Ó Springer-Verlag 1999 Behav Ecol Sociobiol (1999) 45: 293±299 ORIGINAL ARTICLE Klaus Reinhold Paternal investment in Poecilimon veluchianus bushcrickets: bene®cial effects of nuptial feeding on offspring viability Received: 15 May 1998 / Accepted after revision: 11 October 1998 Abstract In most bushcrickets, males transfer a large spermatophore during copulation that is afterwards consumed by the female. In some species this nuptial gift enhances o€spring ®tness and is therefore believed to function as paternal investment. To determine whether this is the case, I examined whether a male's own o€spring bene®t from spermatophore consumption in the bushcricket Poecilimon veluchianus. Females that consumed a spermatophore produced o€spring with increased residuals of dry weight compared to females that were prevented from feeding on the spermatophore. This bene®cial e€ect of spermatophore consumption occurred within the ®rst 4 days after copulation. An increased dry weight indicates higher energy reserves because o€spring dry weight correlates signi®cantly with the lifespan of starved larvae and because spermatophore consumption increased the lifespan of starved o€spring. During egg-laying, females apply a liquid substance to the soil that hardens and probably serves as protection for the egg clutch. The amount of this substance correlated with the number of eggs laid but did not di€er between spermatophore treatments. In P. veluchianus, females mate frequently and there is lastmale sperm precedence. The spermatophore thus only constitutes paternal investment when o€spring produced before female remating bene®t from spermatophore consumption. The dry weight of o€spring increased during the ®rst 4 days after spermatophore consumption and thus within the natural remating interval. This shows that the spermatophore has a paternal investment K. Reinhold (&) Institut fuÈr Zoologie II, UniversitaÈt Erlangen Staudtstraûe 5, D-91053 Erlangen Germany Present address: Institut fuÈr Evolutionsbiologie und OÈkologie UniversitaÈt Bonn, An der Immenburg 1 D-53121 Bonn, Germany e-mail: kreinhold@evolution.uni-bonn.de Tel.: +49-228-735119, Fax: +49-228-735129 function in addition to its already known sperm protection function. Key words Katydids á Nuptial gifts á Spermatophore á O€spring ®tness Introduction During copulation, male bushcrickets transfer large spermatophores as nuptial gifts (for reviews see Gwynne 1990; Gwynne 1997; Vahed 1998). Such a spermatophore consists of the sperm-free spermatophylax and the sperm-containing ampulla. After mating, the female consumes ®rst the spermatophylax and later the ampulla. There are two major hypotheses concerning the function of the spermatophylax: (1) The spermatophylax protects the sperm from being consumed prematurely and may also protect the ejaculatory components that induce a refractory period. According to this hypothesis the spermatophylax serves as mating e€ort. (2) The contents of the spermatophylax increase the ®tness of the male's o€spring. According to this hypothesis the spermatophylax serves as paternal investment. There has been considerable debate over these hypotheses (Gwynne 1986a,b, 1988a,b; Wickler 1985, 1986; Quinn and Sakaluk 1986; Sakaluk 1986; Simmons 1995). These two hypotheses are not exclusive alternatives, and there is evidence for both functions. In some species the spermatophylax seems to serve as a sperm protection device because (a) its size is adjusted in accordance with the time necessary for the sperm to reach the spermatheca (Wedell 1991, 1993b; Reinhold and Heller 1993; Heller and Reinhold 1994), (b) sperm number and spermatophylax size covary (Wedell 1993b), and (c) no e€ects of spermatophylax consumption on o€spring ®tness have been observed (Wedell and Arak 1989; 294 Vahed and Gilbert 1997). In other species, the spermatophylax seems to serve as paternal investment because spermatophore consumption increases o€spring ®tness (Gwynne 1984, 1988a; Simmons 1988, 1990) and because its size is larger than necessary for sperm protection: females take longer to consume the spermatophylax than is necessary for sperm transfer (Gwynne 1986b; but see Simmons 1995). Given that o€spring ®tness increases as an e€ect of spermatophore consumption, the spermatophore can only constitute paternal investment if spermatophore consumption increases the ®tness of a male's own o€spring. There is evidence that bushcricket females remate frequently (Heller and von Helversen 1991), and at least in some species the last male will father the majority of the o€spring (Achmann et al. 1992). Under these conditions, it is crucial to know whether spermatophore consumption increases o€spring ®tness before female remating. If the bene®cial e€ects of spermatophore consumption only appear after female remating, the male will not invest in his own o€spring and the spermatophore thus will constitute ``pseudo-parental investment'' (Wickler 1985). One method to estimate the interval between mating and the occurrence of bene®cial e€ects of spermatophore consumption is to radioactively label the spermatophore and measure the time lag between mating and the incorporation of radioactivity into the eggs (Huignard 1983; Bowen et al. 1984; Wedell 1993a). Another possible method is to measure some components of o€spring ®tness at di€erent intervals after spermatophore consumption (Simmons 1990). This second method has an advantage over radioactive labelling. Female bushcrickets that have recently consumed a spermatophore may invest some additional nutrients from their reserves into the eggs because they can soon replenish these reserves with the nutrients of the spermatophore (Wickler 1994). In this way, the time lag between mating and bene®cial e€ects on o€spring ®tness might decrease markedly. Here, I use the second method and examine whether spermatophore consumption does bene®t o€spring produced within a few days after mating in the bushcricket Poecilimon veluchianus. Females of this species remate frequently, approximately every 2±5 days, and the last male will father the majority of the o€spring (Heller and von Helversen 1991; Achmann et al. 1992). For a spermatophylax to function as paternal investment, the bene®cial e€ects on o€spring ®tness should therefore occur within this period of time. In previous experiments we were able to show that o€spring bene®t from female spermatophore consumption (Reinhold and Heller 1993). Here, in an extension of our previous work, I examine whether o€spring bene®t from spermatophore consumption within the ®rst 4 days after mating. In previous experiments, we showed that spermatophore consumption increases the relative dry weight of P. veluchianus o€spring (Reinhold and Heller 1993). But, there was no signi®cant e€ect of spermatophore consumption on egg number, egg weight or o€spring weight. The increased relative dry weight of o€spring was interpreted as the e€ect of larger energy reserves. To examine whether spermatophore consumption leads to increased energy reserves, the lifespan of starved o€spring was compared between di€erent spermatophore treatments. O€spring of females that consumed a spermatophore showed an increased ability to survive longer periods without food than o€spring of females prevented from consuming a spermatophore. Methods Poecilimon veluchianus Ramme, 1933 is a medium-sized (body weight 0.5±1 g), nocturnal, herbivorous bushcricket endemic to central Greece (Willemse and Heller 1992). During mating, a spermatophore that constitutes approximately 25% of male body weight (Heller and Reinhold 1994) is transferred to the female. This spermatophore consists of the sperm-free spermatophylax and the sperm-containing ampulla. After copulation, it takes a female on average 9 h to consume the spermatophylax (Reinhold and Heller 1993). When the spermatophylax has been consumed completely, the female also eats the ampulla. Around sunset, females deposit their eggs in the soil. Individual females oviposit almost daily, approximately three eggs per day (Reinhold and Heller 1993). The following experiments were conducted with bushcrickets from a meadow 4 km north of the village of Vitoli, Greece (west of Makrakomi, Nomos Fthiotis, Greece, 22°01¢ E 38°58¢ N). There, the reproductive season of the univoltine species lasts approximately from the end of May to the end of June. In May, 40 females were collected as nymphs and caged near their natural habitat. They were fed buds and ¯owers of Spartium junceum ad libitum. The cages with the females were inspected daily for moulted insects. Adult females were separated and transferred into small plastic cages (0.4 l) and supplied with fresh twigs of Spartium as food. Cages were supplied with ®ne sieved sand as oviposition substrate. Six days after the adult moult, a virgin female was paired with a male that had not mated for at least 2 days. Males that remate after a shorter interval would produce spermatophores that are often too small for successful insemination (Reinhold and Heller 1993; Reinhold and von Helversen 1997). Each night, a female was paired randomly with a male until the female mated. Evidence of mating is easy to detect in these insects, because the large spermatophore is conspicuous. Shortly after mating, females were prevented from feeding on the spermatophore by sprinkling ®ne sand on it (Reinhold and Heller 1993). When treated spermatophores had not fallen o€ by the following morning, they were removed with forceps approximately at the time when unmanipulated females ®nished consuming the spermatophore. When a female started ovipositing, a male was again placed into her cage, for approximately 3 h for a maximum of 5 days, until the female remated. These remated females were randomly assigned to two di€erent treatments. Females in the ®rst treatment were again prevented from feeding on the spermatophore, while females in the second treatment were allowed to consume the spermatophore. Sixteen females were assigned to both treatments and the other eight females did not mate a second time. All females in the second treatment consumed their spermatophore. Four females, two in each treatment, were not included because o€spring hatching rates were below 50% or because they died within 5 days after their second copulation. Many females started laying eggs only 4 or more days after their initial copulation. Whether spermatophore consumption increases o€spring ®tness within a short period after copulation can thus only be examined after a subsequent mating. All females were prevented from consuming their ®rst spermatophore, and o€spring bene®ts were thus compared between females that consumed one or no spermatophore. If all females had been allowed to consume their 295 ®rst spermatophore, o€spring bene®ts would have been compared between females that consumed one or two spermatophores. When there is variance in spermatophore contents leading to variance in o€spring bene®ts, the variance of the di€erence between treatments would increase and detection of o€spring bene®ts would have been less likely. Detection of o€spring bene®ts would also have been more dicult if the bene®cial e€ects of additional spermatophores decrease with spermatophore number. During egg laying, females apply a liquid secretion to the soil around the eggs. This secretion hardens, glues soil to the eggs, and probably serves to protect the egg clutch. After a female's ®rst mating, the oviposition substrate in every cage was sieved daily to collect eggs. The eggs from each female were stored separately for each sieve date. The clutches were transferred to the laboratory and handled as described by Reinhold (1996). Oviposition mass The weight of the oviposition mass, including eggs and all attached soil, was determined to the nearest 0.1 mg. The weight of the oviposition mass is used as a measure of the amount of liquid substance applied by the female to the soil around the eggs. The weight of the eggs is small compared to the weight of the attached soil and only contributes approximately 5% to oviposition mass weight (one egg weighs about 3.8 mg, unpublished results). Oviposition mass weight was compared between females that were prevented from spermatophore consumption and females that consumed a spermatophore. The egg shells of P. veluchianus are robust and survive hatching, permitting a determination of the number of eggs each female had laid per clutch. The number of eggs laid within 4 days after spermatophore consumption was included as a covariate in an analysis of variance examining the e€ect of the different spermatophore treatments on the cumulative weight of oviposition masses for the same interval. O€spring dry weight In a previous study, we used the relative dry weight (the ratio between dry and wet weight) of hatched larvae as a measure of o€spring ®tness. The relative dry weight was found to di€er between females that were allowed and prevented from consuming the spermatophore (Reinhold and Heller 1993). However, egg number, egg weight, larva weight and dry weight of larvae did not di€er signi®cantly as an e€ect of treatment. The increased relative dry weight was interpreted as an increased content of reserves. Increased reserves probably also increase egg weight and larval weight, but due to a large variance in larval size, an increased weight is probably dicult to detect. Ratios and indices such as the relative dry weight used in our previous study (Reinhold and Heller 1993) are often used but may be problematic (Packard and Boardman 1988). For this reason, I give values for relative dry weight only for comparison with our previous results. Instead of the relative dry weight, the residuals from the regression between dry weight and wet weight are used as a size-independent measure for o€spring dry weight in all statistical analyses as suggested by Packard and Boardman (1988). Estimated from all available 191 data points, the following regression equation was used to calculate residuals for o€spring dry weight (dry weight ˆ 0.612 + wet weight ´ 0.08115). The dry weight of larvae was measured with the following procedure to examine whether the e€ect of spermatophore consumption occurred within the ®rst days after copulation. Of the larvae that hatched after hibernation, one to two larvae per female and laying date were weighed to the nearest 0.1 mg. After this determination of wet weight, the larvae were dried at 80 °C for 3 h. The dry weights of the larvae were determined to the nearest 0.001 mg with a Cahn electrobalance. Using these dry weight values and the regression between dry weight and wet weight, I calculated dry weight residuals. Because values for dry weight residuals of several o€spring within a family are not independent, I calculated a mean value for each family to avoid pseudoreplication. For each treatment, 44 values for residuals of o€spring dry weight, and thus on average three values per female, were measured. Survival of starved o€spring To examine whether spermatophore consumption leads to higher energy reserves in the o€spring, the lifespan of starved, freshly hatched o€spring was determined. Only those larvae whose hatching date was known accurately, and for which the dry weight of another larva of the same clutch had already been measured, were used in this experiment. To determine the lifespan of starved larvae, a single larva was put into a petri dish and supplied with a drop of water that was replenished if necessary. These dishes were stored at 14 ‹ 0.5 °C and were observed daily for mortality. Larvae hatch in February and March; at that time of the year, comparably low temperatures and high humidity can be expected in Greece. To avoid pseudoreplication, a mean value for lifespan was calculated for each family. In some families, larval lifespan could only be examined in larvae that were produced from 5 to 10 days after copulation. For this reason, family means for larval lifespan were calculated from all o€spring produced within 10 days after copulation. Results Spermatophore consumption did not increase the wet weight of larvae (Tables 1, 2), but females that consumed the spermatophore produced o€spring with a larger relative dry weight and larger dry weight residuals than females that were prevented from consumption (Fig. 1, Tables 1, 2). On the other hand, dry weight residuals of o€spring produced from 5 to 10 days after copulation were not signi®cantly in¯uenced by the female's consumption of the spermatophore (SP) (mean ‹ SE (n), +SP: 24.0 ‹ 11.6 (14), )SP: )6.9 ‹ 16.9 (12); t-test, t ˆ 1.5, P > 0.1). Females that consumed a spermatophore produced o€spring that survived signi®cantly longer than females that were prevented from consuming one (Tables 1, 2). Mean residual dry weight and mean lifespan of starved o€spring were signi®cantly correlated (Fig. 2, Spearman rank-correlation, rs ˆ 0.45, P < 0.02). There was no further signi®cant e€ect of treatment on o€spring lifespan when the e€ect of residual dry weight on o€spring lifespan was eliminated (Table 3). Weight of oviposition mass did not signi®cantly di€er between females that had and had not consumed a spermatophore (Tables 1, 2). The cumulative weight of oviposition masses produced within 4 days after copulation correlated with the number of eggs produced during the same period (Fig. 3). After elimination of the e€ect of egg number on oviposition mass weight, there was still no observable e€ect of treatment (Table 4). Discussion O€spring bene®ts of spermatophore consumption A signi®cant di€erence in the residual dry weight of o€spring was observed between females that consumed a 296 Table 1 Comparison of mean values for o€spring characters between females that consumed a spermatophore (+SP) and females that were prevented from consuming a spermatophore ()SP). Given are means (‹SE) of family means for oviposition mass weight, wet weight, relative dry weight and dry weight residuals of hatched larvae, as well as mean values for lifespan of starved o€spring. Sample sizes are given in parentheses as (number of families, number of o€spring) )SP SP Oviposition mass weight (g) Wet weight (mg) Relative dry weight (%) Residual dry weight (lg) O€spring lifespan (days) 1.03 6.69 17.7 16.9 5.1 ‹ ‹ ‹ ‹ ‹ 0.08 0.16 0.27 20.4 0.17 (14) (14, 44) (14, 44) (14, 44) (14, 66) 1.08 ‹ 6.95 ‹ 16.5 ‹ )50.8 ‹ 4.4 ‹ 0.12 (14) 0.18 (14, 44) 0.25 (14, 44) 16.6 (14, 44) 0.18 (14, 62) Table 2 Analysis of variance (MANOVA) for the e€ect of spermatophore consumption on several measures for o€spring ®tness. Values for oviposition mass weight, family means for o€spring wet weight, o€spring dry weight residuals and for the lifespan of starved o€spring are compared between females that consumed a spermatophore and females that were prevented from consuming a spermatophore (Wilks k = 0.579, P = 0.011) Source of variation df MS F-ratio P Oviposition mass weight Wet weight Residual dry weight O€spring lifespan 1 1 1 1 0.013 0.47 0.032 3.64 0.09 1.1 6.7 8.4 0.769 0.294 0.016 0.007 Fig. 2 Relationship between mean residual dry weight of a female's o€spring and the mean lifespan of her starved o€spring (values from o€spring produced within 10 days after copulation) (black symbols o€spring of females that consumed a spermatophore, open symbols o€spring of females that were prevented from feeding on a spermatophore) Table 3 Analysis (ANCOVA) of family means for the lifespan of starved o€spring produced by females during the ®rst 10 days after copulation. Comparison between females that consumed a spermatophore and females that were prevented from consuming a spermatophore after elimination of residual dry weight by using it as a covariate Fig. 1 Comparison of family means for dry weight residuals for o€spring of females that consumed (black bars, nfamilies,o€spring ˆ 14, 44) and did not consume (open bars, nfamilies,o€spring ˆ 14, 44) a spermatophore. Only values from o€spring produced within 4 days after copulation are shown spermatophore and females that did not. Furthermore, relative dry weight of o€spring increased by 7% (from 0.165 to 0.177) as an e€ect of spermatophore consumption. This corroborates the increase in relative dry weight by 7% (from 0.190 to 0.203) that was found as an e€ect of spermatophore consumption in previous experiments (Reinhold and Heller 1993). Spermatophore consumption may bene®t o€spring, not only byincreasing their residual or relative dry weight, but also by increasing the absolute weight of eggs. Spermatophore consumption has been shown to increase egg weight in the bushcricket Requena verticalis by 2±9% (Gwynne 1984, 1988a) and in the cricket Source of variation df MS F-ratio P Covariate, residual dry weight Treatment Residual 1 1 25 2.70 1.71 0.42 6.44 4.09 0.018 0.054 Gryllus bimaculatus by about 30% (Simmons 1988). In the bushcrickets Decticus verrucivorus, P. veluchianus and Leptophyes laticauda, and in the cricket Gryllodes sigillatus, no signi®cant increase in egg weight was observed as an e€ect of spermatophore consumption (Wedell and Arak 1989; Reinhold and Heller 1993; Will and Sakaluk 1994; Vahed and Gilbert 1997). Whether dry weight of o€spring increases as an e€ect of spermatophore consumption in orthopterans other than P. veluchianus is unknown. An increased egg weight probably translates into larger o€spring that have superior survival and require less time for development (Harvey 1985). It is less obvious whether an increased o€spring dry weight has any 297 Fig. 3 Relationship between the number of eggs laid within 4 days after spermatophore consumption and the weight of the clutches produced during the same interval (black symbols o€spring of females that consumed a spermatophore; open symbols o€spring of females that were prevented from feeding on a spermatophore) Table 4 Analysis (ANOVA) of weight of oviposition masses produced during the ®rst 4 days after copulation by females that consumed a spermatophore and females that were prevented from spermatophore consumption Source of variation df MS F-ratio P Covariate, number of eggs Treatment Residual 1 1 25 2.45 0.03 0.05 47.2 0.5 <0.001 0.5 e€ect on o€spring ®tness when o€spring size is not increased. Such an increase in dry weight without an increase in o€spring size probably means that a larva has a higher content of lipids, proteins or carbohydrates. In Cladocera, small aquatic crustaceans, well-fed mothers produce o€spring with higher lipid reserves, and thus higher relative dry weight, than food-deprived females (Goulden and Henry 1984). The di€erences in dry weight residuals between the o€spring of P. veluchianus females that consumed a spermatophore and females that were prevented from spermatophore consumption so thus probably re¯ect di€erent levels of energy reserves. Larvae with increased energy reserves should be better able to survive periods where food is in short supply. This relationship has been found in Cladocera where o€spring lipid energy reserves are correlated with survivorship under food shortage (Goulden and Henry 1984). In accordance with expectation, spermatophore consumption of P. veluchianus females leads to an increased o€spring lifespan when no food is available. The correlation between dry weight residuals and lifespan of starved o€spring provides additional evidence that larger energy reserves translate into better survival. The e€ects of spermatophore consumption on o€spring ®tness have been examined in other ensiferan species. In G. bimaculatus ®eld crickets and R. verticalis bushcrickets, spermatophore contents seem to increase hatching success (Gwynne 1988a; Simmons 1988) though it is unclear whether energy reserves are increased as an e€ect of spermatophore consumption. Reinhold and Heller (1993) proposed that o€spring may bene®t from the major content of a bushcricket spermatophore ± water ± which constitutes approximately 85% of total spermatophore weight (Heller et al. During the time when P. veluchianus bushcrickets reproduce - from May to July it is usually hot and dry in Greece. Under these conditions, water availability may limit female reproductive success. Females need water for producing the liquid substance they apply to the soil around the eggs that ®nally hardens to protect them. I examined whether females that consumed a spermatophore were able to produce more of this protective substance than females that were prevented from consuming a spermatophore. Weight of oviposition mass did not di€er between treatments. Thus, the water content of the spermatophore does not seem to be important for the production of the liquid substance applied to the soil during egg-laying. At least under the feeding regime used in these experiments, spermatophore consumption does not seem to increase the amount of liquid substance produced. Interval between spermatophore consumption and o€spring bene®ts O€spring dry weight increased as an e€ect of spermatophore consumption within the ®rst 4 days after copulation. O€spring bene®ts within such a short period after spermatophore consumption are surprising because radioactive labelling has shown that nutrients of the spermatophore take about 7 days to be incorporated into eggs in the related species Poecilimon af®nis (D. von Helversen, personal communication). Wickler (1994) proposed a hypothesis that may explain these seemingly contradictionary results. He argued that females may invest some of their reserves into eggs when they have just consumed a nutritious nuptial gift from which they will soon be able to replenish their nutrient reserves. According to this argument, o€spring might bene®t from a spermatophore before any nutrients of the spermatophore can be incorporated into eggs. There may be two alternative explanations for o€spring bene®ts of spermatophore consumption before the incorporation of spermatophore nutrients into eggs. First, males may manipulate female investment into o€spring by in¯uencing female endocrinology (Loher 1979; Stanley-Samuelson et al. 1987; Eberhard 1996). Second, females may increase investment in o€spring after mating with attractive males (de Lope and Mùller 1993). O€spring bene®ts within a short period after mating are not restricted to P. veluchianus. In the katydid Kawanaphila nartee, the number of developing eggs and the weight of mature eggs increase within 2 days after 298 spermatophore consumption (Simmons 1990). It is unknown whether these bene®ts are due to the incorporation of spermatophore nutrients or to the indirect process proposed by Wickler (1994). Using radioactive labelling, Bowen et al. (1984) showed that as early as 3 days after copulation, spermatophore contents were already incorporated into developing eggs in the bushcricket R. verticalis. According to similar experiments, spermatophore contents can even be incorporated into eggs within 24 h in a beetle species having an internal spermatophore (Huignard 1983). In the wartbiter, another bushcricket species, the incorporation of spermatophore contents is relatively slow and starts only 5 days after copulation (Wedell 1993a). Does the spermatophore constitute paternal investment? A male's nuptial gift only constitutes paternal investment when the male's own o€spring bene®t from spermatophore consumption (Wickler 1985, 1986; Gwynne 1986a, 1988b; Quinn and Sakaluk 1986; Sakaluk 1986). In P. veluchianus there is last-male sperm precedence (Achmann et al. 1992), and the male's o€spring thus can only bene®t from spermatophore consumption when the bene®ts occur before remating. In a ®eld population of P. veluchianus, 18.9% of the females mated, on average, per night (Heller and von Helversen 1991). A mean intermating interval of about 5 days can accordingly be calculated. However, the observed median of the remating interval was 3 days and the mode 2 days (Heller and von Helversen 1991). Nevertheless, intermating intervals of 4 and 5 days were still common. The observed increase in residual dry weight of o€spring produced within the naturally occurring intermating interval shows that nuptial feeding functions as paternal investment in P. veluchianus, at least in those cases where females remate after 4 or more days. Thus, paternal investment seems to work in P. veluchianus, even though the incorporation of spermatophore nutrients into eggs probably needs more time than the intermating interval. The indirect mechanism proposed by Wickler (1994) ± females may spend their reserves because these will soon be replenished by spermatophore contents ± may explain how o€spring can bene®t before any spermatophore contents reach the eggs. An even shorter intermating interval than that found in P. veluchianus would probably prevent this indirect mechanism. In ®eld crickets, spermatophore contents seem to increase egg weight and hatching success (Simmons 1988), but females remate several times a day (on average 5.3 times during a 7-h observation period). Simmons (1988) concluded that the spermatophore does not function as paternal investment. Considering the indirect paternal investment mechanism (Wickler 1994) probably would not alter this conclusion. In ®eld crickets, spermatophore donation may thus be labelled ``pseudo-parental investment'', because mainly o€spring of other males bene®t from spermatophore consumption (Simmons 1988). Under these conditions, the bene®ts of spermatophore consumption are probably coincidental side-e€ects of the spermatophore mating e€ort function. In P. veluchianus, protein content and spermatophore size (as a percentage of male body weight) are both more than twice that in D. verrucivorus (Wedell and Arak 1989; Wedell 1993a; Heller and Reinhold 1994; Heller et al. 1998). According to Wedell (1994), spermatophores that function as paternal investment are large and highly nutritious. Protein content and spermatophore size are thus in accordance with the observed paternal investment function of P. veluchianus spermatophores. It is dicult to examine whether the bene®cial e€ect of spermatophore consumption evolved and is maintained because of its paternal investment function. O€spring bene®t may alternatively be an incidental side-e€ect of the male mating e€ort, the production of a large sperm protection device. In P. veluchianus, spermatophylax size seems to be adjusted to its sperm protection, and thus its mating e€ort function, because it is just sucient to allow sperm transfer (Reinhold and Heller 1993; Heller and Reinhold 1994). A male may, however, transfer substances that do not increase spermatophore size but do increase o€spring viability. At least under some circumstances, a male's paternal investment seems to be wasted, e.g. when young females remate before starting oviposition or when females consume a spermatophore prematurely. To compensate for these losses, the bene®cial e€ect of spermatophore consumption has to be substantial. To determine whether paternal investment evolved incidentally or because of its bene®ts to o€spring, it would be necessary to compare the extra costs of production and inclusion of the substance causing the paternal investment function with the bene®ts of paternal investment. If the costs have a larger e€ect on male reproductive success than the bene®ts, the considered substance probably also serves as mating e€ort. In this case, the observed paternal investment function should be considered a side-e€ect of the spermatophore mating e€ort function. As long as it is unknown which spermatophore contents are necessary for its paternal investment function, it is probably impossible to measure the extra cost for the production of these spermatophore constituents. In conclusion, the size of a P. veluchianus spermatophylax seems to be adjusted to its sperm protection function (Heller and Reinhold 1994), whereas its contents might be adjusted to its paternal investment function. This is not a contradiction because the paternal investment and mating e€ort hypotheses are not mutually exclusive (Quinn and Sakaluk 1986). Acknowledgements I am grateful to Klaus-Gerhard Heller and Dagmar and Otto von Helversen for encouragement and stimulating discussion. I thank Georg Seitz for support and Susanne Faltin, Karin Reinhold and Roland Achmann for assistance in the ®eld. Michael Green®eld, Darryl Gwynne, Klaus-Gerhard Heller, Joachim Kurtz, Jay McCartney, and Bernhard Misof provided valuable comments on earlier versions of the manuscript. 299 References Achmann R, Heller K-G, Epplen JT (1992) Last-male sperm precedence in the bushcricket Poecilimon veluchianus (Orthoptera, Tettigonioidea) demonstrated by DNA ®ngerprinting. Mol Ecol 1:47±54 Bowen BJ, Codd CG, Gwynne DT (1984) The katydid spermatophore (Orthoptera: Tettigoniidae): male nutritional investment and its fate in the mated female. Aust J Zool 32:23±31 Eberhard WG (1996) Female choice: sexual selection by cryptic female choice. Princeton University Press, Princeton, NJ Goulden CE, Henry LL (1984) Lipid energy reserves and their role in Cladocera. In: Meyers DG, Strickler JR (eds) Trophic interactions within aquatic ecosystems. AAAS Selected Symposium 85. Westview, Boulder, Colo, pp 167±185 Gwynne DT (1984) Courtship feeding increases female reproductive success in bushcrickets. Nature 307:361±363 Gwynne DT (1986a) Reply to: stepfathers in insects and their pseudo parental investment. Ethology 71:74±77 Gwynne DT (1986b) Courtship feeding in katydids (Orthoptera: Tettigoniidae): investment in o€spring or in obtaining fertilisations? Am Nat 128:342±352 Gwynne DT (1988a) Courtship feeding and the ®tness of female katydids (Orthoptera: Tettigoniidae). Evolution 42:545±555 Gwynne DT (1988b) Courtship feeding in katydids bene®ts the mating male's o€spring. Behav Ecol Sociobiol 23:373±377 Gwynne DT (1990) The katydid spermatophore: evolution of a parental investment. In: Bailey WJ, Rentz DCF (eds) The Tettigoniidae: biology, systematics and evolution. Springer, Berlin Heidelberg New York Gwynne DT (1997) The evolution of edible `sperm sacs' and other forms of courtship feeding in crickets, katydids and their kin. In: Choe JC, Crespi BJ (eds) The evolution of mating systems in insects and arachnids. Cambridge University Press, Cambridge, UK Harvey GT (1985) Egg weight as a factor in the overwintering survival of spruce budworm (Lepidoptera: Tortricidae) larvae. Can Entomol 12:1451±1461 Heller K-G, Helversen D von (1991) Operational sex ratio and individual mating frequencies in two bushcricket species (Orthoptera, Tettigonioidea, Poecilimon). Ethology 89:211±228 Heller K-G, Reinhold K (1994) Mating e€ort function of the spermatophore in the bushcricket Poecilimon veluchianus (Orthoptera, Phaneroptidae): support from a comparison of the mating behaviour of two subspecies. Biol J Linn Soc 53:153±163 Heller K-G, Faltin S, Fleischmann P, Helversen O von (1998) The chemical composition of the spermathophore in some species of phaneropterid bushcrickets (Orthoptera: Tettigonioidea). J Insect Physiol 44:1001±1008 Huignard J (1983) Transfer and fate of male secretions deposited in the spermatophore of females of Acanthoscelides obtectus Say (Coleoptera Bruchidae). J Insect Physiol 29:55±63 Loher W (1979) The in¯uence of prostaglandin E2 on oviposition in Teleogryllus commodus. Entomol Exp Appl 25:107±109 Lope F de, Mùller AP (1993) Female reproductive e€ort depends on the degree of ornamentation of their mates. Evolution 47:1152±1160 Packard GC, Boardman TJ (1988) The misuse or ratios, indices, and percentages in ecophysiological research. Physiol Zool 61:1±9 Quinn JS, Sakaluk SK (1986) Prezygotic male reproductive e€ort in insects: why do males provide more than sperm. Fla Entomol 69:84±94 Reinhold K (1996) Biased primary sex ratio in the bushcricket Poecilimon veluchianus, an insect with sex chromosomes. Behav Ecol Sociobiol 39:189±194 Reinhold K, Heller K-G (1993) The ultimate function of nuptial feeding in the bushcricket Poecilimon veluchianus (Orthoptera: Tettigoniidae: Phaneropterinae). Behav Ecol Sociobiol 32:55± 60 Reinhold K, Helversen D von (1997) Sperm number, spermatophore weight and remating in the bushcricket Poecilimon veluchianus. Ethology 103:12±18 Sakaluk SK (1986) Is courtship feeding by male insects parental investment? Ethology 73:161±166 Simmons LW (1988) The contribution of multiple mating and spermatophore consumption to the lifetime reproductive success of female ®eld crickets. Ecol Entomol 13:57±69 Simmons LW (1990) Nuptial feeding in tettigoniids: male cost and the rates of fecundity increase. Behav Ecol Sociobiol 27:43±47 Simmons LW (1995) Courtship feeding in katydids (Orthoptera: Tettigoniidae): investment in o€spring and in obtaining fertilizations. Am Nat 146:307±315 Stanley-Samuelson DW, Jurenka RA, Blomquist GJ, Loher W. (1987) Sexual transfer of prostaglandin precursor in the ®eld cricket, Teleogryllus commodus. Physiol Entomol 12:347±354 Vahed K (1998) The function of nuptial feeding in insects: a review of empirical studies. Biol Rev 73:43±78 Vahed K, Gilbert FS (1997) No e€ect of nuptial gift consumption on female reproductive output in the bushcricket Leptophyes laticauda Friv. Ecol Entomol 22:479±482 Wedell N (1991) Sperm competition selects for nuptial feeding in a bushcricket. Evolution 45:1975±1978 Wedell N (1993a) Mating e€ort or paternal investment? Incorporation rate and cost of male donations in the wartbiter. Behav Ecol Sociobiol 32:239±246 Wedell N (1993b) Spermatophore size in bushcrickets: comparative evidence for nuptial gifts as a sperm protection device. Evolution 47:1203±1212 Wedell N (1994) Dual function of the bushcricket spermatophore. Proc R Soc Lond B 258:181±185 Wedell N, Arak A (1989) The wartbiter spermatophore and its e€ect on female reproductive output (Orthoptera: Tettigoniidae, Decticus verrucivorus). Behav Ecol Sociobiol 24:117±125 Wickler W (1985) Stepfathers in insects and their pseudo-parental investment. Z Tierpsychol 69:72±78 Wickler W (1986) Mating costs versus parental investment: a reply to Gwynne. Ethology 71:78±79 Wickler W (1994) On nuptial gifts and paternity. Ethology 98:165± 170 Will MW, Sakaluk SK (1994) Courtship feeding in decorated crickets: is the spermatophylax a sham? Anim Behav 48:1309± 1315 Willemse F, Heller K-G (1992) New systematic and faunistic data on Greek species of Poecilimon Fischer, 1853 (Orthoptera: Phaneropterinae). Tijdschr Entomol 135:299±315 Communicated by D.T. Gwynne