Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) live in obligate nutritional relationships with ambrosia fungi (Farrell et al., 2001; Mueller et al., 2005; Biedermann and Vega, 2020). In this mutualistic relationship, the fungi serve as food sources for the beetles, whereas the fungi rely on the beetles for protection, dispersal, and maintenance (Biedermann and Taborsky, 2011; Hulcr and Stelinski, 2017; Nuotclà et al., 2019). Some ambrosia beetles are associated with a single dominant mutualistic fungus, while others are associated with multiple fungal symbionts (Batra, 1963; Gebhardt et al., 2004; Harrington et al., 2010; Kostovcik et al., 2015; Menocal et al., 2017, 2018a; Cruz et al., 2018, 2019, 2021; Saucedo-Carabez et al., 2018; Carrillo et al., 2019). Females in the Xylosandrus and Euwallacea species-complex are commonly associated with Ambrosiella (Microascales: Ceratocystidaceae) and Fusarium (Hypocreales: Nectriaceae) species, respectively, whereas females in the genera Xyleborinus and Xyleborus are strictly associated with Raffaelea species (Ophiostomatales: Ophiostomateacea), suggesting that ambrosia beetles exhibit fidelity toward specific groups of fungal symbionts (Biedermann et al., 2009, 2013; Kasson et al., 2013; Bateman et al., 2015; Mayers et al., 2015, 2020a,b; O’Donnell et al., 2015; Biedermann, 2020).

A few studies have investigated ambrosia beetle symbiont exchange or transition to specific fungi. Carrillo et al. (2014) reported for the first time the lateral transfer of nutritional symbionts between native and exotic ambrosia beetles. Skelton et al. (2019) documented that Xylosandrus compactus Eichhoff can exchange nutritional fungal symbionts among closely related Ambrosiella species, but a shift to more phylogenetically distant fungal species is less likely to occur. In a different study, Carrillo et al. (2020a) demonstrated that various shot hole borers (Euwallacea spp.) can survive and reproduce using each other’s Fusarium fungal symbionts. These studies suggest that transition to other nutritional fungal symbionts could occur more commonly among congeneric ambrosia beetle species that share closely related fungi and in species with relatively small mycangia [pre-oral (Xyleborus spp.) vs. mesonotal mycangia (Xylosandrus spp.)] (Joseph and Keyhani, 2021; Mayers et al., 2022).

Acquisition of new fungal symbionts may occur naturally when one ambrosia beetle species interacts with the fungi inside the gallery system of another species or, when mature females that search for new hosts, encounter fungi present in the environment (Kendra et al., 2011; Carrillo et al., 2014; Rassati et al., 2019). However, only the first mechanism is expected to involve nutritional fungal symbionts. Nutritional ambrosia fungi are not directly transmitted from mother to offspring. Larvae grow feeding on symbiotic fungi, but their gut contents are emptied during the pupal stage. Then, naïve adults acquire their symbionts via feeding (Xyleborus and Euwallacea) or by close contact with the gallery walls (Xylosandrus). This period of independent growth is a critical point for the acquisition of new nutritional fungal symbiont (s) (Six, 2012).

Some invasive ambrosia beetles partner with plant pathogens that can seriously damage forest and agricultural systems (Hanula et al., 2008; Hulcr and Dunn, 2011; Six, 2012; Ploetz et al., 2013; Hughes et al., 2017; Hulcr and Stelinski, 2017). Two new diseases currently threaten avocado production in South Florida. Laurel wilt (LW) and Fusarium dieback (FD) are caused by Harringtonia lauricola (T. C. Harr, Fraedrich & Aghayeva) Z. W. de Beer & M. Procter and Fusarium sp. nov., primary symbionts of Xyleborus glabratus Eichhoff and Euwallacea perbrevis Schedl, respectively (Harrington and Fraedrich, 2010; Eskalen et al., 2013; Smith et al., 2019; de Beer et al., 2022). Although X. glabratus is rarely detected in avocado orchards, other native and exotic ambrosia beetles in these orchards have acquired H. lauricola via lateral transfer (Carrillo et al., 2014; Kendra et al., 2020; Cloonan et al., 2022). The LW pathogen is now associated with the mycangial communities of several Xyleborus, Ambrosiodmus, Xyleborinus, and Xylosandrus species (Carrillo et al., 2014; Ploetz et al., 2017; Cruz et al., 2021). Unlike H. lauricola and its secondary vectors, the FD pathogen (s) are only vectored by Euwallacea spp. However, experiments evaluating the interaction between Fusarium spp. and other Scolytinae species outside the genus Euwallacea are lacking.

Florida avocado orchards house a diverse community of ambrosia beetles that often breed in trees affected by either LW or FD (Carrillo et al., 2012, 2016; Kendra et al., 2017, 2020; Menocal et al., 2018b, 2022; Owens et al., 2019a,b; Rivera et al., 2020; Cloonan et al., 2022). One of these species, Xyleborus bispinatus Eichhoff, is a Florida native beetle that has shown plasticity in its fungal symbiosis (Cruz et al., 2018). This species has become relevant due to its frequent partnership with H. lauricola, and its ability to transmit the pathogen to healthy avocado trees (Carrillo et al., 2014). However, the persistence of this association, the possible additional association with FD pathogen (s), and the effect of these pathogens on the beetle’s fitness are still understudied. To gain insights into this novel partnership, the symbionts of X. bispinatus were removed and the symbiosis was re-established with selected ambrosia fungi species. The specific objectives of this study were to: (1) determine whether X. bispinatus can feed and reproduce on H. lauricola, Fusarium sp. nov., and its original nutritional symbionts R. subfusca and R. arxii, (2) determine how stable these associations could be over several generations, and (3) determine the effect that each fungal symbiont has on offspring production.

The mean number of H. lauricola propagules recovered from X. bispinatus was relatively constant among generations (F = 0.4752; df = 4, 28; p = 0.7218) (Figure 1A). Females fed solely on H. lauricola increased reproduction (i.e., female offspring) in the first three generations and declined drastically in the last two (F = 9.4419; df = 4, 28; p < 0.0001) (Figure 1B). Switching to a H. lauricola diet resulted in significantly fewer female offspring than that recorded from control females with unaltered symbiosis throughout the five generations (F₁: X² = 9.3272, p = 0.0023; F₂: X² = 11.2295, p = 0.0008; F₃: X² = 7.4715, p = 0.0063; F₄: X² = 14.4687, p = 0.0023; F₅: X² = 12.4670, p = 0.0004) (Figure 1B). Notably, no individuals fed exclusively on H. lauricola survived after the fifth generation.

The number of R. subfusca propagules was not significantly different among generations (F = 0.8447; df = 4, 63; p = 0.6969) (Figure 2A). Females fed on R. subfusca diet produced a similar number of female offspring in the first three generations, but reproduction decreased in the last two (F = 6.2366; df = 4, 63; p = 0.0003) (Figure 2B). Females exposed to a R. subfusca diet produced an F₁ with a similar number of females to the unaltered control (X² = 2.5452, p = 0.1106). However, reproduction in subsequent generations was less than in the control (F₂: X² = 18.7822, p < 0.0001; F₃: X² = 18.9397, p < 0.0001; F₄: X² = 21.5985, p < 0.0001; F₅: X² = 25.1267, p < 0.0001) (Figure 2B).

The number of R. arxii propagules recovered from X. bispinatus varied significantly among generations (F = 5.2992; df = 4, 65; p = 0.0254). Raffaelea arxii CFUs were equally constant in the first three generations but increased in the last two (Figure 3A). Xyleborus bispinatus females transitioned to a R. arxii diet produced similar number of female offspring during five generations (F = 0.7125; df = 4, 65; p = 0.5864) (Figure 3B). The F₁ female offspring of R. arxii fed females was similar to the control with unaltered symbiosis (X² = 2.6894, p = 0.1010), but reproduction in subsequent generations was significantly less than in the control (F₂: X² = 12.2685, p = 0.0005; F₃: X² = 11.5424, p = 0.0007; F₄: X² = 19.3982, p < 0.0001; F₅: X² = 24.8068, p < 0.0001) (Figure 3B).

Finally, the number of Fusarium sp. nov. propagules recovered from X. bispinatus varied significantly among generations (F = 4.2888; df = 4, 95; p = 0.0261). CFUs of Fusarium sp. nov. increased from the first to the second generation but decreased in the third generation remaining similar throughout the subsequent generations (Figure 4A). Xyleborus bispinatus females that fed on Fusarium sp. nov. diet produced a higher number of female offspring in the last four generations than in the first one (F = 13.5657; df = 4, 95; p < 0.0001) (Figure 4B). In the first generation, Fusarium sp. nov. fed females produced similar number of female offspring to the control, but reproduction increased in subsequent generations, significantly more than in the control (Figure 4B).

The number of female offspring (F = 4.78; df = 4, 311; p < 0.0001) varied among fungal treatments (Figure 5). Females fed on a Fusarium sp. nov. diet produced the highest number of female offspring (Figure 5). Non-treated beetles (i.e., control) produced fewer individuals than those fed on Fusarium sp. nov., but significantly more than beetles reared on H. lauricola and the two Raffaelea species. There were no significant differences in the number of female offspring among H. lauricola and the Raffaelea treatments (Figure 5). The number of males ranged between 1 or 2 males per colony and did not vary among treatments.

Laboratory reared X. bispinatus exhibits flexibility in its symbiotic associations and can establish stable populations feeding on different fungal diets, including known fungal associates and phylogenetically distant fungi, which are primary symbionts of other ambrosia beetle genera. These changes in fungal diets resulted in persistent mutualistic associations, and some symbionts even increased the beetle’s reproduction. However, ambrosia beetles exhibit high fidelity in their symbiotic relationships with specific groups of fungi under natural conditions. The mechanisms that drive this fidelity have been a matter of active and recent research. In some bark and ambrosia beetles, symbiont fidelity is thought to be mediated by the mycangium (Bracewell and Six, 2015; Mayers et al., 2015, 2020a,b, 2022; Li et al., 2017; Skelton et al., 2019). For instance, Ambrosiella fungi thrive in the mycangium of Xylosandrus beetles, whereas distantly unrelated fungi are suppressed. The mycangium of X. bispinatus does not appear to provide this type of selectivity, allowing mutualistic interactions with distant unrelated fungi. Xyleborus spp. have small pre-oral mycangia (Li et al., 2018; Spahr et al., 2020; Mayers et al., 2020a), which are presumed to be less selective or more permissive than larger, more complex mycangia (Mayers et al., 2015, 2020a; Hulcr and Stelinski, 2017; Joseph and Keyhani, 2021). We infer that species with less mycangial selectivity such as X. bispinatus are more likely to establish mutualisms with new ambrosia fungi. Interactions with other organisms inhabiting beetle galleries can also play an important role in the fidelity of ambrosia symbiosis. Grubbs et al. (2020) and Nuotclà et al. (2021) suggested that bacteria may selectively exclude non-mutualistic fungi from the galleries and mycangia. In addition, the mycangia may provide an environment and specific nutrients that favor the growth of particular nutritional fungi (e.g., biological screening). The host tree physiology may also affect symbiotic fungi and, thus, their association with a given beetle. Some ambrosia beetles target stressed trees producing ethanol while others do not. Ethanol-enriched wood favors the growth of particular nutritional symbionts while suppressing others. This suggests that ethanol content in wood may influence the symbiont composition and thus the likelihood of novel symbiont acquisition (Ranger et al., 2018; Cavaletto et al., 2021, 2022; Lehenberger et al., 2021b). However, such selective effects of substrate were not present in our artificial medium and may influence the effects of particular fungi on beetle fitness in vivo.

Some ambrosia beetles can use one or more closely related fungi (Skelton et al., 2019; Carrillo et al., 2020a) as nutritional food sources. Raffaelea species are common partners of Xyleborus species (Harrington et al., 2010; Campbell et al., 2016; Cruz et al., 2018, 2019; Saucedo-Carabez et al., 2018); therefore, the survival and reproduction of X. bispinatus on R. subfusca and R. arxii diets was expected. By contrast, X. bispinatus reared on H. lauricola declined progressively and stopped reproducing after five generations. This result suggests that H. lauricola was a suboptimal food source for X. bispinatus, which contrasts with previous reports indicating that this fungus enables high fecundity rates and significant brood production in this beetle (Saucedo et al., 2017). Our results also suggest that X. bispinatus benefits from feeding upon multiple ambrosia fungi. Colonies fed singly on H. lauricola or Raffaelea spp. had fewer female offspring than colonies from the control treatment (Figures 1B, 2B, 3B). In agreement with these results, a previous study demonstrated that wild X. bispinatus increased reproduction when H. lauricola was added to their diet (Menocal et al., 2018a). Altogether, the available information suggests that X. bispinatus benefits from the new partnership with H. lauricola, but it is unlikely to use it as its primary symbiont.

Mutualisms between Euwallacea beetles and Fusarium spp. are well recognized (Freeman et al., 2012, 2016; Carrillo et al., 2016, 2020b; O’Donnell et al., 2016; Kendra et al., 2022), but associations between Fusarium spp. and other ambrosia beetles are presumed to be incidental and non-mutualistic (Biedermann et al., 2013; Kostovcik et al., 2015; Bateman et al., 2016; Biedermann, 2020). In our study, X. bispinatus engaged in a persistent mutualism with Fusarium sp. nov. [currently >20 generations – data not shown]. This fungus sustained significantly more female offspring than any other single fungus or the unmanipulated colonies with several fungal symbionts (Figure 5). This result is noteworthy because Fusarium spp. belongs to a distant unrelated taxonomic order (Hypocreales) than most other ambrosia fungi (Ophiostomatales). Interestingly, Fusarium solani (Norris and Baker, 1967, 1968) and Fusarium sp. (AF-9) (Kasson et al., 2013) were reported as mycangial fungal partners of Xyleborus ferrugineus Fabricius. Moreover, X. ferrugineus and X. bispinatus were considered synonyms until Atkinson et al. (2013) recognized them as separate species. These results and our findings suggest that both beetles may exhibit mutualisms with Fusarium spp. and that these symbiotic relationships could be more common than previously presumed.

Our results suggest that exotic ambrosia beetles can induce changes in resident ambrosia beetle communities and their symbionts. In their native range, ambrosia beetles are rooted in a network of interactions with native symbiotic fungi, including endophytes, plant pathogens, and mutualists (Simmons et al., 2016; Carrillo et al., 2019). In invaded areas, ambrosia beetles face novel environments, new host opportunities, and potentially novel symbionts (Rassati et al., 2019). In some cases, invasive beetles may not survive in-or adapt to-a new environment (e.g., X. glabratus in avocado) but their fungi (e.g., H. lauricola) can establish novel partnerships with resident ambrosia beetles (Carrillo et al., 2014; Ploetz et al., 2017; Cruz et al., 2021). The symbionts’ ecology could play a major role in developing novel mutualisms. Most ambrosia beetle symbionts only grow in the xylem-sapwood in close proximity to the beetle galleries and inside the beetle mycangia. Unlike most Raffaelea species, H. lauricola displays systemic growth in its lauraceous hosts (Lynch et al., 2012; Ploetz et al., 2012; Campbell et al., 2017). This could be a significant contributing factor for the lateral transfer of this fungus to multiple ambrosia beetles in areas of recent invasion in the USA. For ambrosia fungi with localized growth, sympatric beetle breeding and natal gallery overlapping may be required for fungal exchange. Under these circumstances, competitive fungi displaying fast growth and the ability to nourish ambrosia beetles can outcompete other symbionts and engage in new partnerships. In our experiment, Fusarium sp. nov. appeared to colonize the rearing substrate faster than other fungi. Fusarium sp. nov. may be more nutritious or better at translocating elements from the rearing plug to its fruiting bodies. Fungal mutualists translocate essential elements such as calcium (Ca), nitrogen (N), phosphorous (P), potassium (K), magnesium (Mg), manganese (Mn), and sulfur (S), through their hyphae from the sapwood to fruiting structures in the galleries (Six and Elser, 2019; Lehenberger et al., 2021a). These elements are essential for fungal and beetle growth (Filipiak et al., 2016). A better understanding of the nutritional quality and growth patterns of ambrosia fungi may help identify species with invasive potential and predict novel ambrosia symbioses.

In conclusion, species such as X. bispinatus with fungus-feeding plasticity are likely to establish new symbiotic relationships with exotic ambrosia fungi like H. lauricola. However, our study was conducted under simplified laboratory conditions. Interactions with multiple abiotic (e.g., temperature, relative humidity) and biotic (plant-secondary compounds and other microbial players) factors under natural conditions may lead to other fitness effects and more complex associations. The ecology of the fungal symbionts may also play an important role in novel ambrosia symbiosis. Further experiments on ambrosia beetle symbiosis may help identify patterns and characteristics that make some species more likely to become invasive.

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors. The data (i.e., fungal sequences) presented in the study were uploaded to the NCBI GenBank (Home - Nucleotide - NCBI (https://www.ncbi.nlm.nih.gov) database and are publicly available (Accession numbers are included in Table 1).

OM, LC, and DC conceived and designed the study. OM and LC performed the experiments and collected the data. OM analyzed the data. PK and MB contributed with reagents/materials/analysis tools. OM wrote the initial draft of the manuscript. LC and DC provided feedback and comments on previous versions of the manuscript. PK and DC reviewed and edited the final version of the manuscript. All authors contributed to the article and approved the submitted version.

This research was funded by NIFA grant 2015–51181-24257 to Daniel Carrillo and by a Non-Assistance Cooperative Agreement between the USDA-ARS and the University of Florida (number: 58-6038–8-004).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.