Introduction

Social wasps (Vespidae), native to Eurasia and Northern Africa, have proved to be prolific invasive species, establishing introduced populations in North and South America, as well as southern Africa and Oceania (Lester and Beggs 2019). The generalist lifestyle of Vespula species, coupled with strong behavioural plasticity, enables invasive wasps to predate upon native insect populations whilst also directly competing with them for food resources, rapidly impacting all trophic levels (Yamane and Yamane 2020). Consequently, Vespula have had significant deleterious impacts on invaded regions, with both environmental and economic consequences (Lester et al. 2014; Cook 2019; Lester and Beggs 2019). For example, in New Zealand, invasive V. vulgaris (common wasp) and V. germanica (German wasp) populations have established in native ecosystems and significantly altered resource flow (Lester et al. 2013; MacIntyre and Hellstrom 2015). With a particular influence on the apiculture and pastoral farming industry, their impact is estimated to cost the New Zealand economy up to $130 million per annum (Clapperton et al. 1989; MacIntyre and Hellstrom 2015). Environmentally, Vespula’s capacity to cause large-scale ecosystem degradation represents one of New Zealand’s greatest invasive threats and is deemed responsible for the degradation of the honeydew beech forest ecosystems of the South Island (Clapperton et al. 1989; Moller and Tilley 1989; Beggs 2001). Such negative impacts are not unique to New Zealand, and are observed in other Vespula invaded regions (e.g. in Argentina Sackmann et al. 2001 or in Australia Wood et al. 2006). In response, strategies have been employed in attempts to control Vespula sp. populations, for example the release of the parasitoid Sphecophaga (Donovan and Read 1987; Donovan et al. 2002), and the use of insecticidal baits (e.g. Vespex) (Edwards et al. 2017), but so far these have proven to be financially and environmentally impractical (Lester et al. 2014; Lester and Beggs 2019; Palmer et al. 2021). For instance, the effectiveness of Sphecophaga was shown to be reduced after a year in the field (e.g. fewer brachypterous adults produced, Beggs et al. 2008) and the use of Vespex, whilst effective after the application, can allow for recolonisation by queens in following years (Lester and Beggs 2019).

The understanding of natural enemies of social wasps can prove very valuable in controlling pest species, in both agricultural and “natural” environments (Lester and Beggs 2019; Palmer et al. 2021). In the native ranges of Vespula vulgaris and V. germanica several parasitoid, predatory, and scavenging species exploit the protected and protein-rich environment of Vespula nests (Ward 2013). Species from diverse taxonomic groups are known to inhabit wasp nests including mites, hoverflies, a beetle and as well as at least one parasitoid wasp (Rupp 1989; Oi et al. 2020), and these insect associates are often collectively referred to as ‘guests’, or ‘sphecophiles’ (Parmentier 2019). Despite their implied negative impact on host colonies, the distribution and prevalence of Vespula sphecophiles within host ranges remains uncertain. Given this void in understanding, and the capability for natural enemies to reduce populations of Vespid wasps, investigations into general parasitoid ecology are important and can lead potentially into the discovery of new or more efficient methods to control invasive Vespula wasps.

Specifically, three species within Volucella (Diptera: Syrphidae), a genus of broad-bodied hoverflies found commonly across Europe (Ball and Morris 2015), have specialised larvae that are obligate sphecophiles of Vespula nests (Speight 2014). The adults of Volucella zonaria, V. pellucens and V. inanis infiltrate Vespula nests and oviposit on the nest-encasing paper envelope. The eggs then hatch and enter the nest cavity to begin foraging (Rupp 1989). Larvae of V. zonaria and V. pellucens migrate to the bottom of the nest and develop in the nest litter, feeding on detritus from the colony above (Rotheray and Gilbert 1998; Speight 2014). Whilst mostly living as scavengers, V. zonaria and V. pellucens larvae can also crawl up into the combs and predate upon live Vespula larvae (Rotheray and Gilbert 1998). Larvae of V. inanis, however, are obligate Vespula ectoparasites (Rotheray and Gilbert 1998; Parmentier 2019) and reside permanently in the nest brood chambers (until ready to pupate); to facilitate this, V. inanis larvae have specialised, dorsoventrally flattened bodies that enable them to enter larval cells alongside Vespula larvae to feed on them (Rupp 1989; Ward 2013). During development, each V. inanis larva consumes multiple Vespula larvae before going to the bottom of the nest to pupate (Rupp 1989; Brown 2021a, b). Volucella females are thought to lay between 60 and 300 eggs in each oviposition (Rupp 1989), and hence infestation by Volucella may impose a substantial impact on Vespula colony development.

Considering the propensity for V. zonaria, V. pellucens and V. inanis to predate Vespula larvae, either being facultative or obligate entomophages (Rotheray and Gilbert 1998), the Volucella genus has been identified as a potential agent of biocontrol against invasive Vespula populations (Ward 2013). Biocontrol research in New Zealand has considered that the release of large numbers of Volucella into native ecosystems could provide suppression of wasp numbers across very large areas and help reduce their invasive impact, without bringing their own deleterious effects (Brown 2021a, b). Of the three aforementioned Volucella species that inhabit Vespula nests, Volucella inanis has been recognised as the best candidate (Brown 2021a, b). Firstly, as the sole obligate ectoparasite (Rotheray and Gilbert 1998), each V. inanis larva is thought to cause more significant and consistent damage to the host wasp nest, as each V. inanis must consume at least two wasp larvae to grow (Brown 2021a, b). Secondly, the more specialised nature of V. inanis’s niche reduces the threat of opportunistic predation of non-target species—trials have shown V. inanis to refuse to parasitise the brood of buff-tailed bumblebees (Bombus terrestris audax; considered the most likely non-target species present in New Zealand), even under no-choice conditions (Brown 2021a, b). Conversely, the more generalist, scavenging niche of V. zonaria and V. pellucens, coupled with reported sightings in non-Vespula nests (Ball and Morris 2004), has driven hypotheses that they are less Vespula-specific than their close relative V. inanis; however, this requires further investigation (Lester et al. 2013). Having been approved by authorities, researchers in New Zealand have begun attempts at introducing V. inanis into Vespula nests in contained trials to assess its efficacy as a biocontrol agent, in situ (Brown 2021a, b). If proven effective, similar biocontrol could be initiated in other invaded regions, both within and beyond New Zealand. However, despite the identification of the potential for Volucella inanis as a biocontrol agent and the commencement of live trials, there are still significant gaps in the understanding of Volucella ecology.

Whilst Volucella species are able to infest nests of both Vespula species that have invaded New Zealand (V. vulgaris and V. germanica) (Rotheray and Gilbert 1998), it is unknown whether they display any host preferences. Could it be that Volucella are predisposed to associate with one species over another, given equal opportunity for both? Additionally, prior exposure has been shown to be able to influence preference in parasitoid species, whereby females prefer to associate with more familiar hosts (Mandeville and Mullens 1990; Turlings et al. 1990; Cortesero et al. 1995; Lentz-Ronning and Kester 2013). Considering this, could Volucella preferentially choose to associate with larvae of the same species as their nest of origin? Knowing if Volucella inanis have a strong preference to the host species from which they were collected could significantly enhance the development of mass rearing methods en route to their releases as biological control agents. Since the two hoverfly species, V. inanis and V. zonaria, can commonly be found in nests of both V. vulgaris and V. germanica, we hypothesised that Volucella larvae will have no preference for either Vespula spp, and that there will be no difference in host prey preference between the parasitoid species. We also hypothesised that Volucella preference will not be dependent on its origin.

Here, we firstly investigated the presence of arthropod parasites in Vespula nests collected in the UK over three years, providing the first exploration of brood parasite prevalence in Vespula nests in southeastern England. We then experimentally test our hypotheses regarding the host preferences of Volucella by conducting two-choice prey preference experiments in petri dish arenas. We compared larvae prey preference of two Volucella species, V. inanis and V. zonaria. Then, we scrutinised prey preference in V. inanis, considering the origin of the parasitic larvae to investigate whether the nest from which V. inanis was sourced (either Vespula vulgaris or V. germanica) had any effect on prey preference.

Material and methods

Vespula and brood parasite field collection

Nests of Vespula vulgaris and V. germanica were collected from the Wallingford area, Oxfordshire, UK, and from around Sutton, London, UK, in September to October of 2018, 2019 and 2021. A total 54 nests were collected (2018, V. vulgaris N = 16, V. germanica N = 4; 2019, V. vulgaris N = 8, V. germanica N = 2; 2021, V. vulgaris N = 21, V. germanica N = 3), whereby all nest material (including detritus that had accumulated at the bottom of the nest cavity) was gathered and brought back to the laboratory at CABI, Egham, UK (2018 and 2019) or UCL, London, UK (September to October 2021). To promote normal parasitoid movement and feeding, nest combs were kept as intact as possible and stacked on top of each other separated by slim foam spacers, and stored in well ventilated plastic cages (Exo Terra Breeding Box). Workers were sedated with CO2 and removed from the combs regularly to avoid attacking the parasites. Cages were stored at room temperature under a natural light regime.

Parasite prevalence

Each nest was then rigorously searched for four species of social parasites: Volucella inanis and V. zonaria (all larval instars); Metoecus paradoxus (adults, and larvae); and Sphecophaga vesparum (both adults and cocoons). Searches were conducted by separating the combs of each nest and examining all cells under white light. The detritus from beneath each nest was also sifted through to identify any individuals that had been residing there, or that had been displaced there during nest extraction. Any parasites discovered were collected using soft forceps and identified, and the presence or absence of each of species was recorded for each nest.

Prey preference experiment

From nests collected in October 2021, we conducted prey preference experiments. Two-choice experiments were conducted to investigate prey preferences of the two hover fly species, Volucella inanis and V. zonaria. Each individual larva was tested 4 consecutive times, alternating the position of the two wasp species (right or left). Test arenas consisted of Petri dishes (9 cm diameter) positioned 50 cm below a LED lamp. All trials were conducted at room temperature under light to encourage movement and to standardise room conditions. Two Vespula vulgaris larvae (5th instar) were placed adjacent to each other at one pole of the arena, and two V. germanica larvae (5th instar) were placed at the opposite pole (Fig. 1). We used two larvae of Vespula to avoid locomotion. All Vespula larvae were placed simultaneously, immediately after removal from their parent nest, and each used in only one trial. The position that each host Vespula species was placed at (left or right pole) was alternated in each trial. A single Volucella second-instar larva (either V. inanis or V. zonaria) was then placed in the centre of the arena, equidistant from all Vespula larvae and orientated to face perpendicular to both V. vulgaris and V. germanica, facing neither. Second instar larvae of Volucella were used in the experiments because in this stage they actively feed and search for prey (Rupp 1989). The Vespula and Volucella larvae used in a given trial were never sourced from the same nest. Trails began with the release of the Volucella larva and lasted for 5 min. All trials were filmed using a webcam (Trust Webcam) for later, blind assessment. Petri dishes were washed with water and detergent after each trial and dried with paper. At the start of trialling, we recorded the body length of each Volucella larva at full extension as a measure of larval size. Overall, 64 Volucella larvae were tested (V. inanis n = 32 individuals, V. zonaria n = 32 individuals), and each larva was tested four consecutive times, providing 256 trials in total. Of the 32 V. inanis larvae used, 16 were extracted from nests of Vespula vulgaris, and 16 from nests of V. germanica. We did not discriminate the nests of origin for V. zonaria due to constraints in the availability of V. zonaria from nests of V. germanica. Any trials in which the Volucella larva did not move from the start position were removed from the dataset before analysis (6 trials, all involving V. inanis), leaving 250 trials for analysis.

Fig. 1
figure 1

Diagram of the test arenas used to investigate prey preference of Volucella inanis and Volucella zonaria between Vespula vulgaris and Vespula germanica larvae. Arenas were split into ‘choice areas’ - ‘V. vulgaris’ in red, ‘V. germanica’ in green. These choice areas were defined by circles of 1 cm radius around each pair of respective wasp larvae

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To discern preference during the choice experiments, the arenas were split into three ‘choice areas’ – ‘V. vulgaris’, ‘V. germanica’, and ‘No choice’. The choice areas for ‘V. vulgaris’ and ‘V. germanica’ were defined by circles of 1 cm radius around each pair of respective wasp larvae, and the ‘No choice’ area comprised of the remainder of the arena (i.e. any of the arena that was more than 1 cm away from any wasp larvae; Fig. 1). An observer blind to the identity of the Vespula species then measured the number of seconds that each parasitic larva spent within each choice area in each trial.

We then used a preference ratio commonly used in dichotomous choice preference tests (Houde 1997) to control for the total time during each trial that the parasitic larvae (V. inanis or V. zonaria) spent in either Vespula choice area. This index was defined as the difference in time spent in each Vespula choice area, standardised by the total amount of time in either of the Vespula choice areas [(time with Vv – time with Vg)/(time with Vv + time with Vg)], in which Vv represents Vespula vulgaris and Vg represents V. germanica. The ratio provides an index of preference between + 1, where all time was spent with V. vulgaris, and -1, where all time was spent with V. germanica, where a 0 value indicates equal time spent with each. The index was then used to decide binomial response results from each trial. Larvae were recognised as preferring V. vulgaris in trials with index values greater than 0, and as preferring V. germanica in trials with index values less than 0.

Statistical analyses

We tested for potential differences in prey preference between the two Volucella species using a generalised linear mixed-effect model (GLMM). The model included Volucella species and larval size as fixed effects, with binomial choice (V. vulgaris or V. germanica) as the response variable. Also, we included individual ID, repeat number (1st, 2nd, 3rd or 4th time each individual was trialled) and the side that V. vulgaris was presented on (left or right) as random factors. We checked the models for overdispersion.

Secondly, we investigated the host preferences of Volucella inanis sourced from nests of different Vespula species (either V. vulgaris or V. germanica), using a similar GLMM approach. Here, the model included the Vespula species from which the V. inanis was sourced as a fixed effect, with binomial choice as the response variable, V. vulgaris larvae or V. germanica larvae. Again, individual ID, repeat number and the side that V. vulgaris was presented on were included as random factors. We did not test Volucella zonaria origin, as the larvae of V. zonaria were kept in the same container without discrimination of nest origin. All statistical analyses and data visualisation were performed in R version 4.0.3. (Team 2012), using the package lme4 (Bates et al. 2015).

Results

Parasite prevalence

Parasitic species that were recorded from Vespula nests in 2018, 2019 and 2021 are displayed in Table 1. In each year, we were able to survey more V. vulgaris nests than of V. germanica. Volucella inanis were the most prevalent species found in 48 (88.9%) of the 54 nests surveyed. The next most prevalent species was V. zonaria which was found in 29 (53.7%) nests overall. Metoecus paradoxus and Sphecophaga vesparum were only collected from Vespula vulgaris colonies. From these nests, M. paradoxus were present in 17 nests or 31.5 % of the total surveyed. Sphecophaga vesparum were the least prevalent, only being found in 7 nests (12.9%). The Volucella larvae for host preference choice test experiments were sourced from Vespula colonies collected in the 2021 nest survey. Volucella inanis and V. zonaria were both present in all three Vespula germanica nests used in the host preference trial. In the 21 V. vulgaris nests, Volucella inanis was present in 81% of nests, V. zonaria in 43%, S. vesparum in 24%, and M. paradoxus in 19% (Table 1).

Table 1 Sampling information of collected wasp nests (n) and the prevalence of  parasitic species (number of nests each species is present and in parentheses the percentage of infested nests/n)
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Preference of Volucella species for Vespula vulgaris versus V. germanica

Across our experiment, Volucella larvae moved to within 1 cm of at least one of the Vespula larvae in all 250 of the trials, and made physical contact with a Vespula larva in 91.6% of trials. Additionally, Volucella larvae moved to within 1 cm of both Vespula species in 47.6% of trials. Firstly, we checked the initial choice, but the model was not significative, and it was left out the main results (P = 0.99, Supplementary material). Using the preference ratio, we found an overall effect of species in the preference for Vespula vulgaris [proportion V. vulgaris, proportion V. germanica: in Volucella inanis (n = 121 trials): 0.587, 0.413; in Volucella zonaria (n = 125 trials): 0.512, 0.488; GLMMpreference: Species: χ2(1) = 3.995, P = 0.0456]. Volucella inanis showed a significant preference for Vespula vulgaris compared to Vespula germanica, whilst Volucella zonaria displayed no significant preference (Table 2A, Fig. 2A).

Table 2 Prey preference of Volucella inanis and Volucella zonaria toward the larvae of Vespula vulgaris and Vespula germanica
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Fig. 2
figure 2

Trials in which Volucella larvae made a prey choice. A Left bars represent the number of trials in which Volucella inanis chose eitherVespula vulgaris (50) or V. germanica (71) as prey. One V. inanis did not make a choice and was excluded. Right bars represent the number of trials in which Volucella zonaria chose Vespula vulgaris (61) and V. germanica (64) as prey. Three V. zonaria larvae did not make a choice and were excluded from analysis. B. Breaks down the 121 prey choices made by V. inanis, presented in Fig 2A, by comparing their choice in the assay with the host species they were collected from. The left bars indicate the choices made by V. inanis larvae that originated from V. germanica colonies and the right bars show choices made V. inanis larvae originally from V. vulgaris nests

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However, it is notable that in 41% of trials the V. inanis larvae chose V. germanica (Fig. 2A), revealing that whilst displaying significant preference for V. vulgaris over V. germanica, V. inanis larvae are not averse to also predating upon V. germanica. Volucella size was also investigated as a fixed effect. Size was found to have no overall effect in host preference [GLMMpreference: Size: χ2(1) = 3.776, P = 0.0520] (Table 2A).

Preference considering nest of origin in Volucella inanis

We found no overall effect of the species of origin in host preference [proportion V. vulgaris, proportion V. germanica: V. vulgaris origin (N = 62 trials): 0.645, 0.355; V. germanica origin (N = 59 trials): 0.525, 0.475; GLMMpreference: Nest of origin: χ2(1) = 1.642, P = 0.200] (Table 2C, Fig. 2B). Volucella inanis showed no significant preference for the Vespula species from which they were collected (and therefore had had prior exposure to), nor any significant preference for a novel Vespula species (Fig. 2B).

Discussion

The prevalence of sphecophiles in Vespula nests showed that the four species, Volucella inanis, Vollucela zonaria, Sphecophaga vesparum and Metoecus paradoxus, were all found in Vespula nests examined in southeast England. Volucella inanis was the most ubiquitous of four parasitoids, with its larvae present in the majority of nests searched. The closely related V. zonaria was found to be less prevalent, inhabiting half of the nests, whilst Sphecophaga vesparum and Metoecus paradoxus were both observed in no more than a quarter of nests. The two Volucella species were found in nests of both Vespula vulgaris and Vespula germanica, but S. vesparum and M. paradoxus were found only in the nests of V. vulgaris. The results of prey preference experiments using Volucella revealed that (1) Volucella inanis larvae have a prey preference for Vespula vulgaris over Vespula germanica, but are nonetheless not averse to predate Vespula germanica, and (2) Volucella zonaria larvae exhibit no preference for either Vespidae species. These results contradict our initial hypothesis that there would be no prey preference in both Volucella sp. We also found that (3) the natal nest of V. inanis larvae (either V. vulgaris or V. germanica) has no effect on the preference of prey species. This corroborates our hypothesis that nest of origin would have no effect on prey preference in V. inanis.

The release of V. inanis has recently been approved to control Vespula populations in New Zealand (Brown 2021a, b), and our results corroborate the choice of Volucella inanis as a promising biocontrol agent. Firstly, the relative high prevalence of V. inanis suggests that it is capable of infiltrating large proportions of nests in inhabited regions, supportive of the notion that the parasitoid could impact Vespula populations at regional scales. Then, although displaying a preference towards V. vulgaris, V. inanis also still predates upon V. germanica—a result that is further supported by the observed presence of V. inanis in nests of both Vespula species. This is reassuring that V. inanis will likely parasitise both Vespula species in areas where invasive populations have established in sympatry (e.g. in New Zealand) and also parasitise V. germanica populations where it is the only invasive Vespidae wasp (e.g. in Argentina, Masciocchi and Corley 2013). Furthermore, our results are indicative that V. inanis larvae reared in nests of either V. vulgaris or V. germanica will predate upon both Vespula species—hence it can be expected that V. inanis extracted from nests of V. vulgaris would predate upon V. germanica brood, and vice versa.

The association between adult and larval host preference is variable among parasitoid species (Giunti et al. 2015). Although there is evidence that pre-imaginal experience can have little bearing on preferences in adulthood (Jaenike 1983; Corbet 1985; Giunti et al. 2015). In some species, the Hopkins host selection principle (Hopkins 1909) supports the conservancy of behaviour of larval holometabolous insects and behaviour observed in adults. Pre-imaginal experience has been found to influence the preferences of several adult holometabolous insects (Hopkins 1909; Tully et al. 1994; Ray 1999; Rietdorf and Steidle 2002; Moreau et al. 2008; Ning et al. 2018). The methodologies of implementing V. inanis as biocontrol agents involve either introducing larvae directly to nests, or releasing cocoons or adults to then naturally increase the density of the parasite’s populations. Requiring less manipulation, the latter approach is likely more attractive in the long term. In the case of conserved larval-adult preference, our results therefore have key implications for the predicted behaviour of released adults of both Volucella species regarding their choice of host species. Further investigation into the behaviour and preference of female adults is crucial for understanding the extent to which the preferences (and lack there-of) observed in V. inanis larvae are conserved into adulthood.

It is of note that the exploration of the four parasite prevalence reported here was a preliminary investigation and that studies over a wider area are needed to confirm our findings. Further investigation would benefit from collecting and searching nests from across the home range of the Vespula species, and across the nest development, as for example V. zonaria is likely to be more frequent in nest decline. Other species are known to inhabit Vespula nests (e.g. Pneumolaelaps mites Felden et al. 2020), which was not included in our survey.

Whilst our findings provide early optimism for the success of V. inanis’s future as an agent of biocontrol, Volucella species and their ecology remain understudied. Given the potential for Vespula biocontrol to deliver strong ecological and economical benefits, we call for further research into Volucella spp. to improve our understanding of Volucella biology, and likely enhance the efficacy of biocontrol efforts. Understanding the prevalence and preferences of proposed bioagents, alongside host-parasitoid compatibility, is evidently valuable to the long-term success of biocontrol programs.

Author contributions

JS and CAO contributed to the study conception and design. Material preparation, data collection and analysis were performed by JS, RLB and CAO. FS analysed the videos. The first draft of the manuscript was written by JS and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.