1981--Final 21 Feb. 1981. Esterase 6 (EST 6) in Drosophila melanogaster is a male reproductive enzyme transferred to females as a component of the seminal ...
Behavior Genetics, Vol. 11, No. 3, 1981
Studies of Esterase 6 in Drosophila melanogaster. VII. Remating Times of Females Inseminated by Males Having Active or Null Alleles D o n a l d G. Gilbert, 1 R o l l i n C. R i c h m o n d , l'z and K a t h y B. S h e e h a n ~
Received 26 Jan. 1981--Final21 Feb. 1981
Esterase 6 (EST 6) in D r o s o p h i l a m e l a n o g a s t e r is a male reproductive enzyme transferred to females as a component o f the seminal fluid [Richmond, R. C., Gilbert, D. G., Sheehan, K. B., Gromko, M. H., and Butterworth, F. W. (1980). S c i e n c e 207:1483-1485]. Here we report investigations into the relation between E S T 6 and remating by females. E S T 6 activity in a strain selected for decreased time to remating is incrdased over control levels. Inseminated females remated to males carrying null or active alleles show no differences in the timing of remating. However, females inseminated by E S T 6-active males remate significantly sooner than females inseminated by null males. Interrupted copulation experiments demonstrate that the remating effect is not due to E S T 6 alone but requires other components o f the ejaculate. Other evidence suggests that sperm stored in the ventral receptacle respond to E S T 6 levels and control remating time. As the first mate o f a female who will remate, null-EST 6 males have, under laboratory conditions, a significantly higher fitness than males carrying active alleles. Thus the absence of null alleleS o f E S T 6 in naturalpopulations presents a dilemma suggesting that the remating effect o f E S T 6 may be balanced by other effects on reproduction. KEY WORDS: Drosophilamelanogaster;esterase 6 activity; esterase 6; null allele; remating; fitness.
This work was supported by NSF Grant BNS 79,21743 and NIH Grant AG 02035 to R.C.R. Computing costs were met in part by the Indiana University Wrubel Computing Center. 1 Department of Biology, Indiana University, Bloomington, Indiana 47405. 2 To whom correspondence should be addressed. 195 0001-8244/81/0500-0195503.00/0 9 1981 Plenum Publishing Corporation
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INTRODUCTION The esterase 6 (Est 6) locus in Drosophila melanogaster is the structural gene for a nonspecific carboxylesterase (EC 3. I. 1.1) which is polymorphic for at least two electromorphs in every natural population investigated (Wright, 1963; Girard et al., 1977). The functional role of the esterase 6 enzyme (EST 6) was entirely unknown until recently (Aronshtam and Kuzin, 1974; Sheehan et al., 1979; Richmond et al., 1980) despite the fact that this system was among the first biochemical polymorphisms described for D. melanogaster (Wright, 1963). EST 6 is a reproductive enzyme that is synthesized in the anterior ejaculatory duct of the adult maleJ reproductive system and is conveyed to the female prior to sperm transfer as a component of the seminal fluid. Since the number of progeny produced by females singly inseminated by males having active or null EST 6 alleles is identical at 25 C (Gilbert et al., 1981), we were led to test the hypothesis that EST 6 might be a part of a male system that affects the remating time of females. Remating by females can have a large effect on the first mate's fitness in Drosophila, since the stored sperm of the female's previous mate are largely "displaced" by the second male's ejaculate (Lefevre and Jonsson, 1962; Prout and Bundgaard, 1977; Gromko and Pyle, 1978). Gilbert and Richmond (1981) have shown that EST 6 has little or no effect on the process of ejaculate competition (i.e., sperm displacement). Manning's (1962, 1967) valuable studies led him to conclude that the sexual receptivity of nonvirgin females is affected by two factors--the presence of stored sperm and an effect associated with copulation. Manning speculated that the copulation effect could be due to a chemical present in the seminal fluid. His results suggested to us that EST 6 might influence the timing of remating by affecting sperm storage and use or by being part of a pheromonal system directly or indirectly affecting female sexual receptivity. Gilbert et' al. (1981) obtained data which indicate that EST 6 influences the rate of sperm release from storage organs. We report here a series of experiments designed to investigate the relationship between EST 6 and the timing of remating in D. melanogaster. The possibility that EST 6 might be invo.lved with the timing of remating was first investigated by measuring enzyme activity in a line selected for decreased times to remating and a similar unselected line. Next, four experiments studying the relationship between EST 6 and the timing of remating in unselected EST 6-active (EST 6 s) and EST 6-null (EST 6~ lines were completed (Table I). (1) The first experiment, denoted SD 1, examined the effect of EST 6 in the ejaculates of second-mating males and was completed in conjunction with other experiments exam-
Est 6 and Remating in Drosophila melanogaster
Table I. Experiment
No. ~?9 a
197
Summary of Remating Experiment Designs Remating effect tested
Remating schemeb
SD 1
40
forked 9 x forked 6 • EST 6 c~
Second male EST 6 type
Stocks
66
EST 6 9 (• EST 6 6) n timesc
EST 6 stock type
IRM 1 IRM 2
75 50
forked 9 x EST 6 c~ x forked 6
First male EST 6 type in absence of sperm
RM 1
59
forked 9 x EST 6 c~ x forked
RM 2
178
(first mating interrupted after 4 min)
First male EST 6 type with sperm, and female type
Ore R or forked $ x E S T 6 g x Ore R or forked
Approximately equal numbers of females were used for each EST 6 group. This column gives the total number. b EST 6 = EST 6~ and EST 6s groups. c See Materials and Methods for a more complete description.
ining the effect of EST 6 on ejaculate competition (Gilbert and Richmond, 1981). (2) The second experiment, STOCKS, compared multiple mating and productivity (number of progeny per female) of the EST 6o and EST 6s stocks. (3) The third experiment with two replicates, IRM 1 and 2, examined the effect of first male ejaculate EST 6 on females remating when the matings were interrupted after EST 6 transfer but before sperm transfer. (4) The final experiment with two replicates, RM 1 and 2, examined the effect of first male EST 6 on females remating with complete first matings. The results support our_ prior assertion (Richmond et al., 1980) that the remating effect observed is due to the direct or indirect action of EST 6 on the rate of sperm release from the female's storage organs. MATERIALS AND METHODS
EST 6 activities were studied in two wild-type strains of D . m e l a n o g a s t e r . Both strains originated from the same base population and are homozygous for the E s t 6 s allele. One strain was selected for reduced time to remating and an unselected but equivalently inbred line was maintained as a control. These two lines were generously provided by Dr. M. H. Gromko, and their origin and selection regime are described by Pyle and Gromko (1981). EST 6 activity was determined in these lines by the use of a spectrophotometric assay described in detail by Cochrane and Richmond (1979) and Sheehan et al. (1979). Protein concentrations were
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determined with the Folin reagent using bovine serum albumin (Schwartz-Mann) as a standard (Lowry et al., 1951). All flies tested for remating were 3- to 4-day-old virgins, raised and stored in groups of 10-20 on a yeasted medium from eclosion to experiment at 25 C, 60% RH, and 12:12 hr lighting. All experimental flies, except those for RM 2, were collected as eclosing virgins using carbon dioxide; RM 2 flies were collected with ether. Matings and rematings were scored visually for single pairs aspirated into yeasted medium vials. Remating tests lasted 2 hr each day during fly morning, and unremated females were transferred to fresh vials at this time. All matings and rematings, except for RM 2, were conducted at room temperature, which ranged between 18 and 25 C; RM 2 matings took place at 25 _ 0.5 C and 60 _+ 10% RH. Remating experiments were conducted with unselected EST 6 variant males derived as described by Sheehan et al. (1979). One strain was homozygous for a null variant producing no detectable EST 6 activity (EST 6~ a genetically similar strain was homozygous for the slow electrophoretic variant (EST 6s). Females were obtained from wild-type Oregon R (Ore R) and forked (forked-bristle, X-linked recessive) stocks. Both of these stocks are homozygous for the EST 6s allele (Sheehan et al., 1979; Gilbert et al., 1981). A summary of the designs for the remating experiments is given in Table I. In experiment SD 1, forked females were first mated to forked males and then were tested daily for remating with EST 6 ~ or EST 60 males. After remating, females were transferred daily for 1 week to allow determination of postremating progeny numbers (productivity). All female progeny were counted and their paternities identified according to whether they were of forked phenotype (forked male paternity) or wild type (EST 6 male paternity). In the STOCKS experiment, females were initially mated to males of their own type (i.e., EST 6 s or EST 6~ and were then transferred and given the opportunity to remate daily for 2 hr, regardless of previous mating, for 8 days. All progeny were counted and sexed for 10 days from first mating. For the IRM 1 and 2 experiments, forked females were mated to EST 60 or EST 6 s males, and copulations were interrupted at 3-4 rain by briefly vibrating the vials on a test tube vortexing machine. Females were then tested for remating at 3, 12, 24, 48, and 72 hr after the interrupted mating for 2-hr periods. Progeny were not counted for the IRM experiments. For the RM experiment (reported briefly by Richmond et al., 1980), forked females were first mated with EST 6 s or EST 60 males and then tested for remating daily with forked males. After remating, females were transferred daily for 1 week to determine productivity and paternity, as for SD 1. For RM 2, forked and
Est 6 and Remating in
Drosophila melanogaster
199
Ore R females were initially mated to EST 6 s or EST 60 males and then tested for remating daily with their respective male types. The number of eggs laid daily and the number hatching by 2 days postlaying were counted until females remated. Females were discarded upon remating. Results are presented below in terms of mean value ___ standard error of the mean (SE), and the 0.05 significance level is used when not explicitly given. The randomization tests for two groups described by Siegel (1956) were used for nonparametric comparisons of group means. Analyses of variance and covariance were performed using the methods described by Bliss (1967, 1970) after appropriate transformation of data.
RESULTS EST 6 Activity in a Line Selected for Early Remating
Pyle and Gromko (1981) achieved a rapid and strong response to selection for earlier female remating in a wild-type strain of D. m e l a n o g a s t e r (realized h 2 = 0.30 for the first five generations of selection). Earlier remating in the selected line was a result of both male and female effects, and analysis of hybrids between selected and unselected lines indicated strong X-chromosome or maternal inheritance. In testing for correlated responses to selection, Pyle and Gromko found a significant reduction in productivity and an increase in male courtship persistence. As the role of EST 6 as a male reproductive enzyme became apparent (Sheehan et al., 1979), we considered control ofremating time as a possible function for EST 6 and predicted that EST 6 activity in the selected and control strains of Pyle and Gromko would be different. If EST 6 exerts its effect on remating by affecting sperm use (Gilbert et al., 1981), increased EST 6 activity would result in an increase in the rate of sperm release from storage and subsequent earlier remating (Manning, 1967), On the other hand, if EST 6 acted to decrease the sexual receptivity of mated females (Tompkins and Hall, 1981), decreased ejacfilate EST 6 would result in earlier remating times. EST 6 activity, measured as optical density units (Sheehan et al., 1979), is significantly greater in the line selected for early remating (1.72 + 0.07 vs. 1.37 _+ 0.09 in selected and unselected lines, respectively). Such activity differences between strains might result from differences in total protein content and rates of enzyme synthesis or degradation with aging rather than selection for variation in EST 6 activity. The potential causes of increased EST 6 activity in the selected line were examined by measuring soluble protein as well as EST 6 levels in homogenates of selected and unselected virgin males aged from 3 to 20 days. Table II
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Gilbert, Richmond, and Sheehan
Table lI. Analysisof Covariance for Male EST 6 Activity (E) Between Strains Selected and Unselected for Faster Remating, Controlling for the Covariates of Male Age (A) and Total Protein Content (T) Sums of squares and cross productsa Term
df
Male group Error
1 32
A2
AT
Tz
AE
TE
E2
0.1176 8.235 576.5 0.3516 24.61 1.051" 1304. -70.38 5480. 45.76 25.50 3.756
Slopesquared 0.3338 1.749
Reduced EST 6 Variance
Male group Error a
df
Mean square
I 30
0.7169 10.72"* 0.0669
F
Significance level of group MS/error MS for EST 6 activity is given; A and T group differences are not significant, although group MS for T approaches significance (P
I
I
A
FORKEFJ
E)
ORE R
I
I
I
I
I
f' ///
UI
'/" SO
O
1
~
I--~
2
3
I 4
5
-
1
S~ERt'q LOSS
I
I
I
I
I
I - -
6
7
1
2
S
4
[DAY POST
MATING
I
5
1
]
6
?
Fig, 1. Cumulative percentage of D. melanogaster females remating (A-C) and of sperm lost from their ventral receptacles (D) for days after first mating. Oregon R and forked females were inseminated by EST 6 s or EST 6o males in A, B, and D. Forked females were first inseminated by forked males, then paired with EST 6 s or EST 6o males for remating
inC.
204
Table IV.
Gilbert, Richmond, and Sheehan
Analysis of Variance for Rernating Time and Associated Preremating Fitness Components Measured in Experiment RM 2 Remating time ~
Egg fertilityb
Progeny~
Term
df
MS
F
MS
F
MS
F
Male type Female type Interaction Error
1 1 1 174
0.6715 0.0054 0.0022 0.1234
5.44* 0.04 0.02
0.2989 0.0046 0.0006 0.0962
3.10 0.05 0.01
83.18 0.07 1.94 9.47
8.78** 0.01 0.20
a Square root transformed remating time and number of progeny (hatched eggs). b Angular transformed proportion of hatched eggs to total eggs laid. *P < 0.05. **P < 0.005.
proportional to the larger difference in mean remating times (38 hr) between experiments. Progeny numbers reported for RM 1 are only females; doubling these numbers indicates that more than twice as many preremating progeny were sired by males of both groups in RM 1 than in RM 2. The basis for the reduction in the number of preremating progeny s~red by EST 6 s males was analyzed in more detail in RM 2. With the analysis of remating time variance reported in Table IV are corresponding analyses of preremating egg fertility and progeny production. These analyses indicate no significant effects on egg fertility, but a significant effect of male type on progeny number, with no effect of female type or the interaction. This significant reduction in progeny number due to EST 6 s males is mirrored by the reduced numbers of eggs laid (Table IIIB). These analyses show that the progeny difference is attributable to fewer eggs laid rather than a lower egg fertility. The earlier remating of EST 6 s group females in RM 1 and RM 2 and the positive correlations of remating time with preremating progeny number suggest that progeny differences are due to remating time differences alone. However, it is possible that EST 6 s inseminated females produced fewer eggs and progeny on a per day basis, as well as remating earlier. This possibility is also supported by the slightly lower initial productivity found for EST 6 ~relative to EST 60 inseminated females in a single-mating design (Gilbert et al., 1981). To test this, an analysis of covariance was performed on preremating progeny, controlling for remating time. The reduced mean square variance for progeny tested over the error mean square gives F ratios of 2.37 (t,53 dO for RM 1 and 3.71 (1,175 df) for RM 2, which are not significant at the 0.05 level. Nonsignificant group by slope interaction terms indicate no difference in the regression of progeny on remating time between male groups. This analysis shows that
Est 6 and Remating in
Drosophila melanogaster
205
there is no male group effect when progeny variance is reduced by remating time covariance. We conclude that preremating progeny differences are due primarily to differences in remating time. Females mated to EST 6~ males produce the same n u of eggs on a per day basis, with the same level of egg fertility, t~ ~y fewer eggs in toto before remating than EST 6o mated females. DISCUSSION These experiments demonstrate that EST 6 influences the timing of female remating and, as a consequence, the number of progeny produced by a female's first mate. Increased EST 6 activity in males causes earlier remating in females they inseminated. As the interrupted copulation experiments demonstrate, this effect is not the result of EST 6 alone but arises from an interaction between the enzyme and other factors transferred to the female. A likely physiological basis for the EST 6 effect on remating is shown in Fig. 1D (data from Gilbert et al., 1981). This figure plots the amount of sperm lost (as a proportion of that stored following mating) by two groups of forked females as a function of days postmating. Females inseminated by EST 6s males initially lose sperm at a significantly greater rate than EST 6o inseminated females. Manning (1962) presented evidence that remating females had lower numbers of sperm stored in their ventral receptacle than nonremating females. Recent observations of ours have confirmed this finding. In particular, there is a strong correlation between numbers of sperm stored in the receptacle and time to remating by females, but no correlation between spermathecal sperm numbers and remating (Gilbert, in preparation). The D. melanogaster ventral receptacle is innervated (Miller, 1950) and sperm stored there remain motile (Fowler, 1973). This evidence and the above results suggest that the remating time depends on stimuli from motile sperm in a female's receptacle, although other factors such as paragonial secretions also affect the remating time (Merle, 1968). We propose that D. melanogaster females respond to levels of sperm motility in their ventral receptacle as the main index of their current fertility rather than a sperm chemical factor suggested by Manning (1962, 1967). The evolutionary significance of our results is problematical. Table V provides a list of relative fitnesses (measured as number of progeny produced) of EST 6 s and EST 60 males under several experimental designs. As mates of a female who will copulate only once at 25 C or as the second and final mate, these two male types have equal fitnesses. At 18 C, EST 6 s male fitness in single matings is greater than that of an EST 6o male. However, the fitness of an EST 6s male is approximately 30%
206
Gilbert, Richmond, and Sheehan
Table V. Relative Fitness of EST 6SMales to EST 6~ Males Measured Under Four Reproductive Fitness Designs Relative fitness Fitness design Single mating At 25 Ca At 18 Cb Multiple female mating Second malec First maled
No. of experiments
Mean
SE
3 2
0.999 1.316
0.011 0.054
3 2
1.000 0.695
0.014 0.004
Gilbert et al. (1981); using total progeny from single matings, Table 2. b Gilbert and Richmond (in preparation). c Gilbert and Richmond (1981); using proportion of postremating progeny sired by second male, Table 2. " d Present report; using preremating progeny, Table III. a
lower than that of an E S T 60 male if the E S T 6 S male is the first of a f e m a l e ' s two mates, simply b e c a u s e the female will r e m a t e sooner. The remating design e m p l o y e d here m a y give a fair approximation of the natural remating f r e q u e n c y (Hardeland, 1972; G r o m k o and Pyle, 1978), and these fitnesses suggest that null alleles at the E S T 6 locus should be strongly f a v o r e d in some environments. H o w e v e r , recent surveys of several natural populations h a v e u n c o v e r e d no nulls at this locus (Voelker et al., 1980; unpublished report). Preliminary results of a population cage e x p e r i m e n t employing the E S T 6 s alleles at 25 C suggest that the null allele m a y be f a v o r e d in s o m e cages. We are therefore faced with a paradox. U n d e r l a b o r a t o r y conditions at 25 C with multiple mating, the E S T 60 allele appears to h a v e a decided advantage o v e r the E S T 6 s allele. H o w e v e r , in natural populations w h e r e multiple mating is frequent (Milkm a n and Zeitler, 1974; G r o m k o e t al., 1980), the null allele is effectively absent. At present we have no solution to this p r o b l e m except to suggest that t e m p e r a t u r e or other factors interact strongly with E S T 6 to influence s p e r m use and productivity. E x p e r i m e n t s are currently in progress to assess this possibility.
ACKNOWLEDGMENTS
W e are indebted to Dr. M a r k G r o m k o for the selected lines. Useful c o m m e n t s on an early draft w e r e provided by two a n o n y m o u s reviewers.
Est 6 and Remating in Drosophila melanogaster
207
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