F the mating activity and fertility in Ilrosophilo melanogaster. NovITSKY

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1 HERE 51-5 MATING AND FERTILITY PATTERN OF DROSOPHILA MELANOGASTER MALES EXPOSED TO DIFFERENT T E M PARATUR ES By INGERID KVELLAND INSTITUTE OF GENERAL GENETICS, UNIVERSITY OF OSLO, BLINDERN, NORWAY (Received June 14th, 1965) INTRODUCTION EW investigators have discussed the influence of cold treatment on F the mating activity and fertility in Ilrosophilo melanogaster. NovITSKY (1949) subjected males and females to low temperature (-5' c) for a longer period and found that as long as the flies survived they would mate and would therefore not show any infertility. IYENGAR and BAKER (1960) subjected young males to -8' C for 15 minutes and found a decrease of 66 percent in the number of offspring produced by the cold treated males conipared to control males. WEDVIK (1962) subjected three day old males to Oo C for 30 minutes and found a marked reduction of male fertility, commencing the second day after the treatment. The present paper presents data and discussion on the mating and fertility patterns of 1)rosophila melanogaster males that were given a cold treatment when respectively 0-2 hours and three days old. Material and methods All males used were unmated wild type taken from a Conton-S stock. The females were taken from a CinnCibrtF brown; ebony'' stock, and were three days old and virgin when used. The males were given a cold treatment immediately prior to mating by placing them for 30 minutes in a thin plastic tube immersed in ice water (0' E! l/2o C). After the cold treatment, the males were mated individually to 10 virgin non-treated females in tubes with ordinary cornmeal, agar-agar, molasse, medium. During several 12 hour periods, the males were inspected every tenth or twelfth minute and all mating pairs were isolated as soon as they were

2 MATING AND FERTILITY PATTERN 89 observed. When a copulation ended the inseminated female was transferred to a tube with fresh medium for egglaying, and the male was transferred back to his original mating tube. During the nights no observations were made, but the individual males were supplied with 10 virgin females at the end of each day s observation period. Every morning these females were transferred individually to tubes with fresh medium for egglaying, and the individual males were given 10 new virgin females in fresh tubes. The offspring from each mating of each individual male were counted on the 17th day after egglaying. The mating procedure used is the same as that described in detail by KVELLAND (1965). The experiments are divided into two groups labelled: Series 00 (males given the cold treatment when 0-2 hours old) and Series 30 (males given the cold treatment when three days old). Two experiments were carried out in Series 00, one over a period of 12 days, the other for eight days. The combined data are given in all figures, while one table gives data for individual males that were kept through the 12 days. One experiment was carried out in Series 30 and it lasted for 49 days. In all figures, data are given for the first 12 days and one table gives data for all 49 days. The control series: Series 022b (males 0-2 hours old, untreated) and Series 322a (males three days old, untreated) done by KVELLAND (1965) are used as control series also in the present investigation. EXPERIMENTAL RESULTS Figure 1 shows that if the cold treatment was applied to the males at an age of 0-2 hours, the treatment had no effect upon male mating frequency. However, if the cold treatment was applied to the males at an age of three days, the treatment had a marked reducing effect upon male mating frequency. Figure 2 shows that the cold treatment of three day old males also resulted in a high frequency of non-mating males; an increase to more than 50 percent on Days 1-5. In the control series (Series 322a) all males mated on Days 1-5. On Days 6 to 12, the proportion of nonmating males increased to more than 90 percent in Series 30, but only to 17 percent in Series 322a. Males exposed to the cold treatment when 0-2 hours old, on the contrary, showed on all days nearly the same proportion of non-mating males as did the young control males in Series 022b.

3 90 INGERID KVELLAND m 5- cn C E Series 022b A- A 4 1 Series 00 A -----A Serles 322a - a Series I n Mating periods in days Fig. 1. Average number of matings per mating male per 12 hour period P ' p----o

4 MATING AND FERTILITY PATTERN 91 i $ E 701 Series 022b A-A Series 00 A-----A Series 322a 0-0 Ser~es 30 O-----O Mating pcriods in days Fig. 3. Percentage of sterile males per 24 hour period. E o L * C Y 40- L * n 30 't - I / = sterility due to sterile matingr 0 I sterility due tenon-mating L Mating periods in days Fig. 4. Percentage of sterile males in Series 30.

5 92 INGERID KVELLAND 100- w- 80- L c 40- U,z M- m Mating periods in days Fig. 5. Percentage of sterile matings per 12 hour period. Series 022b A-A Series00 A-----A Series a Series Mating periods in days Fig. 6. Average number of females fertilized per fertile male per 21 hour period.

6 MATING AND FERTILITY PATTERN 93 Series 022b A- A Series00 A----- A Series 322a 0-0 Series I t Mating periods in days Fig. 7. Average number of offspring produced per fertile male per 24 hour period '\ Lries 022b A-A Seroes 00 A-----A Series 322a 0-0 Series Mating periods in days Fig. 8. Average brood size per 24 hour period.

7 94 INGERID KVELLAND In Figure 3 the curves for Series 00 and for Series O22b show that the cold treatment of 0-2 hour old males had no reducing effect upon male fertility. The curves part slightly only on Days 8 to 10. The cold treatment of three day old males, on the other hand, had a marked reducing effect upon male fertility starting on the second day after the treatment. While nearly all males in Series 322a were fertile on Days 1 to 6, the frequency of sterile males in Series 30 increased on the same days to more than 90 percent. It can be seen from Figure 4 that this high sterility in Series 30 is on Days 2 to 4 due largely to sterile matings, while on later days the sterility is due mainly to non-mating males. Figure 5 shows that the proportion of matings found to be sterile in Series 00 is for all days nearly the same as in Series 022b. In Series 30, on the other hand, the frequency of sterile matings is significantly higher for all days than in the control series (Series 322a). Figure 6 shows that on all days in Series 00 the average number of females fertilized per fertile male per 24 hour period is of almost the same value as that found in Series 022b, while in Series 30 the average number of females fertilized is for all days significantly lower than the frequency found in Series 322a. In Figure 7 the curves for Series 00 and for Series 022b show that the cold treatment of 0-2 hour old males had no reducing effect upon male productivity. The curves part slightly on Days 3 to 10, however, with the cold treated males showing the higher number of offspring produced per fertile male on these days. The cold treatment of three day old males resulted, on the contrary, in a reduced productivity, with the number of offspring produced per fertile male per 24 hour period only one-third of that produced by three day old untreated males. Figure 8 shows that in Series 00, on several days, the average brood size per 24 hour period is noticeably higher than in the control series (Series 022b) ; Series 30, on the other hand, shows for all days a significantly lower brood size than does Series 322a. It can be seen from Table 1 that the total number of matings performed by individual males during a 12 day period varies from 15 to 83 matings in Series 022b and from 12 to 81 matings in Series 00. Total number of offspring produced during the same days varies from 1008 to 4227 in Series 022b and from 618 to 4220 in Series 00. The average number of offspring produced by a male during the 12 day period is considerably higher in the cold treated series than in the control series, likewise, the average percentage of fertile matings is higher in the cold treated series.

8 TABLE 1. Number of matings performed during a 12 day period over number of fertile matings, p of fertile matings, and number of offspring produced during the same 12 days. Series no. 022b 00 Individual male number /60 71/49 76/53 78/47 49/ /45 61/48 64/45 63/47 54/42 53/31 33/ /58 67/59 66/ /49 65/42 58/48 61/47 61/47 41/41 76/49 73/45 67/ : (Table 1 continued) b /29 54/44 61/32 74/29 15/ /44 68/38 57/46 52/43 81/39 56/ /36 51/36 59/37 50/40 57/39 12/

9 96 INGERID KVELLAND

10 MATING AND FERTILITY PATTERN 97 I I I I I I I I I I I +I* + l l l l l l l l l l l+l++ I I * I* ++I I I1+1 I I* I I I I I l l + l l l l l +++* I * I I I I I I I I I I I I I I I * l l l l l l l l 7 - Heredifas 54

11 98 INGERID KVELLAND Table 2 shows that several of the cold treated males in Series 30 stopped mating after a few days, and those males who continued to mate showed long intervals (several days) without mating. A high propoktion of the males in this series died during the 49 day observation period. DISCUSSION NOVITSKI (1949) found that if males and females subjected to low temperatures (-5' C) for a long period survived, they would mate and would also be fertile. NOVITSHI and RUSH (1949) found that males subjected to -10' C for 10 minutes were not affected regarding their subsequent fertility. WEDVIK (1962) subjected three day old males to 0' C for 30 minutes and found a marked reduction in frequency of fertile males by the second day following the treatment. The data from Series 30 in the present investigation is in agreement with the latter author. In this series all males were fertile on Day 1, while on the following days the proportion of sterile males increased highly and reached a value of more than 90 percent on the sixth day of mating. In the control series (Series 322a) nearly all males were fertile on these days. The young cold treated males in Series 00, on the other hand, showed on all days nearly the same proportion of sterile males as did the young control males in Series 022b. BAKER (1960) subjected males to 6' C for 72 hours and mated them to untreated females. A noticeable reduction in the male productivity was found compared to control males. IYENGAR and BAKER (1962) subjected young males to -8'C for 15 minutes and found a decrease of 66 percent in the total number of offspring produced by cold treated males compared to control males. The males in Series 00 were subjected to 0' C for 30 minutes when 0-2 hours old, but this treatment had no reduction effect upon male productivity, in fact, the young cold treated males produced an even higher average number of offspring during a 12 day period than did the young control males. If the same cold treatment were given to three day old males, on the contrary, this resulted in a reduction of the number of offspring to one-third the number found for three day old control males. IYENGAR and BAKER thought that the cold treatment had impaired the physical activity and therefore the fertility of the cold treated males. The males in Series 30 showed no marked reduction in their physical activity the first three days after the cold treatment, however, on the following days a very high proportion of the males stopped mating and those males who continued to mate showed a low mating frequency and very long

12 MATING AND FERTILITY PATTERN 99 intervals (several days) without mating. The young cold treated males, on the contrary, showed no reduction in their mating activity. The average brood size per 24 hour period was found to be significantly lower in Series 30 than in Series 322a, indicating that the cold treatment had either caused the death of some of the mature sperm, or had in some other way made them non-functional. In Series 00, on the other hand, the average brood size was found to be of the same value as in the control series (Series 022b), indicating that the cold treatment had not affected the immature, immotile sperm. KHISHIN (1955) found by cytological examination that the first motile sperm in Drosophila melanogaster males occurred 7 If: 1 hours after eclosion: the young males in Series 00 then, could not have contained any mature, motile sperm at the time of the cold treatment. LEFEVRE and JONSSON (1962) subjected males to -10' C for 10 minutes: for a period of 24 hours after the treatment they found no sperm in the male reproductive organs, nor were any sperm found to be transferred to females during copulation. By dissection of the treated males the authors found a mass of dead sperm in the seminal vesicles. No loss of male sex-drive was found; the treated niales mated repeatedly without transfer of any sperm. The males were found to regain their fertility to a lesser degree, being able to fertilize one or two females during a 24 hour period. The authors thought that less mature, immotile sperm were more resistant to the cold treatment than mature, motile sperm, resulting in a regained fertility. The results from Series 00 support this hypothesis. As mentioned above, the young cold treated males in this series showed no reduction in fertility compared to the young control males. The results from Series 30, on the other hand, indicate that subjecting three day old males to 0' C for 30 minutes has a different effect than subjecting males to -10' C for 10 minutes. While LEFEVRE and JONSSON (1962) found that all males were sterile for the first 24 hour period after the cold treatment, all males in Series 30 were found to be fertile during the first 12 hours after the cold treatment and 37 out of 49 males were found to be fertile during the following 12 hour period. However, in the following mating periods in this series the proportion of sterile males increased highly. None of the niales regained fertility after having been sterile. The proportion of non-mating males in Series 30 increased from 24 percent on Day 3 to 90 percent on Day 7, indicating that the cold treated males in this series lost their sex-drive after a few days. The young cold treated males, on the other hand, showed on all days nearly the same proportion of non-mating males as did the young control males.

13 100 INGERID KVELLAND SUMMARY Two groups of males were given a cold treatment (0' C for 30 minutes) at a male age of respectively 0-2 hours and three days. The cold treated males were mated individually to virgin non-treated females both day and night for several days. Daytime all matings were observed, the inseminated females were isolated and the offspring counted. Likewise, the females inseminated during the nights were isolated every morning and the offspring counted. If the cold treatment was applied to males at an age of 0-2 hours, the treatment was found to have no effect either upon male mating activity nor upon male fertility and productivity. On the other hand, if the cold treatment was applied to males at an age of three days, the treatment was found to have a marked reducing effect both upon male mating activity and upon male fertility, fecundity and productivity. Literature cited BAKER, R. M The effect of cold temperature on fertility. - D. I. S. 34: 70. IYENGAR, S. V. and BAKER, R. M The influence of temperature on the pattern of insemination by Drosophila melanogaster males. - Genetics 47: KHISHIN, A. F. E The response of immature testis of Drosophiln to the mutagenic action of X-rays. - Z. Ind. Abst.-Vererb. lehre 87: KVELLAND, I Some observations on the mating activity and fertility of Drosophila melanoguster males. - Hereditas 53: LEFEVRE, G. (JR.) and JONSSON, U. B The effect of cold shock on Drosophila melanogaster sperm. - D. I. S. 36: NOVITSKI, E Action of X-rays at low temperature on the gamets of Drosophila. - Am. Nat. 83: NOVITSKI, E. and RUSH, G Viability of Drosophila exposed to subzero temperatures. - Biol. Bull. 97: WEDVIK, H The effect of low temperature on the fertility of Drosophila melanogaster males. - D. I. S. 36: 127.