Heredity 70(1993)172—178 Received 5 June 1992 Genetical Society of Great Britain

Sperm competition and melanic polymorphism in the 2-spot ladybird, Adalla bipunctata (Coleoptera, )

PETER W. DE JONG*, MICHELLE D. VERHOOG & PAUL M. BRAKEFIELD Section of Evolutionary Biology, Department of Population Biology, University of Leiden, Schelpenkade 14a, 2313 ZT Leiden, The Netherlands

Spermprecedence was investigated in the 2-spot ladybird, Ada/ia bipunctata by utilizing a di-allelic colour marker gene. Non-melanic (homozygous recessive) virgin females were mated once with a non-melanic male and after subsequent laying of fertile they were mated with a melanic male of known genotype. Frequencies of colour morphs in the offspring provided evidence for almost complete second male sperm precedence, although the data from certain matings do not completely exclude the possibility of first male sperm precedence. The results are discussed in the light of the hypothesis of thermal melanism.

Keywords:Ada/iabipunctata, female rejection, sexual selection, sperm competition, thermal melanism.

Introduction mined by a dominant allele (Lus, 1928, 1932; M. E. N. Majerus & P. M. Brakefield, unpublished data), are Spermcompetition has been defined as the competi- black with red spots. Field data have provided tion within a single female between sperm from two or evidence for different forms of sexual selection as so- more males for the fertilization of the ova (Parker, ciated with the different colour morphs: (i) a female 1970). It is, therefore, of particular significance in preference for melanic males has been described in species in which females mate more than once. Many some British populations (Majerus et at., 1982; different patterns of sperm use in the successful fertili- O'Donald & Majerus, 1988; but see Kearns et al., zation of eggs from different male mating partners have 1990, 1992); (ii) a frequency-dependent mating advan- been described in (Walker, 1980; Thornhill & tage for melanics has been recorded (Muggleton, 1979; Alcock, 1983). There are examples of sperm competi- Kearns et at., 1990); (iii) a general mating advantage to tion resulting in (almost complete) first male or in last melanics over non-melanics was found in The Nether- male sperm precedence, as well as in some degree of lands (Brakefield, 1 984c). An understanding of the sperm mixing (Ridley, 1989). extent of sperm competition in this species is necessary Female ladybird (Coccinellidae) are highly to interpret the consequences of any deviation from polygamous and it is known that they can store sperm random mating. If for example, melanic beetles tend to in a spermatheca (Hodek, 1973; de Gunst, 1978; P. W. mate more frequently but earlier than non-melanics, as de Jong, personal observation). Those of the 2-spot in the Dutch study, then the genetic consequences will ladybird, Ada/ia bipunctata (L.), may mate many times depend on the pattern of sperm precedence and the during the peak spring period of mating which lasts timing of laying. The mating advantage of melanics several weeks; an average of 23.5% of the adult popu- will only increase their fitness relative to non-melanics lation on Rosa shrubs were in copula on any one when eggs are laid at the beginning of the season or sampling occasion in a field study in The Netherlands when sperm transferred in early matings can fertilize (Brakefield, 1 984a). A. bipunctata exhibits genetic eggs laid after later matings. polymorphism for melanism: non-melanics are red This paper describes laboratory experiments to with two black spots while melanics, which are deter- investigate sperm competition after double matings. Paternity was determined by examining the ratio of the melanic morphs in the offspring of the non-melanic * Correspondence:P. W. de Jong. females (the homozygous recessive) which had been 172 SPERM COMPETITION IN THE 2-SPOT LADYBIRD 173 mated initially to a non-melanie male and sub- fertilized. After 4 or 5 days these females were remated sequently, to a melanie. with a melanie male (mating B) and, again, they were allowed to mate only once. No distinction was made between melanie males of the forms quadrimaculata Materialsand methods and sexpustulata with four and six red spots, respect- Adultbeetles were collected in October 1990 on plane ively. The time elapsed until the start and end of mating trees (Platanus x hybrida Brot.) and lime trees (Tilia was scored. Eggs were collected and emerging larvae spp.) in Breda (Netherlands). These were bred for one were raised through to adult. The procedure to reduce generation to obtain virgins and to increase the propor- cannibalism described above was followed. The tion of homozygous melanics. The 180 wild caught morph of each resulting adult F2 offspring was scored. melanie ladybirds were divided into groups of about 15 To discover whether the melanie F1 males used were individuals and kept in 9 cm diameter plastic petri homozygous or heterozygous, they were remated with dishes containing a 7 cm diameter filter paper. Eighty a virgin typica female. They were left together and typica insects were similarly divided up. The whole further remating was possible. Egg batches were col- experiment was carried out under constant conditions lected, and larvae raised as above. It was necessary to (20°C and a L:D ratio of 18:6). Clean petri dishes rear at least seven offspring for each test-male to deter- were provided daily to prevent disease transmission, to mine its genotype with 99% certainty. promote egg-laying and to minimize egg cannibalism Control experiments were carried out to see whether by the adults. Petri dishes containing eggs were kept sperm-exhaustion could play a role over the period of separate until hatching. Ladybirds and larvae were fed 4—5 days between matings A and B. Virgin typica daily with an ample supply of pea (Acyrtho- females were mated once with either typica or melanic siphon pisum Harris) from a laboratory stock. After the males. Conditions were as described above. Numbers second moult, larvae were separated illto groups of 5 and the fertility of daily egg batches were scored over per petri dish, to reduce cannibalism. After the third the female life span. moult, larvae were kept individually in 5.5 cm petri dishes with 4.5 cm diameter filter papers, until - Results tion. Emerging adults were kept separately and fed for 3 days before being held at 9°C. Theexperiment was initiated with 51 lyp Xtyppairs The proportion of melanie ladybirds in the original (mating A). Of these, 22 females produced fertilized Breda sample was 48% (326 typica, 300 melanie). eggs within 5 days and these females were used for the Assuming Hardy-Weinberg equilibrium, the frequency remating with melanie males (mating B). Of these of homozygous melanics in the F1 generation of second matings 12 resulted in fertilized eggs, but one melanics (i.e. those used in this experiment) was 41%, produced only a single offspring so was excluded from more than double that in the field (16.7% of all field the analysis. Mating failures were either due to success- melanics are expected to be melanie homozygotes). ful copulation but failure of fertilization of the eggs, or The use of homozygous insects was preferred since the to no copulation at all. The latter was caused by rejec- results following sperm competition are much easier to tion of the male by the female involving running away, interpret than if a heterozygous parent is involved in a raising the abdomen, kicking the male with the hind cross. legs, rolling over to one side (Majerus & Kearns, 1989) The F1 ladybirds were used for the actual experi- or retracting the abdomen as far as possible under the ment. The ladybirds were sexed using characters elytra (M. D. Verhoog, personal observation). Failure of described in Majerus & Kearns (1989) and de Jong et copulation occurred in eight of 51 mating A pairs and al. (1991). Seven days before the start of the experi- in four of 22 pairs of mating B (Fisher exact test, ment all ladybirds were put at 20°C and their petri P =1.00,two tailed, ns). dishes labelled individually. They were fed daily with The females for which both copulations were suc- pea aphids. Typica males and females were randomly cessful could be divided into two categories: those in selected and placed in pairs in clean petri dishes at time which mating B was with a homozygous melanie male, 0. They were observed continuously and the time and those where it was with a heterozygous melanie elapsed until the start and end of mating was scored male. Details of these pairings are given in Table 1. (minutes) for each pair. Immediately after mating the A successful copulation with a homozygous melanie male was taken out of the petri dish; they were thus male resulted in exclusively melanie offspring in four allowed to mate only once (mating A). The experi- crosses (Table 1: females 4, 5, 6, 7). These crosses mental females were then fed daily and egg batches yielded a total of 104 melanics. A further cross resulted were collected to determine whether they had been in five melanie and one typica offspring (female 8). The 174 P. W. DEJONG ETAL.

crosses involving females 1—3 yielded only lypica a ratio of melanic: typica which was not significantly offspring following mating B. With a heterozygous different from 1:1 (x2= 0.061, ns, and binomial test: melanic male in mating B, female 9 produced only P=0.227, respectively). typica offspring, and females 10 and 11 produced Figure1 shows that those females producing mel- anic offspring, except female 7, did so immediately Table 1 Frequencies of lypica (typ) and melanic (mel) following mating B and continued doing so until they off sprihg produced following mating B with a melanie male ceased producing fertile eggs. (homozygote: horn; heterozygote: het), the duration of The proportion of failed inseminations after suc- copulations and the occurrence of rejection behaviour. cessful copulation did not differ significantly between mating A and B (A= 22successful inseminations, 21 Offspring Duration Rejection unsuccessful;B =8:10;x2= 0.039,ns). The unsuccess- mating behaviour ful inseminations after mating B included four females in in mating Table 1 that continued to produce only typica offspring Male A B and six cases in which only unfertilized eggs were laid FemaleMel B genotype Typ(mm) A after mating B. horn 1 — 10 126 181 — + Using only successful first inseminations with horn 2 — 66 80 118 — + successful rematings in the comparison, copulation horn 3 — 17 121 107 — + times (see Table 1) were not significantly different horn 4 9 — 161 163 — — between matings A and B (mating A: median =122.5 horn 5 51 — 174 144+ — mm, range= 100—174 mm; mating B: median= 148.5 horn 6 35 — 129 161 — — mm, range =88—298mm; Wilcoxon matched-pairs horn 7 9 - 100 150 — — signed-ranks test, T= 11.5; n= 8, ns). Also, no signifi- — horn 8 5 1 111 109 + cant difference was found in copulation times between het 9 — 69 77 136 — + successful and unsuccessful inseminations (Mann— het 10 34 32 116 298 — — Whitney Utest,mating A: successful: median =119.5 het 11 2 5 115 147 — — mm, range =77—174mm; unsuccessful: median =123

9 1 0 00000000** * ** *** * * ** ** **t 2 000000000000000 0*0* 0*****0000O * ** t 3 00 000000 00 4 *0I*3** ** ** *** *E******.** * * 5 0*ISS*S * **. * ** 6 A 0 *0 • ...*. •.S. •• **• 7 0 1S** * * * * * ** ***** * 8 000 0000*** * H1* *** ** ***** * 9 00 *000000000 0 000000*00000000000 0*0* *t 10 00*WG 11 oooocw**** * ** * ****3**** **** f t dayl 10 20 30 40 50 60 t A B Fig. 1 Egg laying history of females 1—11 through time (days). The two successive matings A and B are indicated, as well as death of female (1); if not indicated, date of death unknown. For female 6, mating A took place on day 2. *: unhatched egg batches, 0:typicaoffspring, •: melanic offspring, 0:melanieoffspring and one typica, c: offspring with a ratio of sypica : melanie 1: 1. SPERM COMPETITION IN THE 2-SPOT LADYBIRD 175

mm, range=0.3—172 mm; z0.23; n1=22, n221, there is no evidence from progeny of successful B ns; mating B, preceded by successful mating A: mating (Fisher exact test, P< 005; note all pairs were successful: median =148.5mm, range =88—298 mm; definitely in copula). Of course, the occurrence of first unsuccessful: median= 132.5 mm, range=42—215 male sperm precedence in these matings cannot be mm; U= 27.5; n1 =8,n2 =10,n; preceded by unsuc- ruled out; this might occur, for example, through the cessful mating A: successful: median =151.5mm, deployment of mating plugs, although no direct evi- range= 121—170 mm; unsuccessful: median= 128 dence for such plugs exists in . mm, range= 28—174 mm; U= 26.5; n1 =8,n2 =13,ns). Since the interval between mating A and B was Time until copulation was not significantly different much shorter than the average period of time in which between successful matings A and B (mating A: median the control females laid fertile eggs (5 days versus, on =444 s, range=37—863 s; mating B: median= 140 s, average, 23.5 days), the chance that complete sperm range =54—416s; Wilcoxon matched-pairs signed- exhaustion or sperm depletion played any role in the ranks test, T= 8; n =8, ns), or between successful and chance of sperm transfer is extremely low. However, unsuccessful inseminations (Mann—Whitney U test, examination of Fig. 1 indicates that the four B matings mating A: successful: median=319.5 s, range=37— (females 4-7) with homozygous melanics which 1008 s; unsuccessful: median= 165 s, range=40—1973 produced subsequently only melanie offspring were s; z= 1.13; n1 =22,n2 =21,ns; mating B, preceded by preceded by only single fertile egg batches from the successful mating A: successful: median= 140 s, first mating A (the presence of gaps in the laying of range =54—416 s; unsuccessful: median =221s, fertile egg batches in most of the female histories range=48—2146 s; U=28; n1 =8, n2 10; preceded suggests that the short gaps for females 4—7 prior to the by unsuccessful mating A: successful: median =120.5 second mating are not indicative of complete sperm s, range=51—379 s; unsuccessful: median=92 s, exhaustion). This might indicate that last male sperm range= 59—5228 s; U= 53; n1 =8,n2 =13,ns). precedence and successful insemination is more likely To control for sperm exhaustion, females were to occur when the first mating involves transfer of a mated once to either melanic or Lypica males, and the small amount of sperm. The rejection behaviour period of time during which fertilized egg batches were by females observed prior to unsuccessful second male laid was recorded. For this purpose, the results from copulations might also be indicative of a high remain- Table 1 were also used. The average period of time ing sperm load and, therefore, a low 'motivation' to over which fertilized eggs were laid was 23.5 12.8 remate. This might in turn increase the probability that (S.D.) days (n= 6, range= 5—39 days). a second male cannot successfully transfer sperm. However, the proportion of successful copulations which did not result in fertilization of eggs was not significantly different between mating A (when no Discussion females contained sperm) and B (when all females con- Withthe exception of one female, the results of this tained sperm). study fall into two categories: after having mated Our results provide evidence that the 2-spot lady- initially with a lypica male and then a melanie, either (i) bird shows (almost) complete last male sperm preced- the typica female continues laying eggs fertilized by ence. However, different patterns of sperm precedence only the first (ypica) male, or (ii) after the second may apply under other experimental conditions, for mating she immediately starts laying eggs fertilized example when three or more matings occur or when exclusively by the second (melanie) male. The latter is the time interval between matings is (much) shorter (N. substantiated for four of the homozygous melanic Canham & M. E. N. Majerus, personal ccnmunica- males (100% melanie offspring) and is consistent with tion). The mechanism of last male sperm precedence in the two heterozygous males which produced a 1:1 the 2-spot ladybird is unknown. Several mechanisms ratio of melanics: typica. We found only one exception, have been described for insects (Thornhill & Alcock, where in a family of only six offspring after the second 1983; Birkhead & Hunter, 1990). Probably the most mating with a homozygous melanie male, one lypica prominent among these is sperm displacement. Sperm offspring was found. from previous matings can be repositioned or removed The most likely explanation of the results where by a male, but the female can also influence sperm mating B resulted in 100% lypica offspring is that, precedence. Evidence has been found in some species perhaps due to rejection by the female, the insemina- that a female can, possibly stimulated by a copulating tions failed. Inspection of Table 1 shows that there is a male, remove sperm from her storage organs (Birkhead significantly higher tendency for rejection behaviour by & Hunter, 1990; Villavaso, 1975). In another ladybird the female to occur immediately prior to mating when species, Pallas, sperm is transferred 176 P. W. DE JONG ETAL.

from male to female by a spermatophore, and after uously observing a number of mating pairs of migration of the sperm to the spermatheca, the remains ladybirds, it was noted that usually, when a pair broke of the spermatophore are ejected by the female, and up, the male immediately mounted the female again. eaten (Obata & Hidaka, 1987). If transfer of sperm Although this was not investigated in any detail, this through a spermatophore also occurs in A. bipunctata, observation might indicate mate guarding or remating a mating male may be able to stimulate a female to eject as a mechanism to prevent or reduce the effect of a spermatophore containing sperm from a previous subsequent matings with competing males. mating. If the time interval between the two matings is Knowing that sperm precedence is operating in the short, this could result in sperm displacement. In any 2-spot ladybird, we may ask the question why do females case, a 'last in, first out' mechanism, in which the sperm mate more than once? Females should gain by doing of the first male fertilizes the later eggs, does not seem so, or there would be a selection pressure against to operate in the 2-spot ladybird, since once switched multiple matings. In numerous insects a female receives from typica to melanic offspring, females continue to enough sperm from one insemination to potentially produce melanic offspring until they cease producing fertilize all of her eggs, and given that she can store this fertile eggs. sperm, why would she waste time and energy to mate Interestingly, in six out of 18 successful B copula- again? Several advantages have been suggested tions, the female laid only infertile eggs after mating B, (Thornhill & Alcock, 1983; Halliday & Arnold, 1986). whereas in two of these six copulations she laid ferti- They have been divided into four classes by Thornhill lized eggs until the day before mating B. This is further & Alcock (1983): sperm replenishment, material evidence of the influence of a copulation on previously benefit, genetic benefit and convenience. Although in stored sperm. These data suggest a two-phase copula- most ladybird species one copulation is generally tion, in which previously stored sperm is manipulated reported to be sufficient for the female to fertilize all first, and insemination with new sperm follows. If this is the eggs she lays during her life (Hodek, 1973), evid- true, in the six pairings mentioned above the first phase ence has been found in A. bipunctata that remating could have been successful, but the insemination failed. increases the rate of egg-laying or viability of eggs The copulation times in these six pairings were not (Ellingsen, 1969; Sem'yanov, 1970). In the present significantly different from those successful B matings study, however, ladybirds were able to lay fertilized which followed successful A matings (Mann—Whitney eggs over a relatively long period of time. A second U test: U= 15.5, n1 =6,n2 =8,ns). This suggests that selection pressure favouring remating by females might the insemination phase is very short compared with the be that the male provides the female with nutritional time needed for manipulation of the sperm from benefits while mating. It has already been mentioned previous males. that in at least one ladybird species sperm is trans- No significant differences were found in time until ferred through a spermatophore, which is subsequently copulation or in length of copulation between success- eaten by the female (Obata & Hidaka, 1987). A similar ful first matings and successful rematings. Furthermore, use of nutrients is also known from other insects, for no significant differences were found for these para- example butterflies (Boggs & Watt, 1981) and grass- meters between successful and unsuccessful matings hoppers (Butlin et at., 1987). Spermatophores, or within each class of matings (A or B). No evidence has, quantities of other non-genetic material transmitted therefore, been found for an influence of experience, from the male to the female via the penis may equal as female preference, external disturbances or colour much as 40% of male body weight (Butlin et al., 1987). morph per se on the results. This apparent indepen- However, these quantities apparently do not need to be dence of mating time and colour morph suggests that particularly large to influence female fecundity. This the evidence for non-random mating in the field is not might outweigh disadvantages of remating in terms of an artefact of different mating times (Brakefield, loss of time and energy in relation to feeding activity or 1 984c). However, our data set for rematings is small the risk of predation. and all pairings involving melanic males were B Sperm precedence potentially has important conse- matings. Therefore, an additional experiment involving quences for the population biology of the 2-spot lady- a comparison of first matings of females by typica and bird. An hypothesis about the way in which selection melanic males remains necessary. influences the colour polymorphism involves thermal When sperm precedence occurs in the 2-spot melanism, in which the melanic morph has an advan- ladybird, adaptations are expected to prevent subse- tage relative to the lypica morph under circumstances quent males from successfully inseminating the female. of limited sunshine levels (Lusis, 1961; Muggleton et Mating plugs have already been mentioned as a pos- at., 1975; Brakefield, 1984b; Brakefield & Willmer, sible mechanism. During the experiments, while contin- 1985; Brakefield & Lees, 1987). An essential feature SPERM COMPETITION IN THE 2-SPOT LADYBIRD 177 of this hypothesis is the presence of food (aphids) for BRAKEFIELD, P. M. AND WTLLMER, P. G. 1985. The basis of thermal the ladybirds. Earlier activity (including mating) of the melanism in the ladybird : differences in melanic morph under these conditions increases repro- reflectance and thermal properties between the morphs. ductive success only if food is available at the same Heredity, 54, 9—14. time to produce viable eggs, or if last male sperm BUTLIN, R. K., WOODHATCH, C. W. AND HEWVF, 0, M. 1987. Male precedence does not occur. The synchronization of spermatophore investment increases female fecundity in a grasshopper. Evolution, 41,221—225. food availability and activity of ladybirds becomes even ELLINGSEN, i.-.i. 1969. Fecundity, consumption and more important if complete sperm precedence is survival of the aphid predator Adalia bipunctata L. (Col., operating. A potential advantage for melanic ladybirds Coccinellidae). Norsk ent. Tidsskr., 16,91—95. due to earlier mating would be reduced or could even GUNST, j,H.DE 1978. De Nederlandse Iieveheersbeesjes. Wet. disappear if eggs could only be laid by the time typica med. K.N.N.V. 125. (in Dutch). males have also become active. The typica males would HALLIDAY, T. AND ARNOLD, S. r. 1986. Multiple mating by then have an increasing chance of inseminating females females: a perspective from quantitative genetics. Anini. and thereby displacing melanics' sperm. Behav,, 35, 939—94 1. HODEK, i. 1973. Biology of Coccinellidae. Junk N.y., The Hague/Academia, Prague. lONG, P. W. DE, HOLLOWAY, 0. 1., BRAKEFIELD, P. M. AND VOS, H. DE Acknowledgements 1991. Chemical defence in ladybird beetles (Coccinelli- dae) Ii. Amounts of reflex fluid, the alkaloid adaline and Weare grateful to Graham Holloway and Nicola individual variation in defence in 2-spot ladybirds (Adalia Marples for helpful comments on a previous draft of bipunctata). Chemoecology, 2, 15—19. the manuscript, to Mart Ottenheim for his help in rear- KEARNS, P. W. E., TOMLINSON, I. P. M., O'DONALD, P. AND vELTMAN, C. I. ing all the ladybirds and many stimulating discussions 1990. Non-random mating in the two-spot ladybird and to Michael Majerus for sharing his findings and (Adalia bipunctata): I. A reassessment of the evidence. discussing the results. We thank Els Schlatmann, Bert Heredity, 65,229—240. de Winter and Wini Kanters for helping to keep the KEARNS, P. W. E., TOMUNSON, I. P. M., VELTMAN, C. 3.ANDO'DONALD, aphid-stock going. Henk Heijn and Martin Brittijn i'. 1992. Non-random mating in Adalia bipunctata (the prepared the figure. We are also grateful for the con- two-spot ladybird). II. Further tests for female mating structive comments of two anonymous referees. Peter preference. Heredity, 68, 385—389. LUS, j.i.1928. On the inheritance of colour and pattern in de Jong was supported by the Foundation for Bio- lady beetles Adalia bipunctata L. and Adalia decempunc- logical Research (BION, grant No. 436.031) which is iota L. Izv. Byuro. Genet. 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