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