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Chromosomal principle of radiation-induced F1 sterility in Ephestia kuehniella (Lepidoptera: Pyralidae) Alena Tothová and František Marec

Abstract: A dose-response analysis of chromosomal aberrations was performed in male progeny of gamma-irradiated males in the flour moth, Ephestia kuehniella. For comparison, several female progeny from each dose level were examined. Aberrations were detected on microspread preparations of pachytene nuclei in the electron microscope and classified according to pairing configurations of synaptonemal complexes (SCs). Fragmentation and various translocations were the most numerous aberrations, whereas interstitial deletion and inversion were rare. At 100 Gy, relatively simple multiple translocations were found. Multiple translocations showing complicated configurations occurred at 150 and 200 Gy, and their number increased with the dose. In males, the mean number of chromosomal breaks resulting in aberrations linearly increased with the dose from 8.4 to 16.2 per nucleus. In females, this value achieved a maximum of 11.2 breaks/nucleus at 200 Gy. Three factors were suggested to contribute to the reported higher level of F1 sterility in males than females: (i) survival of males with high numbers of breaks, (ii) crossing-over in spermatogenesis but not in the achiasmatic oogenesis, and (iii) a higher impact of induced changes on the fertility of males than females. It was concluded that translocations are most responsible for the production of unbalanced gametes resulting in sterility of F1 moths. However, F1 sterility predicted according to the observed frequency of aberrations was much higher than the actual sterility reported earlier. This suggests a regulation factor which corrects the predicted unbalanced state towards balanced segregation of translocated chromosomes. Key words: Lepidoptera, Ephestia kuehniella, irradiation, inherited sterility, chromosomal aberrations, synaptonemal complexes. Résumé : Une analyse de la relation entre la dose d’irradiation gamma et les aberrations chromosomiques a été réalisée sur la progéniture mâle d’individus mâles irradiés de la pyrale méditerranéenne de la farine, Ephestia kuehniella. Pour des fins de comparaison, plusieurs femelles de la progéniture ont également été examinées pour chaque dose. Des aberrations ont été décelées sur des étalements préparés à partir de noyaux en pachytène et observés en microscopie électronique. Ces anomalies ont été classifiées en fonction des configurations d’appariement des complexes synaptonémiques (SCs). De la fragmentation et diverses translocations constituaient les aberrations les plus fréquentes, tandis que les délétions ou inversions internes étaient rares. À une dose de 100 Gy, des translocations multiples relativement simples ont été observées. Des translocations multiples montrant des configurations complexes ont été produites à des doses de 150 et 200 Gy, et leur nombre s’est accru avec la dose. Chez les mâles, le nombre moyen de bris chromosomiques produisant des aberrations s’est accru de façon linéaire avec la dose, allant de 8,4 à 16,2 bris par noyau. Par contre, chez les femelles, ce nombre a plafonné à 11,2 bris par noyau à une dose de 200 Gy. Trois facteurs peuvent avoir contribué à la plus grande stérilité en F1 chez les mâles par rapport aux femelles : (i) la survie de mâles avec un grand nombre de bris, (ii) des enjambements lors de la spermatogenèse mais non lors d’une oogenèse achiasmatique, et (iii) un impact plus important des changements induits par irradiation chez les mâles que chez les femelles. Il en a été conclu que les translocations sont les principaux responsables de la production de gamètes déséquilibrés, laquelle entraîne la stérilité des pyrales mâles. Cependant, la stérilité des F1 prédite (en fonction de la fréquence observée des aberrations) était bien supérieure à celle observée dans les faits. Cela suggère l’existence d’un facteur de régulation qui corrige l’état prédit de déséquilibre à la faveur d’une ségrégation équilibrée des chromosomes ayant subi une translocation. Mots clés : Lepidoptera, Ephestia kuehniella, irradiation, stérilité héritée, aberrations chromosomiques, complexes synaptonémiques. [Traduit par la Rédaction]

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Received April 4, 2000. Accepted November 14, 2000. Published on the NRC Research Press Web site March 9, 2001. Corresponding Editor: R.J. Kemble. A. Tothová and F. Marec.1 Department of Genetics, Institute of Entomology, Czech Academy of Sciences, and Faculty of Biological Sciences, University of South Bohemia, Branišovská 31, CZ-370 05 eské Budjovice, Czech Republic. 1

Corresponding author at Department of Genetics, Institute of Entomology, Czech Academy of Sciences, Branišovská 31, CZ-370 05 eské Budjovice, Czech Republic (e-mail: [email protected]).

Genome 44: 172–184 (2001)

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DOI: 10.1139/gen-44-2-172

© 2001 NRC Canada


Tothová and Marec

Introduction Lepidoptera, which are generally considered to be radioresistant, require very high doses of ionizing radiation to be fully sterile (LaChance and Graham 1984). However, if they are exposed to partially sterilizing doses, their progenies (F1 generation) exhibit higher level of sterility than parents (North 1975; LaChance 1985). This phenomenon is termed inherited sterility or F1 sterility. In addition, some other effects arise after irradiation of parents with substerilizing doses. For example, females of the parental generation are more sensitive to irradiation (more sterile) than males. Next, the sex ratio in the F1 generation is heavily skewed toward males. Finally, the level of inherited sterility in F1 females is lower than in F1 males (Anisimov et al. 1989; Seth and Reynolds 1993; Marec et al. 1999). The F1 sterility phenomenon was first described in the silkworm, Bombyx mori L. (Astaurov and Frolova 1935), and later confirmed in the wax moth, Galleria mellonella L. (Ostriakova-Varshaver 1937). Proverbs (1962) rediscovered the F1 sterility in the codling moth, Cydia pomonella (L.), and proposed to use it for the genetic control of this pest. By means of a theoretical model Knipling (1970) showed the potential advantage of F1 sterility over the sterile insect technique (SIT) in pest control strategies against Lepidoptera. Since then the radiation-induced inherited sterility has been considered as the most advantageous genetic technique for the suppression of lepidopteran populations (Knipling and Klassen 1976; LaChance 1985; Mastro and Schwalbe 1988). The method has recently been investigated in many lepidopteran species of economic importance (e.g., Carpenter and Gross 1993; Seth and Reynolds 1993; Makee and Saour 1997; Bloem et al. 1999). The genetic principle of inherited sterility in Lepidoptera was reviewed by LaChance (1985) and discussed by Anisimov et al. (1989). It is generally thought that radiationinduced multiple translocations lead to the production of unbalanced gametes in the F1 generation and thus, cause sterility of their carriers. However, the actual chromosomal mechanisms of inherited sterility are poorly understood. The effects of irradiation on the incidence of visible chromosomal aberrations in Lepidoptera have only been shown in the light microscope (Saifutdinov 1989; Al-Taweel et al. 1990; Carpenter 1991; Carpenter et al. 1997) which, unfortunately, lacks the resolution to cope adequately with lepidopteran chromosomes. Chromosomes of Lepidoptera are very small, numerous (most species have a haploid number around 30; Suomalainen 1969), and uniform in shape during metaphase. They are usually spherical, lack distinct primary constrictions (centromeres), and sister chromatids separate by parallel disjunction at mitotic metaphase. Finally, chromosome fragments are regularly transmitted to next generations. Due to these characteristics, Lepidoptera had been regarded as species with holokinetic chromosomes, and their high radioresistance was attributed to the specific chromosome structure (Murakami and Imai 1974). However, recent findings favor a chromosome type intermediate between the holokinetic and monocentric chromosomes (Wolf 1996). Technical problems when studying lepidopteran chromosomes can be partially overcome when a modified micro-

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spreading technique is used (Weith and Traut 1980). The method permits visualization of the long synaptonemal complexes (SCs; Marec 1996) of spread pachytene nuclei at a high resolution in the electron microscope (EM). It had been used successfully in the analysis of structural mutants in the flour moth, Ephestia kuehniella Zeller (Traut et al. 1986; Marec and Traut 1993a, 1994). In the present study, we have employed the microspreading technique for a dose-response analysis of radiation-induced chromosomal aberrations in male progeny of irradiated males in E. kuehniella. For comparison, several female progeny from each dose were also examined. Results obtained were correlated with the level of F1 sterility for corresponding irradiation doses given in another recent study (Marec et al. 1999).

Materials and methods Insects We used a laboratory wild-type strain WT-C (for its origin, see Marec 1990) of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Insects were reared on milled wheat grains supplemented with a small amount of dried yeast. Experimental cultures were kept in a constant temperature (25 ± 1°C) room 12 h light : 12 h dark without humidity control. Details of rearing and handling methods are given in Marec (1990).

Irradiation The last instar larvae of the WT-C strain were manually separated according to their sex (see Marec 1990). During the pupal stage, males were removed from cocoons and prepared for irradiation, while the female progeny served us as a source of virgin females for crosses. Irradiation was performed at the Entomology Unit of the FAO/IAEA (Food and Agriculture Organization of the United Nations / International Atomic Energy Agency) Laboratories in Seibersdorf (Austria) using 60Co Gammacell 220 (Atomic Energy of Canada Ltd.) at dose rates ranging from 40.6 to 46.0 Gy/min. Late male pupae, 8–10 days old (i.e., black-eyed pupae to pharate pupae), were placed in plastic petri dishes (5 cm in diameter) and treated with gamma rays at doses of 100, 150, or 200 Gy (1 Gy ≈ 100 rad). In Lepidoptera, eupyrene spermiogenesis usually stops after pupation (Friedländer 1997), hence the mature sperm were the only germ line stage treated. Irradiated males were mated to virgin WT-C females and singlepair cultures were established. In the progeny (i.e., F1 generation), male and female larvae were randomly chosen for cytogenetic analysis of chromosomal aberrations inherited from treated male parents. Larvae of the WT-C strain served us as the untreated control.

Electron microscopy preparations of pachytene chromosomes Microspread preparations of late zygotene and pachytene nuclei were created from testes of fourth instar and from ovaries of fifth instar F1 larvae. The microspreading technique was performed essentially following the procedure described in Marec and Traut (1993b). In brief, gonads were dissected out in a saline solution, pretreated in a hypotonic solution (83 mM KCl and 17 mM NaCl) for 10–20 min and disrupted using fine tungsten needles. The swollen nuclei were transferred by micropipette to a spreading solution (0.02% Joy detergent, Procter and Gamble, U.S.A.), allowed to disperse for 6–10 min and centrifuged in a Teflon microcentrifugation chamber (2200 rpm, 4°C, 10 min) onto a carbon-coated, © 2001 NRC Canada



174 200 mesh grid through a 0.1 M sucrose cushion containing 1% formaldehyde. Then the specimen was fixed for 2 min with fresh formaldehyde–sucrose solution, rinsed for 30 s in 0.4% Kodak Foto Flo and air-dried. After routine inspection in a phase-contrast microscope, the specimen was stained for 30 s in 1% ethanolic phosphotungstic acid. Preparations were examined and micrographs taken in a Jeol 1010 transmission electron microscope operated at 80 kV.

Analysis of synaptonemal complexes In microspread preparations, the regular synaptonemal complex (SC) of a pachytene bivalent appears as two parallel lateral elements (LEs) of the same length. Each LE represents a proteinaceous axis of one homologous chromosome. In F1 individuals, a haploid set of LEs originated from untreated females and the other set from treated males. Thus, F1 individuals were heterozygotes for induced chromosome aberrations, and homologous pairing of the normal LE with the aberrant LE (or LEs) resulted in an abnormal configuration of the SC (or SCs). Only complete SC complements were analyzed for induced chromosomal aberrations on EM micrographs of pachytene oocytes and spermatocytes from the control and F1 individuals. In each complement, we recorded types of aberrations and their number, and then estimated the number of chromosomal breaks necessary for the rise of these aberrations. Intrachromosomal aberrations were expected to arise by one break (terminal deletion or fragmentation) or two breaks (interstitial deletion and inversion). In interchromosomal aberrations, we followed the classic hypothesis assuming that two breaks of double-stranded DNA are required for a chromosomal rearrangement involving two chromosomes, three breaks for that involving three chromosomes, etc. (Natarajan 1984; Savage 1993). We assumed that all nuclei of the same F1 larva carry identical aberrations and ignored a possible occurrence of mosaics as well as instability of aberrant chromosomes during formation of multivalent SCs. If a certain multivalent showed different configurations in different nuclei of the same F1 larva, it was classified and recorded according to the nucleus that showed the most compact configuration. Statistical analyses of data were performed with GraphPad PRISM software, version 3. We used the unpaired t-test with two-tailed P values to determine significant differences between the mean values of selected groups. Multiple statistical comparisons were performed using one-way analysis of variance (ANOVA) with the Tukey–Kramer post test at the 5% significance level. Regression analyses were performed to interpret dose-response relationships.

Results Classification of chromosome aberrations The pachytene complement in wild-type males and females of Ephestia consists of 30 bivalents, each forming the SC (Marec and Traut 1993b). In F1 progeny of irradiated males, all main types of chromosome aberrations could be identified according to specific configurations of SCs. A trivalent, consisting of an intact LE and two broken LEs, indicated simple fragmentation (Fig. 1a). Similarly, a bivalent with LEs of unequal lengths together with an unpaired fragment indicated fragmentation or, eventually, deletion when the fragment had been lost (Fig. 1b). Also a univalent plus two paired fragments indicated fragmentation of the original chromosome (Fig. 1c). All these alternative configurations had the same origin (caused by one break) and were classified as fragmentation. A trivalent, consisting of a very long LE and two LEs of normal lengths, was classified as fusion

Genome Vol. 44, 2001

(Fig. 2). A loop between otherwise synapsed LEs indicated inversion (Fig. 3a, 3b) or interstitial deletion (Fig. 3c). As it was difficult to recognize these two aberrations from each other, we interpreted clear loops as consequence of inversion (Fig. 3a) and cases when an additional ring fragment occurred in the SC complement as interstitial deletion (Fig. 3c). Also a bivalent with LEs of different lengths accompanied with a ring fragment was interpreted as interstitial deletion (Fig. 7a). A quadrivalent with LEs forming a closed ring (equivalent to pachytene cross figure) was typical for reciprocal translocation (Fig. 4). Conversely, simple (non-reciprocal) translocation resulted in quadrivalent or quinquivalent with LEs forming a chain (Fig. 5). Finally, a multivalent figure was interpreted as multiple translocation (MT) which was further classified according to the number of translocated chromosomes involved, e.g., MT(3), MT(4), … MT(10) (Fig. 6). A complete analysis of a pachytene nucleus is shown in Fig. 7a. Chromosomal aberrations in F1 larvae Only one male and one female from the control group (WT-C strain) were analyzed simultaneously with treated individuals because of time-consuming procedures and limited funds. However, we inspected preparations of 11 males and 20 females of the WT-C strain made for the study of Marec and Traut (1993b). In over 30 pachytene complements of the WT-C strain examined from each sex, no chromosomal aberrations were found. All homologs were well paired in bivalents. The only exception was frequent incomplete pairing of the sex-chromosome bivalent WZ in female pachytene nuclei. This is, however, a regular phenomenon that is due to the lack of homology between the W and Z chromosomes in Ephestia females (Marec and Traut 1994). A total of 43 F1 individuals (32 males and 11 females) with 174 complete late zygotene and pachytene nuclei were analyzed in the treated series. The F1 individuals showed a variety of radiation-induced chromosomal aberrations from simple fragmentation and deletion to extensive chromosome rearrangements such as multiple translocation (Table 1). No nucleus from F1 males and F1 females examined was without aberrations. In F1 males, fragmentation and various translocations were the most numerous aberrations observed, whereas interstitial deletion (except 150 Gy) and inversion were rare. At each dose, interchromosomal aberrations predominated over intrachomosomal aberrations (Table 1). At 100 Gy, relatively simple multiple translocations were found. Extensive multiple translocations showing complicated configurations occurred at two higher doses, and their number tended to increase with the increasing dose of irradiation. The overall frequencies of chromosomal aberrations did not show a clear dose response. At 100 and 150 Gy, the frequencies were almost identical (4.3 and 4.2 per male, respectively), but multiple translocations at the higher dose involved more chromosomes. This was the reason for estimating the number of induced chromosomal breaks as an alternative measure for radiation damage, discussed further below. A significant increase in the frequency was found only at 200 Gy (6.2/male). This increase was due to the increased fraction of interchromosomal aberrations. © 2001 NRC Canada



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Figs. 1–3. Pachytene configurations of chromosome aberrations in F1 progeny of irradiated Ephestia kuehniella males. Fig. 1. Alternative pairing of chromosome fragments: (a) a trivalent consisting of two broken fragments (F) paired with their normal homolog; (b) a bivalent with lateral elements of unequal lengths indicating terminal deletion (the deleted fragment not shown); (c) two nonhomologously paired fragments (FF) and one autosynapsed univalent (U). Fig. 2. A long trivalent formed by a chromosome fusion and two original homologs (1, 2). Fig. 3. Bivalents with a loop (arrowhead): (a) a typical inversion loop; (b) a loop interpreted as inversion, if a clear inversion loop was observed in another nucleus of the same specimen; (c) a loop formed by two lateral elements of different lengths indicating interstitial deletion; the deleted fragment forms a small ring (arrow). Scale bar = 2 µm.

Translocations in F1 males were classified by using two other criteria that may affect segregation of aberrant chromosomes and consequently, the level of inherited sterility in F1 generation (Table 2). First, we distinguished two basic configurations of translocations: those forming a chain (e.g., Fig. 5) and those forming a ring (e.g., Figs. 4 and 9a). Frequencies of chain figures considerably increased at 200 Gy and highly predominated over ring figures at this particular dose. However, frequencies of ring figures did not statistically differ among treated groups. Second, we distinguished translocations involving at least one chromosome that was broken two or more times (i.e., a chromosome with interstitial translocation or insertion; Fig. 8) from those that arose by one break per chromosome. The former type of

translocations was absent at 100 Gy, but its frequency increased with the increasing dose. In F1 females, similar types and frequencies of chromosomal aberrations were observed (Table 1). Though no conclusion can be drawn because of small sample sizes, the females examined carried less complicated chromosome rearrangements than F1 males and no F1 female carried multiple translocation with more than five chromosomes involved. In most preparations, we could analyze more pachytene nuclei from the same F1 larva. This enabled us to compare pairing configurations of identical aberrations in different nuclei. Some aberrations such as fusion, non-reciprocal and reciprocal translocation showed usually the same configuration in each nucleus. On the other hand, fragmentations reg© 2001 NRC Canada



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Figs. 4–6. Configurations of chromosome translocations in F1 progeny of irradiated Ephestia kuehniella males; numbers indicate structurally normal chromosomes inherited from the untreated mother. Fig. 4. A quadrivalent (1, 2) from a pachytene spermatocyte typical for reciprocal translocation; arrows indicate recombination nodules. Fig. 5. A quinquivalent (1, 2) consisting of five lateral elements typical for nonreciprocal translocation, and a quadrivalent (3, 4) that appears as nonreciprocal translocation from a pachytene spermatocyte. Fig. 6. Multiple translocation in a late zygotene spermatocyte involving 10 normal chromosomes (1–10). Scale bar = 2 µm.

ularly occurred in three alternative configurations (Fig. 1a–c). Multiple translocations frequently showed irregularities in chromosome pairing. Particularly in some male nuclei carrying

complicated figures, chromosomes did not pair completely and tended to create two or more simpler configurations instead of the complete multivalent. In another case, a ring © 2001 NRC Canada



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Fig. 7. Pachytene configurations of chromosome aberrations from one F1 male of Ephestia kuehniella after treatment with 150 Gy; numbers indicate structurally normal chromosomes inherited from the untreated mother. (a) A nucleus showing four chromosome aberrations that arose from a total of 12 breaks: a figure consisting of 13 LEs represents a chain multiple translocation MT(6) that involve six chromosomes (1–6) and was caused by six breaks; a quadrivalent (7, 8) appears here as nonreciprocal translocation that was caused by two breaks; a bivalent with looped lateral elements (9) was interpreted as inversion (caused by 2 breaks), because a typical inversion loop was found in other nucleus of the same specimen; a bivalent with lateral elements of different lengths (10) together with one ring fragment (arrowhead) indicates interstitial deletion (caused by two breaks). (b) A quadrivalent (7, 8) from another nucleus of the same specimen shows complete pairing in a pachytene cross typical for reciprocal translocation. Scale bar = 2 µm.

multiple translocation formed a chain in some nuclei (cf. configuration of chromosomes 7 and 8 in Fig. 7a, 7b). This phenomenon could be caused by lower pairing affinity of short translocated chromosome segments during SC formation or, at least in some cases, by mechanical disruption of pachytene figures during microspreading procedure. Different configurations of the same multiple translocation could significantly influence the probability of formation of balanced gametes (see below). Estimation of radiation-induced chromosome breaks From each F1 larva, one well-spread nucleus displaying all clearly visible aberrations was selected for counting chro-

mosome breaks that were needed to produce the aberrations seen. The mean number of breaks in F1 males increased with the dose (Table 3) and the increase fitted a linear regression curve (r2 = 0.996; P = 0.0019). However, the same value in F1 females did not show a clear dose response. Statistical comparisons of break frequencies revealed a highly significant difference between F1 males and F1 females at 200 Gy (P < 0.001; compared with the unpaired two tailed t-test). Effect of chromosomal aberrations on the frequency and location of recombination nodules Crossing-over between an aberrant chromosome and its normal homolog is, aside from pairing configuration, © 2001 NRC Canada



1 (0.25) 4 (0.80)

2 (0.50) 3 (0.60)

1 (0.50) 1 (0.25) 2 (0.40)

2 (1.00) 5 (1.25) 2 (0.40) 1 (0.50) 3 (0.75) 2 (0.40) 4 (10)

5 (20)

150

200

13 (44) 200

F1 females 100 2 (6)

10 (56) 150

1 (0.10)

1 (0.50)

2 (1.00) 6 (1.50) 11 (2.20)

10 (1.11) 5 (0.50) 19 (1.46) 5 (0.56) 4 (0.40) 4 (0.31) 12 (1.33) 11 (1.10) 17 (1.31) F1 males 100 9 (38)

3 (1.50) 2 (0.50) 5 (1.00) 1 (0.50) 5 (1.25) 9 (1.80)

1 (0.10) 2 (0.15) 1 (0.11) 6 (0.60) 1 (0.08) 11 (1.22) 4 (0.40) 16 (1.23)

ST INV

Total

6 (0.67) 9 (0.90) 14 (1.08)

FUS IDEL FRA

RT

Interchromosomal aberrations Intrachromosomal aberrations

F1 larvae analyzed n (t)

Note: F1 larvae analyzed: n, number of F1 individuals examined; t, total number of analyzed nuclei. Chromosomal aberrations: FRA, fragmentation of a chromosome to two segments (including terminal deletion); IDEL, interstitial deletion; INV, inversion; FUS, chromosome fusion; ST, simple translocation; RT, reciprocal translocation; MT(i), multiple translocation involving i bivalents. Mean aberration frequency: compared using one-way ANOVA with the Tukey–Kramer post test separately for each sex; the identical letter (a or b) indicates no statistical difference between groups.

5.4±1.3a

4.8±1.3a

4.5±0.7a

7 (3.50) 13 (3.25) 16 (3.20)

1 (0.08) 4 (0.44) 2 (0.10) 12 (0.92)

1 (0.11) 5 (0.50) 8 (0.62)

1 (0.11) 2 (0.20) 1 (0.08)

3 (0.30) 3 (0.23)

MT(6) MT(5) MT(4) MT(2)

MT(3)

6.2±1.3b

4.2±1.0a

4.3±1.0a

27 (3.00) 31 (3.10) 64 (4.92)

MT(10)

Total

Mean aberration frequency x ± SD


Dose (Gy)

Table 1. Chromosomal aberrations found in F1 larvae of Ephestia kuehniella after irradiation of male parents. Data indicate numbers and frequencies (in parentheses) of chromosomal aberrations.

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another factor that could affect the production of unbalanced gametes in F1 males but not in F1 females possessing the achiasmatic mode of meiosis (Traut 1977). To clarify the role of crossing-over we paid attention to the distribution and location of recombination nodules (RNs) that indicate positions of cross-over sites along the bivalent (Carpenter 1994) and also positions of chiasma formation (Holm and Rasmussen 1980). In E. kuehniella males, each bivalent is mostly associated with only one RN (Marec and Traut 1993b) and consequently, one chiasma per bivalent is usually seen (Traut 1977). However, pachytene configurations of translocations in F1 males frequently showed more than one RN per chromosome pair. For example, reciprocal translocations involving two original chromosome pairs possessed even four RNs, each located in one homologously paired terminal segment (Fig. 4). Similarly, many multiple translocations possessed one RN in almost every paired segment resulting, for example in MT(4), in seven RNs instead of four expected (Fig. 10). To support this finding we selected 17 nuclei of three F1 males, progeny of males irradiated with 150 Gy, and recorded the total number of RNs. The mean number of RNs in this sample was 32.5 ± 1.1 per nucleus. This value is significantly higher than 30.7 ± 2.2 RNs found by Marec and Traut (1993b) in 25 SC complements of the WT-C strain (P < 0.01; two tailed t-test). The above data indicate the increase of cross-over frequency in F1 males carrying radiation-induced chromosome rearrangements. Crossing-over in F1 males could indirectly influence segregation of aberrant chromosomes via chiasma formation, particularly in ring configurations (Table 2). Distribution of RNs and (or) crossing over in each arm of a ring pachytene figure (Fig. 4) would ensure that the figure was maintained by chiasmata through diplotene and diakinesis till metaphase I (see Fig. 9b). Segregation of chromosomes from such a ring figure might then increase probability of balanced gametes when compared with aberrations forming a chain (Goldman and Hueltén 1993a, 1993b; Tease 1998). Theoretically, crossing-over would increase the production of unbalanced gametes only at the exchange between an aberrant chromosome that arose by two (or more) breaks and its structurally normal homolog, if the cross-over took place within the two breakpoints. In F1 males, this could happen in two types of aberrations, inversions and multiple translocations involving a twice broken chromosome (e.g., cross-over between chromosomes T(1; 2; 3) and 2 in Fig. 8), but not in interstitial deletions. However, only one clear inversion was identified (see Table 1). This implies that crossing-over could contribute to the sterility of F1 males mainly via these complicated multiple translocations that occurred at two higher doses, particularly at 200 Gy (see Table 2). Comparison of expected frequencies of unbalanced gametes with the level of F1 sterility The observed level of radiation-induced F1 sterility in E. kuehniella was taken from our previous study (see Fig. 5 in Marec et al. 1999). The expected percentage of unbalanced gametes, generated by F1 moths, was estimated by using the formula: (1 – PBG) × 100, where PBG is probability of balanced segregation of chromosomal aberrations in meiosis of F1 moths. © 2001 NRC Canada

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Table 2. Comparison of frequencies of specific translocations in F1 males in Ephestia kuehniella after irradiation of male parents. Frequencies with configuration Dose (Gy) 0 100 150 200

Evaluated individuals (n) 12 9 10 13

Total translocations 0 22 27 60

Forming a chain No. 0 16 14 44

x ± SD a

0 1.78±0.97b 1.40±0.52b 3.39±1.26c

Forming a ring

Frequencies with 2 breaks per chromosome

No.

No.

x ± SD

0 0 4 13

0a 0a 0.40±0.52a 1.00±0.82b

0 6 13 16

x ± SD a

0 0.67±0.71ab 1.30±0.67b 1.23±0.93b

Note: The category of ring figures involves all reciprocal translocations and some multiple translocations, the category of chain figures involves all remaining translocations; the two breaks per chromosome category involves a fraction of multiple translocations, in which at least one chromosome was broken two or more times. Statistical analysis was performed using one-way ANOVA with the Tukey–Kramer multiple comparison post test, at the 5% significance level; the identical letter (a, b, or c) within a column indicates no statistical difference between groups.

PBG was estimated separately for each chromosome aberration found in each F1 individual. A total PBG for a particular individual was then a product of probabilities in all independent chromosome aberrations per nucleus. Since some aberrations, especially multiple translocations, showed different configurations in different nuclei of the same F1 larva, we selected for the PBG estimate that nucleus showing the most compact configuration. Only in fragmentations, in which it is possible to estimate PBG directly form pairing configuration, did we calculate a mean value of PBG from all analyzed nuclei of a particular F1 larva. For example, the simplest chromosome aberration in F1 moths was fragmentation. If both chromosome fragments paired with their normal homolog in a trivalent, then PBG = 1 because the intact and the fragmented homolog were expected to segregate to the opposite poles (Marec and Traut 1993a). However, the same aberration formed in some nuclei two optional configurations, each with two figures. If one fragment formed a bivalent with the normal homolog while the other fragment was unpaired and thus segregated randomly, then PBG = 0.5. If the intact homolog formed a univalent and two fragments paired nonhomologously, then balanced gametes could arise only by nondisjunction and, therefore, PBG = 0. For each fusion (forming a trivalent) and inversion (crossing-over was ignored), we counted PBG = 1, for interstitial deletion (the deleted fragment formed a separate ring and segregated randomly) PBG = 0.5. Segregation of chromosomes involved in a translocation depends on threedimensional position of aberrant and normal chromosomes, and on the number and distribution of chiasmata. For simplification, we assumed random segregation of translocated chromosomes (the above factors were ignored). Then, probability of balanced segregation was estimated by using the formula: PBG = (0.5)n–1, where n was the number of translocated chromosomes in a particular pachytene configuration. Comparison of the expected percentage of unbalanced gametes (expected sterility) with the observed sterility revealed considerable disproportion between the two values at each irradiation dose used (Fig. 11a). The observed sterility was much lower than expected. The latter value was about 90% at 100 Gy and achieved almost 100% at 150 and 200 Gy. In other words, almost all gametes produced by F1 moths should be genetically unbalanced and theoretically result in F1 sterility. In addition, there was only a slight difference in the expected sterility between sexes, whereas F1 males exhibited about 10–20% more observed sterility than F1 fe-

males except the dose of 100 Gy (see Marec at el. 1999). Figure 11b shows that mainly random segregation of translocated chromosomes contributed to the high predicted values of unbalanced gametes in F1 males, whereas the contribution of fragmentation plus interstitial deletion was below 50% at 100 Gy and decreased with the increasing dose to 20% at 200 Gy.

Discussion Tolerance of Lepidoptera to chromosome breakage Our study showed that F1 progeny of E. kuehniella males, irradiated with partially sterilizing doses, tolerate an unbelievable number of aberrant chromosomes in their genome. In F1 males after treatment with 200 Gy, for example, about half of the father’s chromosomes were broken at least once. The broken chromosomes constituted aberrations of various types from simple fragmentation up to very complicated chromosome rearrangements such as multiple translocation. These aberrations did not manifest themselves as dominant lethal mutations (DLM), and their F1 carriers not only survived but also retained some fertility (see Marec et al. 1999). How can F1 progeny of irradiated Lepidoptera tolerate so many chromosome breaks? LaChance and Graham (1984) hypothesized that the fate of radiation-induced breaks during mitotic cell cycles is mainly responsible for the tolerance. In organisms with monocentric chromosomes such as Diptera, radiation-induced chromosome breakage is expected to lead to the occurrence of unstable aberrations, acentric fragments and dicentric chromosomes. The former are lost and the latter cause anaphase bridges resulting in cessation of mitosis and consequently in DLM, i.e., in embryonic death (LaChance 1967). However, chromosome breaks in Lepidoptera probably do not lead to the formation of breakage-fusion-bridge cycles because in early embryonic development, chromosome bridges between cleavage nuclei are not seen (see LaChance and Graham 1984). We suggest that this phenomenon is connected with the holokinetic nature of Lepidoptera chromosomes (Wolf 1996). Pairing of aberrant chromosomes In this study, chromosome aberrations in F1 individuals were identified and classified according to pairing configurations observed in late zygotene and pachytene nuclei. However, strictly homologous pairing (homosynapsis) is restricted to zygotene, whereas during pachytene nonhomologous pair© 2001 NRC Canada

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Figs. 8–10. Multiple translocations in F1 progeny of irradiated Ephestia kuehniella males; numbers indicate structurally normal chromosomes inherited from the untreated mother. Fig. 8. A multiple translocation MT(4) involving four normal chromosomes (1–4): pairing of the translocated chromosome T(1; 2; 3) (arrows) indicates two breaks in an original chromosome (2). Fig. 9. Ring translocations: (a) a pachytene figure of six lateral elements formed by multiple translocation MT(3) involving three normal chromosomes (1, 2, 3); (b) a metaphase ring figure (arrowhead) from a spermatocyte nucleus formed by a multiple translocation. Fig. 10. A pachytene figure of multiple translocation MT(4) involving four normal chromosomes (1–4) with seven recombination nodules (arrows). Scale bar = 2 µm.

ing (heterosynapsis) may also occur in heterozygotes for chromosome aberrations (reviewed by John 1990). For example, the LEs in a heterozygous bivalent may attain the same length by synaptic adjustment (Moses 1977). In our preparations, this could happen in bivalents with a deletion or particular inversion that was rarely found. A zygotene inversion loop could disappear at pachytene, since the inverted segment nonhomologously paired in reversed order (Moses et al. 1982) and thus, the inversion was not identified. Rasmussen (1977a) showed in triploid Bombyx mori females that multivalents occurring at zygotene may be corrected during pachytene to produce bivalents and univalents. This indicated that SCs tend to optimize pairing in the form of bivalents. However, this applied only to female meiosis, which is achiasmatic. In tetraploid males, the occurrence of crossing-over prevented to a large extent the transformation of multivalents into bivalents (Rasmussen 1987). Pairing correction, though described in polyploids, might be responsible for the tendency of complicated multiple translocations in F1 males of E. kuehniella to disintegrate into two or more

simpler configurations (this study). Similar disruptions of multivalents were also observed in F1 males of the corn earworm, Helicoverpa armigera (Hubner), after irradiation of male parents (Saifutdinov 1989). Taken together, frequencies of some chromosomal aberrations and, therefore, numbers of chromosome breaks recorded in our study were probably underestimated due to synaptic adjustment and possibly pairing correction. Results obtained in one F1 male after 200 Gy represent an extreme example in favor of the conclusion. In this male, we estimated 19 breaks in a late zygotene nucleus but only 13 breaks in a pachytene nucleus. However, in most F1 larvae examined we could compare nuclei at both the prophase I stages and thus, revise the record of aberrations. Therefore, we suppose that a certain underestimation of our results had only marginal influence on the final data presented here. Comparison of chromosomal aberrations in F1 males and F1 females Distribution of chromosomal aberrations, inherited from © 2001 NRC Canada



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Table 3. Comparison of the mean number of radiation-induced chromosome breaks, observed in F1 individuals, and the level of radiation-induced F1 sterility† in Ephestia kuehniella. Dose (Gy) 100 150 200

F1 males Sterility† % 33.1 56.9 86.9

F1 females Males n 9 10 13

Breaks found‡ x ± SD a

8.4±2.1 11.5±1.8b 16.2±3.4c

Sterility† %

Females n

Breaks found‡ x ± SD

30 37.4 58.3

2 4 5

10.0±1.4a 9.8±2.1a 11.2±1.3a

†

Data taken from Marec et al. (1999). Statistical analysis was performed using one-way ANOVA with the Tukey–Kramer multiple comparison post test, at the 5% significance level; the identical letter (a, b, or c) within a column indicates no statistical difference between groups. ‡

irradiated males, should not be dependent on the sex of F1 progeny, at least during early embryogenesis. Thus, the higher level of induced F1 sterility in males than females must be rooted either in a different fate of the aberrations in both sexes, or in a higher impact of the aberrations on males (see discussion in Marec et al. 1999). The present study revealed a difference between F1 males and F1 females in the relative number of chromosomal breaks only at 200 Gy. At this high dose, breaks were significantly more frequent in F1 males than F1 females. In addition, the break frequency in F1 males was positively correlated with the dose-dependent increase of sterility, whereas clear correlation was absent in F1 females (Table 3). We suppose that the difference between sexes is caused by high mortality of F1 females with high numbers of breaks. In such F1 individuals, there was a high probability that the sex chromosome Z was also hit and any resulting recessive lethal mutation would then kill females but not males. This explanation is in keeping with the known shift of F1 sex ratio in favor of males (Anisimov et al. 1989; Al-Tawell et al. 1990; Seth and Reynolds 1993; Bloem et al. 1999; Marec et al. 1999). We also suggest that recombination between aberrant chromosomes that arose by two breaks, particularly those involved in multiple translocations, and their structurally normal homologs could partially increase a portion of genetically unbalanced gametes in F1 males in comparison with F1 females. These complicated multiple translocations were relatively frequent at 150 Gy (0.4/male) and at 200 Gy (1.0/male) and thus, could explain almost 20% and 30%, respectively, of the difference in sterility between sexes. However, in the previous study on F1 sterility in E. kuehniella (Marec et al. 1999), a dose-dependant increase of the percentage of apparently unembryonated eggs in F1 male crosses was observed together with a high portion of pairs that were fully sterile. However, in F1 female crosses, the percentage of inviable embryonated eggs increased with the dose. This favored an alternative hypothesis that induced genetic changes impair the ability of some F1 males to fertilize females while F1 females remain fertile. This hypothesis is supported by a recent study of Koudelová and Cook (2001) on sperm performance in irradiated E. kuehniella males and their progeny. These authors found a great variability in the number of sperm transferred by F1 males. The mean number of eupyrene (fertile) sperm decreased, whereas the mean number of apyrene (nonfertile) sperm increased, resulting in a high ratio of apyrene to eupyrene sperm. This ratio fluctuated between values similar to untreated males (9.5:1) and very high values (higher than 100:1). Their data implied that chromosomal rearrangements in F1 males may have altered

either mechanisms regulating dichotomous lepidopteran spermiogenesis or mechanisms underlying copulation and sperm transfer. Based on the above results it appears that the sex-specific differences in the radiation-induced F1 sterility cannot be explained by a single mechanism. Most probably there are several factors that can contribute in varying extent to the higher sterility of individual F1 males. We suggest that the main factors are (i) survival of F1 males with high numbers of chromosomal breaks while F1 females with high numbers of breaks die during their ontogenic development, (ii) crossing-over in spermatogenesis but not in the achiasmatic oogenesis, and (iii) a higher impact of induced changes on the fertility of F1 males than F1 females. Chromosomal principle of inherited sterility in Lepidoptera The analysis of SCs complements revealed variability in the number and type of chromosome aberrations in F1 progeny of irradiated E. kuehniella males. This variability was enhanced by different pairing behaviour of aberrant chromosomes during meiosis in individual germ cells; different pairing configurations most probably influenced chromosome segregation in meiosis I, finally resulting either in genetically normal or balanced or unbalanced gametes. Each aberration type is expected to contribute differently to the F1 sterility, depending on its frequency, properties, meiotic pairing, and segregation. Our data suggest that inversion and fusion do not play a significant role in the production of unbalanced gametes. The former were rare and the latter always formed a trivalent, which is expected to lead to balanced segregation (Marec and Traut 1993a). Two similar types of intrachromosomal aberrations, fragmentation and interstitial deletion, obviously increased the frequency of unbalanced gametes in F1 moths, but it is not likely that they are the main chromosomal mechanism of inherited sterility. In addition, unbalanced gametes with an absent or redundant chromosome fragment may not necessarily have a lethal effect for each F2 embryo. Thus, various translocations (nonreciprocal, reciprocal, and multiple), seem to be most responsible for the production of unbalanced gametes in F1 moths. Translocations were most frequent in all treated groups and, particularly at higher doses, tended to create very complicated configurations. Accordingly, theoretical prediction of the frequency of unbalanced gametes caused by translocations was much higher than that caused by fragmentations plus interstitial deletions (Fig. 11b). However, sterility predicted in this study according to the observed frequency of chromosome aberrations was much © 2001 NRC Canada



182 Fig. 11. (a) Comparison of the expected percentage of unbalanced gametes (expected sterility), estimated as the mean probability of unbalanced segregation of all chromosomal aberrations in F1 moths, with the observed sterility in F1 males (M) and F1 females (F) of Ephestia kuehniella after treatment of male parents with gamma-rays. Data on the observed sterility were taken from Marec et al. (1999). (b) Comparison of the expected percentage of unbalanced gametes caused by translocations (trans) and by fragmentation plus interstitial deletion (fra + idel) in F1 males. Bars indicate standard deviation (SD).

higher than the level of inherited sterility found by Marec et al. (1999). Therefore, we suggest that there is a regulation mechanism that somehow enables the moths to correct the predicted unbalanced state towards balanced chromosome segregation during meiosis I. In other words, translocated chromosomes with a high probability do not segregate by chance, and we suppose that homologous pachytene pairing facilitates their balanced segregation to daughter cells. This opinion is in keeping with knowledge about the function of SCs and subsequent chromosome structures in segregation of homologous chromosomes during meiosis I (reviewed by John 1990). Segregation of chromosomes in translocation multivalents depends on the orientation of their kinetochores at metaphase I. At the alternate disjunction, wild-type chromosomes cosegregate to one pole and the translocated products to the other. This leads to the production of two normal and two balanced gametes, whereas two modes of adjacent


disjunction produce four unbalanced meiotic products (reviewed by Rickards 1983). There are a number of examples in various organisms showing that alternate segregation often predominates over the two modes of adjacent disjunction. This has been also shown recently in translocation heterozygotes of yeast (Loidl et al. 1998). The orientation behavior of translocation multivalents is, however, affected by several factors such as the number and size of chromosomes involved, ring versus chain configurations, and also the number and position of chiasmata (Rickards 1983; John 1990, and references therein). Particularly chiasmata may play an important role in correct disjunction as they hold homologous chromosomes together till metaphase I and participate in their orientation to opposite poles of the spindle (Carpenter 1994; Tease 1998). However, there are no universal rules for the role of above factors in the alternate versus adjacent disjunction and, as pointed out by Rickards (1983), each translocation should be considered separately. It is clear from the above that we cannot answer the question of which factor or regulation mechanism could enhance probability of alternate disjunction in translocation multivalents of F1 moths without a detailed study of individual translocations. Nevertheless, the observed increase of RNs in F1 males and distribution of recombination nodules in distal segments of multivalents indicated an increase of cross-over frequency and hence of the increase of chiasma frequency. This implies that the increased number of chiasmata and their proper distribution could facilitate balanced disjunction of chromosomes from translocation multivalents in F1 males of E. kuehniella. The modified SC, which serves a substitute for the lack of crossing-over and chiasma formation to ensure regular disjunction of homologous chromosomes in achiasmatic meiosis of Lepidoptera females (Rasmussen 1977b; see also Marec 1996) could play a similar role in F1 females. However, we lack any supporting argument for the latter hypothesis. In conclusion, the inherited sterility in Lepidoptera is a very complex phenomenon that is based on the induction of a large variety of genetic alterations by irradiation. The alterations reduce fertility of F1 individuals in two ways. Some of them directly prevent the normal course of reproduction in their carriers, i.e., cause physiological sterility. Particular chromosome rearrangements lead to the production of genetically unbalanced gametes, resulting in inviable progeny and thus, cause genetical sterility of their carriers. This study confirmed and extended the hypothesis that multiple chromosomal translocations (LaChance 1985) as well as simple nonreciprocal and reciprocal translocations are most responsible for the genetical sterility of F1 generation. In addition, our results indicate the presence of mechanisms that facilitate balanced disjunction of chromosomes even from complicated translocation multivalents.

Acknowledgements We are very grateful to Dr. Kinsley Fisher (Entomology Unit, FAO/IAEA Laboratories, Seibersdorf, Austria) for permission to use the 60Co Gammacell 220 Unit for moth irradiation. We thank all the staff of the Laboratory of Electron Microscopy at the Institute of Parazitology (eské Budjovice) © 2001 NRC Canada



Tothová and Marec

for their excellent service. Technical assistance of Ms. Ivana Kollárová is also acknowledged. We wish to thank very much Dr. Leo E. LaChance (Fargo, North Dakota) for critical reading of the manuscript and Ms. Katja H. Hora (University of Amsterdam) for her valuable suggestions. This research was supported by Research Contract No. 7161/RB of the International Atomic Energy Agency (Vienna) and by Grant No. A6007609 of the Czech Academy of Sciences (Prague). Finally, we acknowledge the support of the Alexander Humboldt Foundation (Bonn), who gave us some laboratory equipment necessary for the research.

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