Temporal patterns in reproduction may explain variationin mating frequencies in the green-veined white butterfly Pieris napi

There is genetic variation in the female mating rate in the green-veined white butterfly (Pieris napi), and females benefit from male ejaculates that contain both sperm and accessory gland substances. Although polyandry corresponds to higher lifetime fecundity than monandry, some females abstain from remating irrespective of the number of available mates. Explaining genetic variation in mating rates requires that monandrous females perform better than polyandrous ones under some conditions. We experimentally explored the reproductive performance of females either with a low or high intrinsic mating rate by allowing them to mate, feed, and lay eggs freely in a laboratory. Individual females followed different life histories: during the early days of reproduction, females with a low mating rate produced more eggs than females with a high mating rate. Hence, refraining from the benefits of multiple mating may be beneficial, if the time for reproduction is limited, or other female traits associated with polyandry are traded off against longevity. Given the day length of 10 h, a model shows that even if polyandrous females enjoy higher lifetime reproductive success, changeable and unpredictable weather will favor monandry if each period of suitable weather lasts, on average, less than 5 days. Thus, a combination of life history cost and unpredictability of fitness may explain the maintenance of monandry in the wild. Our results are also consistent with the observation that frequency of monandry increases with latitude.     

Preference for male traits in female wolf spiders varies with the choice of available males,female age and reproductive state

Individual variation in female preference for male traits may influence mate choice, especially if benefits and costs of choosiness vary with the range of available males or reproductive timing. We examined variation in female preference for male leg tuft size in Schizocosa ocreata (Hentz) wolf spiders with video playback. Dichotomous (simultaneous) choice test experiments included all possible combinations of four stimuli (i.e., modified versions of the same video male stimulus): average tuft size (control), reduced (−25%), enlarged (+25%), and no tufts (removed). Females exhibited a directional preference for larger tuft size independent of the nature of the choice (except for reduced tufts vs no tufts where no difference was seen). Female preference in the short term (over a period of 4 days) was also highly repeatable for control vs reduced tufts, but not for control vs enlarged tufts. Responses of females in ‘no-choice’ presentations of a single (control) male stimulus varied with age post-maturity; females were less receptive in weeks 1 and 2, highly receptive at week 3, and less thereafter. Mated females were least receptive and most aggressive towards a male stimulus. Females offered choices repeatedly at different ages post-maturity consistently preferred the control male vs reduced tufts over all 3 weeks but varied in their preference for enlarged tufts vs control male. In the first 2 weeks, females preferred the enlarged tuft male stimulus, but showed no preference by the third week. Females tested in week 4 showed no preference in either choice. Results suggest that the potential interaction between female preference for male traits and female reproductive timing may be a critical consideration in mate choice.     

Mating patterns and a test for female mate choice in Etheostoma spectabile (Pisces,Percidae)

Neither size nor breeding color correlated with spawning success of male orangethroat darters, Etheostoma spectabile (Pisces, Percidae), under natural field conditions. When females were presented experimentally with a simultaneous choice they spent no more time in proximity to large than small males, and were subsequently no more likely to spawn with large than with small males. Females also displayed no preference for bright versus dull males. Males and females did not differ significantly in size. Etheostoma spectabile may lack sexual size dimorphism as a result of the lack of female choice for size and the ineffectiveness of male attempts to monopolize females, or selection may be for increased size of females. Males are not dwarfs because of sperm competition. Contest competition among males appeared to be important in initiating spawnings but many males obtained spawnings by participating in ongoing spawning events. Etheostoma spectabile is an example of a sexually dimorphic species with no evident female preference for male size or color.     

Prior encounters with the opposite sex affect male and female mating behavior in a wolf spider (Araneae,Lycosidae)

Mate availability can vary widely in nature depending upon population density and sex ratio and can affect the ability of individuals to be selective in mate choice. We tested the effects of prior encounters with the opposite sex (i.e., exposure to the opposite sex either with or without mating) on subsequent mating behavior in two experiments that manipulated mate availability for both males and females in the wolf spider, Hogna helluo. The probability of mating in the experimental trial depended upon whether the prior encounter involved mating or not, and males and females responded in opposite directions. Exposure without mating resulted in a higher subsequent frequency of mating for females and a lower subsequent frequency of mating for males, while prior mating experience resulted in a lower frequency of female remating and a higher frequency of male remating. Prior exposure without mating did not affect female aggression. However, mated females engaged in precopulatory cannibalism more frequently than virgins. Mated males escaped postcopulatory cannibalism more frequently than virgins. Our results show that males respond to exposure without mating in the expected manner. However, prior mating (1 week earlier) had unexpected effects on males, which may be due to mated males being of higher quality. There were little or no effects of the size of the prior exposure individual or mate on subsequent mating behaviors. Further research is needed to determine why different species use different degrees of prior information in mate choice.     

On the consequences of aggressive male mate-locating behaviour and micro-climate for female host plant use in the butterfly <Emphasis Type="Italic">Lycaena hippothoe</Emphasis>

The distribution of ecological resources and their significance for males and females may vary considerably. Intersexual behavioural interactions may lead, combined with particular resource configurations, to sexual spatial segregation. We investigated this issue relative to host plant use in females of the purple-edged copper butterfly, Lycaena hippothoe. Males exhibited nectar resource-based territoriality, which is an uncommon mate-locating system in butterflies. They perched and patrolled in large territories harassing every passing female. In our study system, the percentage of spatial dimension shared for adult and larval resources was estimated at 50%, and males monopolised 28% of the nectar-rich zones. Under these conditions of harassment, females travelled between nectar-rich zones for feeding and zones with suitable host plants for egg laying, but often without nectar and hence with low male density. This is likely to limit their time budget and, potentially, their realised fecundity as suggested by the low number of eggs found relative to population size. Females were also highly specialised in selecting host plants under particular environmental conditions. Using test choice in experimental cages, we showed that, in the absence of males, only micro-climatic conditions may significantly influencing egg-laying decisions. Moreover, results of egg-rearing experiments under different temperature treatments suggested that eggs were laid in thermally suitable micro-environments. The highly selective egg-laying behaviour can be viewed as a preference-performance choice. Knowledge of individuals' behaviour, including sexual interactions, can be highly significant for our understanding of habitat use, which in turn can be essential for conservation. We discuss this for L. hippothoe, a species of regional conservation concern.     

Varying levels of female promiscuity in four <Emphasis Type="Italic">Apodemus</Emphasis> mice species

Sexual selection in most vertebrates is based on the evolution of fitness optimization strategies such as multiple-male mating (MMM). Several ecological correlates of MMM have been identified in bird and fish populations; however, only few studies have documented the effects of environmental change on promiscuity in mammals. In this study, the 127 pregnant females from four central European and ecologically diverse species of field mice (genus Apodemus) were studied to assess the role of ecological factors that may have shaped the evolution of particular mating systems. MMM was found in all analyzed species: in Apodemus uralensis and Apodemus flavicollis, up to two males could be identified as the fathers of a particular litter, while three males sired 9.1% of analyzed litters of Apodemus sylvaticus and 20.6% of Apodemus agrarius. Furthermore, there were obvious differences between species in relative testes size and the proportion of multiple sired litters during those seasons when the opportunity for multiple mating was high. The species with the smallest testes and the least promiscuous was A. uralensis (only 43.5% of multiple sired litters), while the species with the biggest testes and the most promiscuous was A. agrarius (69.2%). MMM was significantly associated with higher litter size in A. flavicollis, and the probability of MMM strongly increased with season in A. agrarius and with abundance in A. uralensis. These results indicate that ecological factors are associated with MMM rates in Apodemus field mice and more research is needed to fully understand the evolution of mating strategies at different levels of biological resolution.     

Oviposition by butterflies on young leaves: Investigation of leaf volatiles

Summary Various butterflies select young foliage on which to lay their eggs; volatiles emitted by young and old leaves have been compared (by sorption enrichment, followed by GC-MS) to gauge possible qualitative and quantitative differences between the two age groups. The plants investigated are cabbage (Brassica oleracea), two milkweeds (Asclepias syriaca andA. curassavica), the bitter orange (Citrus aurantium) and the lime (C. aurantiifolia). The chemical compounds identified belong to three classes, isoprenoids, fatty acid derivatives and benzenoids. Quantitative differences were found between young and old leaves, of which a few may be characteristic of young leaves only. Thirty-four single trials withDanaus plexippus exposed to volatiles from young and old leaves are recorded.     

Oviposition site guarding by male walnut flies and its possible consequences for mating success

Field studies showed that male Rhagoletis juglandis and R. boycei flies guard egg-laying punctures (and the eggs within) on host walnut (Juglans major) fruit and defend those sites from conspecific and heterospecific males. In field experiments with artificially punctured fruit,as well as field observations on unmanipulated fruit, males were consistently more likely to be sighted and stayed longer on damaged fruit than on undamaged fruit. On artificially punctured fruit, they consistently spent more time in the vicinity of a puncture than expected by chance alone. Males together on damaged fruit were more likely to engage in contests over those fruit than males together on undamaged fruit. Copulations were consistently more frequent for either species on damaged than undamaged fruit, both in observations of unmanipulated fruit and in artificial puncture experiments. Analyses which controlled for the longer male residence time on damaged fruit suggested strongly that copulations were consistently achieved at higher per capita rates on damaged than on undamaged fruit, indicating that puncture-guarding functions to increase access to females. An exception to the pattern in male mating success was noted at a site where both species used host fruit on the same trees. In this case, R. juglandis males were only slightly more common on punctured fruit than on control fruit and male success in copulation did not differ significantly between the two types of fruit. This anomalous result was apparently due to an almost absolute advantage enjoyed by R. boycei males in on-fruit contests with R. juglandis males. A likely basis for improvements in mating success associated with puncture guarding was a propensity for females to deposit eggs into existing punctures. Both in observations of unmanipulated fruit and in artificial puncture experiments, females consistently attempted oviposition more often in damaged than undamaged fruit. In artificial puncture experiments, both species at both sites deposited most clutches in damaged fruit. Mating generally took place as females initiated oviposition. The possible functions of puncture use by females as well as alternative functions of puncture guarding by males are discussed.     

Seminal fluid protein depletion and replenishment in the fruit fly, <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: an ELISA-based method for tracking individual ejaculates

In many species, seminal fluid proteins (SFPs) affect female post-mating behavioral patterns, including sperm storage, egg laying, feeding, and remating. Yet, few studies have investigated the patterns of allocation, depletion, and replenishment of SFPs in male animals, despite the importance of these proteins to male and female reproductive success. To investigate such SFP dynamics, it is necessary to have a sensitive method for quantifying SFP levels in males and mated females. We developed such a method by adapting the enzyme-linked immunosorbent assay (ELISA) using anti-SFP antibodies. Here, we first use two Drosophila melanogaster SFPs (ovulin and sex peptide) to demonstrate that ELISAs provide accurate measures of SFP levels. We find that, consistent with previous data from Western blotting or immunofluorescence studies, levels of both ovulin and sex peptide decline in the mated female with time since mating, but they do so at different rates. We then use ELISAs to show that males become depleted of SFPs with repeated matings, but that previously mated males are able to transfer “virgin” levels of SFPs after 3 days of sexual inactivity. Finally, we demonstrate that ELISAs can detect SFPs from wild-caught D. melanogaster males and, thus, potentially can be used to track mating patterns in the wild. This method of measuring SFP dynamics can be used in a wide range of species to address questions related to male reproductive investment, female mating history, and variation in female post-mating behavioral changes. Norene A. Buehner and Anthony C. Fiumera contributed equally to this work.     

Oviposition stimulants and deterrents control acceptance ofAlliaria petiolata byPieris rapae andP. napi oleracea

Summary Differential acceptance of garlic mustard,Alliaria petiolata byPieris rapae L. andP. napi oleracea is explained by their differential sensitivities to oviposition stimulants and deterrents in the plant. Fractions containing the stimulants and deterrents were isolated by solvent partitioning between water and n-butanol and by open-column chromatography followed by HPLC.P. napi oleracea showed no preference when offered a choice ofA. petiolata or cabbage, but was strongly stimulated to oviposit by post-butanol water extracts ofA. petiolata. The most abundant glucosinolate in this extract was identified as sinigrin, which could explain the high degree of stimulatory activity.P. rapae preferred cabbage plants overA. petiolata, and the relatively low stimulatory activity was also associated with the glucosinolate-containing aqueous extract. However, this species was strongly stimulated by a fraction that contained small amounts of glucotropaeolin along with unknown compounds. Deterrents to both species were found in the butanol extract fromA. petiolata, andP. napi oleracea was more sensitive thanP. rapae to these deterrents. Some HPLC fractions from the BuOH extract were strongly deterrent toP. napi oleracea, but were inactive toP. rapae. The ecological significance of these behavioral differences between the twoPieris species is discussed.     

Unattractiveness of mated females to males in the parasitoid wasp Spalangia endius

Despite common stereotypes, males are not always indiscriminate and eager when it comes to mating. In the parasitoid wasp Spalangia endius, the initial response of males to females was almost always one of apparent excitement; however, this was followed by a clear preference for virgin females over mated females in both no-choice and choice situations. The no-choice data were collected from videotapes of male-female pairs of all possible combinations of mated and virgin individuals. Neither female nor male mating status had a significant effect on likelihood of, or time until, contact or male courtship fanning. However, a males first retreat was sooner when the female was mated than when she was virgin; mated males exhibited their first retreat sooner than did virgin males; and mated females were less likely to be mounted than were virgin females. In addition to the videotapes, male-choice experiments were performed. When given a choice of a virgin and a mated female, both virgin and mated males were more likely to mount and copulate with the virgin. The difference in response to virgin versus mated females seemed to be less in virgin males than in mated males, perhaps due to virgin males greater eagerness to mate: when a virgin male and a mated male were presented with a dead virgin female, the virgin male was usually the first to respond to the female. That males preferentially retreated from and avoided mounting mated females appears to be adaptive given that mated females rarely copulated.Communicated by N. Wedell     

Exogenous testosterone increases female aggression in the European starling (<Emphasis Type="Italic">Sturnus vulgaris</Emphasis>)

In the European starling, Sturnus vulgaris, optimal mating systems differ between males and females. Males gain from polygyny, whereas monogamy increases female fitness. The cost of polygyny to females lead to intense female–female competition, and it has previously been shown that the intensity of female aggression during the pre-breeding period can predict the realised mating system. The physiological regulation of such female aggression in starlings is not yet known. This study examines the role of testosterone in mediating aggressive behaviours involved in intra-specific reproductive competition in female starlings. Testosterone levels were experimentally elevated with testosterone implants in females during the pre-laying period. To simulate a situation in which an additional female tried to mate with the focal female’s mate, a caged female was presented close to a nest-site to which the male could attract a secondary female. Testosterone was significantly related to several behaviours involved in female–female interactions. Females with testosterone implants spent significantly more time close to the caged female and produced more song bouts than control females. In contrast, male behaviour was unrelated to the experimental status of the mate. Females mated to males that attracted a secondary female were less aggressive towards the caged female than those that remained monogamously mated. The effect of exogenous testosterone in this study indicates that androgens may mediate social behaviours in female starlings during the breeding season.     

Dynamics of space use and male vigour amongst wood mice,Apodemus sylvaticus,in the cereal ecosystem

The spatial organisation of male and female wood mice,Apodemus sylvaticus, was investigated in a large-scale radio-tracking study on arable farmland near Oxford, United Kingdom, during the breeding season. Both sexes had significantly larger home ranges in the breeding season than at other times, and the breeding season home ranges of male (X = 1.44 ha) were significantly larger than those of females (X = 0.49 ha). Home range overlap was significantly greater between males, and between males and females, than it was between females. Overlap between males tended to be greatest in heavily utilised areas. Except during sexual consortship, there was minimal evidence of dynamic interaction among individuals. Home range sizes of breeding males varied widely, as did their body weights. There was no relationship between male body weight and home range size or any other movement parameter. However, males with the largest home ranges had the highest scores on all other movement parameters, indicating that they expended more energy in movement. These more vigorous males had access to the home ranges of more females than did males with small home ranges.     

Fertilisation success in the fly Dryomyza anilis (Dryomyzidae): effects of male size and the mating situation

Male fertilisation success in relation to male size and the mating situation (ordinary pair formation with a single, nonvirgin female vs. take overs) was examined in the fly Dryomyza anilis. In ordinary matings, large males achieved higher fertilisation success than small ones when they were the second to mate with the female. Take overs differ from ordinary pair formation in that the second male experiences intensified sperm competition. This is because in take overs the female is not able to discharge any of the sperm inseminated by the first male as she usually does before a new mating. Compared with ordinary matings, take overs reduced the fertilisation success of the second male by 8–10%, whereas that of the first male was 7–14% higher in take overs. Even though the intruder was always larger than the paired male his superior fertilisation success did not compensate for the effect of the sperm already present in the female. In D. anilis, males can increase their fertilisation success by tapping the female's external genitalia with their claspers or having several copulation bouts per mating. Thus, in a take over, the intruder could respond to the intensified sperm competition by performing more tapping sequences per copulation bout or more copulation bouts per mating. In matings observed in the wild, males performed more tapping sequences after a take over than after pair formation with a single female, although the difference was not significant. The results show that there are differences in fertilisation success between males of different size. In addition, different mating situations can result in considerable variation in the fertilisation success of an individual male. Higher fertilisation success for the first male after a take over may be significant, in particular, for the reproductive success of small males, which frequently lose their females to large males.     

Independence, not conflict, characterizes dart-shooting and sperm exchange in a hermaphroditic snail

Although the sexes are united in hermaphrodites, conflict can still occur because the male and female functions have separate interests. We examined the evidence for conflict in the mating system of the terrestrial snail Cantareus aspersus (formerly Helix aspersa) where sharp, calcareous darts are ‘shot’ during courtship. We predicted that the use of the dart would either reflect or create conflict and this would be evident in either the courtship behavior or the transference of sperm. Previous studies demonstrated that the dart functions after sperm transfer to increase sperm survival. Using detailed observations of mating snails, we examined the factors that determine dart shooting order, the behavioral responses after being hit by a dart, the accuracy of dart shooting, and the allocation of sperm resources. We found that each dart was shot independently, and each animal appeared to be interested only in getting off the best possible shot, probably one that penetrates deeply near the genital pore. There is no evidence of mating conflict. Every snail transfers sperm to its partner, and the size of the donation does not depend on the success or failure of either snail’s dart shot. Although the receipt of a dart does not appear to cause harm, it may produce indirect costs due to the partial loss of control over fertilization. We conclude that mating in C. aspersus is a partnership in which independent actors demonstrate unconditional reciprocity during courtship and sperm transfer.     

Population differentiation in female sex pheromone and male preferences in a solitary bee

Population differentiation in female mating signals and associated male preferences can drive reproductive isolation among segregated populations. We tested this assumption by investigating intraspecific variation in female sex pheromone and associated male odour preferences among distant populations in the solitary bee Colletes cunicularius (L.) by using quantitative gas chromatography and by performing field bioassays with synthetic blends of key sex pheromone compounds. We found significant differences in sex pheromone blends among the bee populations, and the divergence in odour blends correlated positively with geographic distance, suggesting that genetic divergence among distant populations can affect sex pheromone chemistry. Our behavioural experiments, however, demonstrate that synthetic copies of allopatric female sex pheromones were cross-attractive to patrolling males from distant populations, making reproductive isolation by non-recognition of mating signals among populations unlikely. Our data also show that patrolling male bees from different populations preferred odour types from allopatric populations at the two sites of bioassays. These male preferences are not expected to select for changes in the female sex pheromone, but may influence the evolution of floral odour in sexually deceptive orchids of the genus Ophrys that are pollinated by C. cunicularius males.     

Sex ratios,mating behavior and sexual size dimorphism of the northern water snake,Nerodia sipedon

Competition among males to mate is generally associated with male-biased size dimorphism. In this study we examine mating behavior in the northern water snake (Nerodia sipedon), a species in which males are much smaller than females despite substantial competition among males to mate. Competition among males was a consequence of a male-biased operational sex ratio due to slightly higher female mortality from a birth sex ratio of 1 : 1, and, in 1 year, more synchronous and longer mating activity by males. Approximately one-third of both males and females appeared not to mate in a given year. Larger males were generally more likely to attempt mating, but size did not explain the variance in the number of aggregations in which individual males participated. Within aggregations, males that were successful at achieving intromission were larger than unsuccessful males in 1 of 2 years. Variation in condition (mass relative to length) and relative tail length were not generally useful predictors of either mating effort or success in males. Because large size was often advantageous to males, sexual size dimorphism appeared not to be a consequence of sexual selection favoring smaller males. Because sexual dimorphism was evident at birth, and both males and females matured sexually at about 4 years, sexual dimorphism was not simply a consequence of one sex growing at the maximum rate for longer. Female fecundity increased with size, and sex differences in size-fecundity relations may underly the pattern of sexual size dimorphism. However, because multiple mating by females is common, sperm competition is likely to be important in determining male reproductive success. Therefore, allocation of energy to sperm rather than growth may also prove to be an important influence on male growth rates and sexual size dimorphism.     

The cost of mating with a non-virgin male in a monandrous butterfly: experimental evidence from the speckled wood, Pararge aegeria

Contrary to vertebrates, sperm production in insects may bear considerable costs for males. This is especially true in species that donate spermatophores containing sperm and nutrient-rich accessory gland products like in butterflies. Hence, spermatophores at first and subsequent copulations can differ in a quantitative and qualitative way. Such effects have particularly been shown in polyandrous species providing large spermatophores. Here we experimentally tested the effect of male mating status (virgin male vs recently mated male) on copulation duration, spermatophore size and females’ fitness components in a monandrous butterfly Pararge aegeria that typically donates small spermatophores. Copulations with non-virgin males lasted on average five times longer than that with virgin males and resulted in a spermatophore which was on average three times smaller. Number of eggs laid and female life span were not affected by the mating status treatment, but there was a significant effect on the number of living caterpillars a female produced, as copulations with virgin males resulted in higher numbers of larval offspring. Interestingly, the difference in spermatophore mass at the first and the second copulation increased with male body size. This suggests differential spermatophore allocation decisions among males of different size. Consequences for females and potential mechanisms influencing female fitness components are discussed. Given the small absolute size of spermatophores in P. aegeria, components other than consumable nutrients (perhaps hormones) should cause the observed effects.     

Extra-pair paternity in tree swallows: why do females mate with more than one male?

Recent studies of monogamous tree swallows (Tachycineta bicolor) suggest that females may receive some type of genetic benefit from extra-pair fertilizations. In this study we attempted to determine what type of genetic benefits might be gained by females. We compared numerous morphological and behavioral traits (Table 1) of every male nesting on one grid of nest-boxes (n = 23) to determine what male traits were correlated with male success at gaining extra-pair fertilizations. DNA fingerprinting revealed an increase in the level of extra-pair paternity from the previous year (50% of broods contained extra-pair young in 1990 vs. 87% of broods in 1991), but no significant correlates of paternity. We found six extra-pair fathers at seven nests (20 nests had extra-pair young). The traits of these extra-pair males did not differ from those of the males they cuckolded. We discuss several reasons for this lack of difference, but argue that our results are not inconsistent with females choosing extra-pair males to enhance the genotypic quality of their offspring. Despite a complete search of the nest-box grid for extra-pair fathers, we were able to explain the paternity of just 21% (13/63) of all extra-pair young. This suggests that extra-pair fathers were either residents off our study grid or non-territorial floaters. Tree swallows are quite mobile and spend only part of the day at their nest prior to laying. In addition, we rarely see swallows visiting other grids of nest-boxes. Therefore, we suggest that most extra-pair copulations occur at some unknown location, possibly at a feeding or roosting area where females may be able to choose from many more potential extra-pair fathers than at their nest-site.     

New insights in analysing parasitoid attracting synomones: early volatile emission and use of stir bar sorptive extraction

Summary. It is well known that feeding by Pieris brassicae caterpillars on cabbage leaves triggers the release of volatiles that attract natural antagonists such as the parasitoid Cotesia glomerata. The temporal dynamics in the emissions of parasitoid attracting volatiles has never been elucidated in this system. In a time course experiment, caterpillar infested leaves attracted the parasitoid within one hour after infestation. At such an early stage of infestation, as much as fifty percent of the parasitoids flew towards the infested plant in a wind tunnel bioassay, while only five percent flew towards the non-infested control plant. Three hours after infestation and later, the response to the volatiles from the infested plant reached its maximum and then continued at a constantly high level for the remaining 14 hours of the experiment. Chemical analyses of volatiles collected from infested leaves at short time intervals during the first 24 hours identified a total of ten compounds, comprising green leaf volatiles, terpenoids, and a nitrile. Significant increase of emission within the first 5 hours following initial herbivory was detected for (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate, cineole and benzylcyanide. Subsequently, a coupled bioassay-chemical analysis procedure was developed allowing for testing and analyzing the same sample for future identification of the bioactive compounds. This was achieved by using stir bar sorptive extraction for the analysis of solvent extracts of caterpillar-damaged leaves.