Discussion

Our observations are largely consistent with other studies on tephritids and show a larval microbiome in Z. cucurbitae dominated by Proteobacteria and to a lesser extent by Firmicutes and Bacteroides. Many of the abundant genera also belonged to the Enterobacterales. The larval core microbiome identified for the F1 Z. cucurbitae samples processed in this study included seven bacterial genera (Acinetobacter , Enterobacter , Klebsiella ,Paenibacillus , Pseudomonas , Stenotrophomonas andSphingobacterium ). Enterobacter , Pseudomonas ,Acinetobacter and Klebsiella were also reported as core bacterial genera of other larval tephritid microbiomes . Enterobacteriaceae such as Enterobacter and Klebsiellaprovide pectinolytic degradation and fixate nitrogen to their tephritid hosts . In contrast, the genera Stenotrophomonas ,Paenibacillus and Sphingobacterium have rarely been reported before in tephritids . Some of these bacterial genera might play a role in plant detoxification as they are known to degrade a variety of toxic compounds . Some of our results are not in line with patterns reported in earlier studies , but this is not surprising considering the high variability already described in the microbiomic diversity of the closely related genera Bactrocera andZeugodacus . Part of the variability reported for the microbiomic patterns of Bactrocera /Zeugodacus, and more in general for frugivorous tephritids can certainly be related to the different lab rearing and experimental conditions reported in the literature . However, discrepancies can also be observed between studies targeting wild- or semiwild- populations (e.g. . For example, previous research on wild populations of Z. cucurbitae suggested a close association with Ochrobactrum , while this bacterial genus has an inconsistent presence across our samples.
One of the objectives of this study was to explore changes in the microbiome composition of a cucurbit-feeder fly feeding on a non-cucurbit host (Solanum melongena , Solanaceae) with the expectation that the shift to a non-cucurbit diet would produce major and consistent changes in its microbiome. This only partially happened. First, changes in the microbiome composition mostly concerned less abundant taxa as differences were observed mainly from the analysis of presence/absence data (Unweighted Unifrac distances) rather than from the weighted abundances (Generalized Unifrac distances). This is consistent with recent studies on termites and wood-eating cockroaches that also found that dietary shifts mainly resulted in changes of rare microbes while abundant microbial taxa remained generally stable . It suggests that low abundant species might have a key role in host plant adaptation. Second, our results show that local factors other than diet, have a deep impact on the microbiome diversity of Z. cucurbitaeand heavily contribute to the variability of patterns observed. Regardless the complex interactions between diet and local-geographical factors, we need to consider that this study mainly focused on F1 larvae. So, one explanation for these results might be that several generations are required before reaching stable and consistent microbiome assemblages in flies shifting to a novel “atypical” diet. A recent study in the whitefly Bemisia tabaci , (Hemiptera, Aleyrodidae) for example, found that an initial host switch from watermelon to the less suitable host pepper did not result in major changes in microbiome composition and structure . Yet, major microbiome changes did occur in subsequent generations. Moreover, the same study also showed that the first generation following the host shift had a lower survival rate, which increased again in subsequent generations, suggesting that the microbiota was involved in longer-term adaptation to the new host plant. Similarly, in the diamondback moth (Plutella xylostella , Lepidoptera, Plutellidae) a host shift to novel pea hosts resulted in major microbiome changes only in later generations, not in the first generation .
Another hypothesis for the lack of straightforward relationships between diet and microbiome composition is that microbiome changes mainly involve bacterial taxa that serve important metabolic functions but which are so rare that they remain undetected. Indeed, rare members of microbial communities sometimes perform key functions in these communities . Desulfosporinus , for example, represents only 0.006% of reads detected in microbial peatland communities and yet it contributes the most to sulfate reduction . Likewise, the capacity of freshwater microbial communities to degrade pollutants is severely reduced when rare taxa disappear . So, rare taxa may support a community with a wide range of metabolic functions that might only be important under specific circumstances such as the use of an unconventional host plant species.
We also need to consider that changes in host plant use might not necessarily translate in compositional changes of microbiome assemblages but rather result in changes of the gene expression patterns of the holobiont (i.e. sensu , i.e. the insect + its microbiota living on the host plant), which is the central unit of symbiogenesis and evolution . Symbiont microbial pectinases complement the insect endogenous cellulases and xylanases in herbivorous beetles (Cassidinae) and the pectinolytic range of symbiotic bacteria of the genusStammera has been associated to the diversity of host plants that can be attacked by these beetles . Accordingly, the flexibility of insect and microbiome gene expression patterns could possibly provide a complementary / alternative explanation to the complex relationships observed between microbiome assemblages, feeding preferences and range expansion of Z. cucurbitae .
Additionally, differences in microbiome composition and structure between larvae feeding on the non-cucurbit host could have also been affected by differences in their parental microbiomes, since significant heterogeneity was detected between the microbiomes of the parental lines and since in tephritids, at least part of the microbiome is vertically transmitted .
This study describes the effects of parent-offspring host switches in a cucurbit feeder fly and reveals complex microbiome responses in wild populations. As in De Cock et al. (2020), our results stress the importance of local effects on microbiome diversity and composition and the important role of processes which don’t seem directly related to host plant diet. We identified the main bacterial genera responsible of the patterns observed and provided a first overview of the metabolic pathways in which they are involved. The high local-scale variability and its interaction with diet shifts reveals the importance of proper spatial replication in microbiomic research targeting wild / semi-wild tephritid flies and provides a cautionary tale on general inferences drawn from laboratory populations.
Acknowledgements
This work was supported by funds from a ‘Bijzonder Onderzoeksfonds’ grant from the University of Antwerp, a ‘Fonds voor wetenschappelijk onderzoek’ PhD fellowship of the FWO and was supported by the International Atomic Energy Agency (IAEA, Vienna) through the technical contract n. 20876 “Comparative Microbiomics of African Fruit Flies” (CMAFF). We thank the Joint Experimental Molecular Unit of RMCA and RBINS for technical and conceptual feedback and stimulating discussion.
References