Introduction
Understanding of the dynamics of competition among species of potential biological control agents, such as parasitoids, that share the same host species and habitat is important for evaluating their efficiency (Mackauer, Reference Mackauer, Mackauer, Ehler and Roland1990; Bogran et al., Reference Bogran, Heinz and Ciomperlik2002; Gurr et al., Reference Gurr, Wratten and Altieri2004; De Moraes & Mescher, Reference De Moraes and Mescher2005; Harvey et al., Reference Harvey, Poelman and Tanaka2013; Orre-Gordon et al., Reference Orre-Gordon, Jacometti, Tompkins, Wratten, Wratten, Sandhu, Cullen and Costanza2013). If individuals of two or more species of parasitoids attack the same single host at the same time, multiple parasitism may occur. Therefore, interspecific competition is expected to occur among these parasitoids (Godfray, Reference Godfray1994; Kato, Reference Kato1996; Paull & Austin, Reference Paull and Austin2006). Little is known about the extent and nature of such competition and its influence at the community level for parasitoids (Force, Reference Force1985; Godfray, Reference Godfray1994; Harvey et al., Reference Harvey, Poelman and Tanaka2013). While competing parasitoid species can co-exist in the same environment (Hawkins, Reference Hawkins, Hochberg and Ives2000; Van Nouhuys & Hanski, Reference Van Nouhuys, Hanski, Holyoak, Leibold and Holt2005; Aluja et al., Reference Aluja, Ovruski, Sivinski, Córdova-García, Schliserman, Nuñez-Campero and Ordano2013), certain circumstances, such as a lack of alternative host species, can lead to a situation where one parasitoid species dominates the parasitism of a host species (Pijls & Van Alphen, Reference Pijls and Van Alphen1996). In some cases, an increase in the number of parasitoid species may result in a decline in the efficiency of biological control (Collier & Hunter, Reference Collier and Hunter2001; Collier et al., Reference Collier, Kelly and Hunter2002). Therefore, it is important to understand how competing parasitoid species interact with each other and which species is superior in particular circumstances. Some studies have investigated competition between parasitoids with differing degrees of host specificity, and in most cases the generalists have a greater likelihood of outcompeting the more specialized species (Iwao & Ohsaki, Reference Iwao and Ohsaki1996; De Moraes & Mescher, Reference De Moraes and Mescher2005). This study investigates competitive interactions that occur in Australian vineyards between two generalist larval parasitoids of a lepidopteran herbivore, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), the light brown apple moth (LBAM).
Interspecific competition between parasitoids can be classified as extrinsic competition, which includes all direct and indirect interactions between adult parasitoids while foraging for hosts, and intrinsic competition, which refers to indirect or direct competition among parasitoid larvae within an individual host (Harvey et al., Reference Harvey, Poelman and Tanaka2013). Extrinsic competition usually occurs between adult conspecifics, and is not common inter-specifically (Boivin & Brodeur, Reference Boivin, Brodeur, Boivin and Brodeur2006). It can involve aggressive behaviour between foraging females or the ability to discriminate against hosts already parasitized by other parasitoids. During intrinsic competition, larvae that share the same host could compete directly or indirectly through physical encounters or indirectly through physiological suppression of one species by another. In solitary parasitoids, intrinsic competition is severe, as it can result in the successful development of a single individual at the expense of others. Such competition has led to the evolution of entomological weaponry. For example, first instars of some parasitoids have large, sickle-like mandibles that are used to fight other larvae or destroy the eggs of competitors (Tian et al., Reference Tian, Zhang, Yan and Wang2008; Wang et al., Reference Wang, Bokonon-Ganta and Messing2008; Harvey et al., Reference Harvey, Gols and Strand2009; Paladino et al., Reference Paladino, Papeschi and Cladera2010). In other cases, physiological suppression occurs when the parasitoid manipulates the physical and chemical environment of the host to create conditions that favour their own survival to the detriment of other parasitoids. This may involve venom, polydnavirus, teratocytes or protein secretions that affect host development (De Moraes et al., Reference De Moraes, Cortesero, Stapel and Lewis1999; Harvey et al., Reference Harvey, Poelman and Tanaka2013).
The present study investigated extrinsic and intrinsic competition between two parasitoids, Dolichogenidea tasmanica (Cameron) (Hymenoptera: Braconidae) and Therophilus unimaculatus (Turner) (Hymenoptera: Braconidae), which attack the LBAM in Australia. D. tasmanica is reported to parasitize Merophyas divulsana (Walker) (Lepidoptera: Tortricidae) (Bishop & McKenzie, Reference Bishop and McKenzie1991) and E. postvittana (Paull & Austin, Reference Paull and Austin2006). T. unimaculatus is known to parasitize hosts in at least three families and six genera of Lepidoptera. These include Myrascia bracteatella (Walker) (Lepidoptera: Oecophoridae), Etiella behrii (Zeller) (Lepidoptera: Pyralidae) and M. divulsana, E. postvittana, Phricanthes asperana (Meyrick) and Acropolitis magnana (Walker) (Lepidoptera: Tortricidae) (Stevens et al., Reference Stevens, Austin and Jennings2011). In addition, we have reared both species from the same species in four genera of tortricids, which were identified using DNA barcodes (unpublished data). Therefore, we consider these species to be generalists in their host associations. The overlap in the host range of these parasitoids indicates that they are competitors, which is likely to influence their abundance in vineyards and in natural habitats. To study extrinsic competition between the two above parasitoid species, a wind tunnel was used to evaluate whether hosts already parasitized by the other species are equally attractive compared to un-parasitized larvae. Intrinsic competition was then studied by investigating the outcome of larval interactions between the two parasitoid species. We conclude by discussing how this information can help to understand co-existence of the two parasitoid species and its implications for their role in the biological control of E. postvittana.
Materials and methods
Study organisms
The LBAM is a native Australian leafroller that attacks a wide range of host plants. It is the most destructive insect pest of wine grapes in Australia (Scholefield & Morison, Reference Scholefield and Morison2010). This species has been introduced to and become a pest in New Zealand, the United Kingdom, Hawaii and California (Suckling & Brockerhoff, Reference Suckling and Brockerhoff2010). There are at least 25 parasitoid species associated with E. postvittana in Australia (Paull & Austin, Reference Paull and Austin2006). Some of these share a range of other host species (Yi Feng, un-published data). Among these, D. tasmanica is considered as the predominant species in many habitats (Charles et al., Reference Charles, Walker and White1996; Suckling et al., Reference Suckling, Burnip, Walker, Shaw, McLaren, Howard, Lo, White and Fraser1998; Paull, Reference Paull2007; Suckling & Brockerhoff, Reference Suckling and Brockerhoff2010), while T. unimaculatus is found in natural habitats in which these two species coexist (T. unimaculatus is described as Bassus sp. in Paull & Austin, Reference Paull and Austin2006; Paull, Reference Paull2007). In a survey conducted in eight vineyards in the Adelaide Hills in South Australia, D. tasmanica was most common in vineyards, while T. unimaculatus was most abundant in adjacent native vegetation (Yi Feng, unpublished data).
Insect rearing
E. postvittana was reared on an artificial diet (Yazdani et al., Reference Yazdani, Feng, Glatz and Keller2014) at 22 ± 2 °C, 12 L:12 D. It has been kept in culture in an insect rearing room for nearly 200 generations, with the annual addition of field-collected individuals to maintain genetic diversity. The colonies of D. tasmanica and T. unimaculatus were originally established from parasitized leafrollers that were collected in vineyards near Adelaide (35°16′S, 138°37′E). Parasitoids were reared on larval E. postvittana that fed on narrow leaf plantain, Plantago lanceolata L. in cages at 23 ± 2 °C, 14 L:10 D. E. postvittana was collected at least once every 2 months from the field (35°58′S, 138°38′E), and adult parasitoids hatching from these larvae were added to the cultures, as was done for the leafroller. To obtain parasitoids for experiments, newly formed parasitoid cocoons were kept in glass vials (18 mm × 50 mm) with a drop of 10% honey in water. All experiments were conducted with 2-to-3-day-old mated females. Pilot observations showed that the two species were most active at oviposition at these ages.
Interspecific host discrimination
An experiment was conducted to determine whether hosts already parasitized by the other species are equally attractive compared to un-parasitized larvae. Individual second-instar E. postvittana feeding on plantain leaves that had been parasitized by one parasitoid species were exposed to the other species in a random order in a variable speed wind tunnel at a wind speed of 20 cm s−1 at 21 ± 2 °C (for details see Keller, Reference Keller1990). For each parasitized larva, there was a control of an un-parasitized larva of the same age that was handled in the same way. Each parasitoid was observed twice, once with a parasitized larva and once with an un-parasitized control, which were presented to wasps in random order. The experimental treatments were larval status (parasitized or not) and the time between ovipositions (<10 min, 24 and 48 h). Larval E. postvittana that were parasitized by either of the two parasitoids were periodically dissected. This revealed that eggs of D. tasmanica hatch after around 48 h, while it took almost twice this time for eggs of T. unimaculatus to hatch. Therefore, 48 h was selected as the maximum interval between ovipositions so neonates of the second parasitoid of either species would encounter hosts containing first instars of the first parasitoid to oviposit.
Larvae parasitized at different time intervals were used randomly in the wind tunnel for each observation. The order of observation for each treatment and control was also randomized. There were ten replicates for each treatment. Parasitoid behaviour was recorded using the Observer software, version XT11 (Noldus Information Technology, Wageningen, Netherlands). Stinging behaviour is directly related to oviposition and interspecific host discrimination (Vet et al., Reference Vet, Meyer, Bakker and Van Alphen1984), so the total duration of stinging, and total time the parasitoid spent on the leaf bearing the host larva were recorded and analysed. Behavioural data were log-transformed to stabilize variances. Statistical analysis was performed using IBM SPSS Statistic 19. A two-way analysis of variance (ANOVA) was followed by a Bonferroni post-hoc test to analyse the influence of time interval and parasitism status on the duration of oviposition and the time spent on the infested leaf.
Intrinsic competition between T. unimaculatus and D. tasmanica
An experiment was conducted to determine whether intrinsic competition occurs within a parasitized host. Individual second-instar E. postvittana were maintained on single plantain leaves for 24 h before the experiment to allow the accumulation of host odour and potentially stimulate parasitoids to oviposit. An individual E. postvittana larva feeding on a plantain leaf was exposed to one mated female parasitoid in a glass vial (see above). Once stinging was observed, the parasitoid was removed and the second species of parasitoid was added at a predetermined time interval (<10 min, 24 and 48 h). Therefore, the variables in this experiment were (1) time interval between stinging and (2) parasitisation order. There were 30 host larvae for each treatment (180 larvae in total). Hosts parasitized by a single parasitoid species (30 for each species) were used as controls. After parasitism, all larvae were reared individually in 100 ml plastic containers with plantain leaves at 23 ± 2 °C, 14 L:10 D. Larvae were checked daily. The presence of parasitoid cocoons, mortality, the emergence of parasitoids, their sex and developmental times from oviposition to cocoon formation and from cocoon formation to adult emergence were recorded.
To investigate the relative hatching times of larval parasitoids, 40 second-instar hosts, each parasitized by a single parasitoid species (80 in total), were dissected at various times. Hosts attacked by D. tasmanica were dissected after 40–60 h, while those attacked by T. unimaculatus were dissected after 80–100 h. Each host larva was placed in water in a glass staining block and dissected with watchmaker's forceps under a 20 × magnification dissection microscope.
Although each parasitoid species can sting the host parasitized by the other species, it is not clear whether both parasitoid species actually lay eggs in host parasitized by the other. To investigate this and explore the possible mechanism of intrinsic competition between parasitoid larvae, 80 second-instar E. postvittana larvae parasitized by both species were prepared. The treatments for parasitisation were (1) time interval between stinging (<10 min and 48 h) and (2) parasitisation order. Therefore, there were 20 larvae for each treatment. Twenty control larvae were parasitized by a single parasitoid species for each species. Parasitized hosts were then dissected using methods described above at a rate of several larvae/h at 90 h following oviposition by T. unimaculatus. Hosts parasitized only by D. tasmanica were dissected 48 h after oviposition.
Differences in parasitoid emergence in this intrinsic competition experiment were analysed using a binominal test, with 0.5 as the null hypothesis. To analyse the significance of the differences in pre-emergent mortality and sex ratio of emerging parasitoids between single parasitized and multiple parasitized hosts, χ2-tests were used. These tests were used to analyse whether the time-interval treatments and order of parasitism resulted in differences in parasitoid emergence rate. Parasitoid developmental times, from egg to cocoon and from cocoon to adult, were analysed using one-way ANOVA followed by an LSD post-hoc test for multiple comparisons (IBM SPSS Statistic 19).
Results
Interspecific host discrimination
The presentation of parasitized and un-parasitized hosts did not elicit any differences in either mean sting duration (fig. 1, D. tasmanica: F = 0.065; df = 1,54; P = 0.800; T. unimaculatus: F = 0.074; df = 1,50; P = 0.786) or mean time the parasitoids spent on the leaf bearing the host larvae (D. tasmanica: F = 0.391; df = 1,54; P = 0.534; T. unimaculatus: F = 0.092; df = 1,50; P = 0.763; fig. 1).
Mean stinging duration and searching time of T. unimaculatus and D. tasmanica searching on a leaf infested with either un-parasitized hosts or hosts parasitized by the other species at three time intervals. Error bars represent standard errors. Different letters above bars indicate significant differences between intervals (P < 0.05). No significant differences in behaviour were found between parasitized and un-parasitized hosts for all analyses.
For T. unimaculatus, there were significant differences in the sting durations (F = 6.116; df = 2,50; P = 0.001) and searching times (F = 7.763; df = 2,50; P = 0.014) between time-interval treatments. Stinging duration was the longest when the interval between first and second attack was 48 h. The time spent on the plant/host complex when the interval was less than 10 min was significantly shorter than at 24 and 48 h. There was no detectable effect of the time interval between ovipositions on the stinging duration (F = 3.044; df = 2,54; P = 0.056) or time spent on the leaf bearing the host larvae by D. tasmanica (F = 0.839; df = 2,54; P = 0.438).
Intrinsic competition between T. unimaculatus and D. tasmanica
Irrespective of the order of parasitization or the interval between the two parasitisation events, the emergence rates of D. tasmanica were significantly higher than for T. unimaculatus (fig. 2). The pre-emergent mortality of multiple-parasitized hosts (31.1%) was significantly higher than for single-parasitized hosts (8.3%) (χ2 = 12.32, df = 1, P < 0.001). For D. tasmanica, the sex ratio of multiple parasitized hosts (42.4% female) did not differ from that in single-parasitized hosts (46.5%) (χ2 = 0.15, df = 1, P = 0.39). The sex ratio of D. tasmanica from hosts that had been parasitized by D. tasmanica first (45.6%) was not significantly different from hosts that had been parasitized by T. unimaculatus first (39.3%) (χ2 = 0.47, df = 1, P = 0.98). In addition, for D. tasmanica, the sex ratio was not significantly different between time-interval treatments: within <10 min (51.4%), 24 h (41.5%) and 48 h (35.7%) (χ2 = 2.56, df = 2, P = 0.56).
Percentage emergence of D. tasmanica and T. unimaculatus when (a) D. tasmanica parasitized E. postvittana first and (b) T. unimaculatus parasitized first. The floating bars at the right indicate the percentage hosts from which an adult wasp did not emerge. The time between first and second oviposition is shown at the left. Asterisks indicate significant differences in parasitoid species emergence within each time interval (binomial test, *P < 0.0005, **P < 0.0001). Numbers between brackets indicate the fraction of female parasitoids that emerged (D. tasmanica on the left, T. unimaculatus on the right). There were no effects of number of parasitizations, time interval between oviposition and order of oviposition on the proportion of female emergence for D. tasmanica (χ2-tests, P > 0.05).
Dissections indicated that eggs of D. tasmanica hatched between 48 and 56 h after oviposition, while those of T. unimaculatus took around 90–96 h to hatch. However, eggs of T. unimaculatus could not be found until they hatched, in which case it was larvae that were detected. It is possible that the eggs of this species may be embedded within the tissues of the host. Both D. tasmanica and T. unimaculatus laid eggs in hosts parasitized by the other species (table 1). In those cases where first instars of both species were found within the same host, the larval T. unimaculatus were all dead, while all larval D. tasmanica were still alive (table 1). The developmental time of male and female larval D. tasmanica did not differ between any treatments. Therefore, data for parasitoid larval stage development for both sexes were pooled. Because in most cases, live T. unimaculatus did not emerge from hosts that had been parasitized by both parasitoids, data for larval-stage development of T. unimaculatus were from single parasitized hosts by T. unimaculatus. For D. tasmanica, the mean developmental time (order of oviposition and time interval between parasitism) did not differ among treatments for both oviposition to cocoon formation and for cocoon formation to adult emergence (table 2). The mean larval development time from oviposition to cocoon formation for T. unimaculatus was significantly longer than D. tasmanica (table 2).
Numbers of larval parasitoids found during dissection of single and multiple parasitized larvae of E. postvittana following exposure to D. tasmanica parasitized and/or T. unimaculatus at two time intervals (<10 min or 48 h).
1 All larvae of T. unimaculatus dead.
Mean (±SE) development time of larval D. tasmanica and T. unimaculatus from oviposition until cocoon formation, and from cocoon formation until adult emergence under different treatments.
Means followed by same letter in columns do not differ statistically (LSD test, P < 0.05).
Discussion
This study demonstrated that neither D. tasmanica nor T. unimaculatus responded differently to hosts parasitized by the other species compared to un-parasitized host (fig. 1). Since multiple parasitism is common among parasitoids (Van Alphen & Visser, Reference Van Alphen and Visser1990), the lack of interspecific host discrimination leads to competition between larval parasitoids inside the same host. In this study system, there is no ‘need’ for the superior intrinsic competitor D. tasmanica to develop interspecific discrimination ability, because its offspring are likely to survive in instances of multiple parasitism (fig. 2). However, for the inferior intrinsic competitor, T. unimaculatus, selection should favour detection of factors that indicate parasitism by another superior competitor if selection pressure is strong. This would enable it to exploit hosts that are free from its superior competitor, D. tasmanica. The data indicate that the chance that its offspring will survive multiple parasitism are virtually nil.
Studies here suggested that coexistence of parasitoids that attack the same host species largely depends on their life history characteristics (Amarasekare, Reference Amarasekare2003; Harvey et al., Reference Harvey, Poelman and Tanaka2013). For example, differences in dispersal ability may enable the co-existence of species that compete for the same hosts, if the relatively poor intrinsic competitor has an advantage in dispersal at the landscape scale (Hanski & Ranta, Reference Hanski and Ranta1983; Yu et al., Reference Yu, Wilson, Frederickson, Palomino, De La Colina, Edwards and Balareso2004). The dispersal ability of D. tasmanica is limited within vineyards (Scarratt et al., Reference Scarratt, Wratten and Shishehbor2008), while the dispersal ability of T. unimaculatus in both natural habitats and agriculture settings is still unknown. It is necessary to further investigate the dispersal ability of these two competing species to determine how they respond to habitats at the local and regional scales.
Resource or niche partitioning is a main factor that facilitates the coexistence of competing species (Amarasekare, Reference Amarasekare2003). There are many possible differences in the characteristics of niches that could allow coexistence of competing parasitoids. T. unimaculatus and D. tasmanica may prefer different host plants, host species and/or stages of hosts. Furthermore, T. unimaculatus has a much bigger body and longer ovipositor than D. tasmanica. Therefore, T. unimaculatus could probably attack hosts in shelters that are unreachable by D. tasmanica. On the other hand, the relatively smaller body size of D. tasmanica enables it to search in niches that are not accessible to the relatively larger T. unimaculatus. Competing species with different competitive abilities that can co-exist through partitioning different niches have been studied in different multispecies parasitoids–host systems. For example, Bogran et al. (Reference Bogran, Heinz and Ciomperlik2002) found that niche partitioning occurred among three parasitoids that share the same whitefly species in cotton. In this instance, the different parasitoids attack hosts located on different parts of plants, and the host suppression reached a maximum when all three parasitoids occurred together. In another study, Van Nouhuys & Punju (Reference Van Nouhuys and Punju2010) found that an inferior competitor can coexist with a superior one by exploiting the small fraction of un-parasitized hosts left by the superior competitor. Moreover, a recent study showed that a parasitoid with a longer ovipositor could attack hosts in larger fruits than those used by its competitors (Aluja et al., Reference Aluja, Ovruski, Sivinski, Córdova-García, Schliserman, Nuñez-Campero and Ordano2013).
Our results suggested substantial disparity when the two larval parasitoid species were present within a host larva. In this study, we found first-instar D. tasmanica has sickle-like mandibles. When both species occurred in the same host, the first-instar T. unimaculatus was always dead while first-instar D. tasmanica was always alive (table 1). Therefore, one mechanism of intrinsic competition between early instars of the two parasitoids could be physical combat, which is common for first-instar parasitoids (Tian et al., Reference Tian, Zhang, Yan and Wang2008; Wang et al., Reference Wang, Bokonon-Ganta and Messing2008; Harvey et al., Reference Harvey, Gols and Strand2009, Reference Harvey, Poelman and Tanaka2013; Paladino et al., Reference Paladino, Papeschi and Cladera2010). In addition to this combat, the competitive superiority of D. tasmanica may be aided by the relatively shorter egg-hatching time compared to T. unimaculatus. Studies have demonstrated that species with shorter egg developmental times have a greater chance to win in intrinsic competition (Mills, Reference Mills2003; De Moraes & Mescher, Reference De Moraes and Mescher2005). Multiple-parasitism can sometimes affect the development of larval parasitoids, which results in longer developmental times in the superior competitor (Pschorn-Walcher, Reference Pschorn-Walcher1971; Reitz, Reference Reitz1996). However, in this study, the development time of larval-stage D. tasmanica in multiple parasitized hosts was not different from single parasitized hosts. Our results indicate that in the presence of D. tasmanica, larvae of T. unimaculatus were killed very soon after hatching (table 1). This could explain why multiple-parasitism did not affect the developmental time of larval D. tasmanica. In this study, we found multiple-parasitized hosts have higher mortality than those single parasitized, which may be caused by physiological factors or physical injury (De Moraes & Mescher, Reference De Moraes and Mescher2005), or parasitized hosts are more vulnerable to infection (Brodeur & Boivin, Reference Brodeur and Boivin2004).
It is well known that there can be advantages and disadvantages of introducing multiple parasitoids for biological control (Turnbull & Chant, Reference Turnbull and Chant1961). Since it is hard to determine the best candidate parasitoids from a number of species, many biological programmes introduce multiple species (Ehler, Reference Ehler, Mackauer, Ehler and Roland1990). However, some studies have indicated that multiple natural enemies might disrupt each other, and hinder the suppression of a pest (Rosenheim et al., Reference Rosenheim, Kaya, Ehler, Marois and Jaffee1995; Murdoch et al., Reference Murdoch, Briggs, Collier, Dempster and McLean1998; Collier & Hunter, Reference Collier and Hunter2001; Collier et al., Reference Collier, Kelly and Hunter2002). Furthermore, evidence suggests that generalist parasitoids normally out-compete the more specialized parasitoids during intrinsic competition (Iwao & Ohsaki, Reference Iwao and Ohsaki1996; De Moraes & Mescher, Reference De Moraes and Mescher2005). In this study, the complete host ranges of these two generalist parasitoid species in Australian vineyard are still unknown. We hypothesize that they could co-exist through exploiting different niches. Therefore, it is important to study the effects of factors such as host plant species, host species and habitat characteristics on the coexistence of the two parasitoid species. More importantly, all of these factors need to be investigated to determine how the competitive interactions between the two species here could influence their efficiency in suppressing E. postvittana. Such knowledge would facilitate ‘ecological engineering’ (Gurr et al., Reference Gurr, Wratten and Altieri2004) that could help to improve parasitism rates by T. unimaculatus in vineyards.
Acknowledgements
This research was supported by a grant from the Australian Grape and Wine Authority (formerly GWRDC), and scholarships from the China Scholarship Council and The University of Adelaide to Yi Feng. Thanks to Dr Nicholas Stevens for identifying Therophilus unimaculatus. Thanks to Dr Katja Hogendoorn for her valuable comments on an early version of the manuscript.
