Mechanisms of Ageing and Development, 10 (1979) 233-240

233

©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

Q U A N T I T A T I V E A N A L Y S I S O F M A T I N G B E H A V I O R IN A G I N G M A L E DROSOPHILA MELANOGASTER

ANGELOS C. ECONOMOS Systems Physiology and Gerontology Group, Life Sciences Division, Technology Incorporated, Mountain View, CA (U.S.A.)

JAIME MIQUEL and ROSEMARIE BINNARD Biomedical Research Division, NASA-Ames Research Center, Moffett FieM, CA (U.S.A.)

SEYMOUR KESSLER School of Medicine, Stanford University, Stanford, CA (U.S.A.]

(Received October 2, 1978)

SUMMARY Dynamic changes in quantitative aspects of mating behavior of the male fruit fly as its life unfolds, from eclosion through maturity (peak performance) and "physiological death" (loss of fertility followed by complete loss of ability to mate), towards actual death, have been identified in this work by observations and measurements on 28 male fruit flies of the Oregon R strain studied individually. At weekly sessions from the first day of their imaginal life until their natural death, each fly was given the opportunity to mate with up to three virgin females during a one-hour period. Length of the latency period of each accomplished mating and duration of copulation were recorded. After mating the females were allowed to lay eggs for 24 hours and the number of offspring was counted 26 days later. The period between I and 4 weeks of age is characterized by peakand fairly constant performance: multiple matings, short latencies, long durations of copulation, and high degree of fertility (number of offspring); then a decline sets in, in some measures slower than in others, as the flies age. Large intra- and inter-individual variabilities were, however, found which obscure possible correlations between individual measures of mating ability and length of life. At the individual level, a preliminary analysis showed a good correlation (r = 0.80 and 0.79) between life span and week of last mating (onset of impotence) or week of last fertile mating (onset of sterility). At the population level it was found that a number of measures, i.e. number of remaining maters at each age, number of remaining fertile maters and total number of matings, had an agecourse similar to survivorship but anticipated it by 4--6 weeks. Other measures, such as number of multiple matings and number of offspring declined with age faster than survivorship.

234 INTRODUCTION Mating behavior of Drosophila as it ages has not been extensively studied. Various investigations over the last several decades have focused on aspects such as effects of mating on longevity of the mating individuals or the relationship between parental age and longevity of offspring [1]. A possible relationship between female fecundity and longevity has also received attention, but it has not been clarified if it is "an exact causeand-effect relationship" [2]. Recently, the effect of aging on mating capacity of male Drosophila was investigated [3] by means of "mass-mating" sessions, in which groups of 50 males were presented with 75 young virgin females; the number of matings accomplished within 11 min was recorded and was reported as an index of mating capacity. This index declined with age towards a zero value reached at about 3/4 of the maximum life span. In the present study we define quantitatively the main features of the age-dynamics of mating ability of male fruit flies by measuring various aspects of mating, (such as latency, duration and number of offspring) in 28 flies tested individually. Because a young male fly placed in a vial with one virgin female generally achieves mating within about 5 min and the duration of mating is of the order of 15-20 min, in each session each male was given three virgin females to mate with within one hour; in this way not only the all-or-none ability to mate was tested but also a measure of the mating vigor was obtained. Furthermore, the fertility of the males at each tested age was checked and quantified in terms of the number of offspring resulting from each mating session after 24-hour laying of eggs by the females.

MATERIALS AND METHODS Inbred Drosophila melanogaster flies of the wild Oregon R strain, raised in an incubator at 21 --+0.5 °C, were used. The 28 males whose mating behavior was studied in this work, (obtained in two groups of 18 and 10 individuals respectively, studied two months apart), eclosed between 4.00 p.m. and 8.00 a.m. the next day; from then on they were placed individually in small glass vials measuring 15 cm in length and 2.5 cm in diameter and kept in the incubator at constant temperature, 21 +- 0.5 °C, and under constant light for the whole of their imaginal life, except during the weekly one-hour mating sessions which took place at room temperature, 23.5 + 0.5 °C. The food medium consisted of corn-rnolasses-agar enriched with brewer's yeast. The males were transferred to clean vials with fresh food at weekly intervals without etherization. The virgin females used each week for studying mating behavior of the males were similarly raised and kept also in the incubator in groups of three in glass vials until they were 8 days old. Because the males were mated weekly with 3 females each, an adequate number of groups of virgin females were collected each week. The mating took place in empty vials, one for each male and 3 females, and lasted for one hour. Latency of each mating and duration of each copulation were recorded. In case a male was still coupled to

235 a female at the end of the hour, removal from the vial was postponed until their spontaneous separation. The males were then separated from the females by careful "shaking" (without resort to etherization) and were placed in clean vials with fresh food and returned to the incubator (21 °C). The three female partners of each male were also placed in clean vials with fresh food but were kept at room temperature (23.5 °C) and allowed to lay eggs for 24 hours; then they were discarded. The vials with the eggs were kept for 26 days and the numbers of offspring were recorded. The whole procedure was repeated once a week until natural death of the individual males, Le. also during the latter period of their life when they no longer had the ability to mate and finally lost interest in mating.

RESULTSAND DISCUSSION The complete set of observations from the two consecutive studies is summarized in Tables I and II. The flies have been identified by serial numbers and for each one of them the characteristics of mating behavior appear in the corresponding column. Data recorded in each session are latency (time until copulation starts) of each mating accomplished in one hour, the corresponding mating durations - appearing in identical sequence as the latencies - and the number of resulting offspring. At those sessions that a mate did not mate, the corresponding part of the column is singly hatched, whereas a double hatching indicates that the male had died. At the bottom of both tables, information deduced from the data has been summarized in order to facilitate further analysis. Two males, numbers 8 and 18, were lost (flew away) after the first mating session, during the difficult procedure of separating each male from the females without etherization; these flies are excluded from the analysis below. Figure 1 shows the survivorship curve of the combined population of 28 flies. Despite the relatively small number of flies, the survivorship curve has the rectangular shape generally observed in large populations of male Drosophila [3] ; this fact shows that the studied population was a random sample. With advancing age, the mating capacity of the flies declines, as the data in Tables I and II show. The decline is not uniform, however; the flies become infertile or are no longer able to mate at ages that show considerable variation. Nevertheless, mating capacity of the total population, expressed as the number of remaining fertile flies ("fertile maters") or as the number of individuals that can still mate ("maters") at each week of age, declines in a rather smooth and uniform way, resembling the age-course of survivorship (see Fig. 2). Both the curve of maters and fertile maters are completed, as expected, before the survivorship curve, respectively within 75 and 60% of the maximum life span. Most male flies mate at least once, and many mate more than once, at each weekly session during the first four weeks of their life. (The first mating session, on day 1, is not considered in the analysis, because it may represent a growth and/or learning effect.) Figure 3 shows the age-course of total number of matings or multiple matings (at least

236

TABLE I EFFECT OF AGING ON INDIVIDUAL CHARACTERISTICS OF MATING ABILITY IN 20 MALE FRUIT FLIES (1ST STUDY)

SER'ALNUMREROFFLY0 ¢ MATING LATENCY.rain*

49 36 481~.~'~~

3

,

~

-?!

9 l0

1 day

MATING DURATION~min:'16-'~7" 71 ~

L="~"

34"32 20 721"~:

4"

"\~

NUMBEROFOFFSFRiNG"-2---6 " 1 6 " 1 ~ MATING LATENCY,min 13 6 '81 5 2 18 6

4 2

~" 74-T6"-2 - ~ "3 4 3 20 :

MATING DURATION, rain 24-21- 72~7''-21"

9"

3

17

4

3 9

6 1tl 2 2 41 34 35 9 22 19 201 7 20 24 161 2 17 18

MATING DURATION, min

19 19 2o 20 20,

21

3

OFOFFSPR,NG-; 3; BT~ 3;

NUMBER MATING LATENCY,min

20 31"32" 20 241"~,'

312O "1i 1 13 5 -5"

2 5

MATING LATENCY,rnin

5 3 131 4 8 21 14 25 21 2 MATING DURATION, min N - 1 9 "1BT6"-3 20 19 18 16 19 NUMBEROF OFFSPRING -36"-38 23"~-E "26 MATING LATENCY,min 8 6 161 5 5 15 241 7 25

8

xl

17

3:; i~ h-T

21 22

3 4

3 6 10 ?:3 17 19 ~9 11

~" 3 B

6

;2 i 4 16 7 ~'~ 4 3

9 9

; 7

2

1~

34i

2 30

18 lgl', 15

21 18

14 6 16 7 7 2Ol

2 34

-24--2:3 ~18 721-~ 16 13 1RI

"2? 16

--(~ - 9" 19 ",:,T" 18 17 6 ~: 15 6 ' -

"

'

3

8

37-~

MATING DURATION,rain

16

?01--20-20 17122 18

I" 7

NUMBER OF OFFSPRING - 2 7 5 ; 31-F~-4i MATING LATENCY,min 12 9 5! 14 14 2 MATING DURATION, rain "2(~-26 "~lBI24 "~-15 20 NUMBER OF OFFSPRING "~E-2.~E 2~1 20~-..~0 MATING LATENCY,min 3 12 61311 23

?4

16- 33" 23 19T

31-

19-~

"t6-

5J fil

20 15 15 "8 1 4 / 5 i ~ 6 7 B 25 29 " " 19 18 12 1~0 -i (~ 24 6 15

'~4"~x 21~:

"-~6 4

-7

~14

35 i5

'13"~

]2-

N

3-6 ~

lal

"29 T

"f

81~

3

8

MATING DURATION, rain 1 3 " 2 ; "111 91-24 20

.'_

,,

MATING DURATION, rain -1 -28 NUMBER OF OFFSPRING "2t" MATING LATENCY,rain 19 1~

-2 %

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MATING DURATION, mien-

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MAT,NGLATENCY. ,o

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MATING LATENCY,~,o

~ ~ ~

MATING DURATION. min

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"NUM'BE-R'-O~'OFF'SP~'N G'-

"4!

AGE,w... e

MAT,NO

IIItlll.

'01" 1121,3I"J'BI ,sl

MATING LATENCY,min I '31 ~111 1,5 ~ i l l MATING DURATION NUMBER OF OFFSPRING |

SERIAL NUMBEROF FLY I Q t @ I WEEK OF LAST FERTILE 5 g

,, ;(~ "O

ol ol B I e k',.',~'..~m

@lcI@l@l@t@l@l l i 3@l5 5 5 8 4 B 4 5 5 6 4 5 4 5 4, ,lllllllf

WEEKOF LASTMATING I 91 ,a[ e I el BI e I e | o l e I 71 sl el B I el a I 71 4 | ~ WEEKOF DEATH I~l~el~ll~ [~sl a I ~ s l i B s I ~ I 9h~l sl~ol'mlHIi:zl~ll4

I sl Iml

*Each male fly was placed in an empty vial with three 8-day old, virgin females and allowed to mate for one hour. **The three females mated with each male were allowed to lay eggs for 24 hours after the mating session and the number of imagoes 26 days later was counted.

237 T A B L E II EFFECT OF AGING ON INDIVIDUAL CHARACTERISTICS OF MATING ABILITY IN l0 MALE FRUIT FLIES (2ND STUDY)

lmO~'~lee"` SERIAL NUMBER OF FLY ~ MATING LATENCY, min* 34 3411 23 0 (1 day)

2 187 13 12 96 1~161 1 MATING DURATION, rain* "2; 11~19" 19 "1; 39- 1{; -~01 23 1,7"18 2116

2, 225f 8~L2

MATING LATENCY, rnin

9

5

14 3

1o12o 148 2

40~..L 5.__L1 4

1

5

-23 -21122 123-

EUM-~o-T;sP-~TnE- MATING LATENCY, rnin

4~ 15 19 22 24 16 1812+6~ 18 2O

5(;

;~13(; ~6 3£3

1022 3

13 6 2

17

s-

87

16 17

-~f37121118-

"':f6+, 17

3

NUMBER OF OFFSPRING-- 16t0-2; --0"-I'~-18"59-3 sMATING LATENCY rain 4 6 5 3 s1111 1 6 4 8 6 11 MATING DURATION, min --2i39 21" 17 2(; ~%12312C 14 20 14 ,8.1_~5 NUMBER OF OFFSPRING 4~-2T TI 7 -1:3 ~%1 81 sMA:'FING LATENCY, min 6 6 13 3 9 5 2 71 1819

4

"MA~'~NG[)U~TI~,~Tn

51 13 21" 20 516 .-NU~ER-OF-OFF-SPR'~G- 13[ 3~ M,~:TING LATENCY rain 16 7 27

i 6

3 115 9 - 1 9 1 2 2 ~ 15 30- 371 I N ] 8 1 3 1 4 1 8 16 ,81~ 12 -16 " E 2~ 13 -~E%I 8l~4 8 B 3 7 51 121 is 10 7 11

19

18

14

1

MATING LATENCY min

33542;

~AT~G~R~I~;o

£ 1~ 2~ 26 ~ ; 6

-2~-211 lOl2C 151 16 7116

21 -I 5

15 "NU-~'E-ROI:O'-FFS-'~IN-'G 12= 6"~ 35 O -0 MATING LATENCY, min 413; 5 27 14 19 I

MATING DURATION, rnin 2, '2E 23 -2T "2; -NUM-BE~O F O'FF'-~RI-N(~ 0 "1] 8 0"22 MATING LATENCY, rnin I01 lJ 1515 ~ 1E f~' M~ATI~GDURAT'ON' min -2~ 2~ 22 20 i "24 1~ ~

~u~Go75~S~N&

r~ 2~ ~;

11 16

1;3 11

long

-1~-231~ 2£ 17 -1~ . 2 9 ~ 2 0 -

SERIAL NUMBEROF FLY ( ~ ( ~ ) ~ ~ 1 ( ~ ~ 1 ( ~ ~ (~) WEEK OF LAST FERTILE 8 9 10 4 10 5 11 10 4 10 MATING WEEKOF LASTMATING 10 9 14 7 12 7 13 12 5 11 WEEKOFDEATH ~1 17 20 B 19 ]( 19 ~6 7 73

*Each male fly was placed in an empty vial with three 8-day old, virgin females and allowed to mate for one hour. **The three females mated with each male were allowed to lay eggs for 24 hours after the mating session and the number of imagoes 26 days later was counted.

238

100

80

~. 6o -r, re

o -> >

40

20

4

8 12 AG E, weeks

16

20

Fig. 1. Survivorship as a function of age in the combined population of 28 male flies.

100

lOO

O MATERS

5 ¸ 80

¢

Z~, MATINGS I'1 MULTIPLE MATINGS

80 r~

A FERTILE MATERS

i

z

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u. 40 0

4o

O¢

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0

~ 20

i

I

4

8 AG E, weeks

-

12

I

16

0

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4

8

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16

AGE, weeks

Fig. 2. Decline o f population mating "potential" with age. The number of flies that are stiJ/able to mate at each age and the number o f the maters that are also fertile, are plotted vz age. The data were obtain6d from Tables I and II. Fig. 3. Decline of population mating "capacity" with age. The number of matings (single or multiple), and the number o f multiple matings, (two or three matings in one session counted as one multiple mating), accomplished by the "combined" group in each weekly session, are plotted v~ age. The data were obtained from Tables I and II,

239 two matings) per session for the entire population of 28 flies. Between 4 and 8 weeks the number of multiple matings decreased sharply while the number of matings decreased more slowly. Although the curve ofmatings resembled the survivorship curve (Fig. 1) and the curves of remaining maters or fertile maters (Fig. 2), the curve of multiple matings exhibited a surprising irregularity after the age of 7 weeks. About 10% of the population accomplished multiple matings until the 11 th week of age. It is not known if large populations of flies generally have as large a number of such vigorous individuals. Figure 4 shows the age-course of number of offspring that resulted from each mating session, together with the curve of multiple matings for comparison. As expected, the "noise" present in the multiple matings-curve at late age is propagated and amplified in the offspring-curve. Finally, the information on lifetime characteristics of mating behavior of individual flies obtained in this study, makes it possible to inquire as to a possible correlation between the values of selected measures of mating ability of individual flies and their length of life. Of course, it cannot be expected that, from information on only the mating characteristics of a fly, a very accurate prediction of its life span could be arrived at; information on other physiological functions is needed as well. However, because of absence in the flies of the mammalian homeostatic plasticity, it is possible that a reasonably good prediction of fly life span could be obtained from a statistical analysis of the quantitative characteristics of just mating behavior. Clearly, if one or more physiological or biochemical variables, (such as neuromuscular coordination measured by means of the "negative geotaxis test" [3] or rate of protein synthesis that could be possibly measured non-invasively through use of radioactive tracer techniques), were

100 r-~ I ~ ~' "~]

0

[] MULTIPLEMATINGS

4 8 AGE,weeks

12

Fig. 4. Decline of fertility and sexual vigor. The number o f offspring resulting from each weekly session for the combined group is plotted v~ age together with the curve of multiple matings from Fig. 3. The data were obtained from Tables I and II.

240 measured longitudinally and individually in the same population in which mating was being studied, then a better statistical prediction o f life span could be obtained. Such experimental studies are in progress in our laboratory, and the general statistical methodology for such a prediction, i.e., a methodology for determination of biological age in both animals and human subjects, is under development (unpublished). However, even without the methodology and additional information, a reasonable statistical prediction o f life span might be obtained, if one used as indices highly "integrated" aspects of the mating behavior. Figure 5 shows two examples. 20-

o 16-

|

0

°/+,++

,.J

oo

8

o

O

0

,,r i,,-

~ =,

O

® / -

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o

0

0 4

0 I

1

4 8 12 WEEK OF LAST MATING

I

I

16

I

I

I

0 4 8 12 WEEK OF LAST FERTILE MATING

Fig. 5. Statistical correlation between an individual's length of life and its mating ability or fertility. Length of life is plotted against the week after which a fly became impotent (top) or the week after which it was irreversibly sterile (bottom), though it usually continued for a few weeks to be able to mate. The data were obtained from Tables I and II.

In this Figure, the duration o f life o f each fly was plotted against the week the fly stopped mating altogether (left) or the week o f its last fertile mating (right). Linear regression lines were constructed in b o t h cases and the correlation coefficient, r, was estimated. The values o f r, 0.80 and 0.79, show that a reasonable correlation exists between life span o f a fly and the week it became i m p o t e n t or sterile.

REFERENCES 1 A. Comfort, Aging: The Biology of Senescence, Holt, Rinehart and Winston, New York, 1964. 2 M. Rockstein and J. Miquel, Aging in insects, in M. Rockstein (ed.), The Physiology of Insecta, Vol. 1, Academic Press, New York, 1973, pp. 371-478. 3 J. Miquel, P. R. Lundgren, K. G. Bensch and H. Arian, Effects of temperature on the life span, vitality and fine structure of Drosophila melanogaster, Mech. Ageing Dev., 5 (1976) 347-370.