How labile are the egg-laying preferences of seed beetles?

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1 Ecological Entomology (4) 29, How labile are the egg-laying preferences of seed beetles? FRANK J. MESSINA Department of Biology, Utah State University, U.S.A. Abstract. 1. Previous studies have produced conflicting results with respect to the genetic lability of host preference in the seed beetle Callosobruchus maculatus. 2. In this study, replicate lines of an Asian population were kept on an ancestral host (mung bean) or switched to a novel host (cowpea). After 4þ generations, lines were assayed for host preference (in choice tests) and host acceptance (under no-choice conditions), and were compared to African lines chronically associated with cowpea. 3. Host preference diverged in the expected direction. When presented a mixture of cowpeas and mung beans, females from the cowpea lines laid a greater fraction of their eggs on cowpea than did females from the mung bean lines. Preference for cowpea was nearly as strong in the cowpea lines as it was in the cowpea-adapted African lines. 4. In contrast, the experimental host shift did not affect long-term host acceptance. African females laid more eggs if given cowpeas than if given mung beans, but realised fecundities in the cowpea and mung bean lines were similar on the two hosts. Females from all lines laid more eggs if they were reared on cowpea than on mung bean, but rearing host had no effect on either relative host acceptance or host preference. 5. Comparisons with earlier studies suggest that the lability of host preference varies among beetle populations, which precludes generalisation at the species level. Because lines were maintained under no-choice conditions, modification of host preference probably occurred via a lower acceptance threshold for the novel host, without a concomitant change in the long-term acceptance of the ancestral host. Key words. Bruchidae, Callosobruchus maculatus, host preference, oviposition behaviour, selection experiment, Vigna. Introduction Although some associations between specialist insects and their host plants are surprisingly old (Becerra, 3), there are many examples of insects rapidly adapting to newly introduced plants (Singer et al., 1993; Camara, 1997). Because colonising a new host plant is akin to invading a Correspondence: Frank J. Messina, Department of Biology, Utah State University, Logan, UT , U.S.A. messina@biology.usu.edu new habitat, host shifts can cause genetic changes in a variety of insect traits (Carroll et al., 1998; Feder & Filchak, 1999) and may promote speciation (Caillaud & Via, ; Nosil et al., 2). Wide variation in the conservatism of plant insect relationships has spurred efforts to identify factors that facilitate or constrain host shifts (Fox & Savalli, ). Of particular interest is the lability of oviposition behaviour, a major determinant of diet breadth among herbivorous insects (Wehling & Thompson, 1997). Several approaches can be used to understand how insects adapt to novel hosts. Phylogenetic or phylogeographic studies provide an historical context by establishing 318 # 4 The Royal Entomological Society

2 Modification of beetle host preference 319 the timing and direction of host shifts (Janz et al., 1; Fordyce & Nice, 3). Breeding designs can estimate genetic variation for host-use traits within populations, and hybridisation experiments can determine the inheritance of traits that differ between conspecific populations on different hosts (Craig et al., 1; Ueno et al., 3). Selection experiments provide a direct measure of insect responses, and are especially useful for detecting fitness trade-offs between ancestral and novel hosts (Fry, 3). This study determined the effect of an experimental host shift on the egg-laying behaviour of a seed beetle, Callosobruchus maculatus (F.). Because of their short generation times and simple life cycles, seed beetles are good subjects for measuring adaptation to novel environments (Mark, 1982; Tucić et al., 1998; Messina & Karren, 3). Natural or experimental host shifts can produce rapid genetic changes in larval performance (Credland, 1987; Desroches et al., 1997) and adult behaviour (Fox et al., 1994; Tucic et al., 1997). For C. maculatus, however, estimates of the lability of host preference have been contradictory. In a classic study, Wasserman and Futuyma (1981) found that host preference (as measured in choice tests) was modified after only 11 generations of selection, both in lines artificially selected to prefer a novel host and in lines simply transferred to the novel host under no-choice conditions. In contrast, the recent results of Kawecki and Mery (3) suggest strong conservatism of host preference in C. maculatus. Four geographic populations (three from Africa, one from Yemen) had been maintained on cowpea [Vigna unguiculata (L.) Walpers] for an indefinite period, and were then reared on either cowpea or a novel host, mung bean [Vigna radiata (L.) Wilczek], for approximately 11 generations. Host preference varied among geographic populations, but was unaffected by recent evolutionary history. Within each population, the proportion of eggs laid on cowpea in choice tests did not differ between the cowpea and mung bean sub-populations, even though the latter group had encountered only mung beans for more than 1 generations (Kawecki & Mery, 3). This study re-examines the lability of host preference in C. maculatus. Replicate lines of an Asian population associated with mung bean (Mitchell, 1991) were either maintained on mung bean or switched to cowpea for 4 or more generations. The direction of the host shift was thus opposite to that described by Kawecki and Mery (3). Both host preference (as measured in choice tests) and host acceptance (as measured under no-choice conditions) were then compared between the cowpea and mung bean lines. Because a female s egg-laying behaviour can be modified by experience (Barron, 1), the effect of rearing host on host preference or acceptance was also examined (Kawecki & Mery, 3). A final experiment considered whether adaptation to one host simultaneously modifies female responses to other hosts (as a kind of cross-adaptation, Agrawal, ). This experiment measured the preferences of females presented cowpea and adzuki bean [Vigna angularis (Willd.) Ohwi & Ohashi], a host more closely related to mung bean than to cowpea (Doi et al., 2). Materials and methods Beetle life cycle Females of C. maculatus lay eggs singly on the surfaces of legume seeds and pods. The hatching larva bores into the seed beneath the oviposition site, and completes its development within a single host. Adults emerging from seeds require neither food nor water, and commence mating and oviposition within several hours after emergence. Laboratory conditions approximate the natural environment of C. maculatus, which has probably infested human stores of grain legumes for thousands of years (Messina, 1998). Host preferences are known to vary genetically within and among beetle populations (Wasserman, 1986; Fox, 1993; Messina & Slade, 1997). Source population and selection experiment A selection experiment was initiated with a beetle strain derived from infested mung beans in Tirunelveli, India (Mitchell, 1991). Even after more than 1 generations in the laboratory, this strain appeared to maintain genetic variation for traits related to host use (Kawecki, 1995). At the start of the experiment, stock cultures had been kept at large population sizes on Berken mung beans for approximately 1 generations. Each generation was formed by adding adults to a 2-l jar containing 75 g of seeds (Messina & Karren, 3). Stock cultures were maintained in a growth chamber at 24 C and constant light. The same conditions were used for the experiments described below. Two generations before the start of the experiment, the stock population was expanded by placing >15 adults into each of three jars containing 75 g of mung beans. In the following generation, six independent replicate lines were established with > newly emerged adults per line. Lines were formed by adding adults to three jars containing mung beans (hereafter, the M lines) and three jars containing cowpeas (C lines). Subsequent generations of each line were formed in the same way as the stock cultures, i.e. by adding newly emerged adults to 75 g of seeds. Adults for each new generation were obtained in the middle of the adult emergence period (28 32 days after the start of the previous generation) to minimise directional selection for development time or other traits (such as body size or fecundity) that are genetically correlated with development time (Møller et al., 199). The California Blackeye 846 cowpeas were about three times heavier than mung beans (mean seed mass ¼ and 65.8 mg respectively; n ¼ 1 seeds). The coefficient of variation for seed mass was similar between hosts: 15.7% for cowpeas and 14.8% for mung beans. Host preference Host preference was first measured 4 generations after the C and M lines were established. To reduce host-related,

3 3 Frank J. Messina non-genetic effects on behaviour, the C lines were switched back to mung bean for one generation before host preference was assessed. This step may have introduced a conservative bias by imposing one generation of selection toward genotypes that readily oviposit on mung bean. To obtain test females, all adults were sieved and discarded from a culture in which adults were actively emerging from seeds, and newly emerged females were collected within two hours. Females had no egg-laying experience because oviposition commences more than 2 h after emergence. Each test female was paired with a newly emerged male and placed in a 6-cm Petri dish containing 15 mung beans and 15 cowpeas. Females were allowed to mate and oviposit for 12 h, after which the number of eggs on each host was recorded. Females were provided equal numbers of the two hosts rather than an equal-mass mixture because only relative host preferences, i.e. potential differences between the C and M lines, were of interest (Messina & Slade, 1997). Providing 3 seeds and limiting the oviposition period to 12 h ensured that host preferences would not be obscured by females switching to the less attractive host because all seeds of the more attractive host already bore eggs (Wasserman & Futuyma, 1981; Messina & Renwick, 1985). More than 45 females were tested per line. A few femalesineachlinefailedtolayeggs,mostlikelybecause of mating failure. Host preference was measured again after 75 generations. This experiment used a similar protocol, but was more complex in two ways. Along with the C and M lines, the experiment included three lines of an African population (A lines) that had no prior exposure to mung beans. The African lines were derived from an infestation of cowpeas near Ouagadougou, Burkina Faso (Messina, 1993) and had been maintained on California black-eyed cowpeas for >14 generations. A second difference was that each line was split and reared on each host (cowpea or mung bean) for one generation prior to the experiment. This experiment could therefore estimate the separate effects of rearing host and recent evolutionary history (selection regime). Any effect of rearing host includes potential effects of larval diet, early adult (pre-oviposition) experience, as well as one generation of potential selection in lines that were switched to a novel host (mung bean for the C and A lines or cowpea for the M lines). At least 25 females were tested from each of the 18 treatments (nine lines two rearing hosts). A final host-preference experiment was conducted after 9 generations. In this experiment, females were offered cowpeas and adzuki beans (15 seeds per host) instead of cowpeas and mung beans. As in the first experiment, the C lines were switched to mung bean for one generation before host preference was assessed, so that all test females developed in mung beans and encountered only mung beans during early adult life. The Dainagon adzuki beans were % lighter than cowpeas (mean seed mass ¼ mg; n ¼ 85 seeds). Thirty-five females were tested from each of the six lines. Host acceptance Long-term host acceptance was estimated as the lifetime realised fecundity of females provided with either cowpeas or mung beans (two test hosts). The first experiment was conducted after 4 generations. As in the first host-preference experiment, test females from both the C and M lines had developed in mung beans and were collected within two hours of adult emergence. Each female was placed in a 6- cm Petri dish with a newly emerged male and approximately 4 cowpeas or 1 mung beans. The total number of eggs was recorded after all females had died (longevity did not differ between the C and M lines, and most eggs are laid within the first 2 days of adult emergence; Credland & Wright, 1989). Twenty-five females were tested from each of the 12 treatments (six lines two test hosts). Host acceptance was again estimated after 75 generations. This experiment resembled the second host-preference experiment by including three African (A) lines in addition to the C and M lines, and by using newly emerged females that had developed in either mung beans or cowpeas. Females were again provided either 4 cowpeas or 1 mung beans; females were tested from each of the 36 treatment combinations (nine lines two rearing hosts two test hosts). Statistical analyses The dependent variable in the three host-preference experiments was the proportion of eggs laid on cowpea. A nested, mixed-model ANOVA was used for the first and third experiments, in which all test females developed in mung bean. Replicate line was treated as a random factor nested within selection regime. The appropriate error term for testing the effect of selection regime was the mean-square for replicate line within selection regime (Table 1). Proportions were subjected to the angular (arcsine-square root) transformation to meet ANOVA assumptions. The same ANOVA model was used to determine whether the total number of eggs laid during the choice tests depended on selection regime. Egg counts were square-root transformed. Females in the second host-preference experiment were reared on cowpea or mung bean. This analysis followed a split-plot design (Wilkinson & Coward, ), in which replicate lines (plots) were again treated as a random factor nested within selection regime. Selection regime was a fixed, between-plot effect; rearing host was a fixed, within-plot effect. Table 1presents error terms for evaluating each main factor and interaction. The angular and square-root transformations were again applied to the proportion of eggs laid on cowpea and the total number of eggs respectively. The dependent variable in the host-acceptance experiments was the number of eggs laid. Analysis of the first experiment followed a split-plot design. Selection regime represented a between-plot effect (with replicate line nested within selection regime), and test host represented a within-plot effect. The second host-acceptance experiment used a split-split-plot design because each line was first split

4 Modification of beetle host preference 321 Table 1. Mixed-model, nested ANOVA for host preferences of Callosobruchus maculatus females from different selection regimes (see text) and presented cowpeas and mung beans, or cowpeas and adzuki beans. Source of variance Error term d.f. F P Experiment 1: females reared on mung bean, and presented cowpeas and mung beans Selection regime (¼ SR) Replicate line (SR) Replicate line (SR) Residual Residual 273 Experiment 2: females reared on cowpea or mung bean, and presented cowpeas and mung beans Selection regime Replicate line (SR) <.1 Rearing host (¼ RH) RH Replicate line (SR) SR RH RH Replicate line (SR) Replicate line (SR) RH Replicate line (SR) RH Replicate line (SR) Residual Residual 483 Experiment 3: females reared on mung bean, and presented cowpeas and adzuki beans Selection regime Replicate line (SR) Replicate line (SR) Residual Residual 188 between two rearing hosts, and was then split between two test hosts. F-tests were performed as described in Table 2. Egg counts were again square-root transformed. Results Host preference Switching beetle populations to cowpea for 4þ generations significantly altered host preferences. In the first experiment, the mean proportion of eggs laid on cowpea was <55% in the three M lines vs. 7% in the C lines (Fig. 1; Table 1). Because the surface area available for oviposition was much greater for cowpea than for mung bean, the egg distributions of M-line females likely reflect an absolute preference for mung bean (see also Messina & Slade, 1997). Variation among replicate lines was not significant (Table 1). Although selection regime had a strong effect on host preference, it did not affect the number of eggs laid. Mean egg number during the 12-h choice test ranged from 5.6 to 8.7 across the six lines, but did not differ between the C and M lines (F 1,4 ¼ 1.83, P ¼.). Table 2. Mixed-model, nested ANOVA for host acceptance (lifetime realised fecundity) of Callosobruchus maculatus females from different selection regimes (see text) and provided with either cowpea or mung bean. Source of variance Error term d.f. F P Experiment 1: females reared on mung bean Selection regime (¼ SR) Replicate line (SR) Test host (¼ TH) TH Replicate line (SR) SR TH TH Replicate line (SR) Replicate line (SR) TH Replicate line (SR) TH Replicate line (SR) Residual Residual 277 Experiment 2: females reared on cowpea or mung bean Selection regime Replicate line (SR) Rearing host (¼ RH) RH Replicate line (SR) <.1 Test host TH Replicate line (SR) SR RH RH Replicate line (SR) SR TH TH Replicate line (SR) RH TH RH TH Replicate line (SR) SR RH TH RH TH Replicate line (SR) Replicate line (SR) RH Replicate line (SR) RH Replicate line (SR) RH TH Replicate line (SR) TH Replicate line (SR) RH TH Replicate line (SR) RH TH Replicate line (SR) Residual Residual 1169

5 322 Frank J. Messina Per cent eggs on cowpea C1 C2 C3 M1 M2 M3 Fig. 1. Percentage of eggs laid on cowpeas (means þ SE) by Callosobruchus maculatus females presented cowpeas and mung beans. Females were reared on mung bean and were from lines switched to cowpea (C1 3) or maintained on mung bean (M1 3). An effect of selection regime on host preference was also evident after 75 generations. Preference for cowpea tended to be higher overall in the second experiment than it was in the first experiment (cf. Figs 1and 2), possibly because of subtle differences in seed quality. Nevertheless, the preference for cowpea was much stronger among C-line females than among M-line females (Fig. 2; Table 1). Females from the African (A) lines (which had no long-term exposure to mung bean) laid virtually all of their eggs on cowpea. In contrast to the strong effect of selection regime, there was no effect of rearing host on host preference (Fig. 2; Table 1). Thus, neither larval diet nor early adult (pre-oviposition) experience influenced the proportion of eggs laid on cowpea. 1 Variation among replicate lines was not significant, and there was no interaction between the effect of rearing host and either selection regime or replicate line (Table 1). Selection regime again had no effect on the number of eggs laid (F 2,6 ¼.66, P ¼.55). The first two experiments suggested that C-line females preferred cowpeas more strongly than M-line females did when all females were presented cowpeas and mung beans. The final experiment (conducted after 9 generations) examined whether an effect of selection regime would also be observed when females were presented cowpeas and adzuki beans. In this case, all lines strongly preferred adzuki beans to cowpeas; the mean percentage of eggs laid on cowpea ranged from 22% to 33% across the six lines (Fig. 3). C-line females placed a slightly higher fraction of their eggs on cowpea than M-line females did, but neither selection regime nor replicate line had a significant effect on host preference (Table 1). Mean egg number varied between 7.4 and 9.2 across the six lines, and again did not depend on selection regime (F 1,4 ¼.67, P ¼.46). Host acceptance Host acceptance was measured as lifetime fecundity on each host. The first experiment was conducted after 4 generations, and all females had developed on mung bean. Mean fecundity ranged between 55 and 7 eggs per female (Fig. 4), but did not differ between the C and M lines (Table 2). Fecundity also did not depend on whether cowpeas or mung beans served as the test host (Fig. 4; Table 2). Variation among replicate lines was not significant, and there were no significant statistical interactions. The selection regime test host interaction was of particular interest because such an interaction would have suggested 5 Per cent eggs on cowpea Per cent eggs on cowpea C1 C2 C3 M1 M2 M3 A1 A2 A3 Fig. 2. Percentage of eggs laid on cowpea (means þ SE) by Callosobruchus maculatus females presented cowpeas and mung beans. Females were reared on mung bean (solid bars) or cowpea (open bars), and were from lines switched to cowpea (C1 3), maintained on mung bean (M1 3), or chronically associated with cowpea (A1 3). C1 C2 C3 M1 M2 M3 Fig. 3. Percentage of eggs laid on cowpeas (means þ SE) by Callosobruchus maculatus females presented cowpeas and adzuki beans. Females were reared on mung bean and were from lines switched to cowpea (C1 3) or maintained on mung bean (M1 3).

6 Modification of beetle host preference 323 Mean number of eggs C1 C2 C3 M1 M2 M3 Fig. 4. Fecundities (means þ SE) of Callosobruchus maculatus females provided mung beans (solid bars) or cowpeas (open bars). Females were reared on mung bean and were from lines switched to cowpea (C1 3) or maintained on mung bean (M1 3). Mean number of eggs (a) (b) C1 C2 C3 M1 M2 M3 A1 A2 A3 that C-line and M-line females responded differently to the two hosts under no-choice conditions. The second experiment simultaneously measured the effects of selection regime, rearing host, and test host. In this case, the effect of selection regime was significant (Fig. 5; Table 2), but was largely due to the inclusion of A-line females, which were more fecund than C-line and M-line females. If the A lines were excluded from the analysis, the effect of selection regime became marginal (F 1,4 ¼ 6.72, P ¼.6), and reflected moderately higher fecundity in the M lines than in the C lines (Fig. 5). Lifetime fecundity also depended on test host (Fig. 5; Table 2), and there was a significant selection regime test host interaction (Table 2). However, these results depended entirely on the inclusion of A-line females, which laid more eggs if given cowpeas than if given mung beans (Fig. 5). When the A lines were removed from the analysis, the effect of test host disappeared (F 1,4 ¼ 1.35, P ¼.31), as did the selection regime test host interaction (F 1,4 ¼.2, P ¼.9). Hence, the second experiment, like the first, provided no evidence that the C and M lines differed in their long-term acceptance of mung bean or cowpea. Rearing host had a strong and uniform effect on fecundity (Table 2). Females from all lines laid more eggs if they developed in cowpeas than if they developed in mung beans (compare Fig. 5a and b). The consistency of this effect is evident from the absence of an interaction between rearing host and either selection regime or test host (Table 2). No other factor interaction was statistically significant in either the full analysis (Table 2) or in the restricted analysis (not shown) that excluded the A lines. Discussion This study suggests that host preference can be a labile character in C. maculatus. In two experiments, conducted 4 C1 C2 C3 M1 M2 M3 A1 A2 A3 Fig. 5. Fecundities (means þ SE) of Callosobruchus maculatus females provided mung beans (solid bars) or cowpeas (open bars). Females were reared on (a) mung bean or (b) cowpea, and were from lines switched to cowpea (C1 3), maintained on mung bean (M1 3), or chronically associated with cowpea (A1 3). 35 generations apart, preference for cowpea was stronger in populations switched to cowpea than in populations maintained on mung bean (Figs 1and 2). This difference was consistent among replicate lines, and therefore unlikely to depend on random genetic drift (Fry, 3). It was also unaffected by rearing host. Preference for cowpea in the C lines converged toward the preference observed in African lines that were collected from cowpea and maintained exclusively on this host (Messina, 1993). At the same time, host preference in the M lines remained similar to that observed in the base population from which all selection lines were derived (Messina & Slade, 1997). Crosses were not performed between the C and M lines, but two studies have found that genetic differences in host preference between natural populations are inherited in a simple, additive way (Messina & Slade, 1997; Fox et al., in press) The lability of host preference in this study is consistent with the results of Wasserman and Futuyma (1981), who found that preference for pigeon pea [Cajanus cajan (L.) Millsp.] increased over 11 generations in C. maculatus lines

7 324 Frank J. Messina switched from adzuki bean to pigeon pea (see also Credland, 1987; Tucić et al., 1995). The present study contrasts that of Kawecki and Mery (3), who found that long-standing geographic variation in host preference was unaffected by recent evolutionary history, i.e. whether populations were reared on cowpea or mung bean for the previous 11 generations. This discrepancy is intriguing because Kawecki and Mery (3) observed conservatism of host preference in four different populations, which implies that it is a species-wide character. A few explanations may be offered to account for the contrasting results. In any study of insect oviposition, subtle differences in experimental protocol may cause differences in the motivational states of females, and hence their level of discrimination (see review by Monks & Kelly, 3). Second, each population used by Kawecki and Mery (3) was maintained on cowpea at population sizes of several hundred individuals for dozens of generations (up to 15 years) before it was divided into sub-populations on cowpea or mung bean (Dick & Credland, 1984). This particular rearing protocol may have failed to preserve genetic variation for behavioural and physiological traits that influence host preference. Several studies, however, have found that even long-term laboratory populations of C. maculatus maintain genetic variation for egg-laying behaviour (Wasserman & Futuyma, 1981; Credland, 1987; Messina & Karren, 3). Third, all populations used by Kawecki and Mery (3) were derived from wild populations in or near Africa, whereas the population in this study was from India. Although available evidence suggests a gradient of decreasing preference for cowpea from West Africa to Asia (Kawecki & Mery, 3), it is not clear why there should be a similar gradient in the lability of host preference. Finally, the direction of the host shift (which included a shift in average seed size) in the present study was opposite to that of Kawecki and Mery (3). Because C. maculatus appears to be of African origin (where it is associated with the wild progenitors of cowpea), the host shift described here may in fact represent a reversion to an ancestral host after thousands of generations on mung beans and related legumes (Berg & Mitchell, 1993). A genetic response to such a reversion may be more rapid than a response to a truly novel host. Regardless of the underlying causes, it appears that the genetic lability of host preference varies among C. maculatus populations. Kawecki and Mery (3) provide evidence of such variation; both of their experiments yielded a statistical interaction between the effects of geographic origin and recent evolutionary history on host preference, i.e. the magnitude or direction of the effect of evolutionary history varied among populations. Population differences in the amount selectable variation would not be surprising in C. maculatus, which is a colonising species frequently associated with human stores of grain legumes. Human transport of infested seeds promotes isolated populations that are established by varying numbers of founders, with subsequent variation in the importance of genetic drift and inbreeding (Tran & Credland, 1995; Bieri & Kawecki, 3; Guedes et al., 3). A robust demonstration of population differences in evolvability would require a selection experiment that incorporates both multiple populations (as in Kawecki & Mery, 3) and multiple replicate lines per population (as in this study). Modification of host preference occurred in lines reared under no-choice conditions, with no selection for preference itself (Wasserman & Futuyma, 1981). It therefore seems likely that switching to cowpea simply lowered the threshold for accepting this host, so that proportionally more eggs were added to cowpea in short-term, choice tests. If so, this change in the acceptance threshold for cowpea did not diminish long-term acceptance of mung bean (Fig. 4). Only African females, whose lines had never encountered mung bean, consistently laid more eggs on cowpeas than on mung beans in the no-choice experiment, and even here the magnitude of the difference was small (Fig. 5). A trade-off associated with increased acceptance of a novel host (in which acceptance of the ancestral host declines) may be more likely if stimuli used to perceive the novel host are very different from those used to perceive the ancestral host. Singer et al. (1993) provided an example in which rapid adaptation to an introduced host by a butterfly did decrease acceptance of the ancestral host. There was no evidence of cross-adaptation of host preference; when females were offered cowpea and adzuki bean, the percentage of eggs laid on cowpea was similar in the C and M lines (Fig. 3). Despite providing less surface area for oviposition, adzuki beans were actually more attractive than cowpeas to females from all lines. The shift to cowpea may have lowered the threshold for accepting cowpea among C-line females, but it apparently had little effect on the already low threshold for accepting adzuki bean. This study did not compare C- and M-line females presented adzuki beans vs. mung beans, but a previous study demonstrated that adzuki bean is also generally preferred over mung bean (Chiu & Messina, 1994). Like mung bean, adzuki bean is part of the Asiatic radiation of the genus Vigna (subgenus Ceratotropis), whereas cowpea is of African origin (subgenus Vigna; Doi et al., 2). Because C. maculatus females may rely on a few specific characters (such as seed coat texture) when choosing oviposition sites, preference hierarchies need not reflect the phylogenetic relationships of potential hosts (Wasserman, 1986; Credland & Wright, 1988). Neither larval food source nor adult pre-oviposition experience modified host preference or relative host acceptance. Chiu and Messina (1994) found that actual egglaying experience could modify subsequent host preference in C. maculatus, but only when discrimination between hosts by inexperienced females is weak. Rearing host did have a consistent effect on realised fecundity, as females laid more eggs if reared on cowpea than if reared on mung bean (Fig. 5a vs. 5b). Because Kawecki and Mery (3) obtained the same result in four different populations, there may indeed be a strong conservatism in the effect of larval host plant on fecundity. Developing in cowpea seeds boosted fecundity even in the M lines, which were derived from a wild population collected on mung bean and maintained

8 Modification of beetle host preference 325 exclusively on mung bean for several hundred generations (Mitchell, 1991). Only a few studies have explicitly compared the relative lability of physiological and behavioural traits in the process by which insects adapt to novel host plants (Berenbaum & Zangerl, 1992; Hawthorne, 1999). This study, like that of Wasserman and Futuyma (1981), suggests that behavioural responses are more labile. Acknowledgements I thank A. J. Bloxham, M. A. Murray, and J. L. Taylor for technical assistance. This study was supported by the Utah Agricultural Experiment Station (paper no. 7571). References Agrawal, A.A. () Host range evolution: adaptation of mites and trade-offs in fitness on alternate hosts. Ecology, 81, Barron, A.B. (1) The life and death of Hopkins host-selection principle. Journal of Insect Behavior, 14, Becerra, J.X. (3) Synchronous coadaptation in an ancient case of herbivory. 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