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Dose-response relationships and R.B.E. values of dominant lethals induced by X-rays and 1.5-MeV neutrons in prophase-1 oocytes and in mature sperm of the two-spotted spider mite Tetranychus urticae Koch (acari, tetranychidae)
361
Mutation Research, 51 (1978) 361--376 © Elsevier/North-Holland Biomedical Press
DOSE--RESPONSE RELATIONSHIPS AND R.B.E. VALUES OF DOMINANT LETHALS INDUCED BY X-RAYS AND 1.5-MeV NEUTRONS IN P R O P H A S E - 1 0 0 C Y T E S AND IN MATURE SPERM OF THE TWO-SPOTTED SPIDER MITE Tetranycbus urticae KOCH (ACARI, TETRANYCHIDAE)
A.M. FELDMANN Association Euratom-ITAL, Postbox 48, Wageningen (The Netherlands) (Received 9 November 1977) (Accepted 23 March 1978)
Summary Mature sperm and prophase-1 oocytes of Tetranychus urticae Koch were irradiated with 250-kVp X-rays or 1.5-MeV fast neutrons. The X-ray doses ranged from 0.5 to 24.0 krad, and those of the fast neutrons from 0.1 to 16.0 krad. The genetic endpoint measured was lethality, expressed in the stages from egg to adulthood in the F, progeny. The frequency of recessive lethals in female germ cells was estimated by comparing survival of fertilized versus unfertilized F1 eggs, after irradiation with the same dosage. X-Rays induce dominant lethals in prophase-1 oocytes by the action of both single hits on single targets and multiple hits on multiple targets. 1.5-MeV neutrons induce these effects predominantly by the action of multiple tracks o n multiple targets. Dominant lethals were induced in mature sperm by X-rays and by fast neutrons by the action of both single hits on single targets and multiple hits on multiple targets. Both for prophase-1 oocytes and for mature sperm the low R.B.E. value corresponded with the relatively large multiple-target component of induction of dominant lethals by fast neutrons. The nature of dominant lethality in relation to the kinetochore organization of the chromosome is discussed. A non-linear trend in the dose--effect relationship was observed for both X-rays and fast neutrons for the estimated frequency of recessive lethals induced in prophase-1 oocytes. X-Rays were more effective than neutrons in inducing recessive lethals in prophase-1 oocytes at doses lower than 3 krad.
Introduction
Tetranychus urticae (Koch) is an economically important pest on a variety of crops. Genetic control has been considered as an alternative to conventional
362 control by pesticides [ 19,26,40]. The release of irradiated males or genetically altered males into a pest population requires a basic knowledge of the radiobiological properties of the species. Some radiobiological work on T. urticae has been published [20,26,28], b u t none of these reports gave adequate dose-response relationships for the induction of dominant and/or recessive lethals. A sterilizing dose of ~/-rays was reported, for both adult males and adult females, of 20--32 krad [20,26]. The F1 progeny from males irradiated with substerilizing doses showed a high extent of heritable partial sterility. The high male-sterilization dose and the inheritance, in a high frequency, of factors that cause meiotic p r o d u c t lethality in the F, progeny are in general observed in insects with holokinetic chromosomes [21]. The chromosomes of T. u rticae are of the holokinetic tpe [32,37], i.e. the kinetic activity of a chromosome is not localized but is spread diffusely over the chromosome. Information on the induction of lethals by ionizing radiation in germ cells of insects with holokinetic chromosomes is meagre compared with that available on species with monokinetic chromosomes. In investigations on the induction of dominant lethals in metaphase-1 oocytes and mature spermatozoa of B o m b y x mori, the dose--effect relationships were different from those observed for similar stages in Drosophila [3,4,15]. The present investigation was designed to examine, in spider mites, the dose--effect relationships for the induction of dominant lethals in prophase-1 oocytes and mature spermatozoa for X-rays and fast neutrons and to assess the relative biological effectiveness of neutrons. Materials and m e t h o d s Strain. The strain of Tetranychus urticae has been reared in the laboratory for a b o u t 5 years. The mites were reared at 28°C and 50--70% relative humidity on detached bean-leaf punches on c o t t o n wool soaked in tap water. Irradiation. The dose rate for both X-rays and fast neutrons was 100 tad per min. X-Rays were produced by a 250/25 Philips deep-therapy apparatus, operating at 250 kVp and 15 mA. No additional filtering was used (HVL = 0.3--0.4 mm Cu; Ee~t in keV = 50--55 [35]. Fast neutrons, with a mean energy of 1.5 MeV, were delivered by the irradiation facility of the Biological and Agricultural Reactor at Wageningen (The Netherlands}. The dose rate was measured by both an acetylene and a muscle-tissue-equivalent ionization chamber; the ~/contamination was 4.4 rad/min, measured with a magnox--argon ionization chamber [14]. Oocyte irradiation and sampling. 100 one-day-old adult virgin females were placed together on 2 bean leaf discs (diam. 3 cm) and irradiated on a rotating table. Immediately after the treatment, 50 females were mated to untreated, 1-day-old adult virgin males in 10 groups of 5 females and 5 males on separate leaf discs. The males were removed after 1 h; and 24 h after the irradiation treatment the females were transferred, in groups of 5 females or individually (indicated in the text where necessary), to fresh leaf punches on which they produced eggs for 24 h° The eggs collected in this way had been irradiated during the diffuse stage of meiotic prophase-1 [ 3 3 , 1 7 ] . The remaining 50 virgin females were also divided into 10 groups of 5 females, b u t were n o t mated.
363
Eggs from irradiated, virgin females were collected in the same way as eggs from irradiated then mated females. In each egg sample, the number of eggs and the number of adult F1 females and/or FI males (males only, concerning progenies from virgin females) developing from the egg sample, were counted. Sperm irradiation and sampling 50 adult virgin males, 0--24 h old, were irradiated in the manner described under "oocyte irradiation". Immediately after irradiation they were mated, in groups of 5, to 5 virgin females for 1 h only. In this way, sperm was collected which had been irradiated as mature sperm [34]. The effect of radiation was studied in progenies produced 24--48 h after mating, from single females, by counting in each sample the number of eggs and the number of resulting adult F1 females and F1 males. The adult F~-survival data were then used for calculating the frequency of germ cells bearing at least one dominant lethal, expressed at any stage of development. Calculation of the effect o f oocyte irradiation on survival (a) Dominant lethals T. urticae reproduces by arrhenotokous parthenogenesis, i.e. fertilized females produce both fertilized (diploid) and unfertilized (haploid) eggs. The haploid eggs develop into males and the diploid eggs develop into females. Unfertilized females produce only haploid eggs [8,12,37]. Such arrhenotokous parthenogenesis offers a special experimental condition for assessing the frequency of both dominant and recessive lethals in the F~ generation after oocyte irradiation [5,38]. The ratio of the number of adult F, males (My) to the number of eggs (Ev) laid by irradiated unmated (virgin) females provides a measure of survival of Fl haploids (Sh): Sh -
Mv
(I)
E~
The number of haploid eggs (Eh) in a mixture of haploid and diploid eggs (E), produced by irradiated then mated females, was calculated from the number of adult FI males (M) and the survival of FI haploids from unmated females irradiated with the same dose: M Eh -
Sh
(2)
Thus the survival of F~ diploids (Sd) was calculated from: F E -- E h F = number of adult F, females (diploids) E = number of eggs produced by irradiated then mated females. Substitution of (2) in (3) results in: F Sd M E---Sh Sd - - -
(3)
(4)
364 This formula allows the use of the numbers of FI eggs, F~ males and F, females, which have been determined in the experiment, and is a simplification of the formula used in the estimation of rates of survival in Habrobracon [5,38].
(b ) Recessive lethals The frequency of genomes with at least one recessive lethal (R), expressed at any stage of development and induced in irradiated oocytes, is derived from the ratio of the survival of F~ diploids (Sd) and F~) haploids (Sh) [5] : R = 1
Sh
(5)
Sd
Calculation o f the effect o f sperm irradiation on survival Formula (4) was used for calculating the survival of F, diploids from irradiated males, in which Sh was given by the survival of F~ progenies from unmated, unirradiated virgin females otherwise collected and treated in the same way as in the irradiation series. Survival curves The survival data were fitted to models of the following types. (1) S = 1 - - ( 1 -- e-KD)N (The model of A t w o o d and Norman [6].} (2) S = e - K I " D ( 1 - - ( 1 - - e - K 2 " D ) N) (The general model of King and Sankaranarayanan [34,36].) S = survival at dose D; KI = sensitivity of the one-target component; K and K2 = the sensitivity of the N-target component. The first model describes a process in which survival is determined by the hit of N targets by different ionization tracks. Model 2 incorporates the action of two processes, one requiring a single hit on a single target and the other requiring one hit on each of N targets. In model 1, two parameters, and in model 2, three parameters, have to be calculated from the experimental data. The statistical analysis of the survival data that were involved in the fitting of the data to models 1 and 2 were performed b y Prof. F.E. Wiirgler at his computer facility in Ziirich [35,2,34]. The best-fit curves, presented in the figures, are drawn according to model 2. This was justified for comparison purposes because: (1) most of the dose-response curves on insects follow this model; (2) the fit of the survival data to model 2 was satisfactory in general; and (3) the conclusions following model 2 were n o t different from those in which model 1 was used. Results
Con trols The experiments were performed during the years 1973--75. Control experiments were done at the beginning and the end o f this period. The survival frequencies of F, haploids, calculated from the different experiments, were independent of the date (0.5 ~ p ~ 0.1). The data on the survival frequency of F1 haploids from the different experiments were pooled, and a mean survival frequency of Fl haploids was calculated (Sh -- 0.961 ± 0.010). This mean sur-
365 viral frequency of F~ haploids was used for the calculation of the survival frequency of F~ diploids in the different control experiments. The calculations resulted in a h o m o g e n e o u s set of data on the survival frequency of F~ diploids (0.9 > p > 0.5). Pooling of all the data from the different control experiments on diploid survival resulted in a mean value for F,. diploid survival of SD = 0.944 + 0.022.
Oocyte radiation with X-rays and fast neutrons (Tables 1, 2 and 3 and Figs. 1 and 2) The survival data, shown in the figures, were corrected by the survival of the control [1].
(a) Survival of F1 haploids and F, diploids The fit of the survival data of F1 haploids to models 1 and 2 was rather poor, both for X-rays and for neutrons, whereas the fit of the survival data of F1 diploids to both models, was rather good (Table 3). The best-fit linear components of induction of lethality were n o t significantly different from zero both for X-rays and for neutrons, except the fitted K1 value of the X-ray-survival data of F1 haploids. The shapes of the curves on a semi-log plot suggest that the responses were non-linear, even for neutrons. The higher effectiveness of neutrons was due to the relative increase of the supralinear component, according to the best-fit parameters of model 2 (see upper part of Table 3). The survival curves of F~ haploids fall more steeply than those for F~ diploids, which is further substantiated by the calculated doses for 80 and 20% survival of F1 haploids and F~ diploids (Table 3). This observation, together with the poor fit of the survival of F~ haploids to a model, suggests that the mortality of F1 haploids has a broader causal base, i.e. dominant and recessive lethals, whereas the mortality of F~ diploids is determined by the frequency of dominant lethals only. The R.B.E. of neutrons was studied by calculating the ratio of t h e dose of X-rays and neutrons at two levels of survival, i.e. 80 and 20% (Table 3). The R.B.E. was independent of the model u s e d and was dose dependent, i.e. 1.3 and 1.7 at 80% surv;.val and 1.5 and 1.4 at 20% survival for F, haploids and diploids respectively. The fitted target numbers, N, of models 1 and 2, for the estimated survival data of F1 haploids, were significantly larger than 1 and n o t significantly different from 2, both for X-rays and fast neutrons. However, the standard errors were large, as a consequence of which the effect of neutrons on the target number, in comparison with X-rays, cannot be studied. The target numbers of Fl-diploid survival for model 1 were significantly larger than 2 and they did n o t differ from each other for the X-ray and neutron data. The target numbers of model 2 for F,
(b) The induction of recessive lethals by X-rays and fast neutrons in prophase-1 oocytes The estimated frequencies of recessive lethals, induced by X-rays and fast neutrons in prophase-1 oocytes, are given in Tables 1 and 2. The standard errors were in general high because of the high number of parameters, having their
6 28 a 52 a
49 a 6 20 a 46 a
2.0 3.0 4.0
• 6.0 8.0 8.0 8.0
515 261 220 431
331 262 531
1500 370 504
133 19 13 23
250 145 255
1441 348 457
0.258 0.073 0.059 0.053
± + ± ±
0.023 0.005 0.013 0.012
0.755 + 0.030 0 . 5 5 3 -+ 0 . 0 5 4 0.480 + 0.026
0.961 + 0.010 0.941 + 0.014 0.907 + 0.017
48 a 10 20 a 50 a
10 48 a 53 a
103 10 10
511 536 215 454
642 483 499
2401 547 735
Number of eggs
i00 64 12 35
378 171 194
1441 361 505
0.340 0.194 0.073 0.442
-+ 0 . 0 5 7 + 0.048 ± 0.023 ± 0.649
0.879 + 0.024 0.874 + 0.170 0.631 + 0.047
0.944 + 0.022 0 . 9 4 7 _+ 0 . 0 3 1 0.932 + 0.018
56 15 3 20
160 159 92
848 156 175
Number of adults
Number of adults
Survival + S
F 1 haploids
F 1 diploids
AS 0--24-h-
0.240 0.625 0.191 0.879
± + ± ±
0.167 0.101 0.341 0.198
0 . 1 4 1 _+ 0 . 0 5 3 0 . 3 6 7 -+ 0 . 1 7 4 0.239 ± 0.084
0.000 + 0.014 0.007 + 0.039 0.027 ± 0.030
Frequency of recessive lethals + S
a Data were collected on progenies from single females, whereas in the other experiments the data were collected on progenies from groups of 5 females.
49 7 9
Survival + S
Number of trials
Number of adults
Number of trials
Number of eggs
Progeny of irradiated and mated females
O F T e t r a n y c h u s urticac ( K o c h ) , I R R A D I A T E D
Progeny of irradiated virgin females
0.0 0.5 1.0
Dose (krad)
THE EFFECTS OF X-RAYS (100 rad/min; 250 kVp) ON THE PROGENY OF UNMATED FEMALES OLD ADULT VIRGINS, EGGS WERE IRRADIATED IN EARLY MEIOTIC PROPHASE 1
TABLE 1
o~
10 9 10
53 a 9 52 a 20 a
0.5 1.0 2.0
3.0 4.0 6.0 8.0
609 465 518 210
509 532 501
1500 659 595
223 116 17 0
468 493 346
1441 636 557
0.366 ± 0.022 0.250 + 0.015 0.033 ± 0.008
0.919 ± 0.017 0 . 9 2 7 -+ 0 . 0 0 8 0.691 ± 0.026
0.961 ± 0.010 0.965 + 0.014 0.936 ± 0.020
53 a 10 46 a 19 a
9 9 10
103 10 10
584 578 410 218
531 506 493
2401 659 624
Number of eggs
190 149 23 1
339 346 249
1441 458 431
0.485 ± 0.037 0.340 ± 0.034 0.138 ± 0.097
0 . 9 4 1 -+ 0 . 0 1 8 0 . 9 2 7 -+ 0 . 0 1 9 0 . 7 0 3 -+ 0 . 0 4 0
0 . 9 4 4 -+ 0 . 0 2 2 0 . 9 6 5 -+ 0 . 0 2 0 0.993 ± 0.020
62 35 8 0
157 123 96
893 178 178
Number of adults
Number of adults
S u r v i v a l +- S
F 2 haploids
F 1 diploids
0.201 ± 0.091 0.267 + 0.093 0.763 ± 0.204
0.023 ± 0.032 0.000 ± 0.023 0.018 + 0.074
0.000 + 0.014 0.000 ± 0.028 0.058 ± 0.032
Frequency of recessive lethals ± S
O F Tetranychus urticae ( K o c h ) ,
a D a t a w e r e c o l l e c t e d o n p r o g e n i e s f r o m single f e m a l e s , w h e r e a s in t h e o t h e r e x p e r i m e n t s t h e d a t a w e r e c o l l e c t e d o n p r o g e n i e s f r o m g r o u p s o f 5 p a r e n t a l f e m a l e s .
49 10 10
S u r v i v a l -+ S
Number of trials
Number of adults
Number o f trials
Number o f eggs
Progeny of irradiated and after that mated females
Progeny of irradiated virgin females
0.0 0.1 0.2
Dose (krad)
THE EFFECT OF FAST NEUTRONS (100 rad/min; 1.5 MeV) ON THE PROGENY OF UNMATED AND MATED FEMALES IRRADIATED AS 0--24-h-OLD ADULT VIRGINS, EGGS WERE IRRADIATED IN EARLY MEIOTIC PROPHASE 1
TABLE 2
0.006 7.8 X 10 -5
OF MATURE
0.5 X 10 -3 0,670
0.3 X 10 -3 0.132
P
0.158 + 0.012 0.251 + 0.027
SPERM
0.777 + 0.061 0.574 + 0.050
0.470 + 0.031 0.488 + 0.052
K
= 1---(I~e--KD) N
PROPHASE-100CYTES
Model 1:8
OF EARLY
1.148 -+ 0.104 1.460 + 0.189
5.346 + 0.976 3.869 + 0.603
3.453 + 0.646 6 . 3 8 7 -+ 1 . 7 2 6
N
1.7 1.6
1.7 1.9
2.1 3.1
10.9 7.8
4.1 5.0
5.9 6.9
0.014 0.052
4 • 10 -4 0.500
0.005 0.239
0.117 _+ 0.039 0.150 -+ 0.012
0.019 + 0.036 4 X 10 -5 -+0.039
0 . 0 6 7 -+ 0 . 0 2 7 0.027 + 0.014
0.063 ± 0.029 0.260 ± 0.088
0 . 7 9 2 -+ 0 . 0 9 1 0.574 + 0.066
0 . 4 7 5 -+ 0 . 0 4 2 0.548 + 0.074
2.044 +- 2.119 10.361 _* 10.074
6.194 + 2,694 3.871 + 1.358
6.129 ± 2.185 1 1 . 7 5 7 _+ 5 . 5 7 5
N
1.7 1.5
1.7 1.9
2.2 3.3
nychus urticae K o c h w i t h X - r a y s o r f a s t n e u t r o n s , o f m o d e l s 1 a n d 2 (see t e x t ) .
Tetra
11.3 8.4
4.1 5.0
6.1 6.9
20%
Dose (krad)
80%
20%
K2
80%
K1
at S =
P
e--KID[1-"(I~e--K2D) N]
at S =
Dose (krad)
M o d e l 2: S =
Best-fit parameters of estimated data points of survival of F 1 haploids and F 1 diploids, after irradiation of early prophase-1 oocytes and mature sperm of
X-rays Neutrons
IRRADIATION
F l haploids F I diploids
Neutrons
F 1 haploids F 2 diploids
X-rays
IRRADIATION
TABLE 3
c.o o~ 00
369 I00- ~-~ -'Li----... i......~ ~ ".. ~
.--. X-rays ~---A neutrons
50
\ k \
2O
\
-S
\ \
>
\
>
10-
\ \
u L i1) e~ 5-
f,g
1
2
3
4 dose ( krad
5
6
7
8
)
Fig. 1. The effect of 250okVp X-rays and of 1.5oMeV fast neutrol~ on the 811ryival of F 1 diploids from irradiated early p r o p h a s e - 1 eggs o f Tetranychus urticae K o c h . F e m a l e s w e r e irradiated as l - d a y - o l d adult virgins and m a t e d , i m m e d i a t e l y after t h e irradiation t r e a t m e n t , t o u n t r e a t e d m a l e s . F e r t i l i z e d eggs w e r e s a m p l e d 24---48 h a f t e r irradiation. D a t a w e r e f i t t e d t o t h e m o d e l o f King and S a n k a r a n a r a y a n a n (see text).
own errors, which were involved in the calculations. A non-linear trend of the dose--effect relationship was observed for both X-rays and fast neutrons. X-Rays were more effective than neutrons in inducing recessive lethals in prophase-1 oocytes at doses lower than 3 krad. The X-ray curve saturated at higher radiation doses, mainly caused by the standard errors of the respective survival frequencies of F, haploids and F~ diploids being large at higher radiation doses. The differences between the survival frequencies of F1 haploids and FI diploids become less significant at higher doses, therefore the ratio of Sd and Sh approaches 1.
Sperm irradiation with X-rays and fast neutrons The effect of X-rays or fast neutrons on the survival of F1 diploids, after irradiation of mature sperm, is given in Table 4. The parameters fitted to the different models and the graphs are shown in the lower part of Table 3 and Fig. 3 respectively. Table 3 shows that the fit of both the X-ray data and of the
370
•- - ,
X- rays
4----A
neutrons
\
\ \
\
_20> >
5-
\
fig
i
\
2
I
l
]
I
1
2
3
4
i
5 dose (krod)
i
i
i
6
7
8
Fig. 2. T h e e f f e c t o f 2 5 0 - k V p X - r a y s a n d of 1 . 5 - M e V fast n e u t r o n s o n t h e survival o f F I h a p l o i d s f r o m i r r a d i a t e d p r o p h a s e - 1 eggs o f Tetranychus urticae. F e m a l e s w e r e i r r a d i a t e d as 1 - d a y - o l d a d u l t virgins a n d r e m a i n e d u n m a t e d a f t e r t r e a t m e n t . Eggs w e r e s a m p l e d 2 4 - - 4 8 h a f t e r i r r a d i a t i o n . D a t a w e r e f i t t e d t o t h e m o d e l of King and Sankaranarayanan.
neutron data to model 2 was better than to model 1. The N values for X-rays and fast neutrons were n o t significantly different from each other either for model 1 or 2. The K factors for X-rays and neutrons of model 1 were significantly different from each other (p < 0.005). The best-fit: parameters o f model 2 show that the sensitivity to the induction of the linear c o m p o n e n t (K,) o f dominant lethality per kilorad X-rays was n o t d i f f e r e n t from the K, value for fast neutrons (0.5 > p > 0.1). The greater effectiveness of fast neutrons for the induction of dominant lethals in sperm is predominantly caused b y the increase o f the supralinear c o m p o n e n t of dominant lethality per kilorad neutrons. The shape of the X-ray curve is non-linear, and the shape of the neutron curve shows the predominance of the supralinear component. The K2 value for neutrons is s i ~ i f i c a n t l y larger than the K2 value for X-rays (0.05 > p > 0.01; cf. m o d e l 2, Table 3}. Table 3 shows that the ratio of the doses for X-rays and neutrons at 80 and 2 0 ~ survival is the same for the different models (i.e. 1.1 at 8 0 % survival and 1.4 at 20% survival). The R.B.E. is dose
ON THE SURVIVAL
OF F 1 DIPLOIDS AFTER IRRADIATION
OF MATURE SPERM
56 10 52
10 10 10
10 54 22
47 52
0.5 1.0 1.0
1.5 2.0 4.0
8.0 8.0 12.0
16.0 24.0
536 705
637 659 241
630 767 655
685 426 510
2401 563 627
Number of eggs a
a Including haploid F l progeny.
103 10 10
Number of trials
0.0 0.2 0.5
Dose . (krad)
31 5
147 160 39
356 404 266
459 247 317
1441 373 370
Number of adults
F 1 diploids
X-rays (100 rad/min; 250 kVp)
0.083 ± 0.015 0 . 0 1 0 -+ 0 . 0 0 6
0.319 ± 0.030 0 . 3 4 2 -+ 0 . 0 2 6 0.229 ± 0.031
0.745 ± 0.022 0 . 6 9 3 -+ 0 . 0 2 8 0.556 ± 0.028
0 . 9 5 4 -+ 0 . 0 1 5 0 . 8 5 9 -+ 0 . 0 2 9 0 . 8 7 5 -+ 0 . 0 2 6
0.944 ± 0.022 0.924 ± 0.029 0.886 ± 0.024
Survival ± S
on the F 1 progeny which was produced 24--48 h after mating.
12.0 16.0
2.0 4.0 8.0
1.0 2.0 2.0
0.5 0.5 1.0
0.1 0.2
Dose (krad)
24 25
55 10 19
50 10 53
10 54 10
10 10
Number of trials
338 355
691 633 239
493 641 711
620 786 535
400 557
Number o f eggs a
F a s t n e u t r o n s ( 1 0 0 r a d / m i n ; 1.5 M e V )
21 2
234 247 29
165 245 336
341 457 280
213 340
Number of adults
F 1 diploids
0.111 ± 0.024 0.008 ± 0.006
0.707 ± 0.029 0.527 ± 0.024 0 . 1 7 8 -+ 0 . 0 3 3
0.676 ± 0.050 0 . 6 9 5 -+ 0 . 0 3 5 0.694 ± 0.025
0.884 ± 0.023 0.876 ± 0.019 0.836 ± 0.049
0.917 ± 0.034 0.958 ± 0.026
Survival frequency ± S
Males w e r e i r r a d i a t e d as 1 - d a y - o l d a d u l t v i r g i n s a n d m a t e d i m m e d i a t e l y a f t e r i r r a d i a t i o n w i t h u n t r e a t e d v i r g i n f e m a l e s d u r i n g 1 h. T h e s u r v i v a l d a t a w e r e e s t a b l i s h e d
O F Tetranychus urticae ( K o c h )
T H E E F F E C T O F 250 k V p X - R A Y S A N D 1.5 MeV F A S T N E U T R O N S
TABLE 4
372
IO0 ~ h ~ "
.I. ~
"ij~
x rays
~---a neutrons
l',
cl 5
\\\
\\\\
2
fig3
\\
\
' l, 5
10
I 20
25
dose ( k r a d )
Fig. 3. The e f f e c t of 2 5 0 - k V p X-rays a n d 1.5-MeV fast n e u t r o n s o n the survival of F 1 d i p l o i d s a f t e r irrad i a t i o n of m a t u r e s p e r m o f Tetranychus urticae. Males were i r r a d i a t e d as 1 - d a y - o l d a d u l t virgins a n d m a t e d i m m e d i a t e l y a f t e r i r £ a d i a t i o n w i t h u n t r e a t e d virgin f e m a l e s d u r i n g 1 h. Survival was e s t a b l i s h e d o n d i p l o i d F 1 p r o g e n y p r o d u c e d 2 4 - - 4 8 h a f t e r m a t i n g . D a t a were f i t t e d to the m o d e l of King a n d Sankaxanarayanan.
Discussion
The induction o f dominant lethals in sperm and oocytes o f T. urticae Regarding the established properties of holokinetic and monokinetic chromosomes, some causes o f dominant lethality can be considered to be related more specifically to one of the t w o types of c h r o m o s o m e than to the other. First, fragmentation of chromosomes, due to radiation-induced breaks, is a major cause of dominant lethality in species with monokinetic chromosomes. This is n o t so in species with holokinetic chromosomes, since broken chromosomes, especially when they are n o t fragmented into very small pieces, can pass through both mitotic [16,11,10,13,23,25,30,22] and meiotic divisions w i t h o u t loss of chromosomal material [30,27,23,17]. It is generally assumed that chromosome breaks are caused by single ionization tracks and these breaks contribute to the linear c o m p o n e n t of induction of dominant lethality. Second, fragmentation of chromosomes, resulting from bridge formation, caused by radiationinduced sister-strand union, is an important cause of dominant lethality in organisms with monokinetic chromosomes [21,9]. Radiation-induced sisterstrand union is rare in cells with holokinetic chromosomes and attributes in a minority to dominant lethality [11,22]. Sister-strand union due to misrepair of a chromosome break is initially caused by single ionization tracks and is part
373 of the contribution to the linear c o m p o n e n t of induction of dominant lethality. Third, the induction of symmetric reciprocal translocations in monokinetic chromosomes is accompanied in equal frequencies by the formation of dicentric and acentric chromosomes which usually cannot regularly pass through cell division and in this way lead to dominant lethality. In species with holokinetic chromosomes all analogous reciprocal reconfigurations are viable, without the complications associated with dicentrics and acentrics [10,7,30,]. The induction of autosomal translocations b y X-rays in mature sperm of Drosophila is based on both single- and multiple-electron tracks, and the relative linear contribution decreases from a b o u t 100X the supralinear contribution at 50 tad to 1X at 5000 rad [ 18]. Neutrons induce autosomal translocations in mature Drosophila sperm b y the action of single p r o t o n tracks [18]. When it is assumed that the reciprocal exchanges leading to the formation of dicentrics and acentrics have the same kinetics of induction, then these causes of dominant lethality are to a large extent induced by single-hit phenomena. In the summarizing of these differences, it m a y be proposed that the induction by X-rays and by fast neutrons of dominant lethality in species with holokinetic chromosomes results in a gross reduction of the linear c o m p o n e n t in comparison with species with monokinetic chromosomes. This was true for the germ cells of T. urticae studied, irrespective of the model used. Both for X-rays and neutron irradiation of prophase-I o o c y t e s of T. urticae, the contribution of the linear c o m p o n e n t to dominant lethality of FI diploids was negligible. For example, the supralinear contribution to dominant lethality for X-ray-irradiated oocytes of T. urticae showed a dose-dependent increase in relative importance: at 1 krad, the supralinear c o m p o n e n t contributed only 0.4% to dominant lethality, b u t at 3, 6 and 9 krad the contribution was 50.2, 86.1 and 91.2% respectively. The shape of the neutron curves shows, for both o o c y t e and sperm irradiation, the predominance of the supralinear components. The radiosensitivity of prophase-1 oocytes of Drosophila was n o t so strikingly different from that of T. urticae as could be expected from the general assumption that species with holokinetic chromosomes are less radiosensitive than species having monokinetic chromosomes [22]. The X-ray doses, resulting in 80% survival, after irradiation of prophase-1 oocytes of Drosophila and T. urticae, were 1.6 krad and 3.3 krad respectively, b u t the doses for giving 20% survival were 6.5 krad and 6.9 krad respectively. The X-ray doses, resulting in 80% survival, after irradiation of mature sperm of Drosophila and T. urticae, were 0.7 krad and 1.7 krad respectively, and the doses for giving 20% survival were 3.6 krad and 11.3 krad respectively. Thus the general assumption that a good correlation exists between the distribution of kinetic properties along the chromosome and differences in species radio-resistance, is at least doubtful. A corresponding conclusion was drawn by LaChance [21], who observed that hemipteran species, having holokinetic chromosomes, can be sterilized by fairly low doses of irradiation to sperm. There is suggestive evidence that low doses of neutrons on prophase-1 o o c y t e s have a positive effect on survival of FI diploids. Up to 1 krad neutrons, no significant negative effect on survival was observed. On the contrary, at 200 rad, the survival was 0.993 + 0.020 in contrast with a control value of 0.944 + 0.022. Corresponding observations were made by Meyer and Abrahamson [24]
374 on sex-linked recessive lethals induced by X-rays in Drosophila oogonia and by N e w c o m b e [29] on 7-ray-induced mortality in sperm of the rainbow trout. The interference of radiation-induced repair [24], over-compensating radiationinduced damage b y repair of natural background damage, may be partially responsible for the non-linear increase of recessive lethals after neutron irradiation of T. urticae oocytes. No similar effect was observed on the survival of F1 haploids after neutron irradiation of oocytes or on the survival of F~ diploids after X-ray or neutron irradiation of T. urticae sperm. R .B.E. Neutrons induce mortality in prophase-1 oocytes of T. urticae, principally by the action of multiple tracks on multiple targets. In agreement with this observation is the low R.B.E. value, indicating that the radiation-induced lesions, which have to interact to produce a dominant-lethal effect, were seld o m produced by the same ionization track. In mature sperm the kinetics of induction of dominant lethality is different from that of prophase oocytes. In mature sperm the relative contribution of the linear c o m p o n e n t for induction of dominant lethality is predominant both for X-rays and for fast neutrons. The higher effectiveness of neutrons compared with X-rays was, according to the best-fit parameters of model 2 (Table 3), caused by the relative increase of the supralinear c o m p o n e n t of the sensitivity to dominant lethality. Thus 1.5 MeV neutrons caused a higher frequency of single hits on single targets, and part of the lesions induced by different proton tracks resulted in dominant lethality after interaction with each other. Since the linear c o m p o n e n t of the sensitivity to dominant lethality of X-rays on sperm was n o t significantly different from that of neutrons, it is concluded that the distance between interacting lesions coincides with the distance between ionizations within an electron track by which denser ionizing radiation is only more effective by the associated production of f-rays with a larger range and LET than many of the electrons produced by X-rays. The induction o f recessive lethals in early prophase-1 oocytes o f T. urticae A numerical approach is used for the assessment of the frequency of recessive lethals induced in prophase-1 oocytes of T. urticae. The percentage of recessive lethals in whole genomes is estimated by comparing the survival of haploids and diploids with formula (5). But only little information is available on possible differential effects of segmental aneuploidy on the survival of the different sexes of T. urticae. There are indications that segmental aneuploidy is as devastating in the male as in the female, since the survival o f haploids and of diploids, produced by a translocation heterozygous female of T. urticae, is the same (Feldmann, unpublished). The trend for the induction of recessive lethals is non-linear, b u t the standard errors of the estimates of recessive lethals, induced in prophase-1 oocytes of T. urticae {Tables 1 and 2), are t o o large to warrant definitive conclusions. More information a b o u t the induction of recessive lethals in oocytes of T. urticae have to await more accurate determinations or the use of other techniques.
375
Acknowledgements The constructive discussions with Drs. Robinson, Leenhouts, Chadwick and Sybenga and the helpful suggestions of Drs. LaChance and Sankaranarayanan are gratefully acknowledged. Prof. Wiirgler and Dr. Barendregt are thanked for their help in the calculations. Elly Broecks-Doorn is gratefully thanked for her enthusiasm and expert technical assistance. References 1 Abbot, W.S., A m e t h o d for c o m p u t i n g the effectiveness of an insecticide, J. Econ. Ent omol ., 18 (1925) 265--267. 2 Alper, T., N.E. Gillies and M.M. Elking, The sigmoid survival curve in radiobiology, Nature (London), 186 (1964) 1062--1063. 3 Astaurov, B.L., Versuche ~ibar experimentelle Androgenese u n d Gynogenese beim Seidenspinner ( B o m b y x mori, L.). Biol. Zhur., 6 (1937) 3--50. 4 Astaurov, B.L., and S.L. Frolowa, Kiinstlich ausgel6ste Mutationen beim Seidenspinner (Bombyx mori, L.). Biol. Zhur., 4 (1935) 861---891. 5 A t w o o d , K.C., R.C. Borstel and A.R. Whiting, An influence of p l o i d y on the t i me of expression of d o m i n a n t lethal m u t a t i o n s in Habrobracon, Genetics, 41 (1956) 804---813. 6 A t w o o d , K.C., an d A. Norman, On the i n t e r p r e t a t i o n of mul t i -hi t survival curves, Proc. Natl. Acad. Sci. (U.S.A.), 35 (1949) 696--709. 7 Bauer, H., Die kinetische Organisation der Lepidoptere n Chromosomen, Chromosoma, 22 (1967) 101--125. 8 Boudreaux, H.B., Biological aspects of some p h y t o p h a g o u s mites, Ann. Rev. Entomol., 8 (1963) 137--154. 9 Borstel, R.C. von, Effects of radiation on germ cells of insects: d o m i n a n t lethals, gamete inactivation and gonial-cell killing, in: R a d i a t i o n and Radioisotopes applied to Insects of Agricultural Import a nc e , IAEA, Vienna, 1963, STI/PUB/74, pp. 367--385. 10 Brown, S.W., and L.J. Wiegmann, Cytogenetics of the me a l ybug Planococcus citri (Risso) (Homoptera: Coccoidea): Genetic markers, lethals and c h r o m o s o m e rearrangements, Chromosoma, 28 (1969) 255--279. 11 Brown, S.W., and W.A. Nelson-Rees, R a d i a t i o n analysis of a lecanoid genetic system, Genetics, 46 (1961) 9 8 3 - - 1 0 0 7 . 12 Cagle, L.R., Life history of the t w o - s p o t t e d spider mite, Virg. Agr. Exp. Stn., Techn. Bull., 113 (1949) 3--31. 13 de Castro, D., A. Camara and N. Malheizos, X-Rays in the c e nt rome re probl e m of Luzula purpurea, Link., Genet. Ib~r., 1 (1949) 48--54. 14 Chadwick, K.H., W.F. Oosterheert and K.J. Puite, Comparison b e t w e e n s p e c t r o m e t r y , ionisation c h a m b e r and threshold d e t e c t o r systems for fast-neutron dos i me t ry in the subeore facility of a swimmin g-p ool r e a c t o r , in: N e u t r o n Irradiation of Seeds II, Technical R e port s Series No. 92, IAEA, Vienna, 1968, pp, 123. 15 Chikushi, H., Fatal action of X-rays on Silkworm eggs, Bull. Sci. Fac. Agr. Kyushu Imp. Univ., 8 (1938) 155--162. 16 Cooper, R.S., E x p e r i m e n t a l d e m o n s t r a t i o n of h o l o k i n e t i c c h r o m o s o m e s and of differential radiosensitivity during oogenesis, in the grass mite, Siteroptes graminum (Reuter), J. Exp. Zoo1., 182 (1972) 69--94. 17 Feiertag-Koppen, C.C.M., Cytological studies of the t w o-s pot t e d spider mi t e Tetranychus urticae Koch (Tetranychidae, Trombidiformes), I. Meiosis in eggs, Genetica, 46 (1976) 445--456. 18 Gonzalez, F.W., Dose--~esponse kinetics of genetic effects induced by 250 kVp X-rays and 0.68 MeV neu tro ns in m a t u r e sperm of Drosophila melanogaster, Mut a t i on Res., 15 (1972) 303--324. 19 Helle, W., New d e v e l o p m e n t s towards biological control of the t w o-s pot t e d spider m i t e by incompatible genes, PubL Org. Eur. PI. Protect. Paris, S~r. A, 52 (1969) 7--15. 20 Henneberry, T.J., Effects of g a m m a r a d i a t i o n on the fertility of the t w o - s p o t t e d spider m i t e and its progeny, J. Econ. E ntomol., 57 (1964) 672--674. 21 LaChance, L.E., Induced sterility in irradiated diptera and lepidoptera, in: Sterility Principle for Insect Control, I.A.E.A., Vienna, 1974, pp. 401--411 (Proc. Ser. STI/PUB: 377, 1975). 22 LaChance, L.E., and J.G. Riemann, D o m i n a n t lethal m u t a t i o n s in insects w i t h h o l o k i n e t i c chromosomes, I. Irradiation of Oncopeltus (Hemiptera: Lygaedae) sperm and oocytes, Ann. E n t o m o l . Soc. Am., 66 (1973) 813--819. 23 La Cour, L.F., The Luzula system analyzed by X-rays, Heredity, 6 (1952) 77--81.
376 2 4 M e y e r , H.W., a n d S. A b r a h a m s o n , P r e l i m i n a r y r e p o r t o n m u t a g e n i c e f f e c t s o f l o w X - r a y d o s e s in i m m a t u r e g e r m cells o f a d u l t D r o s o p h i l a f e m a l e s , G e n e t i c s , 6 8 , S u p p l . ( 1 9 7 1 ) 4 4 . 2 5 M o n t e z u m a - D e - C a r v a l h o , J., X - r a y i n d u c e d b r e a k a g e in c h r o m o s o m e s w i t h d i f f u s e c e n t r o m e r e s , in: E f f e c t s o f i o n i z i n g r a d i a t i o n o n seeds, P r o c . S y m p . K a r l s r u h e , 1 9 6 0 , I . A . E . A . , V i e n n a , 1 9 6 1 , p p . 2 7 1 - 277. 26 Nelson, R., Effects of gamma radiation on the biology and population suppression of the two-spotted s p i d e r m i t e , T e t r a n y c h u s urticae K o c h , P h . D . Thesis, Univ. Calif. Davis, 1 9 6 8 . 2 7 N e l s o n - R e e s , W . A . , N e w o b s e r v a t i o n s o n l e c a n o i d s p e r m a t o g e n e s i s in t h e m e a l y b u g , P l a n o c o c c u s cirri, Chromosoma, 14 (1963) 1--17. 2 8 N e l s o n , R . D . , a n d E.M. S t a f f o r d , E f f e c t s o f g a m m a r a d i a t i o n o n t h e b i o l o g y a n d p o p u l a t i o n s u p p r e s s i o n o f t h e t w o - s p o t t e d s p i d e r m i t e , T e t r a n y c h u s urticae K o c h , H i l g a r d i a , 4 1 ( 1 9 7 2 ) 2 9 9 - - 3 4 1 . 29 Newcombe, H.B., "Benefit" and "harm" from exposure of vertebrate sperm to low doses of ionizing radiation, Health Phys., 25 (1973) 105--107. 3 0 N o r d e n s k i 6 1 d , H., A s t u d y o f m e i o s i s in t h e p r o g e n y o f X - i r r a d i a t e d L u z u l a p u r p u r e a , H e r e d i t a s , 4 9 , (1963) 33--47. 3 1 P i j n a c k e r , L.P., a n d L . J . D r e n t h - D i e p h u l s , C y t o l o g i c a l i n v e s t i g a t i o n s o n t h e m a l e r e p r o d u c t i v e s y s t e m a n d t h e s p e r m t r a c k in t h e s p i d e r m i t e T e t r a n y c h u s urticae K o c h ( T e t r a n y c h i d a e , A c a r i n a ) , N e t h e r l . J. Zool., 2 3 ( 1 9 7 3 ) 4 4 6 - - 4 6 4 . 3 2 P i j n a c k e r , L.P., a n d M . A . F e r w e r d a , D i f f u s e k i n e t o c h o r e s in t h e c h r o m o s o m e s o f t h e a r r h e n o t o k o u s s p i d e r m i t e T e t r a n y c h u s urticae K o c h , E x p e r i e n t i a , 2 8 ( 1 9 7 2 ) 3 5 4 . 33 Pijnacker, L.P., personal communication. 3 4 P o r t e r , E . H . , E l e c t r o n i c c o m p u t e r s a n d s u r v i v a l c u r v e s , Brit. J. R a d i o L , 3 7 ( 1 9 6 4 ) 6 1 0 - - 6 1 5 . 35 Puite, K.J., A thermolumineseence system for the intexcomparison of absorbed dose and radiation q u a l i t y o f X - r a y s w i t h a H V L o f 0 . 1 t o 3 . 0 m m C u , P h y s . M e d . Biol., 21 ( 1 9 7 6 ) 2 1 6 - - 2 2 5 . 3 6 S a n k a r a n a r a y a n a n , K . , T h e e f f e c t o f o x y g e n a n d n i t r o g e n p o s t - t r e a t m e n t s o n t h e m o r t a l i t y of D r o s o p h i l a eggs i r r a d i a t e d as s t a g e 7 o o c y t e s , M u t a t i o n R e s . , 7 ( 1 9 6 9 ) 3 5 7 - - 3 6 8 . 37 Schrader, F., Haploidie bei einer Spinnmflbe, Arch. Mikrosk. Anat. Entwicklungsmech., 97 (1923) 610--622. 3 8 W h i t i n g , A . R . , R . H . S m i t h a n d R . C . V o n B o r s t e l , M e t h o d s f o r r a d i a t i o n s t u d i e s d u r i n g o o g e n e s i s in H a b r o b r a c o n j u g l a n d i s ( A s h m e a d ) , in: E f f e c t s o f R a d i a t i o n o n M e i o t i c S y s t e m s , I . A . E . A . , V i e n n a , 1968, STI/PUB/173, pp. 201--208. 3 9 Wfirgler, F . E . , E l e c t r o n i c c o m p u t e r s a n d s u r v i v a l - c u r v e a n a l y s i s , Int. J. R a d i a t . Biol., 1 4 ( 1 9 6 8 ) 1 9 3 - 196. 4 0 Z o n , A . G . , a n d W.P.J. V a n O v e r m e e r , I n d u c t i o n o f c h r o m o s o m e m u t a t i o n s b y X - i r r a d i a t i o n in Tetran y c h u s urticae ( T e t r a n y c h i d a e ) w i t h r e s p e c t t o a p o s s i b l e m e t h o d o f g e n e t i c c o n t r o l , E n t o m o l . E x p . Appl., 15 (1972) 195--202.
Mutation Research, 51 (1978) 361--376 © Elsevier/North-Holland Biomedical Press
DOSE--RESPONSE RELATIONSHIPS AND R.B.E. VALUES OF DOMINANT LETHALS INDUCED BY X-RAYS AND 1.5-MeV NEUTRONS IN P R O P H A S E - 1 0 0 C Y T E S AND IN MATURE SPERM OF THE TWO-SPOTTED SPIDER MITE Tetranycbus urticae KOCH (ACARI, TETRANYCHIDAE)
A.M. FELDMANN Association Euratom-ITAL, Postbox 48, Wageningen (The Netherlands) (Received 9 November 1977) (Accepted 23 March 1978)
Summary Mature sperm and prophase-1 oocytes of Tetranychus urticae Koch were irradiated with 250-kVp X-rays or 1.5-MeV fast neutrons. The X-ray doses ranged from 0.5 to 24.0 krad, and those of the fast neutrons from 0.1 to 16.0 krad. The genetic endpoint measured was lethality, expressed in the stages from egg to adulthood in the F, progeny. The frequency of recessive lethals in female germ cells was estimated by comparing survival of fertilized versus unfertilized F1 eggs, after irradiation with the same dosage. X-Rays induce dominant lethals in prophase-1 oocytes by the action of both single hits on single targets and multiple hits on multiple targets. 1.5-MeV neutrons induce these effects predominantly by the action of multiple tracks o n multiple targets. Dominant lethals were induced in mature sperm by X-rays and by fast neutrons by the action of both single hits on single targets and multiple hits on multiple targets. Both for prophase-1 oocytes and for mature sperm the low R.B.E. value corresponded with the relatively large multiple-target component of induction of dominant lethals by fast neutrons. The nature of dominant lethality in relation to the kinetochore organization of the chromosome is discussed. A non-linear trend in the dose--effect relationship was observed for both X-rays and fast neutrons for the estimated frequency of recessive lethals induced in prophase-1 oocytes. X-Rays were more effective than neutrons in inducing recessive lethals in prophase-1 oocytes at doses lower than 3 krad.
Introduction
Tetranychus urticae (Koch) is an economically important pest on a variety of crops. Genetic control has been considered as an alternative to conventional
362 control by pesticides [ 19,26,40]. The release of irradiated males or genetically altered males into a pest population requires a basic knowledge of the radiobiological properties of the species. Some radiobiological work on T. urticae has been published [20,26,28], b u t none of these reports gave adequate dose-response relationships for the induction of dominant and/or recessive lethals. A sterilizing dose of ~/-rays was reported, for both adult males and adult females, of 20--32 krad [20,26]. The F1 progeny from males irradiated with substerilizing doses showed a high extent of heritable partial sterility. The high male-sterilization dose and the inheritance, in a high frequency, of factors that cause meiotic p r o d u c t lethality in the F, progeny are in general observed in insects with holokinetic chromosomes [21]. The chromosomes of T. u rticae are of the holokinetic tpe [32,37], i.e. the kinetic activity of a chromosome is not localized but is spread diffusely over the chromosome. Information on the induction of lethals by ionizing radiation in germ cells of insects with holokinetic chromosomes is meagre compared with that available on species with monokinetic chromosomes. In investigations on the induction of dominant lethals in metaphase-1 oocytes and mature spermatozoa of B o m b y x mori, the dose--effect relationships were different from those observed for similar stages in Drosophila [3,4,15]. The present investigation was designed to examine, in spider mites, the dose--effect relationships for the induction of dominant lethals in prophase-1 oocytes and mature spermatozoa for X-rays and fast neutrons and to assess the relative biological effectiveness of neutrons. Materials and m e t h o d s Strain. The strain of Tetranychus urticae has been reared in the laboratory for a b o u t 5 years. The mites were reared at 28°C and 50--70% relative humidity on detached bean-leaf punches on c o t t o n wool soaked in tap water. Irradiation. The dose rate for both X-rays and fast neutrons was 100 tad per min. X-Rays were produced by a 250/25 Philips deep-therapy apparatus, operating at 250 kVp and 15 mA. No additional filtering was used (HVL = 0.3--0.4 mm Cu; Ee~t in keV = 50--55 [35]. Fast neutrons, with a mean energy of 1.5 MeV, were delivered by the irradiation facility of the Biological and Agricultural Reactor at Wageningen (The Netherlands}. The dose rate was measured by both an acetylene and a muscle-tissue-equivalent ionization chamber; the ~/contamination was 4.4 rad/min, measured with a magnox--argon ionization chamber [14]. Oocyte irradiation and sampling. 100 one-day-old adult virgin females were placed together on 2 bean leaf discs (diam. 3 cm) and irradiated on a rotating table. Immediately after the treatment, 50 females were mated to untreated, 1-day-old adult virgin males in 10 groups of 5 females and 5 males on separate leaf discs. The males were removed after 1 h; and 24 h after the irradiation treatment the females were transferred, in groups of 5 females or individually (indicated in the text where necessary), to fresh leaf punches on which they produced eggs for 24 h° The eggs collected in this way had been irradiated during the diffuse stage of meiotic prophase-1 [ 3 3 , 1 7 ] . The remaining 50 virgin females were also divided into 10 groups of 5 females, b u t were n o t mated.
363
Eggs from irradiated, virgin females were collected in the same way as eggs from irradiated then mated females. In each egg sample, the number of eggs and the number of adult F1 females and/or FI males (males only, concerning progenies from virgin females) developing from the egg sample, were counted. Sperm irradiation and sampling 50 adult virgin males, 0--24 h old, were irradiated in the manner described under "oocyte irradiation". Immediately after irradiation they were mated, in groups of 5, to 5 virgin females for 1 h only. In this way, sperm was collected which had been irradiated as mature sperm [34]. The effect of radiation was studied in progenies produced 24--48 h after mating, from single females, by counting in each sample the number of eggs and the number of resulting adult F1 females and F1 males. The adult F~-survival data were then used for calculating the frequency of germ cells bearing at least one dominant lethal, expressed at any stage of development. Calculation of the effect o f oocyte irradiation on survival (a) Dominant lethals T. urticae reproduces by arrhenotokous parthenogenesis, i.e. fertilized females produce both fertilized (diploid) and unfertilized (haploid) eggs. The haploid eggs develop into males and the diploid eggs develop into females. Unfertilized females produce only haploid eggs [8,12,37]. Such arrhenotokous parthenogenesis offers a special experimental condition for assessing the frequency of both dominant and recessive lethals in the F~ generation after oocyte irradiation [5,38]. The ratio of the number of adult F, males (My) to the number of eggs (Ev) laid by irradiated unmated (virgin) females provides a measure of survival of Fl haploids (Sh): Sh -
Mv
(I)
E~
The number of haploid eggs (Eh) in a mixture of haploid and diploid eggs (E), produced by irradiated then mated females, was calculated from the number of adult FI males (M) and the survival of FI haploids from unmated females irradiated with the same dose: M Eh -
Sh
(2)
Thus the survival of F~ diploids (Sd) was calculated from: F E -- E h F = number of adult F, females (diploids) E = number of eggs produced by irradiated then mated females. Substitution of (2) in (3) results in: F Sd M E---Sh Sd - - -
(3)
(4)
364 This formula allows the use of the numbers of FI eggs, F~ males and F, females, which have been determined in the experiment, and is a simplification of the formula used in the estimation of rates of survival in Habrobracon [5,38].
(b ) Recessive lethals The frequency of genomes with at least one recessive lethal (R), expressed at any stage of development and induced in irradiated oocytes, is derived from the ratio of the survival of F~ diploids (Sd) and F~) haploids (Sh) [5] : R = 1
Sh
(5)
Sd
Calculation o f the effect o f sperm irradiation on survival Formula (4) was used for calculating the survival of F, diploids from irradiated males, in which Sh was given by the survival of F~ progenies from unmated, unirradiated virgin females otherwise collected and treated in the same way as in the irradiation series. Survival curves The survival data were fitted to models of the following types. (1) S = 1 - - ( 1 -- e-KD)N (The model of A t w o o d and Norman [6].} (2) S = e - K I " D ( 1 - - ( 1 - - e - K 2 " D ) N) (The general model of King and Sankaranarayanan [34,36].) S = survival at dose D; KI = sensitivity of the one-target component; K and K2 = the sensitivity of the N-target component. The first model describes a process in which survival is determined by the hit of N targets by different ionization tracks. Model 2 incorporates the action of two processes, one requiring a single hit on a single target and the other requiring one hit on each of N targets. In model 1, two parameters, and in model 2, three parameters, have to be calculated from the experimental data. The statistical analysis of the survival data that were involved in the fitting of the data to models 1 and 2 were performed b y Prof. F.E. Wiirgler at his computer facility in Ziirich [35,2,34]. The best-fit curves, presented in the figures, are drawn according to model 2. This was justified for comparison purposes because: (1) most of the dose-response curves on insects follow this model; (2) the fit of the survival data to model 2 was satisfactory in general; and (3) the conclusions following model 2 were n o t different from those in which model 1 was used. Results
Con trols The experiments were performed during the years 1973--75. Control experiments were done at the beginning and the end o f this period. The survival frequencies of F, haploids, calculated from the different experiments, were independent of the date (0.5 ~ p ~ 0.1). The data on the survival frequency of F1 haploids from the different experiments were pooled, and a mean survival frequency of Fl haploids was calculated (Sh -- 0.961 ± 0.010). This mean sur-
365 viral frequency of F~ haploids was used for the calculation of the survival frequency of F~ diploids in the different control experiments. The calculations resulted in a h o m o g e n e o u s set of data on the survival frequency of F~ diploids (0.9 > p > 0.5). Pooling of all the data from the different control experiments on diploid survival resulted in a mean value for F,. diploid survival of SD = 0.944 + 0.022.
Oocyte radiation with X-rays and fast neutrons (Tables 1, 2 and 3 and Figs. 1 and 2) The survival data, shown in the figures, were corrected by the survival of the control [1].
(a) Survival of F1 haploids and F, diploids The fit of the survival data of F1 haploids to models 1 and 2 was rather poor, both for X-rays and for neutrons, whereas the fit of the survival data of F1 diploids to both models, was rather good (Table 3). The best-fit linear components of induction of lethality were n o t significantly different from zero both for X-rays and for neutrons, except the fitted K1 value of the X-ray-survival data of F1 haploids. The shapes of the curves on a semi-log plot suggest that the responses were non-linear, even for neutrons. The higher effectiveness of neutrons was due to the relative increase of the supralinear component, according to the best-fit parameters of model 2 (see upper part of Table 3). The survival curves of F~ haploids fall more steeply than those for F~ diploids, which is further substantiated by the calculated doses for 80 and 20% survival of F1 haploids and F~ diploids (Table 3). This observation, together with the poor fit of the survival of F~ haploids to a model, suggests that the mortality of F1 haploids has a broader causal base, i.e. dominant and recessive lethals, whereas the mortality of F~ diploids is determined by the frequency of dominant lethals only. The R.B.E. of neutrons was studied by calculating the ratio of t h e dose of X-rays and neutrons at two levels of survival, i.e. 80 and 20% (Table 3). The R.B.E. was independent of the model u s e d and was dose dependent, i.e. 1.3 and 1.7 at 80% surv;.val and 1.5 and 1.4 at 20% survival for F, haploids and diploids respectively. The fitted target numbers, N, of models 1 and 2, for the estimated survival data of F1 haploids, were significantly larger than 1 and n o t significantly different from 2, both for X-rays and fast neutrons. However, the standard errors were large, as a consequence of which the effect of neutrons on the target number, in comparison with X-rays, cannot be studied. The target numbers of Fl-diploid survival for model 1 were significantly larger than 2 and they did n o t differ from each other for the X-ray and neutron data. The target numbers of model 2 for F,
(b) The induction of recessive lethals by X-rays and fast neutrons in prophase-1 oocytes The estimated frequencies of recessive lethals, induced by X-rays and fast neutrons in prophase-1 oocytes, are given in Tables 1 and 2. The standard errors were in general high because of the high number of parameters, having their
6 28 a 52 a
49 a 6 20 a 46 a
2.0 3.0 4.0
• 6.0 8.0 8.0 8.0
515 261 220 431
331 262 531
1500 370 504
133 19 13 23
250 145 255
1441 348 457
0.258 0.073 0.059 0.053
± + ± ±
0.023 0.005 0.013 0.012
0.755 + 0.030 0 . 5 5 3 -+ 0 . 0 5 4 0.480 + 0.026
0.961 + 0.010 0.941 + 0.014 0.907 + 0.017
48 a 10 20 a 50 a
10 48 a 53 a
103 10 10
511 536 215 454
642 483 499
2401 547 735
Number of eggs
i00 64 12 35
378 171 194
1441 361 505
0.340 0.194 0.073 0.442
-+ 0 . 0 5 7 + 0.048 ± 0.023 ± 0.649
0.879 + 0.024 0.874 + 0.170 0.631 + 0.047
0.944 + 0.022 0 . 9 4 7 _+ 0 . 0 3 1 0.932 + 0.018
56 15 3 20
160 159 92
848 156 175
Number of adults
Number of adults
Survival + S
F 1 haploids
F 1 diploids
AS 0--24-h-
0.240 0.625 0.191 0.879
± + ± ±
0.167 0.101 0.341 0.198
0 . 1 4 1 _+ 0 . 0 5 3 0 . 3 6 7 -+ 0 . 1 7 4 0.239 ± 0.084
0.000 + 0.014 0.007 + 0.039 0.027 ± 0.030
Frequency of recessive lethals + S
a Data were collected on progenies from single females, whereas in the other experiments the data were collected on progenies from groups of 5 females.
49 7 9
Survival + S
Number of trials
Number of adults
Number of trials
Number of eggs
Progeny of irradiated and mated females
O F T e t r a n y c h u s urticac ( K o c h ) , I R R A D I A T E D
Progeny of irradiated virgin females
0.0 0.5 1.0
Dose (krad)
THE EFFECTS OF X-RAYS (100 rad/min; 250 kVp) ON THE PROGENY OF UNMATED FEMALES OLD ADULT VIRGINS, EGGS WERE IRRADIATED IN EARLY MEIOTIC PROPHASE 1
TABLE 1
o~
10 9 10
53 a 9 52 a 20 a
0.5 1.0 2.0
3.0 4.0 6.0 8.0
609 465 518 210
509 532 501
1500 659 595
223 116 17 0
468 493 346
1441 636 557
0.366 ± 0.022 0.250 + 0.015 0.033 ± 0.008
0.919 ± 0.017 0 . 9 2 7 -+ 0 . 0 0 8 0.691 ± 0.026
0.961 ± 0.010 0.965 + 0.014 0.936 ± 0.020
53 a 10 46 a 19 a
9 9 10
103 10 10
584 578 410 218
531 506 493
2401 659 624
Number of eggs
190 149 23 1
339 346 249
1441 458 431
0.485 ± 0.037 0.340 ± 0.034 0.138 ± 0.097
0 . 9 4 1 -+ 0 . 0 1 8 0 . 9 2 7 -+ 0 . 0 1 9 0 . 7 0 3 -+ 0 . 0 4 0
0 . 9 4 4 -+ 0 . 0 2 2 0 . 9 6 5 -+ 0 . 0 2 0 0.993 ± 0.020
62 35 8 0
157 123 96
893 178 178
Number of adults
Number of adults
S u r v i v a l +- S
F 2 haploids
F 1 diploids
0.201 ± 0.091 0.267 + 0.093 0.763 ± 0.204
0.023 ± 0.032 0.000 ± 0.023 0.018 + 0.074
0.000 + 0.014 0.000 ± 0.028 0.058 ± 0.032
Frequency of recessive lethals ± S
O F Tetranychus urticae ( K o c h ) ,
a D a t a w e r e c o l l e c t e d o n p r o g e n i e s f r o m single f e m a l e s , w h e r e a s in t h e o t h e r e x p e r i m e n t s t h e d a t a w e r e c o l l e c t e d o n p r o g e n i e s f r o m g r o u p s o f 5 p a r e n t a l f e m a l e s .
49 10 10
S u r v i v a l -+ S
Number of trials
Number of adults
Number o f trials
Number o f eggs
Progeny of irradiated and after that mated females
Progeny of irradiated virgin females
0.0 0.1 0.2
Dose (krad)
THE EFFECT OF FAST NEUTRONS (100 rad/min; 1.5 MeV) ON THE PROGENY OF UNMATED AND MATED FEMALES IRRADIATED AS 0--24-h-OLD ADULT VIRGINS, EGGS WERE IRRADIATED IN EARLY MEIOTIC PROPHASE 1
TABLE 2
0.006 7.8 X 10 -5
OF MATURE
0.5 X 10 -3 0,670
0.3 X 10 -3 0.132
P
0.158 + 0.012 0.251 + 0.027
SPERM
0.777 + 0.061 0.574 + 0.050
0.470 + 0.031 0.488 + 0.052
K
= 1---(I~e--KD) N
PROPHASE-100CYTES
Model 1:8
OF EARLY
1.148 -+ 0.104 1.460 + 0.189
5.346 + 0.976 3.869 + 0.603
3.453 + 0.646 6 . 3 8 7 -+ 1 . 7 2 6
N
1.7 1.6
1.7 1.9
2.1 3.1
10.9 7.8
4.1 5.0
5.9 6.9
0.014 0.052
4 • 10 -4 0.500
0.005 0.239
0.117 _+ 0.039 0.150 -+ 0.012
0.019 + 0.036 4 X 10 -5 -+0.039
0 . 0 6 7 -+ 0 . 0 2 7 0.027 + 0.014
0.063 ± 0.029 0.260 ± 0.088
0 . 7 9 2 -+ 0 . 0 9 1 0.574 + 0.066
0 . 4 7 5 -+ 0 . 0 4 2 0.548 + 0.074
2.044 +- 2.119 10.361 _* 10.074
6.194 + 2,694 3.871 + 1.358
6.129 ± 2.185 1 1 . 7 5 7 _+ 5 . 5 7 5
N
1.7 1.5
1.7 1.9
2.2 3.3
nychus urticae K o c h w i t h X - r a y s o r f a s t n e u t r o n s , o f m o d e l s 1 a n d 2 (see t e x t ) .
Tetra
11.3 8.4
4.1 5.0
6.1 6.9
20%
Dose (krad)
80%
20%
K2
80%
K1
at S =
P
e--KID[1-"(I~e--K2D) N]
at S =
Dose (krad)
M o d e l 2: S =
Best-fit parameters of estimated data points of survival of F 1 haploids and F 1 diploids, after irradiation of early prophase-1 oocytes and mature sperm of
X-rays Neutrons
IRRADIATION
F l haploids F I diploids
Neutrons
F 1 haploids F 2 diploids
X-rays
IRRADIATION
TABLE 3
c.o o~ 00
369 I00- ~-~ -'Li----... i......~ ~ ".. ~
.--. X-rays ~---A neutrons
50
\ k \
2O
\
-S
\ \
>
\
>
10-
\ \
u L i1) e~ 5-
f,g
1
2
3
4 dose ( krad
5
6
7
8
)
Fig. 1. The effect of 250okVp X-rays and of 1.5oMeV fast neutrol~ on the 811ryival of F 1 diploids from irradiated early p r o p h a s e - 1 eggs o f Tetranychus urticae K o c h . F e m a l e s w e r e irradiated as l - d a y - o l d adult virgins and m a t e d , i m m e d i a t e l y after t h e irradiation t r e a t m e n t , t o u n t r e a t e d m a l e s . F e r t i l i z e d eggs w e r e s a m p l e d 24---48 h a f t e r irradiation. D a t a w e r e f i t t e d t o t h e m o d e l o f King and S a n k a r a n a r a y a n a n (see text).
own errors, which were involved in the calculations. A non-linear trend of the dose--effect relationship was observed for both X-rays and fast neutrons. X-Rays were more effective than neutrons in inducing recessive lethals in prophase-1 oocytes at doses lower than 3 krad. The X-ray curve saturated at higher radiation doses, mainly caused by the standard errors of the respective survival frequencies of F, haploids and F~ diploids being large at higher radiation doses. The differences between the survival frequencies of F1 haploids and FI diploids become less significant at higher doses, therefore the ratio of Sd and Sh approaches 1.
Sperm irradiation with X-rays and fast neutrons The effect of X-rays or fast neutrons on the survival of F1 diploids, after irradiation of mature sperm, is given in Table 4. The parameters fitted to the different models and the graphs are shown in the lower part of Table 3 and Fig. 3 respectively. Table 3 shows that the fit of both the X-ray data and of the
370
•- - ,
X- rays
4----A
neutrons
\
\ \
\
_20> >
5-
\
fig
i
\
2
I
l
]
I
1
2
3
4
i
5 dose (krod)
i
i
i
6
7
8
Fig. 2. T h e e f f e c t o f 2 5 0 - k V p X - r a y s a n d of 1 . 5 - M e V fast n e u t r o n s o n t h e survival o f F I h a p l o i d s f r o m i r r a d i a t e d p r o p h a s e - 1 eggs o f Tetranychus urticae. F e m a l e s w e r e i r r a d i a t e d as 1 - d a y - o l d a d u l t virgins a n d r e m a i n e d u n m a t e d a f t e r t r e a t m e n t . Eggs w e r e s a m p l e d 2 4 - - 4 8 h a f t e r i r r a d i a t i o n . D a t a w e r e f i t t e d t o t h e m o d e l of King and Sankaranarayanan.
neutron data to model 2 was better than to model 1. The N values for X-rays and fast neutrons were n o t significantly different from each other either for model 1 or 2. The K factors for X-rays and neutrons of model 1 were significantly different from each other (p < 0.005). The best-fit: parameters o f model 2 show that the sensitivity to the induction of the linear c o m p o n e n t (K,) o f dominant lethality per kilorad X-rays was n o t d i f f e r e n t from the K, value for fast neutrons (0.5 > p > 0.1). The greater effectiveness of fast neutrons for the induction of dominant lethals in sperm is predominantly caused b y the increase o f the supralinear c o m p o n e n t of dominant lethality per kilorad neutrons. The shape of the X-ray curve is non-linear, and the shape of the neutron curve shows the predominance of the supralinear component. The K2 value for neutrons is s i ~ i f i c a n t l y larger than the K2 value for X-rays (0.05 > p > 0.01; cf. m o d e l 2, Table 3}. Table 3 shows that the ratio of the doses for X-rays and neutrons at 80 and 2 0 ~ survival is the same for the different models (i.e. 1.1 at 8 0 % survival and 1.4 at 20% survival). The R.B.E. is dose
ON THE SURVIVAL
OF F 1 DIPLOIDS AFTER IRRADIATION
OF MATURE SPERM
56 10 52
10 10 10
10 54 22
47 52
0.5 1.0 1.0
1.5 2.0 4.0
8.0 8.0 12.0
16.0 24.0
536 705
637 659 241
630 767 655
685 426 510
2401 563 627
Number of eggs a
a Including haploid F l progeny.
103 10 10
Number of trials
0.0 0.2 0.5
Dose . (krad)
31 5
147 160 39
356 404 266
459 247 317
1441 373 370
Number of adults
F 1 diploids
X-rays (100 rad/min; 250 kVp)
0.083 ± 0.015 0 . 0 1 0 -+ 0 . 0 0 6
0.319 ± 0.030 0 . 3 4 2 -+ 0 . 0 2 6 0.229 ± 0.031
0.745 ± 0.022 0 . 6 9 3 -+ 0 . 0 2 8 0.556 ± 0.028
0 . 9 5 4 -+ 0 . 0 1 5 0 . 8 5 9 -+ 0 . 0 2 9 0 . 8 7 5 -+ 0 . 0 2 6
0.944 ± 0.022 0.924 ± 0.029 0.886 ± 0.024
Survival ± S
on the F 1 progeny which was produced 24--48 h after mating.
12.0 16.0
2.0 4.0 8.0
1.0 2.0 2.0
0.5 0.5 1.0
0.1 0.2
Dose (krad)
24 25
55 10 19
50 10 53
10 54 10
10 10
Number of trials
338 355
691 633 239
493 641 711
620 786 535
400 557
Number o f eggs a
F a s t n e u t r o n s ( 1 0 0 r a d / m i n ; 1.5 M e V )
21 2
234 247 29
165 245 336
341 457 280
213 340
Number of adults
F 1 diploids
0.111 ± 0.024 0.008 ± 0.006
0.707 ± 0.029 0.527 ± 0.024 0 . 1 7 8 -+ 0 . 0 3 3
0.676 ± 0.050 0 . 6 9 5 -+ 0 . 0 3 5 0.694 ± 0.025
0.884 ± 0.023 0.876 ± 0.019 0.836 ± 0.049
0.917 ± 0.034 0.958 ± 0.026
Survival frequency ± S
Males w e r e i r r a d i a t e d as 1 - d a y - o l d a d u l t v i r g i n s a n d m a t e d i m m e d i a t e l y a f t e r i r r a d i a t i o n w i t h u n t r e a t e d v i r g i n f e m a l e s d u r i n g 1 h. T h e s u r v i v a l d a t a w e r e e s t a b l i s h e d
O F Tetranychus urticae ( K o c h )
T H E E F F E C T O F 250 k V p X - R A Y S A N D 1.5 MeV F A S T N E U T R O N S
TABLE 4
372
IO0 ~ h ~ "
.I. ~
"ij~
x rays
~---a neutrons
l',
cl 5
\\\
\\\\
2
fig3
\\
\
' l, 5
10
I 20
25
dose ( k r a d )
Fig. 3. The e f f e c t of 2 5 0 - k V p X-rays a n d 1.5-MeV fast n e u t r o n s o n the survival of F 1 d i p l o i d s a f t e r irrad i a t i o n of m a t u r e s p e r m o f Tetranychus urticae. Males were i r r a d i a t e d as 1 - d a y - o l d a d u l t virgins a n d m a t e d i m m e d i a t e l y a f t e r i r £ a d i a t i o n w i t h u n t r e a t e d virgin f e m a l e s d u r i n g 1 h. Survival was e s t a b l i s h e d o n d i p l o i d F 1 p r o g e n y p r o d u c e d 2 4 - - 4 8 h a f t e r m a t i n g . D a t a were f i t t e d to the m o d e l of King a n d Sankaxanarayanan.
Discussion
The induction o f dominant lethals in sperm and oocytes o f T. urticae Regarding the established properties of holokinetic and monokinetic chromosomes, some causes o f dominant lethality can be considered to be related more specifically to one of the t w o types of c h r o m o s o m e than to the other. First, fragmentation of chromosomes, due to radiation-induced breaks, is a major cause of dominant lethality in species with monokinetic chromosomes. This is n o t so in species with holokinetic chromosomes, since broken chromosomes, especially when they are n o t fragmented into very small pieces, can pass through both mitotic [16,11,10,13,23,25,30,22] and meiotic divisions w i t h o u t loss of chromosomal material [30,27,23,17]. It is generally assumed that chromosome breaks are caused by single ionization tracks and these breaks contribute to the linear c o m p o n e n t of induction of dominant lethality. Second, fragmentation of chromosomes, resulting from bridge formation, caused by radiationinduced sister-strand union, is an important cause of dominant lethality in organisms with monokinetic chromosomes [21,9]. Radiation-induced sisterstrand union is rare in cells with holokinetic chromosomes and attributes in a minority to dominant lethality [11,22]. Sister-strand union due to misrepair of a chromosome break is initially caused by single ionization tracks and is part
373 of the contribution to the linear c o m p o n e n t of induction of dominant lethality. Third, the induction of symmetric reciprocal translocations in monokinetic chromosomes is accompanied in equal frequencies by the formation of dicentric and acentric chromosomes which usually cannot regularly pass through cell division and in this way lead to dominant lethality. In species with holokinetic chromosomes all analogous reciprocal reconfigurations are viable, without the complications associated with dicentrics and acentrics [10,7,30,]. The induction of autosomal translocations b y X-rays in mature sperm of Drosophila is based on both single- and multiple-electron tracks, and the relative linear contribution decreases from a b o u t 100X the supralinear contribution at 50 tad to 1X at 5000 rad [ 18]. Neutrons induce autosomal translocations in mature Drosophila sperm b y the action of single p r o t o n tracks [18]. When it is assumed that the reciprocal exchanges leading to the formation of dicentrics and acentrics have the same kinetics of induction, then these causes of dominant lethality are to a large extent induced by single-hit phenomena. In the summarizing of these differences, it m a y be proposed that the induction by X-rays and by fast neutrons of dominant lethality in species with holokinetic chromosomes results in a gross reduction of the linear c o m p o n e n t in comparison with species with monokinetic chromosomes. This was true for the germ cells of T. urticae studied, irrespective of the model used. Both for X-rays and neutron irradiation of prophase-I o o c y t e s of T. urticae, the contribution of the linear c o m p o n e n t to dominant lethality of FI diploids was negligible. For example, the supralinear contribution to dominant lethality for X-ray-irradiated oocytes of T. urticae showed a dose-dependent increase in relative importance: at 1 krad, the supralinear c o m p o n e n t contributed only 0.4% to dominant lethality, b u t at 3, 6 and 9 krad the contribution was 50.2, 86.1 and 91.2% respectively. The shape of the neutron curves shows, for both o o c y t e and sperm irradiation, the predominance of the supralinear components. The radiosensitivity of prophase-1 oocytes of Drosophila was n o t so strikingly different from that of T. urticae as could be expected from the general assumption that species with holokinetic chromosomes are less radiosensitive than species having monokinetic chromosomes [22]. The X-ray doses, resulting in 80% survival, after irradiation of prophase-1 oocytes of Drosophila and T. urticae, were 1.6 krad and 3.3 krad respectively, b u t the doses for giving 20% survival were 6.5 krad and 6.9 krad respectively. The X-ray doses, resulting in 80% survival, after irradiation of mature sperm of Drosophila and T. urticae, were 0.7 krad and 1.7 krad respectively, and the doses for giving 20% survival were 3.6 krad and 11.3 krad respectively. Thus the general assumption that a good correlation exists between the distribution of kinetic properties along the chromosome and differences in species radio-resistance, is at least doubtful. A corresponding conclusion was drawn by LaChance [21], who observed that hemipteran species, having holokinetic chromosomes, can be sterilized by fairly low doses of irradiation to sperm. There is suggestive evidence that low doses of neutrons on prophase-1 o o c y t e s have a positive effect on survival of FI diploids. Up to 1 krad neutrons, no significant negative effect on survival was observed. On the contrary, at 200 rad, the survival was 0.993 + 0.020 in contrast with a control value of 0.944 + 0.022. Corresponding observations were made by Meyer and Abrahamson [24]
374 on sex-linked recessive lethals induced by X-rays in Drosophila oogonia and by N e w c o m b e [29] on 7-ray-induced mortality in sperm of the rainbow trout. The interference of radiation-induced repair [24], over-compensating radiationinduced damage b y repair of natural background damage, may be partially responsible for the non-linear increase of recessive lethals after neutron irradiation of T. urticae oocytes. No similar effect was observed on the survival of F1 haploids after neutron irradiation of oocytes or on the survival of F~ diploids after X-ray or neutron irradiation of T. urticae sperm. R .B.E. Neutrons induce mortality in prophase-1 oocytes of T. urticae, principally by the action of multiple tracks on multiple targets. In agreement with this observation is the low R.B.E. value, indicating that the radiation-induced lesions, which have to interact to produce a dominant-lethal effect, were seld o m produced by the same ionization track. In mature sperm the kinetics of induction of dominant lethality is different from that of prophase oocytes. In mature sperm the relative contribution of the linear c o m p o n e n t for induction of dominant lethality is predominant both for X-rays and for fast neutrons. The higher effectiveness of neutrons compared with X-rays was, according to the best-fit parameters of model 2 (Table 3), caused by the relative increase of the supralinear c o m p o n e n t of the sensitivity to dominant lethality. Thus 1.5 MeV neutrons caused a higher frequency of single hits on single targets, and part of the lesions induced by different proton tracks resulted in dominant lethality after interaction with each other. Since the linear c o m p o n e n t of the sensitivity to dominant lethality of X-rays on sperm was n o t significantly different from that of neutrons, it is concluded that the distance between interacting lesions coincides with the distance between ionizations within an electron track by which denser ionizing radiation is only more effective by the associated production of f-rays with a larger range and LET than many of the electrons produced by X-rays. The induction o f recessive lethals in early prophase-1 oocytes o f T. urticae A numerical approach is used for the assessment of the frequency of recessive lethals induced in prophase-1 oocytes of T. urticae. The percentage of recessive lethals in whole genomes is estimated by comparing the survival of haploids and diploids with formula (5). But only little information is available on possible differential effects of segmental aneuploidy on the survival of the different sexes of T. urticae. There are indications that segmental aneuploidy is as devastating in the male as in the female, since the survival o f haploids and of diploids, produced by a translocation heterozygous female of T. urticae, is the same (Feldmann, unpublished). The trend for the induction of recessive lethals is non-linear, b u t the standard errors of the estimates of recessive lethals, induced in prophase-1 oocytes of T. urticae {Tables 1 and 2), are t o o large to warrant definitive conclusions. More information a b o u t the induction of recessive lethals in oocytes of T. urticae have to await more accurate determinations or the use of other techniques.
375
Acknowledgements The constructive discussions with Drs. Robinson, Leenhouts, Chadwick and Sybenga and the helpful suggestions of Drs. LaChance and Sankaranarayanan are gratefully acknowledged. Prof. Wiirgler and Dr. Barendregt are thanked for their help in the calculations. Elly Broecks-Doorn is gratefully thanked for her enthusiasm and expert technical assistance. References 1 Abbot, W.S., A m e t h o d for c o m p u t i n g the effectiveness of an insecticide, J. Econ. Ent omol ., 18 (1925) 265--267. 2 Alper, T., N.E. Gillies and M.M. Elking, The sigmoid survival curve in radiobiology, Nature (London), 186 (1964) 1062--1063. 3 Astaurov, B.L., Versuche ~ibar experimentelle Androgenese u n d Gynogenese beim Seidenspinner ( B o m b y x mori, L.). Biol. Zhur., 6 (1937) 3--50. 4 Astaurov, B.L., and S.L. Frolowa, Kiinstlich ausgel6ste Mutationen beim Seidenspinner (Bombyx mori, L.). Biol. Zhur., 4 (1935) 861---891. 5 A t w o o d , K.C., R.C. Borstel and A.R. Whiting, An influence of p l o i d y on the t i me of expression of d o m i n a n t lethal m u t a t i o n s in Habrobracon, Genetics, 41 (1956) 804---813. 6 A t w o o d , K.C., an d A. Norman, On the i n t e r p r e t a t i o n of mul t i -hi t survival curves, Proc. Natl. Acad. Sci. (U.S.A.), 35 (1949) 696--709. 7 Bauer, H., Die kinetische Organisation der Lepidoptere n Chromosomen, Chromosoma, 22 (1967) 101--125. 8 Boudreaux, H.B., Biological aspects of some p h y t o p h a g o u s mites, Ann. Rev. Entomol., 8 (1963) 137--154. 9 Borstel, R.C. von, Effects of radiation on germ cells of insects: d o m i n a n t lethals, gamete inactivation and gonial-cell killing, in: R a d i a t i o n and Radioisotopes applied to Insects of Agricultural Import a nc e , IAEA, Vienna, 1963, STI/PUB/74, pp. 367--385. 10 Brown, S.W., and L.J. Wiegmann, Cytogenetics of the me a l ybug Planococcus citri (Risso) (Homoptera: Coccoidea): Genetic markers, lethals and c h r o m o s o m e rearrangements, Chromosoma, 28 (1969) 255--279. 11 Brown, S.W., and W.A. Nelson-Rees, R a d i a t i o n analysis of a lecanoid genetic system, Genetics, 46 (1961) 9 8 3 - - 1 0 0 7 . 12 Cagle, L.R., Life history of the t w o - s p o t t e d spider mite, Virg. Agr. Exp. Stn., Techn. Bull., 113 (1949) 3--31. 13 de Castro, D., A. Camara and N. Malheizos, X-Rays in the c e nt rome re probl e m of Luzula purpurea, Link., Genet. Ib~r., 1 (1949) 48--54. 14 Chadwick, K.H., W.F. Oosterheert and K.J. Puite, Comparison b e t w e e n s p e c t r o m e t r y , ionisation c h a m b e r and threshold d e t e c t o r systems for fast-neutron dos i me t ry in the subeore facility of a swimmin g-p ool r e a c t o r , in: N e u t r o n Irradiation of Seeds II, Technical R e port s Series No. 92, IAEA, Vienna, 1968, pp, 123. 15 Chikushi, H., Fatal action of X-rays on Silkworm eggs, Bull. Sci. Fac. Agr. Kyushu Imp. Univ., 8 (1938) 155--162. 16 Cooper, R.S., E x p e r i m e n t a l d e m o n s t r a t i o n of h o l o k i n e t i c c h r o m o s o m e s and of differential radiosensitivity during oogenesis, in the grass mite, Siteroptes graminum (Reuter), J. Exp. Zoo1., 182 (1972) 69--94. 17 Feiertag-Koppen, C.C.M., Cytological studies of the t w o-s pot t e d spider mi t e Tetranychus urticae Koch (Tetranychidae, Trombidiformes), I. Meiosis in eggs, Genetica, 46 (1976) 445--456. 18 Gonzalez, F.W., Dose--~esponse kinetics of genetic effects induced by 250 kVp X-rays and 0.68 MeV neu tro ns in m a t u r e sperm of Drosophila melanogaster, Mut a t i on Res., 15 (1972) 303--324. 19 Helle, W., New d e v e l o p m e n t s towards biological control of the t w o-s pot t e d spider m i t e by incompatible genes, PubL Org. Eur. PI. Protect. Paris, S~r. A, 52 (1969) 7--15. 20 Henneberry, T.J., Effects of g a m m a r a d i a t i o n on the fertility of the t w o - s p o t t e d spider m i t e and its progeny, J. Econ. E ntomol., 57 (1964) 672--674. 21 LaChance, L.E., Induced sterility in irradiated diptera and lepidoptera, in: Sterility Principle for Insect Control, I.A.E.A., Vienna, 1974, pp. 401--411 (Proc. Ser. STI/PUB: 377, 1975). 22 LaChance, L.E., and J.G. Riemann, D o m i n a n t lethal m u t a t i o n s in insects w i t h h o l o k i n e t i c chromosomes, I. Irradiation of Oncopeltus (Hemiptera: Lygaedae) sperm and oocytes, Ann. E n t o m o l . Soc. Am., 66 (1973) 813--819. 23 La Cour, L.F., The Luzula system analyzed by X-rays, Heredity, 6 (1952) 77--81.
376 2 4 M e y e r , H.W., a n d S. A b r a h a m s o n , P r e l i m i n a r y r e p o r t o n m u t a g e n i c e f f e c t s o f l o w X - r a y d o s e s in i m m a t u r e g e r m cells o f a d u l t D r o s o p h i l a f e m a l e s , G e n e t i c s , 6 8 , S u p p l . ( 1 9 7 1 ) 4 4 . 2 5 M o n t e z u m a - D e - C a r v a l h o , J., X - r a y i n d u c e d b r e a k a g e in c h r o m o s o m e s w i t h d i f f u s e c e n t r o m e r e s , in: E f f e c t s o f i o n i z i n g r a d i a t i o n o n seeds, P r o c . S y m p . K a r l s r u h e , 1 9 6 0 , I . A . E . A . , V i e n n a , 1 9 6 1 , p p . 2 7 1 - 277. 26 Nelson, R., Effects of gamma radiation on the biology and population suppression of the two-spotted s p i d e r m i t e , T e t r a n y c h u s urticae K o c h , P h . D . Thesis, Univ. Calif. Davis, 1 9 6 8 . 2 7 N e l s o n - R e e s , W . A . , N e w o b s e r v a t i o n s o n l e c a n o i d s p e r m a t o g e n e s i s in t h e m e a l y b u g , P l a n o c o c c u s cirri, Chromosoma, 14 (1963) 1--17. 2 8 N e l s o n , R . D . , a n d E.M. S t a f f o r d , E f f e c t s o f g a m m a r a d i a t i o n o n t h e b i o l o g y a n d p o p u l a t i o n s u p p r e s s i o n o f t h e t w o - s p o t t e d s p i d e r m i t e , T e t r a n y c h u s urticae K o c h , H i l g a r d i a , 4 1 ( 1 9 7 2 ) 2 9 9 - - 3 4 1 . 29 Newcombe, H.B., "Benefit" and "harm" from exposure of vertebrate sperm to low doses of ionizing radiation, Health Phys., 25 (1973) 105--107. 3 0 N o r d e n s k i 6 1 d , H., A s t u d y o f m e i o s i s in t h e p r o g e n y o f X - i r r a d i a t e d L u z u l a p u r p u r e a , H e r e d i t a s , 4 9 , (1963) 33--47. 3 1 P i j n a c k e r , L.P., a n d L . J . D r e n t h - D i e p h u l s , C y t o l o g i c a l i n v e s t i g a t i o n s o n t h e m a l e r e p r o d u c t i v e s y s t e m a n d t h e s p e r m t r a c k in t h e s p i d e r m i t e T e t r a n y c h u s urticae K o c h ( T e t r a n y c h i d a e , A c a r i n a ) , N e t h e r l . J. Zool., 2 3 ( 1 9 7 3 ) 4 4 6 - - 4 6 4 . 3 2 P i j n a c k e r , L.P., a n d M . A . F e r w e r d a , D i f f u s e k i n e t o c h o r e s in t h e c h r o m o s o m e s o f t h e a r r h e n o t o k o u s s p i d e r m i t e T e t r a n y c h u s urticae K o c h , E x p e r i e n t i a , 2 8 ( 1 9 7 2 ) 3 5 4 . 33 Pijnacker, L.P., personal communication. 3 4 P o r t e r , E . H . , E l e c t r o n i c c o m p u t e r s a n d s u r v i v a l c u r v e s , Brit. J. R a d i o L , 3 7 ( 1 9 6 4 ) 6 1 0 - - 6 1 5 . 35 Puite, K.J., A thermolumineseence system for the intexcomparison of absorbed dose and radiation q u a l i t y o f X - r a y s w i t h a H V L o f 0 . 1 t o 3 . 0 m m C u , P h y s . M e d . Biol., 21 ( 1 9 7 6 ) 2 1 6 - - 2 2 5 . 3 6 S a n k a r a n a r a y a n a n , K . , T h e e f f e c t o f o x y g e n a n d n i t r o g e n p o s t - t r e a t m e n t s o n t h e m o r t a l i t y of D r o s o p h i l a eggs i r r a d i a t e d as s t a g e 7 o o c y t e s , M u t a t i o n R e s . , 7 ( 1 9 6 9 ) 3 5 7 - - 3 6 8 . 37 Schrader, F., Haploidie bei einer Spinnmflbe, Arch. Mikrosk. Anat. Entwicklungsmech., 97 (1923) 610--622. 3 8 W h i t i n g , A . R . , R . H . S m i t h a n d R . C . V o n B o r s t e l , M e t h o d s f o r r a d i a t i o n s t u d i e s d u r i n g o o g e n e s i s in H a b r o b r a c o n j u g l a n d i s ( A s h m e a d ) , in: E f f e c t s o f R a d i a t i o n o n M e i o t i c S y s t e m s , I . A . E . A . , V i e n n a , 1968, STI/PUB/173, pp. 201--208. 3 9 Wfirgler, F . E . , E l e c t r o n i c c o m p u t e r s a n d s u r v i v a l - c u r v e a n a l y s i s , Int. J. R a d i a t . Biol., 1 4 ( 1 9 6 8 ) 1 9 3 - 196. 4 0 Z o n , A . G . , a n d W.P.J. V a n O v e r m e e r , I n d u c t i o n o f c h r o m o s o m e m u t a t i o n s b y X - i r r a d i a t i o n in Tetran y c h u s urticae ( T e t r a n y c h i d a e ) w i t h r e s p e c t t o a p o s s i b l e m e t h o d o f g e n e t i c c o n t r o l , E n t o m o l . E x p . Appl., 15 (1972) 195--202.