushroom product. University Park, Pennsylvania: Proc 2nd Int Conference 1996. p 57–66.
———, Hocquart S, Imbernon M, Desmerger C, Olivier JM. 1998. Bsn-t allele from French field strains of Agaricus bisporus. Appl Env Microbiol 64:2105–2110.
———, Jacobe de Haut I, Imbernon M, Guinberteau J, Theochari I. 2003. A novel homothallic variety of Agaricus bisporus comprises rare tetrasporic isolates from Europe. Mycologia 95:222–231.
Challen MP, Kerrigan RW, Callac P. 2003. A phylogenetic reconstruction and emendation of Agaricus section Duploannulatae. Mycologia 95:61–71.
Chang ST. 1978. Nuclear behavior utilizing light microscopy. In: Chang ST, Nayes WA., eds. The biology and cultivation of edible mushrooms. New York: Academic Press. p 35–51.
Couture C, Michel A, Imbernon M, Callac P. 2004. Inheritance of the haploid fruiting ability in Agaricus bisporus. Mush Sci 16:45–52.
Dunkan EJ, Galbraith MN. 1972. Postmeiotic events in the Homobasiodiomycetes. Trans Br Mycol Soc 58(3):387–392.
Evans HJ. 1959. Nuclear behaviour in the cultivated mushroom. Chromosoma 10:115–135.[CrossRef][Medline]
Hawksworth LD, Kirk PM, Sutton BC, Pegler DN. 1995. Ainsworth and Bisby’s dictionary of the fungi. 8th ed. Cambridge, UK: University Press.
Hou HH, Elliott TJ. 1978. Comparative cytology in the genus Agaricus. Mush Sci 10(1):51–62.
Imbernon M, Callac P, Granit S, Pirobe L. 1995. Allelic polymorphism at the mating type locus in Agaricus bisporus var. burnettii and confirmation of the dominance of its tetrasporic trait. Mush Sci 14(1):11–19.
———, ———, Gasqui P, Kerrigan RW, Velcko Jr AJ. 1996. BSN, the primary determinant of basidial spore number and reproductive mode in Agaricus bisporus, maps to chromosome I. Mycologia 88:749–761.[CrossRef]
Kamada T, Tanabe S. 1995. The role of the cytoskeleton in the movement and positioning of nuclei in Coprinus cinereus. Can J Bot 73(I):S364–S368.[CrossRef]
Kamzolkina OV. 1996. Cytological study of homokaryotic and heterokaryotic strains of Agaricus bisporus (J. Lange) Imbach. Microbiologia 2:228–234.
Kerrigan RW, Royer JC, Baller LM, Kohli Y, Horgen PA, Anderson JB. 1993. Meiotic behavior and linkage relationships in the secondarily homothallic fungus Agaricus bisporus. Genetics 133:225–236.[Abstract]
———, Imbernon M, Callac P, Billette C, Olivier JM. 1994. The heterothallic life cycle of Agaricus bisporus var. burnettii and the inheritance of its tetrasporic trait. Exp Mycol 18:193–210.
Korhonen K. 1983. Observations on nuclear migration and heterokaryotization in Armillaria. Cryptog Mycol 4:79–85.
Kuhner R. 1977. Variation of nuclear behaviour in the Homobasidiomycetes. Trans Br mycol Soc 68(1):1–16.
Lamoure D. 1989. Indices of useful in formations for incompatibility tests in basidiomycetes. V. Agaricales sensu lato. Cryptog Mycol 10:41–80.
Lange M. 1952. Species concepts in the genus Coprinus. Dansk Bot Ark 14:1–140.
Miller RE, Kananen DL. 1972. Bipolar sexuality in the mushroom. Mush Sci 13:713–718.
Ota Y, Fukuda K, Suzuki K. 1998. The nonheterothallic life cycle of Japanese Armillaria mellea. Mycologia 90:396–405.[CrossRef]
Pelham J. 1967. Techniques for mushroom genetics. Mush Sci 6:49–64.
Raper CA, Raper JR, Miller RE. 1972. Genetic analysis of the life cycle of Agaricus bisporus. Mycologia 64:1088–1117.[CrossRef]
Reynolds ES. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Biophys Biochem Cytol 17:208.
Ross IK, Margalith P. 1987. Nuclear behavior in the basidia of the secondarily homothallic Coprinus bilanatus. Mycologia 79:595–602.[CrossRef]
Saksena KN, Marino R, Haller MN, Lemke PA. 1976. Study on development of Agaricus bisporus by fluorescent microscopy and scanning electron microcopy. J Bact 126:417–428.
Sonnenberg SM, Fritsche G. 1989. Cytological observation in Agaricus arvensis. Mush Sci 12(1):101–107.
Stepanova AA, Vasilyev AE. 1994. Ultrastructural bases of mushroom morphogenesis. Ashghabat: Ylym Publishing House. 264 p.
Summerbell RC, Castle ZJ, Horgen PA, Anderson JB. 1989. Inheritance of restriction fragment length polymorphisms in Agaricus brunnescens. Genetics 123:293–300.
Xu J, Kerrigan RW, Horgen PA, Anderson JB. 1993. Localization of the mating-type gene in Agaricus bisporus. Appl Env Microbiol 59:3044–3049.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
