- Email: [email protected]
Sperm storage and utilisation and egg fertility in the sheep blowfly, Lucilia cuprina
J. Insecr Physiol. Vol. 34, &;o. 2, pp. 125-129,
1988
0022-1910/8853.00+O.OO Copyright 0 1988Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
SPERM STORAGE AND UTILISATION AND EGG FERTILITY IN THE SHEEP BLOWFLY, LUCILIA CUPRINA PETER H.
SMJTH,
LINDSAY BARTON BROWNE and A. C. M.
VAN
GERWEN
Divisiou of Entomology, CSIRO, G.P.O. Box 1700, Canberra, A.C.T. 2601, Australia (Received 22 April 1987; revised 10 July 1987)
Abstract-Makr of the sheep blowfly, Luciliu cuprina (Diptera: Calliphoridae), in their first mating, transferred an average. of 3019 sperm (n = 80) [range t&10,104] into the spermathecae of the female. When the number of sored sperm was greater than 1000, for each batch of about 250 eggs laid a constant 38% of the sperm stclre of the female was lost. The efficiency of sperm utilisation therefore varied from 15 to 1.5 sperm lost per egg fertilised across the range of 10,000 to 1000 sperm stored. Egg masses had high fertility (> 80%.1 if the sperm store was above 800 sperm, below this value fertility declined markedly. No loss of sperm from the spermathecae occurred in females held with no opportunity to lay for 9 days after
mating. Key Word Index: Luciliu cuprina, sperm storage, sperm
utilisation. fertility
MATERIALS AND METHODS
IN’I’RODIJCTION Studies of quantitative aspects of sperm transfer and the efficiency of sperm utilisation in insects have rarely proceeded beyond estimation of the number of sperm transferred at mating. An exception is the detailed study of the efficiency of sperm utilisation, that is, the number of sperm lost from storage for each egg fertilised, in Drosophila melanogaster Meig. which was initiated because of its relevance to genetic phenomena such as segregation distortion (Peacock and Erickson, 1965; Zimmering et al., 1970). Sperm transfer in this species is very wasteful. Of 4000 to 6000 sperm passed into the uterus of the female, only about 1100 enter the seminal receptacle and spermathecae, a process which takes 7 h to complete. Leakage from these stores occurs continuously thenceforth and the efficiency elf sperm utilisation varies depending on how rapidly the female lays her eggs (Fowler, 1973; Gilbert, 1981). The maximal efficiency of utilisation is about 2 sperm per egg (Gilbert, 1981). In contrast to Drosophila, in Lucilia cuprina (Wied.) [Merritt, 19881 as in L. sericata Meig. (Lewis and Pollock, 1975) sperm are pumped directly into the spermathecae, being observed there within 2 min of the initiation of mating. Sperm transfer into the spermathecae is complete by the end of copulation which takes about 11.min. Females of L. cuprina are anautogenous and have synchronised development of their ovarioles followed by laying in about 15 min of all mature eggs (aboat 250 in flies of the size used in these experiments-Vogt et al., 1985) in a single egg mass. First egg messes are of high fertility and therefore provide an opportunity for detailed investigation of the ethciency of sperm utilisation. We have studied the number of sperm transferred by males in their first mating, the efficiency of sperm utilisation and the relationship of the sperm store to egg fertility in this species.
Flies and rearing
The strain of L. cuprina (strain G) was collected near Canberra and had been in laboratory culture for between 86 and 123 generations at the time of the experiments. Larvae were reared on sheep liver at an initial larval density of 9000 per kg of liver at 27°C under 12 h light-12 h dark with lights on at 0600 h. Matings
All mating occurred in clear plastic containers (4 cm dia x 6.5 cm) with a wire gauze lid. One female and two males were placed in the mating pot. Mating duration under these conditions is not very variable; mean = 10.8 min, SD = 1.8 min (n = 100) [at 27”C]. Only matings of 10-12 min duration were used. At the time of mating, all males were 5-7 days after eclosion, had been segregated from females soon after emergence, had had access to liver on days 3 and 4 (emergence on day 1) and were mating for the first time. Females were 5-7 days after eclosion and had received similar treatment. They were thus gravid at the time of mating. Holding conditions
After mating females were kept singly in containers as above and fed sugar and water except when liver was presented for oviposition or protein feeding as indicated in the text. No eggs were laid by females when liver was not present. Egg collection and fertility determination
For collected egg masses and protein feeding for ovarian development fresh sheep liver was provided for each female from 1000 to 1600 h in a glass tube (14 mm dia x 48 mm) inserted into a hole in the side 125
126
PETERH. SMITHetal.
of the mating pot near to the base. Egg masses were lifted from the liver on a moist camel hair brush and spread onto moist filter paper. Fertility was determined by scoring the egg hatch 24 h later. Sperm counting
Table I. Number of sperm transferred into spermathecae of virgin females in first mating of male Number of sperm IS200 20&400 40&600 600-800 SO& IO00
Frequency 4
I 3 16 4 41
To determine the number of sperm stored, the three (occasionally four) spermathecae were dissected 100&2000 18 from a female under saline and placed on a slide in 2000-3000 15 300G4000 IO a small drop of saline. The preparation was observed 400&5000 9 under phase contrast and the cover slip tapped to 5000-6000 2 break the spermathecal capsules and spread the 600&7000 2 sperm. Tapping was continued until the sperm were 7OOt%8000 I 800&9000 3 well spread though not extending to the edge of the 9000-10000 3 cover slip. 1000&11000 I When fewer than 200 sperm were present a direct count was made. For larger numbers of sperm we used a variant of a method which estimates the length of thread in a plane to measure the total length of of mating (Table 1). The number of sperm transferred ranged from 0 to 10,104 with a median of about 2375, sperm on the slide. The length of thread in a plane can be determined by counting the number of occa- and a mean of 3019. These data are composed of counts made on five sions the thread intersects a grid of parallel lines according to the equation: Total length = n/2 x I x d occasions with males from different rearings. The mean sperm numbers obtained on these occasions where I = number of intersections, and d = distance (ranked) were 1723 (n = 18), 1963 (n = 20) 3853 between lines (Wiebel, 1969). (n = 20), 3950 (n = 10) and 4560 (n = 12). MannUnder 100 x phase contrast or dark-field illumination, a sperm-squash slide was moved across the Whitney U tests demonstrated significant (P < 0.05) field of view and the number of intersections of sperm differences in all comparisons of the two lowest with an eyepiece graticule line counted. The stage was means with the three highest. These differences are moved forward 1 mm and a similar traverse scored. presumably the result of rearing itself, all other This was repeated until the entire area of the cover conditions being strictly controlled, with all matings l&l2 min in duration. An implication for the experislip had been scanned. The sperm are of constant length (0.21 mm) and ments that follow is that for all comparisons equal the distance between grid lines (d) was 1 mm, there- numbers of sperm estimates had to be made within fore from the equation the number of sperm on a each rearing used. In our sperm-counting procedure the spermathecae slide was 7.32 x the number of intersections counted. were observed while being broken and the spermaSperm were rarely intersected twice, therefore thecae could be readily distinguished since the ducts where sufficient spreading was achieved so that intersections of different sperm were independent, the of one pair adhere. Though we made no counts of variance of an estimate is (from the binomial the- sperm in individual spermathecae, our impression was that approximately equal numbers of sperm were orem): Var(N) = N(l -p)/p where N = estimated present in each spermatheca throughout the range of number of sperm, and p = probability of intersecting any particular spermatozoan (= l/7.32 = 0.14). The sperm numbers observed. Sperm counts were carried out on 11 nulliparous 95% confidence limits of an estimate is f 5.03 x ,/N. Since clumping of sperm increases the variance of an females hand-caught in the field. The values ranged estimate, where clumping was noticed, scoring was from 589 to 6797 with a mean of 2610. Females rarely carried out at 0.5 mm intervals. Excluding these mate more than once (Smith ef al., 1988). Therefore, cases, the effect of clumping on the variance was these data suggest that field matings transfer similar estimated by regarding each sperm count as two numbers of sperm to those observed in the laboratory. independent counts at 2mm intervals. The variance of the difference between the square roots of each pair Loss of sperm with time of counts was significantly higher than the variance Cohorts of females from two rearings were mated expected from the binomial theorem. However removing 5% of the most clumped cases from the data then divided in two. Sperm counts were carried out removed this difference. Therefore, the effect of on 40 (20 + 20) females within 2 days of their mating. clumping can be disregarded in most of the data and The other females remained in individual pots after their mating with no opportunity to lay or mate. the 95% confidence limits from the binomial theorem as displayed in Figs 1 and 2 are good estimates of the Sperm numbers of 40 (20 + 20) of these females were likely errors in estimation. determined 9-10 days after mating. The two sets of counts were ranked and counts of the same rank plotted against each other (Fig. 1). The data closely RESULTS follow a 45” line indicating there is no difference between sperm numbers immediately after mating Number of sperm transferred at first mating and those 8 days later. The Mann-Whitney U-test Eighty counts of the number of sperm transferred also indicates no significant difference between the by males in their first mating were made within 2 days two sets of counts (U = 732, P = 0.52).
Sperm
F
10000
z E -
9000
storage
and utilisation
in Lucid
cup-inn
127
there were more than 1000 sperm before oviposition lie along a line parallel to the 45” line, indicating that above this value an approximately constant proportion of the sperm store is lost while fertilising an egg mass. The estimate of the proportion of the store lost is 38%. The sperm utilisation when the store is less than 1000 sperm at the beginning of laying is difficult to determine. The shape of the curve describing the points in this region in Fig. 2 is radically affected by statistical variation such as removal of low values
T
i
i
from either series of counts. Also (see below) the fertility of egg masses laid is reduced when the store
is in this range. Relationship between sperm store and reduction in fertility of egg masses
Fig. 1. Sperm numbers In spermathecae of females 1-2 days and 9-10 days after ma’:ing. The numbers were ranked then values of similar rank plotted against each other. The error bars indicate the 95% confidence limits of an estimate from our sperm scoring method across a range of sperm numbers.
Sperm lost while fertilising an egg mass
Cohorts of females from three rearings were mated then divided into two. Sperm counts were carried out on a sample of 40 1.18+ 12 + 10) females within 2 days of mating. The other females were provided with liver and those that laid egg masses retained and sperm counts carried out on a sample of 40 (18 + 12 + 10) females within 2 days of having laid. Since 89% of the females given the opportunity laid, there is little possibility of bias between the samples taken before and after laying. The two sets of counts were ranked then plotted against each other, as before, and with the axes transformed [Fig. 21. In the log plot the points where
An experiment was carried out to estimate the number of sperm in storage at which a reduction in the fertility of egg masses begins to occur. Females were mated and provided with liver on the next day. The fertilities (percentage hatch) of the 83 egg masses laid were determined. The 77 females whose egg masses were > 80% fertile were retained and exposed to a routine which maximised their rate of ovarian development and egg laying. Liver was presented to the females for a day of oviposition and on the next day, thus providing 2 days of protein feeding. After a day without liver feeding, liver for oviposition was again presented. The fertilities of all egg masses were determined. Sixteen females whose egg fertility had shown a marked decline from the fourth egg mass onwards were dissected and the number of sperm in their spermathecae determined. Seven females whose fourth to sixth egg mass was greater than 90% fertile were also dissected and their sperm stores determined. The fertility of eggs collected from a female remained high (i.e. >80%) through a series of egg masses then declined. Of the 83 females that laid at least one egg mass 35% laid a sequence of high fertility (> 80%) egg masses then one or more egg masses of low fertility. The rest ceased laying before 10000
‘Oooor9000
T
ET8ooQ i .L I
7000
i %
6000
i i
Sperm
number
before
laymg
Fig. 2. Two representations (linear scale, log scale) of sperm numbers in spermathecae of females before laying their first egg mass and after laying their first egg mass. The numbers were ranked then values of similar rank plotted against each other. The error bars indicate the 95% confidence limits of an estimate from our sperm scoring method across a range of sperm numbers.
PETER H. SMITH et al.
128
Fig. 3. Sperm numbers in the spermathecae of females that had laid a number of egg masses plotted against the percentage hatch of their last egg mass. The arrows indicate values exceeding the ordinate scale.
laying a low fertility egg mass. The proportions of females from these two categories that laid four or more highly fertile egg masses were 59 and 61%, that laid five or more were 31 and 44%, that laid six or more were 14 and 17% and that laid seven or more were 3 and 4%. One female laid nine highly fertile egg masses. Once substantial reduction in fertility occurred in an egg mass, fertility did not recover in subsequent egg masses. Instead, it declined rapidly reaching less than 30% within two or three egg masses. This suggests that the decline in fertility was a result of the sperm store being reduced to a level where it could no longer supply sufficient sperm. The number of sperm stored in the 23 females dissected is plotted against the fertility of their last egg mass in Fig. 3. Females whose last egg mass was less than 60% fertile retained fewer than 400 sperm in their spermathecae. Those whose last egg mass was between 60 and 75% fertile had up to 1200 with a mean of 559. All of those whose 4th to 6th egg mass was still greater than 90% fertile had sperm stores in excess of this mean value. This indicates that sperm stores that are reduced to below about 600 by laying an egg mass are below a threshold for achieving high fertility. DISCUSSION
In their first mating male Luciliu cuprina transferred an average of 3019 sperm (n = 80) into the spermathecae of the female. The range of numbers transferred by individual males was wide, from 0 to 10,104, and significant differences occurred between males from different rearings with means ranging from 1723 (n = 18) to 4560 (n = 12). Mean numbers of sperm transferred into the female’s storage organs have been estimated for a number of other Diptera including the nematocerans Simulium decorum Walker with 4048 (n = 43) (Linley and Simmons, 1983), Aedes aegypti L. with 1146 (n = 13) (Jones and Wheeler, 1965), and Culicoides melleus (Coq.) with 1156 (n = 26) (Linley and Hinds, 1974). Linley and Simmons (1983) report a wide range of values (572-6892) transferred by individual S. decorum. With regard to the higher Diptera, Gilbert (1981) reports that D. melanogaster males transfer about 1100 sperm into the female’s storage organs; Cunningham et al. (1971) estimated the number of
sperm transferred in the tephritid Ceratitis capitata (Wied.) as “many thousands”. No loss of sperm was evident from the spermathecae of females who were held with no opportunity to lay for a 9-day period (at 27’C) after mating. This is a considerable portion of a female’s lifetime, sufficient time for the laying of three egg masses, when the laying of even two egg masses is probably unusual for females in the field (Kitching. 1977). By contrast, continuous leakage of sperm from storage has been demonstrated in D. melunogaster (Gilbert. 1981) and gradual loss of sperm from storage in C. cupitata (Cunningham et al., 1971), though in this case, a rating system was used for sperm numbers and significant reduction in the sperm store could only be demonstrated after 44 days of isolation. When more than about 1000 sperm were present in the spermathecae a constant proportion of the sperm store, estimated as 38%, was lost while fertilising the first egg mass. The data give little indication of the sperm loss when the store is less than 1000 because the ranking method of analysis used is very sensitive to variation at its extremes. Since first egg masses are highly fertile, estimates of the efficiency of sperm utilisation (i.e. the number of sperm lost for each egg fertilised) can be made. The efficiency ranges from 15 sperm per egg with 10,000 stored to 1.5 sperm per egg with 1000 stored. There seem to be no other measurements of sperm utilisation across a wide range of stored sperm numbers. In D. melunogaster efficiency of sperm utilisation has been measured by a number of workers (Peacock and Erickson, 1965; Zimmering et al., 1970). It has been found to differ among strains and among methods of egg collection (Fowler, 1973). Gilbert (1981) claims that there is continuous loss of sperm from storage in this species and that the utilisation efficiency is therefore determined by the rate of egg laying and the degree of sperm motility. He considers two sperm per egg as the maximal efficiency of utilisation in this species. When females lay a series of egg masses, a variable number of highly fertile (> 80%) egg masses is laid before any egg mass of low fertility. Once an egg mass of less than 80% fertility is laid, subsequent egg masses show a rapid reduction in fertility. This suggests that the sperm store determines the fertility of egg masses. The data show that on average, when a sperm store is reduced to below about 600 by laying an egg mass, the egg mass has substantially reduced fertility. Assuming a utilisation efficiency of 1.5 sperm per egg, the value measured with 1000 sperm in store, this indicates that only a female with a sperm store of greater than about 800 will lay egg masses of high fertility. A single mating generally provides sufficient sperm for the female’s lifetime egg production. In the experiment in which the females were called upon to lay a series of egg masses the most fecund female produced 9 egg masses all highly fertile; a total of 1703 fertile eggs. Twenty-three of the 70 females who laid produced more than 1000 fertile eggs, and 51 of the 70 produced more than 500 fertile eggs. A female rarely lays more than two egg masses, amounting to about 500 eggs, under field conditions (Kitching, 1977). We have only demonstrated proportional sperm
Sperm
storage
and utilisation
loss when fertilising the first egg mass. However a wide range of stored sperm numbers (lOOO-10,000) were involved so it seems likely that a similar relationship would apply to all egg masses of high fertility. This can be checked from the data of the last experiment by extrapolating the relationship backwards through each female’s sequence of egg masses to predict the original distribution of sperm numbers. In 30 females a sequence of highly fertile egg masses was followed by one or more egg masses of low fertility. Using the minimal figure of 800 stored sperm required to produce 1he last highly fertile egg mass and our estimate of 311%loss of sperm in laying each egg mass, the predicted original sperm numbers for these 30 females are distributed with a mean of 3732. If all of the females in the experiment are included (assuming that if they had continued laying their next egg mass would have had low fertility) the mean is 4196. The mean original sperm number would be somewhat higher than this value because some females may have had more than a minimal store at laying their final highly fertile egg mass, while others may have had a sufficient store when they stopped laying to produce furlher egg masses of high fertility. However, the value would be in reasonable accord with the measurements of sperm number directly after mating in which the cohort with the highest sperm number had a mean of 4560. Average fertility was high in this experiment compared to that in other similar experiments we carried out (Smith et al., 1988), so a high sperm number would be expected. The estimated original sperm number for the most fecund female is 36,640 considerably higher than the maximal sperm number we have scored. This may be due to accumulating an approximate multiplying factor over several egg masses. Using 30% sperm lost for each egg mass le.id reduces this sperm number estimate to 13,877, quite a likely value, but the means then are 2383 and 2588, respectively, values that are smaller than expected. Proportional utilisation of the sperm store at all high fertility egg masses with a loss rate in the range 3@?8% thus readily accommodates these data. Gilbert et al. (1981) have successfully applied a mathematical model for progeny production in D. melanogaster whit n incorporates continuous exponential decline of the sperm store. Their sperm counts confirm that such an exponential decline is a major feature until eggs begin to lose fertility. Lucilia’s habit of laying discrete egg masses has enabled us to give a direct demonstration of the phenomenon of exponential utilisation in this species, though the system differs in that release of sperm only occurs during short periods iassociated with egg laying, while at other times no release of sperm occurs. Therefore the basic mechanism of sperm release while fertil-
in Lucilia cuprina
129
isation is occurring seems to have similar stochastic properties in L. cuprina to those accorded to D. melanogaster by Gilbert et al. (1981). The release of each sperm can be regarded as a probabilistic event independent of other sperm release or of egg release. Acknowledgement-We thank Mr R. Delves who reared the flies used in these experiments.
1988
0022-1910/8853.00+O.OO Copyright 0 1988Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
SPERM STORAGE AND UTILISATION AND EGG FERTILITY IN THE SHEEP BLOWFLY, LUCILIA CUPRINA PETER H.
SMJTH,
LINDSAY BARTON BROWNE and A. C. M.
VAN
GERWEN
Divisiou of Entomology, CSIRO, G.P.O. Box 1700, Canberra, A.C.T. 2601, Australia (Received 22 April 1987; revised 10 July 1987)
Abstract-Makr of the sheep blowfly, Luciliu cuprina (Diptera: Calliphoridae), in their first mating, transferred an average. of 3019 sperm (n = 80) [range t&10,104] into the spermathecae of the female. When the number of sored sperm was greater than 1000, for each batch of about 250 eggs laid a constant 38% of the sperm stclre of the female was lost. The efficiency of sperm utilisation therefore varied from 15 to 1.5 sperm lost per egg fertilised across the range of 10,000 to 1000 sperm stored. Egg masses had high fertility (> 80%.1 if the sperm store was above 800 sperm, below this value fertility declined markedly. No loss of sperm from the spermathecae occurred in females held with no opportunity to lay for 9 days after
mating. Key Word Index: Luciliu cuprina, sperm storage, sperm
utilisation. fertility
MATERIALS AND METHODS
IN’I’RODIJCTION Studies of quantitative aspects of sperm transfer and the efficiency of sperm utilisation in insects have rarely proceeded beyond estimation of the number of sperm transferred at mating. An exception is the detailed study of the efficiency of sperm utilisation, that is, the number of sperm lost from storage for each egg fertilised, in Drosophila melanogaster Meig. which was initiated because of its relevance to genetic phenomena such as segregation distortion (Peacock and Erickson, 1965; Zimmering et al., 1970). Sperm transfer in this species is very wasteful. Of 4000 to 6000 sperm passed into the uterus of the female, only about 1100 enter the seminal receptacle and spermathecae, a process which takes 7 h to complete. Leakage from these stores occurs continuously thenceforth and the efficiency elf sperm utilisation varies depending on how rapidly the female lays her eggs (Fowler, 1973; Gilbert, 1981). The maximal efficiency of utilisation is about 2 sperm per egg (Gilbert, 1981). In contrast to Drosophila, in Lucilia cuprina (Wied.) [Merritt, 19881 as in L. sericata Meig. (Lewis and Pollock, 1975) sperm are pumped directly into the spermathecae, being observed there within 2 min of the initiation of mating. Sperm transfer into the spermathecae is complete by the end of copulation which takes about 11.min. Females of L. cuprina are anautogenous and have synchronised development of their ovarioles followed by laying in about 15 min of all mature eggs (aboat 250 in flies of the size used in these experiments-Vogt et al., 1985) in a single egg mass. First egg messes are of high fertility and therefore provide an opportunity for detailed investigation of the ethciency of sperm utilisation. We have studied the number of sperm transferred by males in their first mating, the efficiency of sperm utilisation and the relationship of the sperm store to egg fertility in this species.
Flies and rearing
The strain of L. cuprina (strain G) was collected near Canberra and had been in laboratory culture for between 86 and 123 generations at the time of the experiments. Larvae were reared on sheep liver at an initial larval density of 9000 per kg of liver at 27°C under 12 h light-12 h dark with lights on at 0600 h. Matings
All mating occurred in clear plastic containers (4 cm dia x 6.5 cm) with a wire gauze lid. One female and two males were placed in the mating pot. Mating duration under these conditions is not very variable; mean = 10.8 min, SD = 1.8 min (n = 100) [at 27”C]. Only matings of 10-12 min duration were used. At the time of mating, all males were 5-7 days after eclosion, had been segregated from females soon after emergence, had had access to liver on days 3 and 4 (emergence on day 1) and were mating for the first time. Females were 5-7 days after eclosion and had received similar treatment. They were thus gravid at the time of mating. Holding conditions
After mating females were kept singly in containers as above and fed sugar and water except when liver was presented for oviposition or protein feeding as indicated in the text. No eggs were laid by females when liver was not present. Egg collection and fertility determination
For collected egg masses and protein feeding for ovarian development fresh sheep liver was provided for each female from 1000 to 1600 h in a glass tube (14 mm dia x 48 mm) inserted into a hole in the side 125
126
PETERH. SMITHetal.
of the mating pot near to the base. Egg masses were lifted from the liver on a moist camel hair brush and spread onto moist filter paper. Fertility was determined by scoring the egg hatch 24 h later. Sperm counting
Table I. Number of sperm transferred into spermathecae of virgin females in first mating of male Number of sperm IS200 20&400 40&600 600-800 SO& IO00
Frequency 4
I 3 16 4 41
To determine the number of sperm stored, the three (occasionally four) spermathecae were dissected 100&2000 18 from a female under saline and placed on a slide in 2000-3000 15 300G4000 IO a small drop of saline. The preparation was observed 400&5000 9 under phase contrast and the cover slip tapped to 5000-6000 2 break the spermathecal capsules and spread the 600&7000 2 sperm. Tapping was continued until the sperm were 7OOt%8000 I 800&9000 3 well spread though not extending to the edge of the 9000-10000 3 cover slip. 1000&11000 I When fewer than 200 sperm were present a direct count was made. For larger numbers of sperm we used a variant of a method which estimates the length of thread in a plane to measure the total length of of mating (Table 1). The number of sperm transferred ranged from 0 to 10,104 with a median of about 2375, sperm on the slide. The length of thread in a plane can be determined by counting the number of occa- and a mean of 3019. These data are composed of counts made on five sions the thread intersects a grid of parallel lines according to the equation: Total length = n/2 x I x d occasions with males from different rearings. The mean sperm numbers obtained on these occasions where I = number of intersections, and d = distance (ranked) were 1723 (n = 18), 1963 (n = 20) 3853 between lines (Wiebel, 1969). (n = 20), 3950 (n = 10) and 4560 (n = 12). MannUnder 100 x phase contrast or dark-field illumination, a sperm-squash slide was moved across the Whitney U tests demonstrated significant (P < 0.05) field of view and the number of intersections of sperm differences in all comparisons of the two lowest with an eyepiece graticule line counted. The stage was means with the three highest. These differences are moved forward 1 mm and a similar traverse scored. presumably the result of rearing itself, all other This was repeated until the entire area of the cover conditions being strictly controlled, with all matings l&l2 min in duration. An implication for the experislip had been scanned. The sperm are of constant length (0.21 mm) and ments that follow is that for all comparisons equal the distance between grid lines (d) was 1 mm, there- numbers of sperm estimates had to be made within fore from the equation the number of sperm on a each rearing used. In our sperm-counting procedure the spermathecae slide was 7.32 x the number of intersections counted. were observed while being broken and the spermaSperm were rarely intersected twice, therefore thecae could be readily distinguished since the ducts where sufficient spreading was achieved so that intersections of different sperm were independent, the of one pair adhere. Though we made no counts of variance of an estimate is (from the binomial the- sperm in individual spermathecae, our impression was that approximately equal numbers of sperm were orem): Var(N) = N(l -p)/p where N = estimated present in each spermatheca throughout the range of number of sperm, and p = probability of intersecting any particular spermatozoan (= l/7.32 = 0.14). The sperm numbers observed. Sperm counts were carried out on 11 nulliparous 95% confidence limits of an estimate is f 5.03 x ,/N. Since clumping of sperm increases the variance of an females hand-caught in the field. The values ranged estimate, where clumping was noticed, scoring was from 589 to 6797 with a mean of 2610. Females rarely carried out at 0.5 mm intervals. Excluding these mate more than once (Smith ef al., 1988). Therefore, cases, the effect of clumping on the variance was these data suggest that field matings transfer similar estimated by regarding each sperm count as two numbers of sperm to those observed in the laboratory. independent counts at 2mm intervals. The variance of the difference between the square roots of each pair Loss of sperm with time of counts was significantly higher than the variance Cohorts of females from two rearings were mated expected from the binomial theorem. However removing 5% of the most clumped cases from the data then divided in two. Sperm counts were carried out removed this difference. Therefore, the effect of on 40 (20 + 20) females within 2 days of their mating. clumping can be disregarded in most of the data and The other females remained in individual pots after their mating with no opportunity to lay or mate. the 95% confidence limits from the binomial theorem as displayed in Figs 1 and 2 are good estimates of the Sperm numbers of 40 (20 + 20) of these females were likely errors in estimation. determined 9-10 days after mating. The two sets of counts were ranked and counts of the same rank plotted against each other (Fig. 1). The data closely RESULTS follow a 45” line indicating there is no difference between sperm numbers immediately after mating Number of sperm transferred at first mating and those 8 days later. The Mann-Whitney U-test Eighty counts of the number of sperm transferred also indicates no significant difference between the by males in their first mating were made within 2 days two sets of counts (U = 732, P = 0.52).
Sperm
F
10000
z E -
9000
storage
and utilisation
in Lucid
cup-inn
127
there were more than 1000 sperm before oviposition lie along a line parallel to the 45” line, indicating that above this value an approximately constant proportion of the sperm store is lost while fertilising an egg mass. The estimate of the proportion of the store lost is 38%. The sperm utilisation when the store is less than 1000 sperm at the beginning of laying is difficult to determine. The shape of the curve describing the points in this region in Fig. 2 is radically affected by statistical variation such as removal of low values
T
i
i
from either series of counts. Also (see below) the fertility of egg masses laid is reduced when the store
is in this range. Relationship between sperm store and reduction in fertility of egg masses
Fig. 1. Sperm numbers In spermathecae of females 1-2 days and 9-10 days after ma’:ing. The numbers were ranked then values of similar rank plotted against each other. The error bars indicate the 95% confidence limits of an estimate from our sperm scoring method across a range of sperm numbers.
Sperm lost while fertilising an egg mass
Cohorts of females from three rearings were mated then divided into two. Sperm counts were carried out on a sample of 40 1.18+ 12 + 10) females within 2 days of mating. The other females were provided with liver and those that laid egg masses retained and sperm counts carried out on a sample of 40 (18 + 12 + 10) females within 2 days of having laid. Since 89% of the females given the opportunity laid, there is little possibility of bias between the samples taken before and after laying. The two sets of counts were ranked then plotted against each other, as before, and with the axes transformed [Fig. 21. In the log plot the points where
An experiment was carried out to estimate the number of sperm in storage at which a reduction in the fertility of egg masses begins to occur. Females were mated and provided with liver on the next day. The fertilities (percentage hatch) of the 83 egg masses laid were determined. The 77 females whose egg masses were > 80% fertile were retained and exposed to a routine which maximised their rate of ovarian development and egg laying. Liver was presented to the females for a day of oviposition and on the next day, thus providing 2 days of protein feeding. After a day without liver feeding, liver for oviposition was again presented. The fertilities of all egg masses were determined. Sixteen females whose egg fertility had shown a marked decline from the fourth egg mass onwards were dissected and the number of sperm in their spermathecae determined. Seven females whose fourth to sixth egg mass was greater than 90% fertile were also dissected and their sperm stores determined. The fertility of eggs collected from a female remained high (i.e. >80%) through a series of egg masses then declined. Of the 83 females that laid at least one egg mass 35% laid a sequence of high fertility (> 80%) egg masses then one or more egg masses of low fertility. The rest ceased laying before 10000
‘Oooor9000
T
ET8ooQ i .L I
7000
i %
6000
i i
Sperm
number
before
laymg
Fig. 2. Two representations (linear scale, log scale) of sperm numbers in spermathecae of females before laying their first egg mass and after laying their first egg mass. The numbers were ranked then values of similar rank plotted against each other. The error bars indicate the 95% confidence limits of an estimate from our sperm scoring method across a range of sperm numbers.
PETER H. SMITH et al.
128
Fig. 3. Sperm numbers in the spermathecae of females that had laid a number of egg masses plotted against the percentage hatch of their last egg mass. The arrows indicate values exceeding the ordinate scale.
laying a low fertility egg mass. The proportions of females from these two categories that laid four or more highly fertile egg masses were 59 and 61%, that laid five or more were 31 and 44%, that laid six or more were 14 and 17% and that laid seven or more were 3 and 4%. One female laid nine highly fertile egg masses. Once substantial reduction in fertility occurred in an egg mass, fertility did not recover in subsequent egg masses. Instead, it declined rapidly reaching less than 30% within two or three egg masses. This suggests that the decline in fertility was a result of the sperm store being reduced to a level where it could no longer supply sufficient sperm. The number of sperm stored in the 23 females dissected is plotted against the fertility of their last egg mass in Fig. 3. Females whose last egg mass was less than 60% fertile retained fewer than 400 sperm in their spermathecae. Those whose last egg mass was between 60 and 75% fertile had up to 1200 with a mean of 559. All of those whose 4th to 6th egg mass was still greater than 90% fertile had sperm stores in excess of this mean value. This indicates that sperm stores that are reduced to below about 600 by laying an egg mass are below a threshold for achieving high fertility. DISCUSSION
In their first mating male Luciliu cuprina transferred an average of 3019 sperm (n = 80) into the spermathecae of the female. The range of numbers transferred by individual males was wide, from 0 to 10,104, and significant differences occurred between males from different rearings with means ranging from 1723 (n = 18) to 4560 (n = 12). Mean numbers of sperm transferred into the female’s storage organs have been estimated for a number of other Diptera including the nematocerans Simulium decorum Walker with 4048 (n = 43) (Linley and Simmons, 1983), Aedes aegypti L. with 1146 (n = 13) (Jones and Wheeler, 1965), and Culicoides melleus (Coq.) with 1156 (n = 26) (Linley and Hinds, 1974). Linley and Simmons (1983) report a wide range of values (572-6892) transferred by individual S. decorum. With regard to the higher Diptera, Gilbert (1981) reports that D. melanogaster males transfer about 1100 sperm into the female’s storage organs; Cunningham et al. (1971) estimated the number of
sperm transferred in the tephritid Ceratitis capitata (Wied.) as “many thousands”. No loss of sperm was evident from the spermathecae of females who were held with no opportunity to lay for a 9-day period (at 27’C) after mating. This is a considerable portion of a female’s lifetime, sufficient time for the laying of three egg masses, when the laying of even two egg masses is probably unusual for females in the field (Kitching. 1977). By contrast, continuous leakage of sperm from storage has been demonstrated in D. melunogaster (Gilbert. 1981) and gradual loss of sperm from storage in C. cupitata (Cunningham et al., 1971), though in this case, a rating system was used for sperm numbers and significant reduction in the sperm store could only be demonstrated after 44 days of isolation. When more than about 1000 sperm were present in the spermathecae a constant proportion of the sperm store, estimated as 38%, was lost while fertilising the first egg mass. The data give little indication of the sperm loss when the store is less than 1000 because the ranking method of analysis used is very sensitive to variation at its extremes. Since first egg masses are highly fertile, estimates of the efficiency of sperm utilisation (i.e. the number of sperm lost for each egg fertilised) can be made. The efficiency ranges from 15 sperm per egg with 10,000 stored to 1.5 sperm per egg with 1000 stored. There seem to be no other measurements of sperm utilisation across a wide range of stored sperm numbers. In D. melunogaster efficiency of sperm utilisation has been measured by a number of workers (Peacock and Erickson, 1965; Zimmering et al., 1970). It has been found to differ among strains and among methods of egg collection (Fowler, 1973). Gilbert (1981) claims that there is continuous loss of sperm from storage in this species and that the utilisation efficiency is therefore determined by the rate of egg laying and the degree of sperm motility. He considers two sperm per egg as the maximal efficiency of utilisation in this species. When females lay a series of egg masses, a variable number of highly fertile (> 80%) egg masses is laid before any egg mass of low fertility. Once an egg mass of less than 80% fertility is laid, subsequent egg masses show a rapid reduction in fertility. This suggests that the sperm store determines the fertility of egg masses. The data show that on average, when a sperm store is reduced to below about 600 by laying an egg mass, the egg mass has substantially reduced fertility. Assuming a utilisation efficiency of 1.5 sperm per egg, the value measured with 1000 sperm in store, this indicates that only a female with a sperm store of greater than about 800 will lay egg masses of high fertility. A single mating generally provides sufficient sperm for the female’s lifetime egg production. In the experiment in which the females were called upon to lay a series of egg masses the most fecund female produced 9 egg masses all highly fertile; a total of 1703 fertile eggs. Twenty-three of the 70 females who laid produced more than 1000 fertile eggs, and 51 of the 70 produced more than 500 fertile eggs. A female rarely lays more than two egg masses, amounting to about 500 eggs, under field conditions (Kitching, 1977). We have only demonstrated proportional sperm
Sperm
storage
and utilisation
loss when fertilising the first egg mass. However a wide range of stored sperm numbers (lOOO-10,000) were involved so it seems likely that a similar relationship would apply to all egg masses of high fertility. This can be checked from the data of the last experiment by extrapolating the relationship backwards through each female’s sequence of egg masses to predict the original distribution of sperm numbers. In 30 females a sequence of highly fertile egg masses was followed by one or more egg masses of low fertility. Using the minimal figure of 800 stored sperm required to produce 1he last highly fertile egg mass and our estimate of 311%loss of sperm in laying each egg mass, the predicted original sperm numbers for these 30 females are distributed with a mean of 3732. If all of the females in the experiment are included (assuming that if they had continued laying their next egg mass would have had low fertility) the mean is 4196. The mean original sperm number would be somewhat higher than this value because some females may have had more than a minimal store at laying their final highly fertile egg mass, while others may have had a sufficient store when they stopped laying to produce furlher egg masses of high fertility. However, the value would be in reasonable accord with the measurements of sperm number directly after mating in which the cohort with the highest sperm number had a mean of 4560. Average fertility was high in this experiment compared to that in other similar experiments we carried out (Smith et al., 1988), so a high sperm number would be expected. The estimated original sperm number for the most fecund female is 36,640 considerably higher than the maximal sperm number we have scored. This may be due to accumulating an approximate multiplying factor over several egg masses. Using 30% sperm lost for each egg mass le.id reduces this sperm number estimate to 13,877, quite a likely value, but the means then are 2383 and 2588, respectively, values that are smaller than expected. Proportional utilisation of the sperm store at all high fertility egg masses with a loss rate in the range 3@?8% thus readily accommodates these data. Gilbert et al. (1981) have successfully applied a mathematical model for progeny production in D. melanogaster whit n incorporates continuous exponential decline of the sperm store. Their sperm counts confirm that such an exponential decline is a major feature until eggs begin to lose fertility. Lucilia’s habit of laying discrete egg masses has enabled us to give a direct demonstration of the phenomenon of exponential utilisation in this species, though the system differs in that release of sperm only occurs during short periods iassociated with egg laying, while at other times no release of sperm occurs. Therefore the basic mechanism of sperm release while fertil-
in Lucilia cuprina
129
isation is occurring seems to have similar stochastic properties in L. cuprina to those accorded to D. melanogaster by Gilbert et al. (1981). The release of each sperm can be regarded as a probabilistic event independent of other sperm release or of egg release. Acknowledgement-We thank Mr R. Delves who reared the flies used in these experiments.