AbstractParasitoid wasps serve as natural enemies of numerous insect species; therefore, knowledge of host-parasitoid interactions is fundamental for understanding ecosystems. Each endoparasitoid wasp taxon exhibits a specific host range. Female parasitoids, however, occasionally oviposit into non-host species. Since the survival probability of eggs in non-host species is virtually zero, these behaviors have long been considered maladaptive. However, in the present study, we found that eggs of a specialist parasitoid, Cotesia kariyai (Hymenoptera: Braconidae), oviposited in unsuitable host caterpillars, Mythimna loreyi (Lepidoptera: Noctuidae), successfully complete larval development in the non-host when these caterpillars are simultaneously oviposited by another naturally sympatric parasitoid wasp, Meteorus pulchricornis (Hymenoptera: Braconidae), for which My. loreyi is the usual host. This observation suggests that the seemingly maladaptive behavior of ovipositing in unsuitable host insects can be adaptive, allowing them to maintain reproductive potential in environments where their ordinary hosts are absent. We propose a new term, “pirate parasitism”, for this type of obligatory multiparasitism. Understanding detailed mechanisms of this phenomenon may provide deeper insights into parasitoid-host dynamics and evolution of host use strategies by parasitoids.
IntroductionParasitoid wasps comprise one of the most species-rich animal groups. They serve as natural enemies of numerous insect species, and are widely recognized as important in both natural and agricultural ecosystems1,2,3. Each parasitoid species exhibits a more or less specific host range; thus, in the context of ecological, evolutionary, and agricultural science, host-parasitoid associations are essential to understand biological networks in natural and agricultural ecosystems1,2,4,5,6,7.Parasitoids, which deposit their eggs in bodies of host insects and develop inside them, known as endoparasitoids, must overcome host immunity to complete their development1. Endoparasitoid wasps employ various maternal strategies to protect their eggs and larvae from host immune systems. For instance, females of ichneumonoid endoparasitoids (Hymenoptera: Braconidae and Ichneumonidae) utilize venom, a mixture of proteinaceous and non-proteinaceous components, and/or polydnaviruses, symbiotic, large, double-stranded DNA viruses, to suppress host immunity8,9,10,11,12,13. Anti-host venoms and viruses exhibit specificity depending on host species or strain; therefore, such specificity restricts host ranges of endoparasitoids14,15,16. In fact, parasitoid eggs are encapsulated and are usually killed by host cells when deposited in non-host species17,18,19. Consequently, female parasitoids may have evolved host-location and host-acceptance strategies using physical and chemical cues to oviposit in suitable host species20,21,22,23.However, female parasitoids encounter numerous sympatric non-host species (species unsuitable for the parasitoid to complete development). Occasionally, parasitoid females are attracted to cues derived from non-host species, either chemically or physically24,25,26,27,28,29, so that they accept the non-host species, and oviposit onto or into them24,30,31,32,33,34,35. Since the survival probability of parasitoid eggs in non-host species has been regarded as essentially zero, these behaviors have been considered as “misunderstandings of information”28, “accidents”2 or “mistakes”36 by parasitoid females. However, in the context of parasitoid host range evolution, theory predicts that these “mistakes” may lead to host shifts or host range expansion at the population level when parasitoids fortuitously attack potential new host species that are suitable for parasitoid development. This occurs through associative learning by newly emerged female parasitoids, resulting in a preference for oviposition in the same species in which they developed2,37,38,39. However, despite the potentially significant role of such “mistakes” in evolution of parasitoid host range, their adaptive significance has been little discussed.Kraaijeveld31 hypothesized that oviposition by female parasitoids into non-host species could potentially increase the fitness of parasitoids in the absence of their ordinary host species. The scenario proposed was as follows. Eggs of the parasitoid oviposited in a non-host species might be able to complete their development if the non-host insects had previously been parasitized by a sympatric parasitoid species that uses the same insect species as its ordinary host. In other words, the endoparasitoid might engage in kleptoparasitism by exploiting anti-host immunity conferred by another parasitoid species via multiparasitism in unsuitable host species. To test this hypothesis, Kraaijeveld31 used larvae of Drosophila simulans (Diptera: Drosophilidae), an endoparasitoid, Asobara tabida (Hymenoptera: Braconidae), and another endoparasitoid, Leptopilina boulardi (Hymenoptera: Figitidae). The study revealed that even if A. tabida accepted D. simulans larvae and oviposited into them, its offspring could not complete development in those larvae. However, when the host larva had previously been oviposited by L. boulardi, approximately 15% of Asobara larvae survived in D. simulans larvae for 4 or 6 days after oviposition. Based on these results, the author concluded that the response and oviposition of female parasitoids into non-host species could indeed benefit parasitoids because it could result in reproductive success in the context of multiparasitism31,40. Similarly, Vinson41 also conducted multiparasitism experiments using the parasitoid, Cardiochiles nigriceps (Hymenoptera: Braconidae), its non-host caterpillar, Heliothis zea (Lepidoptera: Noctuidae), and a parasitoid Microplitis croceipes (Hymenoptera: Braconidae), which uses H. zea as its ordinary host. The author demonstrated that parasitism by M. croceipes inhibits the immune response of H. zea to C. nigriceps eggs/larvae.As far as we know, successful parasitism in unsuitable hosts through multiparasitism with other parasitoids has been reported in several insect systems32,42,43,44, such as Cotesia flavipes (Hymenoptera: Braconidae)32 and Psyttalia fletcheri (Hymenoptera: Braconidae)44. In all of these cases, parasitoids showed successful parasitism in non-host species only when the host had been previously oviposited by another parasitoid species. However, all the previous examples involved interactions between parasitoid species, at least one of which was introduced into the host range32,42,43. Even if the ecological significance of such systems may apply in the context of biological invasions, Kraaijeveld’s hypothesis has never been examined in herbivore-parasitoid systems that are naturally sympatric and share a long evolutionary history. To test Kraaijeveld’s hypothesis, successful parasitism in non-host species via multiparasitism should be examined in herbivore-parasitoid systems that satisfy the following conditions: (1) a potential host species and two parasitoid species are naturally sympatric and occur simultaneously in the field, (2) both parasitoids have the capacity to search for and oviposit into the potential host species, (3) the potential host species is unsuitable for one of the parasitoids due to its immune response when parasitized by that species alone, while being a suitable host for the other parasitoid.Here, we tested Kraaijeveld’s hypothesis using the following naturally sympatric insects in laboratory experiments: two endoparasitoids, Cotesia kariyai (hereafter, Ck) (Hymenoptera: Braconidae) and Meteorus pulchricornis (hereafter, Mp) (Hymenoptera: Braconidae), and larvae of a moth, Mythimna loreyi (hereafter, Myl) (Lepidoptera: Noctuidae).Ck is a monophagous specialist, and a gregarious endoparasitoid of Mythimna separata (hereafter, Mys) (Lepidoptera: Noctuidae) caterpillars45. However, Ck females respond to cues related to sympatric unsuitable host lepidopteran larvae, including Myl, and oviposit into them, but no offspring successfully develop and emerge24,28,34,46. The other parasitoid, Mp, is a generalist, solitary larval endoparasitoid of numerous species of exophytic lepidopteran caterpillars, including Myl47,48.These two parasitoids are comprehensively studied for biological characteristics, such as general bionomics, host searching behavior, oviposition behavior, and immune suppression mechanisms46,48,49,50,51,52,53,54,55. In addition, multiparasitism of Ck and Mp in Mys, a suitable host for both parasitoids, is also well documented56,57,58.Caterpillars of both Myl (an unsuitable host for Ck, but a suitable host for Mp) and Mys (a suitable host for both parasitoids) are external leaf feeders that feed primarily on grasses. Myl and Mys are frequently sympatric and frequently co-occur in maize or sorghum fields in South, Southeast, and East Asian countries59,60,61,62. They are morphologically, behaviorally, and ecologically very similar; in terms of body size, color pattern, range of host plants, and diurnal larval behavior. However, they can be distinguished by morphology of adults and hairs on the larval head capsule59,62,63,64. To the best of our knowledge, there are no records of Ck emerging from Myl. However, reports of parasitoids from Myl in Japan are extremely scarce, and as mentioned earlier, the external morphologies of Myl and Mys are very similar. Consequently, there is a possibility that records of Ck emerging from Myl may have been mistakenly attributed to Mys.In this study, we tested Kraaijeveld’s hypothesis by addressing the following questions: (i) Do females of both parasitoid species accept unparasitized Myl caterpillars or Myl caterpillars previously oviposited by the heterospecific parasitoid species as targets for oviposition? (ii) Do Ck eggs oviposited in Myl caterpillars show successful parasitism, when multiparasitism with Mp occurs? (iii) Does the order and interval of oviposition by the two parasitoid species affect the outcome of multiparasitism? (iv) Can successful parasitism in non-hosts occur through multiparasitism in simultaneous free-oviposition tests in cages? Additionally, larvae of both parasitoids in multiparasitized Myl caterpillars were observed by dissection to ensure that hyperparasitism by both parasitoid species (cf.65) did not occur.Here, we report successful parasitism by a specialist parasitoid in an unsuitable host species via multiparasitism in a naturally sympatric insect system. Based on our results, we discuss the ecological significance of erroneously responding and ovipositing in unsuitable host species. We propose a new term, “pirate parasitism”, for this type of parasitism.ResultsExperiment 1: host acceptance and ovipositionRegardless of the host instar or previous oviposition by the heterogeneous parasitoid, all Ck females exhibited oviposition behavior towards Myl caterpillars, similar to their behavior toward their usual host, Mys (Supplementary Figure S1a). The number of Ck eggs in oviposited Myl caterpillars, ranged from 53.7 ± 17.8 to 59.0 ± 18.3 (mean ± SD, n = 20 for each), and did not vary significantly among host instars or in relation to previous parasitism. The number of Ck eggs deposited did not differ significantly from those in the usual host, Mys (Supplementary Figure S1b, ANOVA, F6, 133 = 0.19, p > 0.05). Similarly, over 85.0% (n = 30 for each) of Mp females accepted Myl caterpillars for oviposition, regardless of the host instar or previous oviposition by the heterogeneous parasitoid, and the difference in the host acceptance rate did not differ significantly among Myl instars or in relation previous parasitism (χ2 = 3.41, df = 5, p > 0.05, Supplementary Figure S1c). A single Mp egg was consistently oviposited in Myl caterpillars without exception (Supplementary Figure S1d).Experiment 2: single-parasitism experimentsRegardless of the host instar, Ck never emerged from Ck-oviposited Myl caterpillars (Supplementary Figure S2a, n = 200 for each). Among Ck-oviposited Myl caterpillars, 83.0–86.5% of larvae survived until pupation, whereas the remainder died without emergence of Ck (Supplementary Figure S2a). In contrast, Mp successfully emerged from 76.7 to 86.7% of Mp-oviposited Myl caterpillars (Supplementary Figure S2b, n = 30 for each). The difference in the Mp emergence rate did not differ significantly among Myl instars (χ2 = 1.25, df = 2, p > 0.05, Supplementary Figure S2b).Experiment 3: multiparasitism experiments in different host instarsAmong multiparasitized caterpillars, parasitoid wasps emerged from 50.0 to 90.0% of the caterpillars (Fig. 1a). Regardless of host instar or order of oviposition, no instances were observed in which both parasitoid wasps emerged from a single larva. Interestingly, Ck emerged from 5.0 to 15.0% of Myl caterpillars (Fig. 1a). The successful parasitism rate of Ck did not differ among treatments (χ2 = 4.56, df = 5, p > 0.05). The number of emerged Ck adult per Myl caterpillar, ranging from 25.7 ± 13.8 to 45.6 ± 21.4 (mean ± SD) on average, was not significantly different among caterpillar instars or order of oviposition. However, significantly fewer Ck adults emerged from Myl caterpillars than from their usual host, Mys (Fig. 1b, ANOVA, F6, 47 = 7.028, p < 0.05, Tukey-Kramer, p < 0.05).Regardless of the host instar or order of oviposition, the successful parasitism rate of Mp (31.7–73.3%) was significantly higher than that of Ck (Fig. 1a, binomial test, p < 0.05). For Ck emerging from Myl, all 30 Ck females (5 per treatment) showed oviposition behavior toward Mys caterpillars, and their offspring emerged from the caterpillars. The number of Ck adults that emerged from those caterpillars did not differ significantly among treatments (Supplementary Figure S3, ANOVA, F5, 24 = 0.256, p < 0.933).Fig. 1Outcome of multiparasitism by Cotesia kariyai (Ck) and Meteorus pulchricornis (Mp) in Mythimna loreyi (Myl) caterpillars. The successful parasitism rate of each parasitoid in 3rd, 4th or 5th instar Myl caterpillars (a), and number of emerged wasps per caterpillar when Ck emerged from the caterpillars (b). Mys = My. separata. (∗P < 0.05; ∗∗P < 0.01 and ∗∗∗P < 0.001 by binomial test; N.S.: no significant differences by Chi-square test, Bars labeled with the same letter are not significantly different on the basis of Tukey’s HSD test after ANOVA)Full size imageExperiment 4: multiparasitism in different sequences and intervalsSuccessful parasitism by Ck occurred when Ck oviposited from 24 h before until 12 h after Mp (Fig. 2a). Beyond these times, no successful parasitism by Ck was observed. Under experimental conditions in which successful parasitism by Ck was observed, the success rate of Ck ranged from 5.0 to 20.0%. The latter occurred when Ck oviposited 12 h before Mp, but the success rate was significantly lower when Ck oviposited 24 h before Mp (Fig. 2a, χ2 = 38.04, df = 7, p < 0.05; Tukey’s WSD, p < 0.05). The number of emerged Ck adults per Myl caterpillar, ranging from 26.0 ± 17.1 to 38.4 ± 11.7 (mean ± SD) on average, was not significantly different among conditions, but they were significantly fewer than the number of Ck adults that emerged from their usual host, Mys (78.1 ± 16.7) (Fig. 2b, ANOVA, F5, 36 = 16.05, p < 0.05, Tukey-Kramer, p < 0.05). In all conditions, the successful parasitism rate of Mp was significantly higher than that of Ck (Fig. 2a, binomial test, p < 0.001).Fig. 2Effects of order and interval of oviposition by two parasitoids on outcomes of multiparasitism. The successful parasitism rate of each parasitoid in 4th instar Myl caterpillars (a), and numbers of wasps per caterpillar when Ck emerged from the caterpillars in different orders and intervals of oviposition by two parasitoids (b). Myl = Mythimna loreyi, Mys = My. separata, Ck = Cotesia kariyai, and Mp = Meteorus pulchricornis. (∗P < 0.05; ∗∗P < 0.01 and ∗∗∗P < 0.001 by binomial test; bars labeled with the same letter are not significantly different on the basis of (a) Tukey’s WSD teat after Chi-square test and (b) Tukey’s HSD test after ANOVA.)Full size imageExperiment 5: observations of parasitoid larvae in multiparasitized caterpillarsMyl caterpillars were dissected in four conditionsFig. 3a) and parasitoid larvae were observedFig. 3b). No Ck larvae were observed in Ck-oviposited Myl caterpillars 8 days post-ovipositionFig. 3c). In the case of day 8-multiparasitized Myl caterpillars, living Ck larvae were observed in 83.3% of caterpillars. Among these, 70.0% of caterpillars contained both living Ck and living Mp larvae whereas 13.3% contained only Ck larvaeFig. 3c). No Ck larvae were observed in 16.7% of the caterpillars, whereas 3.3% contained only Mp larva and the remaining 13.3% contained no parasitoid larvaeFig. 3c). In dead multiparasitized Myl caterpillars from which Ck emerged, dead Ck larvae were observed in 66.7% of the caterpillars, whereas the remaining caterpillars contained no Ck larvae. No Mp larvae were observed in these cases. In dead multiparasitized Myl caterpillars from which Mp emerged, dead Ck larvae were observed in 86.7%, while the others contained no parasitoid larvae. Hyperparasitism, in which Ck larvae parasitize in Mp larvae, was not observed (Fig. 3b).Fig. 3Observation of parasitoid larvae in Mythmna loreyi caterpillars. Preparation and dissection timing of parasitized caterpillars used for observation (a) and observed larvae of two parasitoids in multiparasitized Myl caterpillar (b). Proportion of observed parasitoid larvae is shown in (c). Myl = Mythimna loreyi, Ck = Cotesia kariyai, and Mp = Meteorus pulchricornis.Full size imageExperiment 6: simultaneous free-oviposition rearing in cagesOf 10 replicates, 5 showed successful parasitism of Ck in Myl caterpillars (Fig. 4). Overall, Ck emerged from 6.0% of Myl caterpillars. The number of Ck adults emerging from Myl caterpillars was 21.6 ± 9.3 (mean ± SD). Mp emerged from 48.0% of Myl. 22.0% of them pupate and another 22.0% died during experiments. Additionally, 2.0% of Myl caterpillars disappeared during cage experiments.Fig. 4Outcome of simultaneous free-oviposition rearing experiments in cages. Results of each 10 replications and their total are shown.Full size imageDiscussionSuccessful parasitism by C. kariyai in non-host My. loreyi parasitized by Me.
pulchricornis
Attraction of parasitoid wasps to sympatric unsuitable host species and subsequent oviposition onto or into unsuitable hosts is widely documented. Females of the specialist endoparasitoid wasp, C. kariyai (Ck), also respond to chemical cues from unsuitable host species, including sympatric My. loreyi (Myl), and exhibit oviposition behavior toward them24,28,34,46. Our results demonstrate that Ck females recognize Myl caterpillars and oviposit into them as into their usual host, My. separata (Mys) caterpillars (Experiment 1). However, Myl caterpillars parasitized solely by Ck never produced Ck adults (Experiment 2).Remarkably, our multiparasitism experiments revealed that Ck can complete larval development in Myl caterpillars when these caterpillars are also parasitized by the naturally sympatric endoparasitoid wasp, Me. pulchricornis (Mp), which uses Myl as its usual host (Experiments 3 and 4). In essence, Ck can utilize unsuitable host Myl caterpillars as hosts when multiparasitism with Mp occurs. According to observations of parasitoid larvae in multiparasitized Myl caterpillars, no Ck larvae were found parasitizing Mp larva (Experiment 5). Therefore, hyperparasitism (cf.65), does not explain this phenomenon.Ck females protect their eggs and their larvae from the immune response of their usual hosts, Mys caterpillars, by injecting polydnavirus (CkPDV) and venom46,50,55,66. These maternal anti-immunity factors are effective against Mys caterpillars, but not against other caterpillars46, which restricts the range of potential hosts for Ck. In fact, although Ck females oviposited 53.7–59.0 eggs into Myl caterpillars (Experiment 1), no Ck larvae were observed in hemocoels of Myl caterpillars eight days post-oviposition (Experiment 5), and successful parasitism never occurred (Experiment 2). Similarly, Mp females introduce virus-like particles (MpVLPs) into host caterpillars along with a single egg, which then regulate the immune response of the host insects51,52,53. Since 83.3% of multiparasitized Myl caterpillars contained Ck larvae eight days after oviposition, it is possible that maternal factors of Mp protect not only Mp eggs and larvae, but also Ck eggs and larvae, allowing Ck larvae to survive in Myl caterpillars. However, identifying physiological mechanisms that allow Ck to survive in unsuitable host caterpillars in multiparasitism remains a future challenge.A series of studies by Magdaraog’s group56,57,58 clarified intrinsic competition between Ck and Mp in Mys caterpillars, which are ordinary hosts for both parasitoids. They demonstrated that multiparasitized Mys produce either Ck or Mp when the interval between the first and second oviposition is between 1 and 96 h, but the parasitoid species ovipositing first generally prevails over the other56. For example, when Ck oviposited into Mys 1 h before Mp, the successful parasitism rate of Ck was approximately 35%, which is significantly higher than that of Mp56. In contrast, in our study, the successful parasitism rate of Ck in multiparasitized Myl is significantly lower than that of Mp, regardless of oviposition order or host instar, with the maximum successful parasitism rate of Ck being only 20.0%, when Ck oviposited into 4th instar Myl caterpillars 12 h before Mp (Experiment 4).The outcome of intrinsic competition between Ck and Mp in Mys was also mutually exclusive56, as observed in Myl in this study, and is determined by three factors: resistance to the host immune response, direct conflict among larvae, and toxic effects of maternal anti-immunity factors (CkPDV, MpVLP or venom) on heterospecific parasitoid larvae58. Our results showed that multiparasitized-Myl have the ability to produce Ck when female Ck oviposited Myl caterpillars < 24 h before Mp, or < 12 h after Mp. In cases in which Ck oviposited more than 48 h before Mp, Ck eggs may have been killed by the Myl immune response before Mp oviposition. Conversely, when Ck oviposited more than 48 h after Mp, Ck could not complete larval development because Myl caterpillars die soon after Mp emergence67. This assumption is supported by observation of dead Ck larvae inside Myl caterpillars from which Mp had emerged67.Kraaijeveld31 hypothesized that oviposition into sympatric, unsuitable host species by parasitoids could increase survival probability when the ordinary host species is absent, acting as a kleptoparasitoid by exploiting the anti-host immunity of another parasitoid species via multiparasitism. However, successful parasitism of a parasitoid in unsuitable host species when multipatrasitised with a naturally sympatric parasitoid had not been demonstrated previously31,32,42,43,44. Our results present successful parasitism by a wasp in an unsuitable host through multiparasitism with a naturally sympatric parasitoid. Although the parasitism success rate by Ck in Myl caterpillars multiparasitized with Mp (< 20.0% ) was lower than that in the ordinary host Mys (> 70%24,68), oviposition by Ck females in unsuitable host Myl caterpillars is clearly possible. Therefore, as Kraaijeveld31 predicted, oviposition of parasitoid wasps in unsuitable hosts can be adaptive, especially when females cannot find their ordinary hosts. For example, in the case of Ck in Japan, the ordinary host, Mys, cannot overwinter when the average winter temperature falls below 4 °C, so the Mys population in spring is typically low. However, their numbers increase during summer and autumn due to mass migrations from China (Koyama & Matsumura69 and its references). Under these conditions, temporary use of “non-host” species like Myl, which occur stably throughout the year, may enhance the reproductive potential of Ck. Given that the parasitism rate of Mp on Myl in the wild can reach 42.9%47, it is likely that multiparasitism with Ck occurs. However, the emergence rate of Ck from Myl in cage experiments (Experiment 6) was extremely low (6%), and currently, there are no records of Ck emerging from Myl in the wild. The frequency of this phenomenon occurring in natural environments is likely extremely low, and further field investigations are required to document this interaction.Pirate parasitism: introducing a new termOver 100 years ago, Pemberton and Willard43 discovered that the larval parasitoid wasp Tetrastichus giffardianus (Hymenoptera: Eulophidae), introduced into Hawaii from West Africa70, could use a local, unsuitable host, the Hawaiian fruit fly, Bactrocera cucurbitae (= Dacus cucurbitae) (Diptera: Tephritidae), only when the fruit fly larva was previously parasitized by another parasitoid, Psyttalia fletcheri (= Opius fletcheri) (Hymenoptera: Braconidae), also introduced to Hawaii from India71. Based on this study, Askew72 called this phenomenon “obligatory multiparasitism”, without providing a definition. On the other hand, today, the term “obligatory multiparasitism” (or obligate multiparasitism) is also used to describe a phenomenon observed in species such as Pseudorhyssa (Hymenoptera: Ichneumonidae) or Eurytoma (Hymenoptera: Eurytomidae)73,74. These wasps parasitize insects hidden in hard materials, such as timber or thick cocoons, as their hosts. However, they cannot drill through these materials themselves to reach the hosts. Therefore, these parasitoids attack hosts previously oviposited by another parasitoid by following an oviposition hole in the material created by the previous parasitoid. This behavior is often termed “obligate multiparasitism”73,74. Consequently, the term “obligatory parasitism” currently connotes parasitism that cannot be completed without the help of oviposition by another parasitoid.To avoid confusion, we propose a redefinition of the term “obligatory parasitism” and introduce a new term, “pirate parasitism” (Fig. 5). We redefine “obligatory multiparasitism” as kleptoparasitic parasitism by a parasitoid that requires oviposition by another parasitoid species to attack the host and/or complete development in/on the host body post-oviposition. In the latter case, as a subclass of obligatory multiparasitism, we propose a new term, “pirate parasitism”, defined as parasitism by parasitoids that necessitates prior parasitism by another parasitoid species, resulting in complete development through multiparasitism. We also propose referring to parasitoids that require multiparasitism to utilize an “unsuitable host” as “pirate parasitoids.” We call those parasitoid wasps that facilitate pirate parasitoids as “mediators” (Fig. 5).Fig. 5Pirate parasitism: Introducing a new term. Obligatory multiparasitism refers to kleptoparasitic parasitism by a parasitoid that requires oviposition by another parasitoid species either to (a) reach the host, or (b) complete its development in the host after oviposition. In the latter case, termed “pirate multiparasitism”, parasitism is defined as occurring when a parasitoid necessitates prior or subsequent parasitism by another parasitoid species and can complete its development through multiparasitism.Full size imageIn the case of the host/parasitoid system in our study, C. kariyai is a specialist parasitoid on My. separata, but can utilize My. loreyi through “pirate parasitism” when multiparasitism with Me. pulchricornis occurs. In this case, C. kariyai is the “pirate parasitoid”, and Me. pulchricornis is the “mediator.” Meteorus pulchricornis, suffers a disadvantage due to pirate parasitism. However, there may also be cases of pirate parasitism in which the mediator suffers no disadvantage.The ecological and evolutionary significance of the response and oviposition in unsuitable host speciesThe concept of pirate parasitism indicates that attraction to, acceptance of, and oviposition by parasitoids on unsuitable host species increases reproductive potential. If parasitoid wasps were only attracted to their customary hosts, their reproductive success would be reduced to zero whenever their usual host was absent. However, with pirate parasitism, parasitoids can oviposit in unsuitable hosts, thereby maintaining some reproductive potential and increasing their fitness. Specialist parasitoid wasps, in particular, are more likely than generalist parasitoids to encounter environments in which their hosts are absent. Therefore, reproducing via pirate parasitism might significantly increase their reproductive potential. This suggests that the seemingly maladaptive behavior of parasitoid wasps ovipositing in non-hosts may actually represent an adaptive behavior. In fact, many species of parasitoids are attracted to and oviposit not only in sympatric unsuitable hosts, but also in introduced unsuitable hosts (Kruitwagen et al.,75 and references).The concept of pirate parasitism is also important from the perspective of evolution of parasitoid host ranges. Host shifts and host range expansions of parasitoid wasps have been thought to occur through a process in which wasps accidentally oviposit in unsuitable host that they had not previously used2,37,38,39. If development is successful in unsuitable hosts, newly emerged wasps learn the smell of that new host or its habitat, and subsequently show a preference for oviposition on it. The fact that our research results indicate that oviposition in unsuitable hosts can increase reproductive success implies that such oviposition behavior can serve as preadaptation in the context of host shifts and host range expansions in parasitoid wasps. This could increase the likelihood of the aforementioned “happy accident.”Multiparasitism is also generally considered maladaptive for parasitoids as it reduces the likelihood of successful parasitism due to direct or indirect competition between parasitoid species76. However, in the case of pirate parasitism, a pirate parasitoid increases its fitness through multiparasitism. This suggests that female pirate parasitoids can identify and preferentially oviposit in unsuitable hosts that have already been parasitized by mediators, but this possibility remains to be verified.Even though multiparasitism is thought to occur frequently in the field, it is difficult to detect by host collection and rearing because in most cases, only one parasitoid species emerges from the host76. Future studies need to investigate the presence, frequency, and generality of pirate parasitism in parasitoid-host systems by detecting multiparasitism using molecular tools with species-specific primers, as recently used for detection of hyperparasitism77,78 and laboratory multiparasitising experiments.Materials and methodsInsectsMythimna separata and C. kariyai were obtained from stock cultures in the Laboratory of Applied Entomology and Zoology, University of Tsukuba, Japan. Caterpillars of Mythimna loreyi were collected from maize and sorghum fields in Kimotsuki-cho, Kanoya City and Kagoshima City, Kagoshima Prefecture, Kyushu, Japan, between July 28 and 30, 2020. The original colony of Meteorus pulchricornis was obtained from M. separata caterpillars collected in the field, as described above. Both Mys and Myl colonies were reared on an artificial diet (Silkmate® 2 S, Nosan Corporation, Yokohama, Japan) in the laboratory (25 ± 1 °C, 16 L: 8D photoperiod and 60 ± 10% RH) following methods for Mys described in Fukushima et al.79 and Magdaraog et al.56 under the same conditions as the herbivores. Populations of both parasitoid species have been maintained using 3rd instar Mys caterpillars as hosts under laboratory conditions. For Ck, three to five-day-old mated, naïve females were used for all experiments. Because the Mp strain we used was an apomictic thelytokous strain (cf. Fujie et al.,80; Wachi et al.,81), unmated, 7-10-day-old females were used for all experiments.Experiment 1: host acceptance and ovipositionFemale parasitoids with no oviposition experience were used for the experiment. Each female parasitoid was placed individually in a Petri dish (5.3 cm diameter, 1.5 cm height) containing wet cotton 30–60 min prior to the experiment. A single caterpillar was then introduced into the Petri dish, and parasitoid’s behavior was observed for 10 min. Whether the parasitoid attacked the caterpillar was recorded. Throughout experiments in this study, oviposition was confirmed by observing a single insertion and withdrawal of the ovipositor from the caterpillar. For Ck, 3rd, 4th or 5th instar unparasitized Myl caterpillars or Myl caterpillars previously oviposited by Mp were offered. Similarly, 3rd, 4th or 5th instar unparasitized Myl caterpillars or Myl caterpillars previously oviposited by Ck were offered to Mp. Parasitized Myl caterpillars used for these experiments were prepared by single oviposition by parasitoids ca. 1 h before experiments. As a positive control for Ck oviposition, 4th instar, unparasitized Mys caterpillars (ordinary host for Ck) were also offered.To confirm the presence of parasitoid egg(s) in the caterpillar body, caterpillars were dissected within 30 min after oviposition using the method of Aikawa et al.34. Numbers of parasitoid eggs in the caterpillar were recorded. Dissected caterpillars were observed under a binocular stereo microscope (SMZ 1270, Nikon, Tokyo, Japan).Experiment 2: single-parasitism experimentsHost suitability of Myl caterpillars for the two parasitoids when each parasitoid oviposited alone was determined. Parasitoids with no prior oviposition experience were allowed to oviposit into 3rd, 4th or 5th instar Myl caterpillar in a Petri dish. The oviposited caterpillar was then transferred into a plastic container (100 mm in diameter, 40 cm in height, Sansho, Tokyo, Japan) and reared individually with artificial diet until the parasitoid emerged, the caterpillar pupated, or the caterpillar died.Experiment 3: multiparasitism experiments in different host instarsTo examine successful parasitism of Ck in Myl caterpillars parasitized with Mp, the outcome of multiparasitism with both parasitoids in 3rd, 4th or 5th instar Myl caterpillars was observed. Multiparasitized Myl caterpillars, with oviposition by Ck and Mp in either order in < 15 min, were prepared using methods described for Experiment (1). Parasitized caterpillars were reared individually, and outcomes were recorded as described for Experiment (2). When Ck emerged from Myl caterpillars, to verify their reproductive ability, five newly emerged female adults from each treatment were allowed to oviposit into their usual host, Mys, after mating. Parasitized Mys caterpillars were reared individually as described above, and outcomes were recorded using the methods described in the single-parasitism experiment section.For positive control for Ck parasitism, 4th instar Mys was also offered to Ck and oviposited Mys caterpillars were reared individually as same as Myl caterpillars. Then, the number of parasitoids per caterpillar was counted.Experiment 4: multiparasitism in different sequences and intervalsTo examine effects of the order and timing of oviposition by the two parasitoid species on outcomes of multiparasitism in Myl caterpillars, 4th instar caterpillars parasitized by the two parasitoid species in different sequences and intervals were reared. Both parasitoids were allowed to oviposit into caterpillars in either order of oviposition, at intervals of < 10 min, 12 h, 24 h, and 48 h. Parasitized caterpillars were reared individually, and outcomes were recorded using methods described above.Experiment 5: observation of parasitoid larvae in multiparasitized caterpillarsBased on results of multiparasitism experiments, multiparasitized Myl caterpillars produced not only Mp, but also Ck. To ascertain whether hyperparasitism occurred involving the two parasitoid species in the same Myl caterpillars, multiparasitized Myl caterpillars were dissected and parasitoid larvae in the caterpillars were observed. Multiparasitized 4th instar Myl caterpillars, which were oviposited by Ck followed by Mp within < 10 min, were prepared using methods described previously. Parasitized caterpillars were reared on artificial diet. Caterpillars were dissected in 70% ethanol-water solution before parasitoid emergence (on day 8 after oviposition) or within 12 h after parasitoid emergence to observe the presence or absence of parasitoid larvae, and whether hyperparasitism occurred between the two parasitoid species. Additionally, Myl caterpillars oviposited by Ck alone were also dissected on day 8 after oviposition to determine whether Ck larvae survived in Myl. All observations were conducted under a binocular stereo microscope (SMZ 1270, Nikon, Tokyo, Japan).Experiment 6: simultaneous free-oviposition rearing in cagesTo examine whether Ck could emerge from Myl in an environment in which it could freely oviposit, we created a rearing cage in which Ck, Mp, and Myl coexisted and evaluated the emergence rate of Ck from Myl. This experiment was conducted in transparent plastic containers (17 × 19 cm, 29 cm high). Maize (“Honey-Bantam Peter 610”, Sakata Seed Co., Japan) was utilized as the host plant for Myl caterpillars. Maize plants were cultivated from seed in plastic flowerpots (14.0 cm diameter, 11.5 cm height) in a greenhouse (25 ± 1 °C, 16 L:8D photoperiod)82. Stems of 3- to 4-week-old maize plants containing 3–4 leaves, approximately 25 cm high, were cut and placed in a 50-mL beaker filled with water. Maize leaves were placed at the center of the container, and ten 4th instar Myl caterpillars were placed on the leaves. Three females each of both Ck and Mp were released into the container. Honey and wet cotton were provided in the container for the wasps. These containers were maintained in a rearing room (25 ± 1 °C, 16 L: 8D photoperiod and 60 ± 10% RH) for 48 h. All caterpillars were then collected from the container and reared individually, and outcomes were recorded using the same methods described in the singleparasitism experiment section.Statistical analysisFor experiment 1, differences in host acceptance rates of parasitoids among treatments were evaluated with Chi-square tests, and differences in numbers of eggs oviposited in caterpillars among caterpillar conditions were evaluated using analysis of variance (ANOVA). For experiment 2, the successful parasitism rate among host instars was evaluated with Chi-square tests. In experiment 3, the significance of the emergence percentage of the two wasps in each host instar and order of oviposition was analyzed using binominal tests. The successful parasitism rate of Ck among host instars and the order of oviposition were evaluated with Chi-square tests. Mean numbers of Ck adults that emerged from caterpillars were evaluated using Tukey-Kramer honestly significant difference (HSD) tests after ANOVA. In experiment 4, the successful parasitism rate of Ck by order and interval of oviposition was evaluated with Tukey’s wholly significant difference (WSD) tests after Chi-square tests, and mean numbers of Ck adults emerging from caterpillars were evaluated using Tukey-Kramer HSD tests after ANOVA. All analyses were performed in R v. 4.0.3 software83 and Tukey-Kramer HSD tests and Tukey WSD tests were performed using an open-source package (http://aoki2.si.gunma-u.ac.jp/R/m_multi_ comp.html). Since experiments 5 and 6 were qualitative experiments, statistical analyses were not conducted.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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R Core Team. R: A Language and environment for statistical computing. R Foundation Stat. Comput. Vienna Austria. https://www.R-project.org/ (2023).Download referencesAcknowledgementsWe are grateful to Dr. Katsuo Tsuda, Dr. Yositaka Sakamaki, and members of the Laboratory of Entomology, Kagoshima University, Japan, for their cooperation in insect collecting. This study was supported in part by a JSPS KAKENHI, Grant-in- Aid for JSPS Fellows (20J00497) and Grant-in-Aid for Early-Career Scientists (22K14897) to KK. We thank Dr. Steven D. Aird for editing the manuscript.Author informationAuthors and AffiliationsFaculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki, 305-8572, JapanKazumu Kuramitsu & Yooichi KainohInstitute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), 1‑2 Owashi, Tsukuba, Ibaraki, 305‑8634, JapanKazumu Kuramitsu & Kotaro KonnoJapan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo, 102-0083, JapanKazumu KuramitsuAuthorsKazumu KuramitsuView author publicationsYou can also search for this author in
PubMed Google ScholarYooichi KainohView author publicationsYou can also search for this author in
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PubMed Google ScholarContributionsK. Ku., Y. K. and K. Ko. conceived research. K. Ku. performed experiments and wrote the first draft. Y. K and K. Ko reviewed the paper and checked all the details. All authors read and approved the final manuscript.Corresponding authorCorrespondence to
Kazumu Kuramitsu.Ethics declarations
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Reprints and permissionsAbout this articleCite this articleKuramitsu, K., Kainoh, Y. & Konno, K. Multiparasitism enables a specialist endoparasitoid to complete parasitism in an unsuitable host caterpillar.
Sci Rep 15, 8361 (2025). https://doi.org/10.1038/s41598-025-91403-3Download citationReceived: 28 November 2024Accepted: 20 February 2025Published: 11 March 2025DOI: https://doi.org/10.1038/s41598-025-91403-3Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard
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KeywordsKleptoparasitismPirate parasitism
Cotesia kariyai
Meteorus pulchricornis
Multiparasitism
Mythimna loreyi