Introduction
The natural population of insect herbivores can be controlled using various natural enemies. Typically, natural enemies share hosts with minimal competition because of temporal or spatial separation. However, field surveys have revealed that more than one natural enemy species can exist simultaneously, and that these coexisting natural enemies might compete for limited resources (Harvey et al., Reference Harvey, Poelman and Tanaka2013). Predators are predominantly generalists and can switch to alternative prey to avoid competition. Parasitoids tend to be specialists with a narrow host range. Competition can occur when more than one parasitoid species occupies the same host population, particularly when two solitary endoparasitoid species occupy similar niches simultaneously. The co-occurring endoparasitoid species might oviposit successively on the same host but, typically, only one endoparasitoid offspring can survive and become cocooned (De Moraes et al., Reference De Moraes, Cortesero, Stapel and Lewis1999; Tamò et al., Reference Tamò, Roelfstra, Guillaume and Turlings2006; Cingolani et al., Reference Cingolani, Greco and Liljesthrom2013). Therefore, past studies have suggested some substantial competition that might occur between endoparasitoid species.
In endoparasitoid species, two types of competition can occur: intrinsic and extrinsic competition. Intrinsic competition occurs inside the host body at the parasitoid larval stage. By contrast, extrinsic competition occurs between adult parasitoids outside the host body (Mills, Reference Mills, Brodeur and Boivin2006; Harvey et al., Reference Harvey, Poelman and Tanaka2013). Competitive interaction can affect the population size, development time, survival rate, and individual size of the competing parasitoid species (Price et al., Reference Price, Westoby and Rice1988; Reitz, Reference Reitz1996; Bogran et al., Reference Bogran, Heinz and Ciomperlik2002; Tian et al., Reference Tian, Zhang, Yan and Wang2008). Moreover, competition among the natural enemies might even decrease the control efficiency of one specific species or the sum efficiency. For example, when Campoletis chlorideae (Uchida) and Eriborus argenteopilosus (Cameron) (Hymenoptera: Ichneumonidae) are released concurrently, the parasitism rate of E. argenteopilosus is lower than it is when they are released individually (Bajpai et al., Reference Bajpai, Ballal, Rao, Singh and Bhaskaran2006). Tamò et al. (Reference Tamò, Roelfstra, Guillaume and Turlings2006) similarly reported that Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae) and Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) exhibit higher parasitism rates when released separately than when released concurrently.
Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is a polyphagous insect pest of several crops. A study recorded 389 plant species belonging to 109 families that are host plants of S. litura (Qin et al., Reference Qin, Wang, Ding, Huang and Ye2006). Although this insect has caused substantial economic damage in numerous countries, it reportedly has several natural enemy species. Ranga Rao et al. (Reference Ranga Rao, Wightman and Ranga Rao1993) indicated that S. litura may contain as many as 71 species of parasitic enemy species worldwide. Field surveys have identified 14 parasitoid species of S. litura in Taiwan (Chiu & Chou, Reference Chiu and Chou1976; Chen et al., Reference Chen, Wang and Chen2009). Because parasitoid species are highly diverse, they can become valuable resources for the biological control of S. litura.
Snellenius manilae (Ashmead) (Hymenoptera: Braconidae) is the most common and widely distributed solitary endoparasitoid of S. litura larvae in Taiwan (Chiu & Chou, Reference Chiu and Chou1976). All recorded hosts of S. manilae belong to the Spodoptera genus (Rajapakse et al., Reference Rajapakse, Ashley and Waddill1985; Lu, Reference Lu1989; Ando et al., Reference Ando, Inoue, Maeto and Tojo2006; Sun & Huang, Reference Sun and Huang2010; Qiu et al., Reference Qiu, Zhou, Luo and Xu2012), in which the S. manilae parasitoid is the dominant species. In addition, Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae) is a solitary S. litura endoparasitoid species first reported to parasitize S. litura in Taiwan in 1976 (Chiu & Chou, Reference Chiu and Chou1976). One study identified two strains of M. pulchricornis: an arrhenotokous (biparental) group from Europe and a thelytokous (uniparental) group from Asia (Fuester et al., Reference Fuester, Taylor, Peng and Swan1993). The host range of M. pulchricornis consists of 11 families of Lepidoptera (Huddleston, Reference Huddleston1980; Okada, Reference Okada1989; Maetô, Reference Maetô1990; Berry, Reference Berry1997).
S. manilae and M. pulchricornis are both solitary endoparasitoids of S. litura. During a field survey, both species can be identified in the same location at the same time. Studies have indicated that S. manilae and M. pulchricornis prefer to parasitize second- or third-instar S. litura larvae (Qiu & Tang, Reference Qiu and Tang2010; Chen et al., Reference Chen, Li and Meng2011). Therefore, because of niche overlap, a competitive relationship may exist between the two species. However, according to our research, no study has reported a competitive interaction between these two parasitoid species. Therefore, the objective of this study was to evaluate the competition between S. manilae and M. pulchricornis when S. litura was used as the host.
Materials and methods
Plant
Brassica oleracea was used as the host plant in all experiments. Seeds were purchased from Know-You Seed Co., Ltd, and commercial soil (Stender, Germany) was used to cultivate these plants. To sterilize the seed surface and accelerate germination, the seeds were placed in a water bath at 45°C for 30 min before they were sown (Yadav et al., Reference Yadav, Tan and Hwang2010). After warm-water treatment, the seeds were sown into a 30 × 20 × 5 cm3 plastic plate. After 1 week, the plantlets were separately transplanted to 4-inch pots. All plants were cultivated in a greenhouse at 25 ± 2°C in a light-dark (LD) 16:8 h cycle.
Host
The initial population of S. litura was collected from a field in Caohu, Dali District, Taichung City, Taiwan. The population was maintained in incubators at 25 ± 2°C, in an LD 12:12 h cycle at 70 ± 5% relative humidity (RH). The S. litura larvae were provided an artificial diet adopted from Kao (Reference Kao1995), reared in plastic cups (8 cm diameter × 6 cm height) until the sixth instar, and then transferred to separate wells in 30-well plates until pupation. Pupae were collected and placed into plastic cups and were then sterilized using a 25% bleaching liquid (Clorox). Adult S. litura were placed in a plastic cylinder (14.9 cm diameter × 21 cm height), and tissue paper was used to cover the inside to facilitate the collection of eggs. The plastic cylinders were maintained in laboratory conditions and the adult moths were provided a sugar solution (Kao, Reference Kao1995; Yadav et al., Reference Yadav, Tan and Hwang2010). The egg masses were collected, placed into plastic cups, and sterilized using 1.5% sodium hypochlorite (Riedel-de Haën). According to previous studies (Ando et al., Reference Ando, Inoue, Maeto and Tojo2006; Qiu & Tang, Reference Qiu and Tang2010; Chen et al., Reference Chen, Li and Meng2011), S. manilae and M. pulchricornis prefer to parasitize early instars of S. litura; therefore, second-instar larvae were used in the bioassays.
Parasitoids
S. manilae and M. pulchricornis were obtained from parasitized S. litura larvae collected from the field. Both parasitoids were reared in incubators at 25 ± 2°C in an LD 12:12 h cycle at 70 ± 5% RH. Second-instar S. litura larvae were used as the host for the parasitoids. Parasitized S. litura were provided an artificial diet and kept in plastic cups. After parasitoid larvae emerged from the hosts and became cocooned, the cocoons were collected and placed in petri dishes (5.5 cm diameter × 1.5 cm height; Alpha Plus Scientific Corp). Adults were transferred to a plastic cup and provided a 15% (w/w) sugar solution . According to the previous studies about the fecundity peak of the two parasitoids (Hwang et al., Reference Hwang, Byeon, Lee, Kim, Choi, Kim, Kim, Park, Lee, Lee and Lee2010; Ting, Reference Ting2011), S. manilae aged 3–5 days and M. pulchricornis aged 13–15 days were used in bioassays.
Intrinsic competition
Various sequences and time intervals were used to evaluate the intrinsic competition between S. manilae and M. pulchricornis inside S. litura body. Second-instar S. litura larvae were fed on 29-day-old B. oleracea for 24 h and then prepared for the test. The larvae were first parasitized by one parasitoid species and then, after the specific time intervals, were parasitized by the other parasitoid species. The specific time intervals were 0, 6, 12, 18, 24, and 48 h. Overall, 12 treatments were tested. The S. litura larvae were verified to have been successfully parasitized when they were stung by the parasitoid ovipositor. Each replication of treatments comprised 30 trials, and each treatment was replicated five times. Parasitized S. litura larvae were maintained in individual petri dishes and provided an artificial diet. When the parasitoids emerged to cocoon, the species of parasitoids were recorded.
Cage experiments
In the cage experiments, different numbers of the two parasitoid species were evaluated. Second-instar S. litura larvae (n = 120) were transferred into a 30 × 30 × 30 cm3 cage (MegaView Science Co, Ltd) and fed 29-day-old B. oleracea for 24 h. Different numbers of two different parasitoid species were then released into the test cage. In each treatment, four or eight parasitoids, either S. manilae or M. pulchricornis, or a combination of both species, were used. After 24 h of exposure, the S. litura larvae were transferred into individual petri dishes and provided an artificial diet until the parasitoids emerged. For each treatment, the parasitoid species of cocoons and the parasitism rate were recorded and five replications were conducted.
Data analysis
The cocoons collected from each treatment of intrinsic competition were separated by parasitoid species and the cocoon ratios of S. manilae and M. pulchricornis were calculated. In cage experiments, the parasitism rates were estimated based on the number of cocoons and the number of unparasitized hosts. Furthermore, the cocoons collected from the treatments involving both parasitoids were separated by parasitoid species and calculated to ratio values. All data collected from each experiment were analyzed by SAS version 9.00 and were subjected to χ2 test. In the cage experiments, pairwise comparisons with Bonferroni corrections between treatments were conducted in the case of significance.
Results
Intrinsic competition
Our results indicated that, when M. pulchricornis was the first parasitoid to parasitize, significantly greater M. pulchricornis than S. manilae cocoons were collected regardless of the ovipositional intervals between the two parasitoids (fig. 1). M. pulchricornis cocoon ratios were consistently higher than 75% in all treatments. However, when S. manilae was the initial parasitoid, the competitive outcomes depended on the interval of time between two parasitization (fig. 2). The cocoon ratios of S. manilae increased as the time between two parasitization increased, whereas the cocoon ratios of M. pulchricornis decreased as the time between two parasitization increased. In the treatments involving 0 and 6 h time intervals, the cocoon ratios of M. pulchricornis were 71 and 69%, respectively. The cocoon numbers of M. pulchricornis were significantly higher than those of S. manilae (χ2 = 19.59, P < 0.0001; χ2 = 16.04, P < 0.0001). When the time intervals between the first and second parasitization were 12 and 18 h, no significant difference between the two parasitoids was observed (χ2 = 0.43, P = 0.5102; χ2 = 2.06, P = 0.1508). When the time intervals were longer than 24 h, S. manilae cocoons were significantly more than M. pulchricornis cocoons. The cocoon ratios of S. manilae were 66 and 84% for 24 and 48 h intervals, respectively.
Cocoon ratios (Mean + SE) of M. pulchricornis (Mp) and S. manilae (Sm) when M. pulchricornis was the first to oviposit in host larvae of S. litura, followed by S. manilae at different time intervals. The numbers between Mp and Sm are the time intervals (h) between the two parasitizations. The asterisks indicate a significant difference between the two parasitoids within each time interval (χ2: *** P < 0.0001).
Cocoon ratios (Mean + SE) of M. pulchricornis (Mp) and S. manilae (Sm) when S. manilae was the first to oviposit in host larvae of S. litura, followed by M. pulchricornis at different time intervals. The numbers between Mp and Sm are the time intervals (h) between the two parasitizations. The asterisks indicate a significant difference between the two parasitoids within each time interval (χ2: **P < 0.01 and ***P < 0.0001).
Cage experiments
The results of cage experiments indicated that parasitism rates increased with parasitoids density. The parasitism rates were significantly higher in the treatments of eight parasitoids than that in the treatments of four parasitoids whether M. pulchricornis only, S. manilae only, or both species were released together (χ2 = 25.55, P < 0.0001; χ2 = 35.33, P < 0.0001; χ2 = 76.51, P < 0.0001). The parasitism rates by M. pulchricornis only were not significantly different from that by S. manilae only in both densities (fig. 3). When both parasitoid species were released together, the total parasitism rates reduced at each parasitoid density. Following the release of four parasitoids, the parasitism rates by only one parasitoid species were significantly higher than that by two species released together (χ2 = 50.97, P < 0.0001, fig. 3a). Similarly, following the release of eight parasitoids, the parasitism rate by M. pulchricornis only was significantly higher than that by two species released together (χ2 = 8.81, P = 0.0122, fig. 3b). When both parasitoid species were released together, the cocoon numbers of M. pulchricornis were significantly higher than that of S. manilae (fig. 4). In the treatment involving two M. pulchricornis and two S. manilae parasitoids, M. pulchricornis cocoon ratio accounted for 57% of all cocoons (χ2 = 5.65, P = 0.0174, fig. 4a). In the treatment in which four M. pulchricornis and four S. manilae parasitoids were released, M. pulchricornis cocoon ratio accounted for 75% of all cocoons (χ2 = 66.50, P < 0.0001, fig. 4b). More M. pulchricornis cocoons emerged than those of the S. manilae indicated the superiority of M. pulchricornis. Furthermore, the difference of the cocoon numbers between M. pulchricornis and S. manilae increased with parasitoids density.
Parasitism rates (Mean + SE) obtained from different treatments when the total number of female parasitoids was (a) four or (b) eight in cage experiments. Mp: M. pulchricornis; Sm: S. manilae. The same letter above bars indicates no significant difference among treatments (χ2 with Bonferrini correction, P < 0.05).
Cocoon ratios (Mean + SE) of M. pulchricornis (Mp) and S. manilae (Sm) in (a) the treatment released two M. pulchricornis and two S. manilae and (b) the treatment released four M. pulchricornis and four S. manilae. Different letters above bars indicate significant differences between the ratios of M. pulchricornis and S. manilae (χ2: P < 0.05).
Discussion
Our results indicated that competition occurred between S. manilae and M. pulchricornis when these two wasps parasitized S. litura simultaneously. In the majority of the tested conditions, M. pulchricornis was the dominant parasitoid. However, these results contrast the findings of previous field surveys in which S. manilae was the dominant of the two species (Chiu & Chou, Reference Chiu and Chou1976).
Previous studies have indicated that the duration of the egg stage may be the principal factor determining the success of a parasitoid in intrinsic competition because the first-hatching species can use the host's resources immediately and attack the eggs of the other parasitoids (Tillman & Powell, Reference Tillman and Powell1992; Bajpai et al., Reference Bajpai, Ballal, Rao, Singh and Bhaskaran2006; Tian et al., Reference Tian, Zhang, Yan and Wang2008). Qiu et al. (Reference Qiu, Zhou, Luo and Xu2012) reported that the eggs of S. manilae hatched in approximately 2.7 days (26°C, LD 14:10 h) when second-instar Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) was the host. When M. pulchricornis was observed to parasitize Mythimna separata (Walker) (Lepidoptera: Noctuidae), the egg period was approximately 3.8 days (25 ± 1°C, LD 16:8 h) (Magdaraog et al., Reference Magdaraog, Harvey, Tanaka and Gols2012). The observation of our study similarly indicated that M. pulchricornis egg-larva stage required a longer developmental period than S. manilae. According to the hatching time, S. manilae should be the prevailing parasitoid when S. manilae and M. pulchricornis exist inside a host body concurrently. However, our results indicated that, when S. manilae and M. pulchricornis parasitized S. litura larvae simultaneously, M. pulchricornis was the dominant species unless S. manilae parasitized S. litura at least 12 h prior to M. pulchricornis. Therefore, traits other than the hatching sequence may play major roles in determining the fates of the competing parasitoids.
To protect the eggs from encapsulation of the host immune system, parasitoid females will inject either the venom, calyx fluid, polydnavirus (PDV), non-PDV symbiotic viruses, or virus-like particles into the host body (Pennacchio & Strand, Reference Pennacchio and Strand2006). Adult M. pulchricornis has been reported to release virus-like particles into the host body during oviposition. These chemical particles can protect the parasitoid egg from host encapsulation by inhibiting the spread of granulocytes and plasmatocytes as well as the induction of apoptosis (Suzuki & Tanaka, Reference Suzuki and Tanaka2006; Suzuki et al., Reference Suzuki, Miura and Tanaka2008, Reference Suzuki, Miura and Tanaka2009). In addition, S. manilae was reported having polydnsvirus symbiont which could prevent the encapsulation from the host (Sri Ratna et al., Reference Sri Ratna, Buchori and Santoso2011). The abilities of these two parasitoids to avoid encapsulation can affect not only the success of parasitization but also the results of interspecific competition. Furthermore, the virulence released by parasitoid females may also have the effect on the parasitic competitors. Besides chemical effects, Magdaraog et al. (Reference Magdaraog, Harvey, Tanaka and Gols2012) and Chau & Maeto (Reference Chau and Maeto2008) indicated that M. pulchricornis larvae possess large mandibles and retain a caudal appendage. M. pulchricornis larvae might use the mandibles to attack competitors and the caudal appendage to move in the host body. Suzuki & Tanaka (Reference Suzuki and Tanaka2007) indicated that the M. pulchricornis embryo is enclosed in a serosal cell membrane that might protect the parasitoid from defensive reactions of the host. These physical characteristics might also contribute to the superiority of M. pulchricornis in intrinsic competition.
The cage experiments results indicated that treatment combining S. manilae and M. pulchricornis is associated with the lowest parasitism rate. This observation indicated that direct competition or interference occurs when the two parasitoid species exist in the same place at the same time. Similarly, Tamò et al. (Reference Tamò, Roelfstra, Guillaume and Turlings2006) identified lower parasitoid cocoon numbers following the concurrent release of C. marginiventris and C. sonorensis in comparison with the separate release of the species.
Following the simultaneous release of M. pulchricornis and S. manilae in the bioassay, we collected a significantly higher number of M. pulchricornis cocoons than S. manilae cocoons from the parasitized S. litura larvae. This result was expected because M. pulchricornis dominated the intrinsic competition and, thus, cocoons emerging from the host body were more likely to be M. pulchricornis than S. manilae. Previous studies similarly observed that C. chlorideae and C. sonorensis were the dominant parasitoids in intrinsic competition and contributed higher numbers of cocoons when they were released with other parasitoids (Bajpai et al., Reference Bajpai, Ballal, Rao, Singh and Bhaskaran2006; Tamò et al., Reference Tamò, Roelfstra, Guillaume and Turlings2006). Our study results indicated that the parasitism rate increased as the number of parasitoids released increased, and might increase by up to 100% following the release of more than eight parasitoids.
According to our results, M. pulchricornis is substantially more competitive than S. manilae in intrinsic competition and cage experiments. However, previous studies have indicated that S. manilae is the prevailing parasitoid of S. litura in Taiwan (Chiu & Chou, Reference Chiu and Chou1976). The reason for the discrepancies in results is unclear. M. pulchricornis may have several properties that facilitate the control of S. litura, such as thelytoky, ease of mass propagation, and the longer adult longevity (Ando et al., Reference Ando, Inoue, Maeto and Tojo2006; Hwang et al., Reference Hwang, Byeon, Lee, Kim, Choi, Kim, Kim, Park, Lee, Lee and Lee2010). Therefore, additional studies are required to fully elucidate the potential of using M. pulchricornis as a biological agent for controlling S. litura.
Acknowledgements
We would like to thank Dr Chi Feng Lee of Taiwan Agricultural Research Institute, Taiwan for helping to identify the parasitoids. We also thank members in Insect-Plant Interaction Lab, National Chung Hsing University, Taiwan for their assistances.
