Mortality of native and invasive ladybirds co-infected by ectoparasitic and entomopathogenic fungi

Harmonia axyridis is an invasive alien ladybird in North America and Europe. Studies show that multiple natural enemies are using Ha. axyridis as a new host. However, thus far, no research has been undertaken to study the effects of simultaneous infection by multiple natural enemies on Ha. axyridis. We hypothesized that high thallus densities of the ectoparasitic fungus Hesperomyces virescens on a ladybird weaken the host’s defenses, thereby making it more susceptible to infection by other natural enemies. We examined mortality of the North American-native Olla v-nigrum and Ha. axyridis co-infected with He. virescens and an entomopathogenic fungus—either Beauveria bassiana or Metarhizium brunneum. Laboratory assays revealed that He. virescens-infected O. v-nigrum individuals are more susceptible to entomopathogenic fungi, but Ha. axyridis does not suffer the same effects. This is in line with the enemy release hypothesis, which predicts that invasive alien species in new geographic areas experience reduced regulatory effects from natural enemies compared to native species. Considering our results, we can ask how He. virescens affects survival when confronted by other pathogens that previously had little impact on Ha. axyridis.


INTRODUCTION
In nature, hosts may be exploited by more than one natural enemy. These organisms interact with each other and with their hosts (Furlong & Pell, 2005). Complex interactions shape the population structure and dynamics of all organisms in the system. Natural enemies also compete with one another, and the impact on the host can be either synergistic, additive, or antagonistic (Shapiro-Ilan, Bruck & Lacey, 2012). These interactions can be manifested in various aspects of host fitness or mortality. For example, biological control of Drosophila suzukii (Diptera, Drosophilidae), an important pest of fruit and berry crops, can be improved by treatments combining multiple natural enemies, which have an additive effect (Renkema & Cuthbertson, 2018). At the same time, dual infections (even if causing an increase in host mortality) may be deleterious to one or both pathogens in terms of pathogen growth, fecundity, or other fitness parameters.
Hesperomyces virescens (Ascomycota, Laboulbeniomycetes, Laboulbeniales) is a common obligate ectoparasite of ladybirds (Roy et al., 2016;Haelewaters et al., 2017). Although known since 1891, it was shown only recently that He. virescens is in reality a complex of multiple host-specific species (Haelewaters, De Kesel & Pfister, 2018). Contrary to most multicellular fungi, He. virescens as well as other members of the order Laboulbeniales lack hyphae; instead they form 3-dimensional multicellular thalli by determinate growth (Blackwell, Haelewaters & Pfister, in press). Laboulbeniales, including He. virescens, cannot be grown in axenic culture and no asexual stages are known, which makes their study challenging (Haelewaters, Blackwell & Pfister, 2021). Given locally high prevalence of He. virescens on ladybird hosts (Riddick & Cottrell, 2010;Haelewaters et al., 2017) and the abundance of entomopathogenic fungal strains in the environment (Roy & Cottrell, 2008), we examined mortality of native and invasive He. virescens-infected ladybirds exposed to either Beauveria bassiana or Metarhizium brunneum (Ascomycota, Sordariomycetes, Hypocreales) (sensu Cottrell & Shapiro-Ilan, 2003;Cottrell & Shapiro-Ilan, 2008). Because He. virescens forms a branched, non-septate, rhizoidal haustorium (Weir & Beakes, 1996) that penetrates the host's exoskeleton and makes contact with the body fluid for nutrient uptake, we hypothesized that high thallus densities with concomitant haustorial formation by He. virescens weaken host defenses, thus increasing the host's susceptibility to infection by other natural enemies. With this experiment, we assess how He. virescens affects ladybird survival when exposed to other natural enemies that alone have little impact on Ha. axyridis populations, and we compare results with a North American-native ladybird of similar body size, Olla v-nigrum. If He. virescens-on its own and in combination with other natural enemies-significantly impacts survival of the invasive ladybird but not the native one, then the results of this work could have consequences toward a pest management strategy to control infestations of vineyards and agroecosystems by Ha. axyridis.

Field collections and laboratory colonies
Harmonia axyridis and Olla v-nigrum ladybirds were collected for the purpose of establishing laboratory colonies of Hesperomyces-infected and non-infected ladybirds. Specimens were collected at overwintering sites at the 485-ha USDA-ARS, Southeastern Fruit and Tree Nut Research Laboratory, located in Byron, Georgia, USA (32.657792, -83.7383580). Sex and age of field-collected specimens were not determined to reduce dispersal of fungal propagules (Cottrell & Riddick, 2012). All specimens were brought to the laboratory and housed in individual Petri plates (10 cm diam.) with 1/3 of a piece of a cotton dental wick (Deerpack Products, LLC, Miami, Florida) drenched in water for hydration. Ladybirds were housed in environmental chambers at 25 ± 1 • C and photoperiod of 14:10 (L:D) h. Food was provided 3 × per week in the form of Ephestia kuehniella eggs (Lepidoptera, Pyralidae) and an artificial meat-based diet (Beneficial Insectary, Redding, California). Olla v-nigrum and Ha. axyridis ladybirds were maintained within the Petri plates for 14d and 21d (Cottrell & Riddick, 2012), respectively, at which time ladybirds were visually examined for presence of Hesperomyces using a dissecting microscope at 50× magnification. Eggs were harvested from ovipositing ladybirds and used to establish clean (free from fungal growth) laboratory-reared colonies of ladybirds with known age.

Laboratory rearing of ladybirds
During examination for presence/absence of Hesperomyces, ladybirds were divided into two groups, infected and non-infected. Both groups of ladybirds were placed into plastic rearing containers of 19 × 13.5 × 9 cm (Pioneer Plastics, North Dixon, Kentucky), which were modified with two 3-cm diameter circular openings, one that was covered by 1 × 1 mm mesh to allow for air flow; and the second that was covered with a removable #7 rubber stopper to allow for feeding routinely as well as adding newly emerged laboratoryreared ladybirds. Routine maintenance included transferring ladybirds into fresh rearing containers at the end of each 7d period, which included nutrient supplementations of laboratory-reared yellow pecan aphids, Monelliopsis pecanis (Hemiptera, Aphididae).
The first laboratory generation of adults emerged about one month after placement in rearing containers. Emerging adults were placed into fresh rearing containers and stored into a separate incubator (25 ± 1 • C, 14:10 (L:D) h) for 7 days. Similar to field-captured O. v-nigrum and Ha. axyridis, M. pecanis aphids were used as a diet augmentation. As the study progressed, we also incorporated black pecan aphids, Melanocallis caryaefoliae (Hemiptera, Aphididae), in the ladybird diet (3 × per week).

Artificial transmissions of Hesperomyces
Not only did we need the ladybirds for our experiments to be of the same age, we also needed to artificially infect a subset of these ''clean'' laboratory-grown, adult ladybirds with Hesperomyces virescens. Exposure to Hesperomyces was conducted via tumbling of field-captured 'source' ladybirds (infected with Hesperomyces) with randomly selected laboratory-reared 'target' ladybirds (Cottrell & Shapiro-Ilan, 2008). A total of 25 target ladybirds were mixed with 5 Hesperomyces-infected source ladybirds in a 1.6 × 5.8 cm glass tube, which was placed on a hot-dog roller (Nostalgia Electrics, Green Bay, Wisconsin) for 5 min. This procedure was repeated for at least 160 target ladybirds of both species. We only performed intra-specific artificial transmissions of Hesperomyces, meaning from source Ha. axyridis to target Ha. axyridis and from source O. v-nigrum to target O. v-nigrum. Both Hesperomyces-exposed target ladybirds and clean (unexposed) ladybirds were fed a diet of M. pecanis aphids for 24 h. We did a second tumbling experiment using randomly selected emerged adults from the second cohort of laboratory-reared colonies. More tumbling experiments were performed to increase quantities of Hesperomyces-infected ladybirds, but source/target numbers were changed to 100/40.
To reduce competition for food, ladybirds from all laboratory colonies were transferred from the plastic rearing containers to 14-cm diameter Petri plates. Ladybirds were provided with water ad libitum, E. kuehniella eggs, and artificial meat-based diet. Finally, for assay preparation, the ladybirds were transferred back to clean 19 × 13.5 × 9 cm plastic rearing containers by species.

Dual fungal infections assay
Within 24 h preceding the assay, 160 non-infected and 160 Hesperomyces-infected ladybirds of each species (Ha. axyridis and O. v-nigrum) were each placed into sterile test tubes, one individual per test tube. Test tubes were then closed with a sterile foam stopper to prevent ladybirds from escaping while allowing for air flow. Infected ladybirds were divided into categories according to numbers of thalli per specimen. Because the assay would assess potential interactions between fungal infections, we aimed at selecting heavily Hesperomyces-infected ladybirds; as a baseline, we only used specimens in our bioassays with 14 or more thalli each.
The assay started by pipetting a 1 mL of 2.5 × 10 5 conidia/mL suspension to each test tube (Cottrell & Shapiro-Ilan, 2003;Cottrell & Shapiro-Ilan, 2008). Treatments included native B. bassiana (native Bb), a commercial B. bassiana strain (GHA Bb; Mycotrol ES, Mycotech, Butte, Montana), M. brunneum strain F52 (Mb, isolated from a tortricid moth, Austria 1971; Novozymes, Franklinton, North Carolina), and double-distilled water (ddH 2 O) as a control treatment. Ladybirds were submerged and swirled for 5 s, after which the suspension was removed again using a pipette and each ladybird was placed into a 6 cm-diameter Petri plate.
Any remaining droplets of excess suspension was removed by touching only the droplet with a Kimwipe tissue (Kimtech Science Brand, Kimberly-Clark Worldwide, Roswell, Georgia). Petri plates with treated ladybirds were placed into an incubator (25 ± 1 • C, 14:10 (L:D) h). Food and cotton rolls drenched in water were provided ad libitum, and Petri plates were replaced as needed in all treatments and replications simultaneously. Ladybirds were observed for mortality and entomopathogen-induced mycosis at day 14. During assay #1, we made daily observations for ladybird mortality and mycosis. Upon death of a given ladybird, ample water was added to the cotton roll to provide moisture for entomopathogen growth and Parafilm was applied around the Petri plate to prevent spreading of the fungus. Deaths of ladybirds and visual confirmations of mycosis were recorded.
We performed 8 different treatments for each ladybird species: (1)  . In a single assay, we replicated every treatment 3 or 4 times. We performed the entire assay with all treatments and replicated 3 times, using 6-10 ladybirds for each treatment. Note that M. brunneum treatments were used only in assay #3 (Table S1). Over all assays done during this study, we used 1,289 specimens of ladybirds (667 O. v-nigrum and 622 Ha. axyridis) (Table S2).

Statistical analyses
All statistical analyses were performed in the R language and open-access environment for statistical computing (version 3.5.0; R Core Team, 2018). We used generalized linear mixed models (function glmer(), R-package lme4; Bates et al., 2015) to analyze the effect of the different treatments (GHA Bb, native Bb, Mb) on the survival of Ha. axyridis and O. v-nigrum in relation to prior infection with Hesperomyces. We modeled the binary response variable survival (alive/dead) of each ladybird individual for both host species separately, and used Hesperomyces infection status as well as the interaction of Hesperomyces infection status with treatment as explaining variables. Further, to correct for variation within replicates and assays, we included the random effect of treatment nested in replicate nested in assay. We compared our candidate models to a respective Null-model using likelihood ratio tests and, furthermore, calculated pseudo R 2 -values (function r2(), R package sjstats; Lüdecke, 2018) to evaluate model fit. To visualize the modeling results and obtained model estimates as forest plots, we used the function plot_model() implemented in the R package sjstats (Lüdecke, 2018). For assay #1, we further fitted Kaplan-Meier curves to daily mortality data and tested for significant differences in mortality between ladybird species using the function survfit() of the R package survival (Therneau & Lumley, 2019).

RESULTS
Our candidate models for both host species Ha. axyridis and O. v-nigrum were significantly better at explaining survival relative to chance variation (Chi-squared test, χ 2 = 156.7, P < 0.001; χ 2 = 153.0, P < 0.001, respectively). The overall model fit was high for both candidate models (Ha. axyridis: Nagelkerke's R 2 = 0.40; O. v-nigrum: Nagelkerke's R 2 = 0.53) suggesting the variance is well described by our applied models. We found a significant negative effect on ladybird survival of the M. brunneum treatment on He. virescens-negative Ha. axyridis (Fig. 1, Table 1), whereas B. bassiana treatments did not affect the survival of He. virescens-negative individuals. Infection with He. virescens significantly affected Ha. axyridis survival over all treatments (Fig. 1). However, there was no additional effect detectable among co-infection treatments for He. virescens-positive ladybirds (Table 1). Each treatment applied to O. v-nigrum had a significantly negative effect on the survival for both He. virescens-negative and -positive ladybirds (Fig. 1, Table 1). Finally, we found an additional negative effect of all co-infection treatments on the survival of He. virescens-positive O. v-nigrum (Fig. 1, Table 1). These results suggest that there is no effect of dual infections on Ha. axyridis, whereas O. v-nigrum is highly affected by simultaneous exposure to both He. virescens and an entomopathogenic fungus. Percentages of ladybird mortality by treatment are also presented in tabulated form in Table S3.
When comparing the daily survival of Ha. axyridis and O. v-nigrum, no significant differences were found in Hesperomyces-positive only treatments (log rank test, P = 0.4). However, when co-infected O. v-nigrum showed a significantly lower survival compared to Ha. axyridis for GHA and native B. bassiana strains (log rank test, P = 0.0014 and P <0.001, respectively). Figure 2 shows how survival is significantly different between the two ladybird species when co-infected with both Hesperomyces and B. bassiana (GHA and native).

DISCUSSION
Research on the additive effects of multiple natural enemies on a given host is rare, likely because of the complexity involved in designing robust bioassays that include all partners of the system. Combining the natural enemies Orius insidiosus (Hemiptera, Anthocoridae) and Heterorhabditis bacteriophora (Rhabditida, Heterorhabditidae) resulted in the largest decline in larvae of Drosophila suzukii (Renkema & Cuthbertson, 2018). This fruit fly causes major economic losses to fruit crops in its invasive range, spanning North and South America and Europe (Lee et al., 2011). The addition of O. insidiosus resulted in 50% fewer D. suzukii larvae compared to treatment with only H. bacteriophora. Plutella xylostella (Lepidoptera, Plutellidae), an important cosmopolitan pest of brassicaceous crops, offers another example. This organism shows resistance to almost all chemical insecticides (Sarfraz, Keddie & Dosdall, 2005). Pandora blunckii and Zoophthora radicans (Zoopagomycota, Entomophthoromycetes, Entomophthorales) both infect P. xylostella in the field. In co-inoculation studies with Pa. blunckii and Z. radicans in P. xylostella larvae, larval cadavers (three days post-mortality) were most frequently found with conidia of a single entomopathogen, usually the one that had been inoculated first (prior ''residency'')meaning that the other species was excluded (Sandoval-Aguilar et al., 2015). In general, the presence of competing species in the same host resulted in a decreased proportion of P. xylostella larvae that were infected compared to single inoculations.   Regarding Ha. axyridis, the following co-infections of natural enemies have been observed in nature: He. virescens + Coccipolipus hippodamiae mites (Acarina, Podapolipidae) in the USA, Austria, and the Netherlands (Christian, 2001;Riddick, 2010;Raak-van den Berg et al., 2014) and He. virescens + Parasitylenchus bifurcatus nematodes (Nematoda, Allantonematidae) in the Czech Republic, Germany, and the Netherlands (Raak-van den Berg et al., 2014;Haelewaters et al., 2017;Herz & Kleespies, 2012). Given the status of Ha. axyridis as an invasive alien species, these findings demand a better understanding of interactions among the different natural enemies and their potential role in limiting populations of Ha. axyridis. To date, bioassays to determine mortality of ladybirds induced by infection by one or more natural enemies have not yet been performed. Likewise, bioassays including Laboulbeniales have only been carried out in one study (Konrad et al., 2015).
When we started this study, He. virescens was considered a single species with many ladybird hosts, potentially with strains that infect only a single species or closely related ones (Cottrell & Riddick, 2012). However, it was recently shown that He. virescens is a complex of multiple species, each with its own ladybird host (Haelewaters, De Kesel & Pfister, 2018), which calls for caution in reviewing reports from the extensive body of literature on Hesperomyces findings (summarized in Haelewaters & De Kesel, 2017). This also means that isolates of He. virescens from Ha. axyridis and O. v-nigrum in fact represent two different species of Hesperomyces. In other words, the experiments in the current study allow us to make comparisons between two host species, each with their own specific fungal parasite. Future experiments are needed to further disentangle these interactions. Even though horizontal transmission of Hesperomyces among ladybird species is rare (Cottrell & Riddick, 2012), we should try to infect Ha. axyridis and O. v-nigrum ladybirds with the species of Hesperomyces specific to Olla and Harmonia, respectively, perform bioassays, and compare mortality rates under different treatments with our current results. Analyzing interactions among natural enemies only make sense when the taxa considered represent single biological species.
We found a significant negative effect of Hesperomyces-only infection on the survival of both ladybird hosts (Fig. 1A, Table S3). Previous work has shown Hesperomyces infections to result in decreased mating frequency of female ladybirds, lower (male) survival rates in winter, and impeded sensing ability and flexibility of legs in heavily infected ladybirds (Nalepa & Weir, 2007;Riddick, 2010;Haelewaters et al., 2017). One study implicated parasitism by He. virescens as the cause of late summer mortality of Chilocorus bipustulatus ladybirds (Kamburov, Nadel & Kenneth, 1967) but this was later disputed based on controlled laboratory experiments (Applebaum et al., 1971). Our research is the first to explicitly link Hesperomyces infection with increased ladybird mortality.
Our findings on the effects of Hesperomyces on ladybird survival provided a unique opportunity for setting up dual infection assays-the first such experiments to be conducted on ladybirds. When first infected with He. virescens and then exposed to either B. bassiana or M. brunneum, Ha. axyridis mortality was not increased. This result was unexpected. We had hypothesized that Ha. axyridis with high thallus densities of He. virescens would have lowered host defenses against other pathogens. In contrast, the mechanism fostering low susceptibility of Ha. axyridis to entomopathogenic fungi (Cottrell & Shapiro-Ilan, 2003;Knapp et al., 2019) is not compromised by infection with He. virescens. Similarly, infection of O. v-nigrum by He. virescens-only increased mortality but-in contrast to Ha. axyridis-there was significantly higher mortality when co-infected by entomopathogenic fungi. Differential susceptibility to entomopathogenic fungi was reported by Cottrell & Shapiro-Ilan (2003), who showed that native B. bassiana was pathogenic to O. v-nigrum but not to Ha. axyridis. We confirm these results regarding the native strain but we also found the same differential pattern for the GHA strain of B. bassiana, whereas in the earlier study this strain was reported to be pathogenic to neither ladybird species (Cottrell & Shapiro-Ilan, 2003). It is perhaps surprising that we detect the GHA strain to be pathogenic to native ladybirds in contrast to the previous results, but ladybird populations may become more susceptible over time for various reasons and natural enemies also become better adapted (Knapp et al., 2019). We note that differential susceptibility has also been reported for entomopathogenic nematodes-again, Ha. axyridis was less susceptible compared to O. v-nigrum (Shapiro-Ilan & Cottrell, 2005).
In addition, our data are the first account of differential susceptibility to M. brunneum between the invasive Ha. axyridis and the native O. v-nigrum. Whereas infection with M. brunneum had a significantly negative effect on the survival of He. virescens-negative Ha. axyridis, this effect was not visible in the dual infection treatment. The infection with Laboulbeniales probably decreased the susceptibility of Ha. axyridis to infection by M. brunneum, similar to the findings of Konrad et al. (2015). These authors found that Laboulbenia-infected Lasius neglectus ants (Hymenoptera, Formicidae) showed a decreased susceptibility to M. brunneum. This protection against Metarhizium was positively correlated with parasite load. Information on the parasite load of He. virescens on ladybirds in nature is nonexistent. In our bioassays, we selected ladybirds bearing 14 or more fungal thalli as He. virescens-positive specimens. Previous work from a long-term ATBI project in the Netherlands (van Wielink, 2017) points at an average of 19.8 ± 4.9 thalli and a maximum of 129 thalli per Ha. axyridis specimen (n = 270). No such data are available for O. v-nigrum. In other words, based on the available information, the artificial parasite load in our bioassays seems to closely mimic the natural conditions.
Our results provide direct support for the enemy release hypothesis (Jeffries & Lawton, 1984). This hypothesis is illustrative for the success of invasive alien species and stipulates that an invasive species in new geographic regions will experience reduced regulatory effects from natural enemies compared to native species, resulting in increased population growth of the invasive species (Colautti et al., 2004;Roy et al., 2011). However, invasions are dynamic and this escape-from-enemies could be lost as invasive species acquire new enemies over time (Hokkanen & Pimentel, 1989;Schultheis, Berardi & Lau, 2015;Haelewaters et al., 2017). Support for enemy release explaining the success of Ha. axyridis has come from two studies that reported decreased susceptibility of Ha. axyridis to entomopathogenic fungi (Cottrell & Shapiro-Ilan, 2003) and entomopathogenic nematodes (Shapiro-Ilan & Cottrell, 2005) compared to the native American ladybird species. Our work adds another level of complexity by the addition of a second natural enemy to the interactions. Again, we find differential susceptibility between the invasive and native ladybird species-with a reduced regulatory effect of the tested natural enemies on Ha. axyridis.

CONCLUSIONS
In this paper, we show a negative effect of infection by Hesperomyces virescens on the survival of both Harmonia axyridis and Olla v-nigrum ladybirds (Fig. 1A). This is the first study to unequivocally link Hesperomyces infection with increased host mortality and only the second to perform bioassays with hosts co-infected with Laboulbeniales and a second entomopathogenic fungus (Konrad et al., 2015). However, the susceptibility to a secondary entomopathogenic fungus was only elevated in the native American ladybird species (O. v-nigrum), whereas the globally invasive Ha. axyridis showed no significant increase in mortality when co-infected with either Beauveria bassiana or Metarhizium brunneum (Figs. 1 and 2). These findings are consistent with the enemy release hypothesis (Jeffries & Lawton, 1984) and highlight the difficulty in controlling this invasive alien species. Future studies are needed to elaborate population-specific effects on native and commercial strains of entomopathogenic fungi used in pest control.