# Invasive Vespa velutina nigrithorax hornets are more susceptible to entomopathogenic fungus than two other hymenopteran species, the wasp Vespa vulgaris and the bumblebee Bombus terrestris.

Mathilde Lacombrade, Naïs Rocher, Blandine Mahot-Castaing, Fanny Vogelweith, Mathieu Lihoreau, Denis Thiéry

► To cite this version:

Mathilde Lacombrade, Naïs Rocher, Blandine Mahot-Castaing, Fanny Vogelweith, Mathieu Lihoreau, et al.. Invasive Vespa velutina nigrithorax hornets are more susceptible to entomopathogenic fungus than two other hymenopteran species, the wasp Vespa vulgaris and the bumblebee Bombus terrestris .. 2024. hal-04806532

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Preprint submitted on 27 Nov 2024

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1 Invasive *Vespa velutina nigrithorax* hornets are more susceptible to

2 entomopathogenic fungus than two other hymenopteran species, the wasp

3 *Vespa vulgaris* and the bumblebee *Bombus terrestris*.

4

5 Mathilde Lacombrade¹,²*, Naïs Rocher³, Blandine Mahot-Castaing¹, Fanny Vogelweith⁴, Mathieu

6 Lihoreau¹⁺, Denis Thiéry³⁺

7

8 ¹ Research Center on Animal Cognition (CRCA), Center for Integrative Biology (CBI); CNRS,

9 University Paul Sabatier – Toulouse III, France

10 ² M2i Biocontrol, 370 route de Cauzenil, 46140 Parnac, France

11 ³ UMR 1065 Santé et Agroécologie du Vignoble, INRAe, F-33883 Villenave d'Ornon, France

12 ⁴ Entomo-Logik, 46000 Cahors, France

13

14 * Corresponding author: lacombrademathilde@gmail.com

15 + These authors contributed equally to the work

16

17

18
19 Abstract

Entomopathogenic fungi are commonly used as biological control agents. Recently, a strain of
*Metarhizium robertsii* (Sorokin, 1883) (Hypocreales: Clavicipitaceae) collected in France was
identified as a good candidate for controlling invasive populations of yellow-legged hornet, *Vespa
velutina nigrithorax* (Buysson, 1905) (Hymenoptera: Vespidae) in Europe. Since *M. robertsii* is
generalist, its use as bioagent could be damaging for non-targeted species. Here, we compared the
lethal effects of three concentrations of this *M. robertsii* strain on the invasive hornet and two non-
targeted species commonly found in Europe, the common wasp *Vespa vulgaris* (Linnaeus, 1758)
(Hymenoptera: Vespidae) and the buff-tailed bumblebee *Bombus terrestris* (Linnaeus, 1758)
(Hymenoptera: Apidae). Exposure to *M. robertsii* spores altered the survival of all three species but at
different degrees. Hornets had consistently lower survival, even under low concentrations of spores,
when compared to wasps and bumblebees. Such lower susceptibility of beneficial Hymenoptera is
encouraging in the perspectives of using *M. robertsii* as biocontrol agent against invasive hornets.

32

33 **Keywords:** Hymenoptera, Vespidae, Hypocreales, Clavicipitaceae, biological control, invasive
34 species

35

36 Statements and Declarations

37 The authors declare no competing interests. This study was funded by the ADEME (No. 2082C0061)
38 and the ANRT (CIFRE No. 2020_0399). MLa, FV, MLi and DT designed the study. MLa, NR, BM-C
39 performed the experiments. MLa wrote the first draft. MLa, FV, MLi and DT revised the manuscript.

40

41 Acknowledgments

42 We thank Mael Rannou for helping during experiments.
43 **Introduction**

44 The yellow-legged hornet (Vespa velutina nigrithorax) (Buysson, 1905) (Hymenoptera: Vespidae) is
45 an invasive social insect species throughout the world, especially in Europe (Haxaire et al. 2006),
46 Korea (Kim et al. 2006) and Japan (Sakai and Takahashi 2014) (Thiery and Monceau 2024). Their
47 geographic expansion poses major concerns to beekeeping activities and the environment (Monceau
48 et al, 2014). Current methodologies to control hornet populations are expensive and use pesticides
49 harmful for the biodiversity (Kishi and Goka 2017; Goulson 2019). Recently, however, several
50 biocontrol options have been explored (Turchi and Derijard 2018) including the use of an
51 entomopathogenic fungus, *Metarhizium robertsii* (Sorokin, 1883) (Hypocreales: Clavicipitaceae)
52 (Poidatz et al. 2018). This fungus pertains to the complex of species *M. anisopliae* that is already
53 commercialized for biocontrol and known to be efficient against Orthopterans, Isopterans and
54 Thysanopterans (i.e. *M. anisopliae* strain F52 approved in Europe, USA, Brazil, Columbia and China
55 for the use against various insects; *M. anisopliae* strain FI-985 approved in Australia, India and China
56 for the use against grasshoppers and locusts). In particular, Poidatz et al. (2018) have demonstrated
57 that the exposure to spores of four isolates of *M. robertsii* (EF2.5(1), EF3.5(1), EF3.5(2), EF3.5(4)) at
58 10⁷ sp/ml reduced by half the survival of yellow-legged hornets after only 5 to 6 days.
59

59

60 From a biological control perspective, entomopathogenic fungi like *M. robertsii* constitute good
61 candidates to target a social insect such as hornets. On the one hand, sociality favors parasite
62 transmission between genetically close individuals that are spatially aggregated, and engaged in
63 numerous behavioral interactions, through social contacts, food exchange, or proximity with nest
64 materials (Dimbi et al. 2013; Khun et al. 2021). However, the potential infection of non-targeted
65 species should also carefully be taken into consideration if infected hornets leave their nest and fly,
66 disseminating spores from their body surface (Smagghe et al. 2013). In particular, there is a risk that
67 wild and managed pollinators get contaminated through direct contact with spores when contaminated
68 hornets hunt unsuccessfully and release their prey (Ueno 2015), or land on a honeybee hive flight
69 board, or contaminate flowers on which their collect nectar (Kapongo et al. 2008).
Several studies on the complex species of *M. anisopliae* (including *M. robertsii*) (Rehner and Kepler 2017) have reported negligible (Smagghe et al. 2013) to strong negative impact (Shaw et al. 2002) on several beneficial insects such as pollinators (e.g. honey bees, bumblebees), depending on the doses (Kanga et al. 2003; Smagghe et al. 2013), the formula (Ball et al. 1994), and the methods of application used (Rodríguez et al. 2009). For instance, the contact with a powder concentrate of 10⁷ sp/g decrease the survival of bumblebees by almost 25% within 6 weeks (Smagghe et al. 2013). The decrease of survival reaches 100% at a concentration of 10⁹ sp/g. The survival of honey bees was lower when the fungus was sprinkled on and between the frames of a hive (decreased by around 55%) than when conidia were stamped on filter papers and placed on half the frames (decreased by 5%) (Rodríguez et al. 2009). Given these potential advantages and disadvantages for hornet biocontrol, there is a need to better characterize the impact of *M. robertsii* exposure with comparable methodologies both on invasive hornets and non-target species before it can be further considered for biocontrol.

Here we compared the lethal effect of a *M. robertsii* strain EF3.5(1) on *V. v. nigrithorax* and two non-target pollinator species commonly found in the invaded area, the common wasp *Vespula vulgaris* (Linnaeus, 1758) (Hymenoptera: Vespidae) and the buff-tailed bumblebee *Bombus terrestris* (Linnaeus, 1758) (Hymenoptera: Apidae). We hypothesized that *M. robertsii* would have different effects depending on the ecology and the natural history of the host species. Specifically, we expected that the virulence of the parasite would be stronger in hornets than in the two other species, because these invasive insects only build aerial nests which could greatly limit the exposition to *M. robertsii*, whereas the local non-target species may have co-evolved with it since long by nesting in soil. We tested this hypothesis by exposing hornets, wasps, and bumblebees to different concentrations of *M. robertsii* and monitoring their survival for eight days.
## 97 Methods

### 98 Insects

All the experiments were performed in Bordeaux (France) during summers 2021 to 2023. We used four wild *V. v. nigrithorax* nests, one wild *V. vulgaris* nest, and four commercial *B. terrestris* nests (see Table 1). Although commercially reared since about 30 years, these bumblebee populations originate from wild populations sampled in Europe and are frequently mixed with freshly caught wild individuals (Velthuis and Doorn 2005). For each species, we collected the adults from the different nests and grouped them into microcolonies of 10±3 individuals in plastic boxes (72 x 26.5 x 25 cm). Each microcolony was provided sucrose solution (40% v/v) and water *ad libitum*. Bumblebee microcolonies were also supplied with a commercial mixture of pollen (Icko-apiculture company, France).

### 108 Entomopathogen fungus

We used the strain EF3.5(1) of *M. robertsii* sampled in 2015 in an experimental INRAe vineyard (Villenave d'Ornon, France) (see Poidatz et al. 2018 for details). We selected this particular isolate because of its high virulence against hornets as shown by the direct inoculation and inoculation by transfer between hornets (Poidatz et al. 2018). *M. robertsii* was cultivated on a Petri dish (9 cm diameter) containing oat agar media (Oat flour 40g and Potatoes Dextrose Agar (Sigma Aldrich), Chloramphenicol (Sigma Aldrich), and permuted water) in a dark room. The fungus was transplanted every 2-3 months to ensure its healthy growth through nutrient resupplying.

### 116 Infections

We prepared spore solutions by mixing *M. robertsii* spores (extracted from culture media), distilled water, and 0.02% Tween80 (Sigma Aldrich) (Poidatz et al. 2018; Ponchon et al. 2022). The concentration of the solution was adjusted at 10³ spores/ml (identified in a preliminary experiment as the sublethal concentration for bumblebees, named *low concentration*) or 10⁷ spores/ml (named *high concentration*).
concentration) by counting spores in 10µl using a Neubauer cell. The control treatment was composed of distilled water and 0.02% Tween80. Each insect was exposed to one of these treatments by immersion of their entire body in the solution for 1 second. We used *M. robertsii* exposure by immersion because of its higher efficacy on hornet survival compared to exposure by contact (infected support or individual) or by food (Poidatz et al. 2018).

## Survival assays

We started the survival survey immediately after exposure to spores and the formation of microcolonies. From then on, we recorded the number of daily-dead insects and removed them from each microcolony for 8 consecutive days. We also monitored food consumption by weighing the containers of aqueous solutions (sucrose solution and water) every day.

## Statistical analysis

We ran all the analyses in R 4.3.3. We measured the effect of *M. robertsii* spore concentrations on survival for each species using a mixed-effect Cox model (*coxme* function, *survival* package, Therneau 2023) with treatment as a fixed factor and microcolony as a random effect. We ran a Cox proportional hazards regression model (*coxph* function, *survival* package, Therneau 2023) with the treatment as a fixed factor for wasps as they came from a single mother nest. We measured species sensibility differences by comparing the survival of species when exposed to a similar treatment. We used a mixed Cox model with species as a fixed factor and microcolony as a random factor. We calculated an estimate of the survival curve for each treatment within species using the *survfit* function (*survival* package, Therneau 2023) with the treatment as a fixed factor.

141

142

143

144
145

**Results**

146

**Hornets were the most impacted by spore exposure.** We first tested the influence of *M. robertsii*

147

concentrations on the survival of hosts (Fig. 1). For all three species, individuals exposed to high

148

concentrations of spores showed reduced survival compared to unexposed controls of the same

149

species (see Table 2 for statistics). Lowly-exposed hornets showed a significantly decreased survival

150

by 3.16 times (Cox, 95% confidence interval [CI]: 1.21-8.24, p=0.048) compared to controls. By

151

contrast, in bumblebees, there was no difference between lowly-exposed individuals and controls

152

(Table 2). Highly-exposed hornets showed a decreased survival by 36.83 times (Cox, CI: 14.58-93.08,

153

p<0.001) compared to controls. This reduction was much higher than the reduction by 6.43 times

154

(Cox, CI: 3.32-12.45) observed in wasps or by 1.61 times (Cox, CI: 0.76-3.44) in bumblebees at the

155

same spore concentration. Overall, the difference in survival between exposure doses was larger in

156

hornets than in wasps and in bumblebees (ANOVA, hornets: Treatment: X²=73.43, df=2, p<0.001;

157

wasps: Treatment: X²=40.67, df=1, p<0.001; bumblebees: Treatment: X²=7.88, df=2, p=0.019)

158

indicating that non-targeted species (wasps and bumblebees) were less susceptible to spore exposure

159

than hornets.

160

161

**Hornets and wasps lived longer than bumblebees at all spore concentrations.** We then compared

162

the effects of the parasite on host survival between species and for each treatment. In unexposed

163

controls, hornets and wasps showed a similar lifespan (Table 3 for statistics) and had a higher

164

probability of survival than bumblebees (Table 3). When they were exposed to the parasite at low or

165

high concentrations, hornets, and wasps also lived longer than bumblebees. At high concentrations,

166

wasps survived longer than hornets (Table 3).

167
168 **Discussion**

169 We exposed three Hymenoptera species to different concentrations of a strain of entomopathogenic
170 fungus recently reported to affect invasive yellow-legged hornets (Poidatz et al. 2018). Overall, for all
171 spore concentrations tested, hornets were more susceptible to spore exposure than wasps and
172 bumblebees, comforting the idea that *M. robertsii* is a good candidate for biocontrol against yellow-
173 legged hornets with negligible impact on non-targeted species.

174

175 Our parasite strain EF3.5(1) harmed differently the survival of the three insect species depending on
176 the exposure concentration. In particular, bumblebees had a shorter lifespan than the two other species
177 but seemed less sensitive to entomopathogenic concentrations than the hornets. Since *B. terrestris*
178 nests are found underground (Meyling and Eilenberg 2007), it is possible that they have already been
179 in contact with closely related strains of *M. robertsii* during their evolution. Cases of natural exposure
180 of bumblebees to *Metarhizium spp.* have already been recorded (Macfarlane et al. 1995; Schmid-
181 Hempel 2001). In the context of host-parasite coevolution, bumblebees could have developed greater
182 resistance to this parasite (Bérénos et al. 2009) which should less (or not) be the case for yellow-
183 legged hornets (invasive species, aerial niche) and wasps (local species but subterranean, or aerial
184 niche). Our observations that bumblebees are less sensitive to the parasite support this hypothesis of
185 an evolutionary adaptation. Note however that the fact that we observed a low survival of bumblebees
186 in our experiments could be explained by the health of the colonies we received. It would be
187 interesting to replicate these experiments on wild bumblebees although if this bias had been present, it
188 would have influenced our results in the direction of a greater susceptibility to the parasite.

189

190 Interestingly, the effects of *M. robertsii* were the strongest on the yellow-legged hornets. Our
191 observation that only half of the hornets survived four days after the exposition to spores is
192 comparable to the results in Poidatz et al. (2018), thereby confirming the efficiency of *M. robertsii*
193 against hornets. From a biocontrol perspective, the application method of *M. robertsii* seems key to
194 enhancing selectivity: the direct application of spores into hornets' nests could be interesting as it
allows a selective application and the reduction of potential non-targeted species contamination.

Moreover, it is possible that *M. robertsii* causes a range of early post-infection sublethal effects that impair hornet activity before they die. Indeed, many parasites and pathogens can alter the behavior of insects (Gómez-Moracho et al. 2017). *M. anisopliae* can alter the reproduction of cockroaches (*Blatella germanica*) (Quesada-Moraga et al. 2004), the nutrition of fruit flies (*Drosophila melanogaster*) (Toledo-Hernández et al. 2018) and also the locomotion activity of termites (Hassan et al. 2021). It is, for instance, possible that contaminated hornets are less able to leave their nest and forage, thus greatly limiting the opportunities of contaminating non-targeted species through contact with water, flowers, or honey bee hives. However, even when intraspecific contamination occurs, if the quantity of spores deposited on the surface is lower enough, it should be harmless to non-targeted species as our results demonstrate that bumblebees and wasps were not impacted by a low concentration of spores. The sublethal effects of *M. robertsii* on hornets, their ability to spread spores in the environment, and the realistic spores' transmission to other pollinators through contaminated surfaces should therefore be further taking into consideration to establish the safety of this biocontrol agent. Further studies are also necessary to assess the actual spores' quantity to inject in hornets' nests which will be dependent on the parasite transmission between nestmates. Many social insects use behavioral strategies to limit the transmission of parasites or pathogens in the colony and if so, to limit the establishment of parasites and pathogens in the nest such as grooming or reduced contact with infected individuals (Cremer et al. 2007). A preliminary experiment suggests hornets increase the duration of their grooming behavior when infected by *M. robertsii* (personal observations). These hygienic behaviors and potential others could be restrictive in an entomopathogen use context.

Our study suggests entomopathogenic fungi, and particularly *M. robertsii* strains, are good candidates for biocontrol of invasive yellow-legged hornets as the higher mortality found compared to the two non-target species reinforced its potential. However, as the effect on non-targeted species is concentration-dependent, there is a need to quantify the real risk of spores spreading in the environment by an infected individual to better assess the safety of this biological control agent on local biodiversity.
223

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<table><caption>318 Table 1: Details of all the insect colonies used.</caption><thead><tr><th></th><th>Hornet</th><th>Wasp</th><th>Bumblebee</th></tr></thead><tbody><tr><td>European evolution history</td><td>Invasive</td><td>Non-invasive</td><td>Non-invasive</td></tr><tr><td>Nest</td><td>Aerial</td><td>Underground<br>Occasionally aerial</td><td>Underground</td></tr><tr><td>Origin</td><td>Wild</td><td>Wild</td><td>Commercial</td></tr><tr><td>Number of nests</td><td>4</td><td>1</td><td>4</td></tr><tr><td>Origin<br>(Department code)</td><td>Laburgade (46)<br>Bouliac (33)<br>Bouliac (33)<br>Cahors (46)</td><td>Saucats (33)</td><td>Netherlands<br>Netherlands<br>Netherlands<br>Netherlands</td></tr><tr><td>Collection date</td><td>2021-10-12<br>2021-10-18<br>2021-10-25<br>2021-11-28</td><td>2021-12-10</td><td>2021-06-08<br>2021-06-08<br>2021-06-08<br>2021-06-08</td></tr><tr><td>Approximate nest size<br>(cm)</td><td>&lt;30<br>30-80<br>30-80<br>&gt;80</td><td>60-70</td><td>&lt;20<br>&lt;20<br>&lt;20<br>&lt;20</td></tr><tr><td>Number of microcolonies</td><td></td><td></td><td></td></tr><tr><td>Control</td><td>9</td><td>6</td><td>6</td></tr><tr><td>10<sup>3</sup> sp/ml</td><td>9</td><td></td><td>6</td></tr><tr><td>10<sup>7</sup> sp/ml</td><td>9</td><td>6</td><td>6</td></tr></tbody></table>

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**Table 2:** Comparison of survival probability across treatment and for each species using Cox model.

Treatments: C (control), L (low spore concentration), H (high spore concentration). HR (Hazard Ratio): the risk of an event occurring (in this case, the death of an individual) when comparing one treatment with another, CI (Confidence Interval): 95% confidence interval and p-value (the probability of this risk occurring at random).

<table><thead><tr><th>Species</th><th>Compared treatment</th><th>HR</th><th>CI</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="3">Hornet</td><td>C v.s. L</td><td>3.16</td><td>1.21-8.24</td><td>0.0484</td></tr><tr><td>C v.s. H</td><td>36.83</td><td>14.58-93.08</td><td>&lt;0.001</td></tr><tr><td>L v.s. H</td><td>11.66</td><td>5.50-24.72</td><td>&lt;0.001</td></tr><tr><td>Wasp</td><td>C v.s. H</td><td>6.43</td><td>3.32-12.45</td><td>&lt;0.001</td></tr><tr><td rowspan="3">Bumblebee</td><td>C v.s. L</td><td>1.92</td><td>0.87-4.21</td><td>0.238</td></tr><tr><td>C v.s. H</td><td>4.01</td><td>2.23-7.21</td><td>0.014</td></tr><tr><td>L v.s. H</td><td>1.61</td><td>0.76-3.44</td><td>0.431</td></tr></tbody></table>

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345 **Table 3:** Comparison of survival probability across species and for each treatment using Cox models.
346 Treatments: C (control), L (low spore concentration), H (high spore concentration). HR (Hazard Ratio): the risk of an event occurring (in this case, the death of an individual) when comparing one species with another, CI (Confidence Interval): 95% confidence interval and p-value (the probability of this risk occurring at random).
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<table><thead><tr><th>Treatment</th><th>Compared species</th><th>HR</th><th>CI</th><th>p-value</th></tr></thead><tbody><tr><td rowspan="3">Control</td><td>H v.s. W</td><td>1.38</td><td>0.23-8.46</td><td>0.935</td></tr><tr><td>H v.s. B</td><td>10.62</td><td>2.07-54.44</td><td>0.0128</td></tr><tr><td>W v.s. B</td><td>7.69</td><td>1.28-46.26</td><td>0.0666</td></tr><tr><td>Low concentration</td><td>H v.s. B</td><td>8.35</td><td>3.08-22.67</td><td>&lt;0.001</td></tr><tr><td rowspan="3">High concentration</td><td>H v.s. W</td><td>0.44</td><td>0.25-0.72</td><td>0.004</td></tr><tr><td>H v.s. B</td><td>1.75</td><td>1.05-2.92</td><td>0.083</td></tr><tr><td>W v.s. B</td><td>4.13</td><td>2.31-7.40</td><td>&lt;0.001</td></tr></tbody></table>

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<figure><img src="image_2.png" /></figure>

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<caption>**Fig. 1:** Survival probability of the three Hymenoptera tested during eight days: a) hornet, b) wasp, c) bumblebee. We exposed the insects to three treatments: control (green and upper line), low concentration of spores (10³ sp/ml, orange and middle line when three lines are present), high concentration of spores (10⁷ sp/ml, red and lower line). Solid lines represent experimental data and colored areas, the 95% confidence interval. Stars represent significant differences between groups calculated with a mixed-effect Cox model (a,c) or a Cox proportional hazards regression model (b).</caption>