Ecological Entomology (1995) 20, 191 - 194

SHORT COMMUNICATION

Paternity and female remating in Requena verticalis (Orthoptera: Tettigoniidae) DARRYL T. GWYNNE and A N D Y W . SNED,DEN Zoology Department, Erindale College, University of Toronto, Mississauga, Ontario, Canada

Key words. Tettigoniidae, sperm competition, courtship feeding, sperm- atophore.

A study of sperm competition in the tettigoniid Requena verticalis showed a pattern that differs from the sperm mixing noted in most insects (Parker, 1970; Gwynne, 1984). The paternity for the first of two male R.verticalis to mate was virtually complete (Gwynne, 1988a). The lack of paternity of the second males was of particular interest because the study used radiolabelling and genetic markers to show that these males provided a material investment in the first male’s offspring. The investment is a large spermatophylax, a part of the spermatophore eaten by the female, and enhances the fitness of her offspring (Gwynne, 1988b).

The question arises as to why second males mate, especially when spermatophyalx nutrients are costly to male R.verticalis (Davies & Dadour, 1989). Perhaps the 4-5-day female refractory period (Gwynne, 1986) used as the remating interval in Gwynne (1988a) does not reflect the actual remating interval in nature. Recent studies of R. verticalis support this contention. First, the fact that virgins outcompete already-mated females in the struggle for mates (Lynam et al., 1992) and are probably preferred by males (Simmons etal . , 1994) provides a mechanism for delayed female remating. Second, there is direct field evidence of such a delay: Simmons et al. (1994) showed a 13-day interval between two very distinct modes of adult ages of thirty-nine female observed mating; and, by monitoring a population of marked females for 15 days, K.-G. Heller (unpublished) noted an 11-day and a 13-day remating interval in the two rematings he observed during the study period. Evidence that female remating interval can influence paternity in orthopterans was found in Locusta migratoria (Acrididae). If female remating is delayed until after oviposition, paternity of the second male to mate (P2) more than doubles (Parker & Smith, 1975).

Here we examine the relative paternity of two R.verti- calk males when remating is delayed until after oviposition.

Correspondence: Dr D. T. Gwynne, Department of Zoology, Erindale College, University of Toronto, Mississauga, Ontario, LSL lC6. Canada.

We show that the average paternity of the second male (P2) is significantly higher when compared to the P2 of virtually zero found in the previous work. We discuss the possible relevance of the variation in paternity in R. verticalis in the light of the large male investment in the spermatophore.

R. verticalis is a listroscelidine katydid endemic to south- western Australia. Insemination follows the termination of copulation: the female removes and eats the spermato- phylax immediately after copulation. This meal is followed by removal and consumption of the empty sperm ampulla. Spermatophore size was obtained by determining male weight loss during mating (to the nearest 0.1 mg). Female katydids can mate and oviposit multiply. Details of the reproductive behaviour of this species are in Gwynne et al. (1984) and Gwynne (1986).

Nymphal males and females were collected from gardens in Perth, Western Australia, and shipped by air to Toronto. They were raised to adulthood in individuals jars in an environmental chamber at 12:12 L:D and 75% r.h., misted daily with water and fed a mixture of pollen, seeds and Tetra-min tropical fish food.

Sperm competition was determined using the sterile male technique (reviewed by Parker, 1970). Each mating replicate consisted of a female mated to two males. No individual was used more than once in any experiment.

Experimental males were normal (N) or sterilized (S) with 10krad of radiation (66.7min at 0.15 kad/min). The main treatments consisted of the two male mating-orders (NS and SN). In the two other treatments (SS and NN) the female was mated to two males of the same type. Two cases in which no eggs were laid, and two in which no eggs hatched after an N mating (probably because no sperm were transferred; Gwynne et al., 1984), were excluded from the analysis. Four NS, six SN, four NN and six SS matings remained.

The percentage fertilized (x) by the N male in NS and SN matings was estimated from the proportion of eggs hatching ( a ) following mating using mean viabilities in NN (b) and SS ( c ) matings using the formulax = (a -c ) / (b -c ) (Sillen-Tullberg, 1981). From these estimations we deter-

191

192 D . T. Gwynne and A . W . Snedden

mined the proportion of progeny sired by the second male (P2) for each mating. The two negative P2 values were adjusted to zero. Females were first mated when 1- 2 weeks old as adults, i.e. at a stage when few eggs have matured. Each mated female was given dry sand and this oviposition substrate was checked for eggs every 2-3 days. On the day after the first observation of eggs the female was paired with the second male and given a different male daily until she mated. After the second mating she was again given access to oviposition substrate. Eggs were hatched by incubating at 13-14°C for 12 weeks then warming to 25°C. The sperm competition analysis considered only the first clutch of eggs after the second mating.

All statistical procedures followed Systat (SYSTAT, 1992) and Winer (1971). Percentage data were arcsin- transformed before analysis. Measures of central tendency are presented as .t*SE or median and range (in the case of non-normal data). If data were non-normal (Kolmogorov-Smirnov (Lillifors), P < 0.05), or variances were heterogenous (Cochran’s test, P<O.O5), I used non- parametric tests. All tests of probability are two-tailed.

There were no significant differences between the four mating treatments in the number of eggs laid following the second mating: Kruskal-Wallis H = 7.38, P = 0.06 (medians and ranges: NS= 18, 5-48 (n=4); SN=24, 19-33 (n=6); SS=15, 11-37 ( n = 6 ) ; and NN=14, 11- 16 (n = 4)). Females laid a clutch of eggs at a median of 13 days following the first mating (range, 6-33 days (n = 20)) and, as they were then given access to males, they exhibited a median 14.5-day remating interval (range 11-37). The similarity of this value to 11-13-day remating interval noted in the field (see introduction) suggests that, in nature, females may remate after ovipositing.

Irradiation was effective in sterilizing males as the mean percentage of eggs hatching in the clutch of eggs laid after the second mating from six SS matings was 0.45k0.45 (range 0-2.7%). The mean percentage hatch following four NN matings was 71.8 f 3.6 (range 64-81%).

The percentage of eggs fertilized by the second male (P2) showed considerable variation around a median of 19.1% (n = 10) (Fig. 1). For the four NS matings the median was 13.3% (range 0-94.4%) and for the six SN matings 19.1% (range 0-69.4%). When the second of two males mated with female R. verticalis before she first ovi- posited, median P2 was 0 (n = 11) (data from Gwynne, 1988b), a value significantly lower than the 19.1% value reported here when the second mating occurs after ovi- position (Mann-Whitney U = 27, P = 0.019) (Fig. 1). However, the tendency to first-male paternity remains even though the second males probably transferred more sperm to the (older) females (Simmons et al . , 1993). It remains possible that the differences between Gwynne (1988b) and the present results are caused by differences in methodology, but this seems unlikely. A study com- paring genetic and sterile-male estimates of sperm com- petition in a single species found no differences in mean P2 (Eady, 1991).

What factors might determine the variation that we now

PROPORTION FERTILIZED BY 2ND MALE

Fig. 1. Frequency distribution of P2 values when second matings occur before oviposition (solid bars are data from Gwynnc. 1988a) and after oviposition (hatched bars, from the prcscnt study).

find in P2 (Fig. 1) (see Lewis & Austad, 1!490)? Although our sample size is small, some preliminary conclusions can be drawn. A longer remating interval doeq not appear to cause the variation (Spearman rank correlation of P- 9 versus remating interval = 0.19, P = 0.56 ( n = 10)). Another possibility is the relationship between male mating invest- ment and paternity. In the tettigoniid Decticrrs verruci- vorous a male’s paternity is positively related to the relative size of his spermatophylax meal (Wedell, 1991). The cause of this pattern is that in this species, and mqst other insects showing male courtship-feeding, a male which provides a small meal transfers fewer sperm because insemination is terminated by the female when the meal is complete (Thornhill, 1976; Sakaluk, 1984; Wedell & Arak, 1989; Reinhold & Heller, 1993). In contrast, in R. verticalis, consumption times of even the smallest spermatophylax meal was longer than necessary to transfer a full ejaculate (the average size of the spermatophylax meal was more than twice the size necessary for ejaculate transfer) (Gwynne, 1986). This argument is, however, based on data for matings with young females. The paternity of male R. verticalis producing below-average ,meals could be compromised in our second matings because males mating older females reduce spermatophylax size tiy an average of about 25% (Simmons et al . , 1993).

We did not estimate spermatophylax size, but did deter- mine total spermatophore mass. About 75% of spermato- phore mass is spermatophylax and the remainder, the mass of the sperm ampulla, is not influenced by female age (Simmons et al., 1993). A preliminary analysis suggested that spermatophylax size might be influencing paternity; there was a positive ( r = 0.54), although non-significant (P = 0.108) correlation between the second male’s

Paternity and female remating in Requena verticalis 193

0 0 0 0

0 0

0

0

0

40 50 60 70 80

SPERMATOPHORE MASS OF FIRST MALE (mg)

Fig. 2. Relationship between the mass of the first male’s sperm- atophore and his paternity. Open circles are SN matings; filled circles are NS matings.

paternity and the mass of his spermatophore relative to the first male ( n = 10). However, this relationship does not appear to be caused by female interference with the second insemination due to a reduced spermatophylax size. Such a mechanism predicts a correlation between P2 and the absolute mass of the second spermatophore which we did not find ( r = -0.18, P = 0.6, n = 10). Instead, the relationship between P2 and relative spermatophore size is due to a significant positive relationship between the absolute mass of the first male’s spermatophore and his paternity (r = 0.79, P = 0.006, n = 10) (Fig. 2).

The body mass of the first mating male also correlated with his paternity (post-mating mass used to exclude spermatophore mass: r = 0.74, P = 0.015, n = 10) (Fig. 3). Interestingly, post-mating body mass of the first male to mate does not correlate well with his spermatophore mass ( r = 0.27, P = 0.37, n = 20), yet both body and spermato- phore mass are positively related to paternity (partial correlation: spermatophore mass T = 2.8, P = 0.025, post- mating body mass, T = 2.3, P = 0.053), explaining 79% of the variation in P2 (F2,, = 13.2, P = 0.004). There are, however, no significant correlations between P2 and these two variables in the twenty second matings (partial cor- relation: spermatophore mass T = 0.4, P = 0.71, post- mating body mass, T = 1.8, P = O . l l ) , explaining 35% of the variance in P2 (F2,, = 1.8, P = 0.23).

What factors might explain correlations with body and spermatophore size in the first mating? First, spermato- phylaxes received by virgin females in this experiment did not appear to be smaller than in previous work (in which the smallest spermatophylax was still large enough to ensure full sperm transfer to virgins (Gwynne, 1986)): spermatophore masses were 65.9 -t 1.8 ( n = 20) and 60.0 L 3.0mg ( n = 18) in the two studies respectively.

1.2 w

3 B + 0.8

[ 0.6

01

0.4 z

m

0 0 0

0 0

0

0

0

100 200 300 400 500 BODY MASS OF FIRST MALE (mg)

Fig. 3. Relationship between the body mass of the first male to mate and his paternity. Open circles are SN matings; filled circles are NS matings.

Secondly, there is no evidence that the effect of spermato- phore size on paternity is due to larger sperm ampullae containing more sperm; there is no positive correlation between ampulla mass and sperm numbers in R.verticalis (Simmons et al., 1993). It is possible, however, that larger males transfer more sperm, independent of ampulla size.

A final suggestion is that virgin females mating with large males that provide large spermatophores prefer- entially use this male’s sperm following remating (Lloyd, 1979), because these phenotypic characters are indicators of male genetic quality (Thornhill & Alcock, 1983, discuss body size and male quality). The fact that we found significant patterns only in first matings may be due to chance effects due to sample size. Alternatively, these differences between matings may reflect the fact that the greater number of sperm inseminated into older females (our second matings) (Simmons et al., 1993) somehow conceals these patterns.

In conclusion, our results compared to Gwynne (1988b) (Fig. 1) argue strongly for detailed study of the natural remating interval of females as a prerequisite to any inves- tigation of paternity. Such studies are probably rare in investigations of sperm competition patterns in insects.

Acknowledgments

Thanks to Ian Dadour for collecting and shipping insects, Bill Cade and Brock University for allowing us access to the gamma cell used to irradiate males and to John Alcock, M. Andrade, Bill Brown, K.- G. Heller, G. A. Parker, L. Simmons and N. Wedell for comments. The research was supported by a grant from the NSERC (Canada) to D. T. Gwynne.

104 D. T. Gwyririe arid A. W . Stiedderi

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Accepted 21 November 1994