Mycobiomes of two distinct clades of ambrosia gall midges (Diptera: Cecidomyiidae) are species-specific in larvae but similar in nutritive mycelia

ABSTRACT Ambrosia gall midges (AGMs) are mostly host plant-specific. In their galls, they harbor fungal symbionts on which they feed. Therefore, they represent unique steps in the evolution of the gall-forming Cecidomyiidae (Diptera). Gall-associated fungi have been studied predominantly by cultivations, and potential larval endosymbionts have been completely neglected. Using ITS2 rRNA metabarcoding, we characterized the mycobiomes of individual gall compartments (gall surface, gall interior, and larva) of six species from two phylogenetically separated tribes (Asphondyliini and Lasiopterini). Compared to the gall surface and interior, the larvae harbored significantly higher fungal richness and taxonomic diversity, and a larger pool of indicator taxa. Larval mycobiome composition was more species-specific; however, the fungal genera Fusarium, Filobasidium, Tilletiopsis, Alternaria, and Aureobasidium were indicator taxa shared among species. Overall, the larvae harbored 29% of unique taxa that can play a functional role in the host (e.g., initiation of gall development or selection of the mycelia composition). The mycobiome of the gall interior was assembled least stochastically, and its composition was the least species-specific, being dominated by Botryosphaeria dothidea (except for Lasioptera arundinis). Therefore, the interior of ambrosia galls offers a unique environment that supports the growth of similar fungi, regardless of the host plant species and the phylogenetic distance between the AGM tribes. Our study illustrates a range of fungal microorganisms indicative of individual gall compartments, but their potential function, especially in larvae, remains to be solved. IMPORTANCE Ambrosia gall midges are endophagous insect herbivores whose larvae live enclosed within a single gall for their entire development period. They may exhibit phytomycetophagy, a remarkable feeding mode that involves the consumption of plant biomass and mycelia of their cultivated gall symbionts. Thus, AGMs are ideal model organisms for studying the role of microorganisms in the evolution of host specificity in insects. However, compared to other fungus-farming insects, insect–fungus mutualism in AGMs has been neglected. Our study is the first to use DNA metabarcoding to characterize the complete mycobiome of the entire system of the gall-forming insects as we profiled gall surfaces, nutritive mycelia, and larvae. Interestingly, larval mycobiomes were significantly different from their nutritive mycelia, although Botryosphaeria dothidea dominated the nutritive mycelia, regardless of the evolutionary separation of the tribes studied. Therefore, we confirmed a long-time hypothesized paradigm for the important evolutionary association of this fungus with AGMs.

2. The gall surfaces, gall interiors, and larvae should be displayed as a figure .3.The raw data obtained by sequencing should be submitted to the public repository.4.Why the authors used ITS3 and ITS4 primers to amplify the ITS2 rDNA?5.The Figure 5 should be revised, the horizontal coordinate is chaotic.

Reviewer #2 (Comments for the Author):
This manuscript reports original data on fungi associated with a few species of cecidomyid gall midges.Although the authors could not demonstrate a clear symbiotic role by any of the identified species, their findings represent a valuable contribution for improving our understanding of this unique biological association.The manuscript is well written and offers an exhaustive introductory overview of the current knowledge on the subject.I only observe that the discussion section should be revised by removing some uncircumstantial considerations.Particularly, concerning text at lines 394-398 I remark that Fusarium spp.are very diverse in their occurrence and ecological interactions; in fact, they are often mentioned as entomopathogens, and their mycotoxins could have a noxious effect on the larvae rather than protecting them from bacterial infections.Some statements in this section also require revision.At line 366, it is incorrect that Radulidium subulatum had been previously identified as Macrophoma sp.; indeed, Macrophoma is an old name for Botryosphaeria dothidea, and it is very likely that fungi identified with this name in the old papers corresponded to the latter species.At line 368-369 it is incorrect that Cercospora (Dothideomycetes) is similar to Sarocladium (Sordariomycetes).Finally, at lines 370, 'plant' should be deleted; in fact, the saprobic aptitude is not intended to be exerted on living organisms, and ref. 89 generically qualifies Myrmecridium as 'saprobes'.

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This manuscript reports original data on fungi associated with a few species of cecidomyid gall midges.Although the authors could not demonstrate a clear symbiotic role by any of the identified species, their findings represent a valuable contribution for improving our understanding of this unique biological association.The manuscript is well written and offers an exhaustive introductory overview of the current knowledge on the subject.I only observe that the discussion section should be revised by removing some uncircumstantial considerations.Particularly, concerning text at lines 394-398 I remark that Fusarium spp.are very diverse in their occurrence and ecological interactions; in fact, they are often mentioned as entomopathogens, and their mycotoxins could have a noxious effect on the larvae rather than protecting them from bacterial infections.

## Thank you for this comment; we incorporated this important information to the manuscript (lines 253-255).
Some statements in this section also require revision.At line 366, it is incorrect that Radulidium subulatum had been previously identified as Macrophoma sp.; indeed, Macrophoma is an old name for Botryosphaeria dothidea, and it is very likely that fungi identified with this name in the old papers corresponded to the latter species.

## Thank you for this comment; we agree. This statement was misinterpreted due to incorrect translationwe corrected it (line 223).
Finally, at lines 370, 'plant' should be deleted; in fact, the saprobic aptitude is not intended to be exerted on living organisms, and ref. 89 generically qualifies Myrmecridium as 'saprobes'.

## Deleted (now line 225).
October 24, 2023 1st Revision -Editorial Decision Re: Spectrum02830-23R1 (Mycobiomes of two distinct clades of ambrosia gall midges (Diptera: Cecidomyiidae) are speciesspecific in larvae but similar in nutritive mycelia) Dear Dr. Petr Pyszko: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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