Insight into the bacterial communities of the subterranean aphid Anoecia corni.

Many insect species are associated with bacterial partners that can significantly influence their evolutionary ecology. Compared to other insect groups, aphids harbor a bacterial microbiota that has the reputation of being poorly diversified, generally limited to the presence of the obligate nutritional symbiont Buchnera aphidicola and some facultative symbionts. In this study, we analyzed the bacterial diversity associated with the dogwood-grass aphid Anoecia corni, an aphid species that spends much of its life cycle in a subterranean environment. Little is known about the bacterial diversity associated with aphids displaying such a lifestyle, and one hypothesis is that close contact with the vast microbial community of the rhizosphere could promote the acquisition of a richer bacterial diversity compared to other aphid species. Using 16S rRNA amplicon Illumina sequencing on specimens collected on wheat roots in Morocco, we identified 10 bacterial operational taxonomic units (OTUs) corresponding to five bacterial genera. In addition to the obligate symbiont Buchnera, we identified the facultative symbionts Serratia symbiotica and Wolbachia in certain aphid colonies. The detection of Wolbachia is unexpected as it is considered rare in aphids. Moreover, its biological significance remains unknown in these insects. Besides, we also detected Arsenophonus and Dactylopiibacterium carminicum. These results suggest that, despite its subterranean lifestyle, A. corni shelter a bacterial diversity mainly limited to bacterial endosymbionts.

Introduction

Insects maintain a variety of symbiotic relationships with heritable bacteria that can deeply influence their evolutionary ecology [1-3]. Thanks to their well-studied associations with a wide range of heritable symbiotic bacteria, aphids (Hemiptera: Aphididae) are valuable model systems for studying the evolution of bacterial mutualism in insects [4-6]. Like many insect species that feed on nutrient-deficient diets, aphids typically harbor an ancient nutritional obligate endosymbiont, Buchnera aphidicola, confined in specialized cells called bacteriocytes and stably maintained in host populations by vertical transmission [5,7]. In addition to their obligate partner, aphids can also host various facultative endosymbionts that can positively or negatively affect a variety of host phenotypes, depending on the ecological context [4,6].

The functional diversity of facultative symbionts associated with aphids includes γ-proteobacteria [e.g. Arsenophonus sp. [8,9], Regiella insecticola [10,11], Serratia symbiotica [12,13], Hamiltonella defensa [14,15], Rickettsiella viridis [16,17] and Candidatus Fukatsuia symbiotica [18,19]], α-proteobacteria [e.g. the genus Rickettsia [20,21] and Wolbachia [22,23]] and Mollicutes of Spiroplasma genus [24,25]. Ecological effects associated with these bacterial partners include defense against parasites [26-30], body color modification [31,32], heat stress tolerance [33], host plant use and nutrition [34,35] and host reproductive manipulation [36].

In addition to intracellular endosymbiotic bacteria, the aphid microbiota may also include bacterial partners involved in less lasting interactions, which may be transient or even antagonistic, and include gut bacteria, plant associates, pathogens, and environmental contaminants [1,2,37-44]. In aphids, these microorganisms have received limited consideration, notably because of the virtual absence of bacteria in the plant-phloem [45]. However, high-throughput sequencing approaches provide the opportunity to get more complete pictures of the bacterial communities associated with these insects. In this regard, recent studies suggest that the microbiota of some aphid species may be more diverse than previously thought, involving a wider a bacterial diversity that includes members of the genera Pseudomonas, Erwinia, Acinetobacter, Staphylococcus and Pantoea among others [40,42,46]. Tackling this diversity is crucial to understanding how heritable bacterial endosymbioses are established from free-living lineages. Indeed, recent studies suggest that heritable endosymbionts derive from free-living bacteria, which can sometimes reside in the host plant and in the digestive tract of aphids [39,47-51].

The diversity of the microbiota associated with aphids is linked to their living environment, and the evolutionary acquisition of certain symbionts is probably due to particular habitats. In this context, the soil is an extraordinary reserve of microbiological diversity whose functions are essential to the functioning ecosystems [52]. We therefore hypothesize that aphid species living in close association with the soil, such as the dogwood-grass aphid Anoecia corni, are likely to harbor a more diverse and original microbiome than the aphid species strictly present in the areal parts of plants. This particular species notably has access to the xylem tissues of the roots, the first gateway through which many soil-borne bacteria, sometimes pathogenic, transit [53-56].

A. corni is a holocylic dioecious species belonging to a genus that includes about twenty aphid species widely distributed in the Holarctic, many whose ecological and taxonomic position remain largely unknown [57,58]. In temperate areas, overwintering eggs hatch on dogwood (Cornus sanguinea) during spring, giving rise to a generation of fundatrices. In summer, the alates leave dogwood and migrate onto the roots of grasses and sedges (Poaceae, Cyperaceae) where they are often attended by ants [59]. The microbiota associated with A. corni is unknown, and its subterranean lifestyle makes it an ideal candidate to test the hypothesis that close contact with the vast microbial community of the rhizosphere could promote the acquisition of a richer bacterial diversity compared to other aphid species. In this study, we sampled A. corni colonies on wheat roots in two regions of Morocco. The 16S rRNA amplicon Illumina sequencing approach was used to examine the composition of the microbiota associated with aphid samples and to clarify the relationship between these two organisms. For this, the evolutionary history was inferred using the Neighbor Joining (NJ). Our results are discussed in light of previous studies on the microbiota associated with other aphid species.

Materials and methods

Sample collection and DNA extraction

Apterous adults of A. corni were collected during August 2014 on roots of wheat plants (Triticum sp.) in Morocco with the kind permission of the landowners. A total of 16 colonies were sampled in two important regions in terms of cereal crops: eight colonies were collected in the locality of Béni Mellal-Khénifra and eight colonies were collected in the locality of Casablanca-Settat (Fig 1; S1 Table in the Supporting Information). Aphid collection consisted of three wingless parthenogenetic adult females per colony, which were immediately immersed in 95% ethanol during collection and preserved at 4°C until use.

Fig 1

Geographical location of collection sites of A. corni colonies analyzed in this study (for details, see S1 Table in the supporting information).

DNA extraction, PCR amplification, library preparation and sequencing

Prior to DNA extractions, aphid samples, each comprising three adult aphids of the same colony, were surface-sterilized with 99% ethanol, 10% bleach and rinsed with sterile water to remove surface contaminants. The genomic DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN) following the instruction of the manufacturer. The quantity and quality of the DNA extractions were measured with a NanoDrop spectrophotometer (Thermo Scientific, USA). Extractions were then stored at -20°C. After extraction, the genomic 16S rRNA has been diluted using sterilized ultrapure water in equal concentration (5 ng/μl) from each sample for further steps.

PCR amplification, library preparation and sequencing

Sequencing libraries were prepared according to the Illumina MiSeq system instructions 16S workflow and as described previously [40]. First, DNA was amplified using universal primers with overhang adapters attached (F: 5’ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and R: 5΄GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) and which targets the V3-V4 variable region of the 16S bacterial rRNA gene. This first PCR step (PCR1) was carried out using a kit 2x KAPA HiFi HotStart ReadyMix in a mixture total volume of 25 μl. The conditions of this first PCR were 95°C for 3 min (1 cycle); 95°C for 30 s, 55°C for 30 s and 72°C for 30 s (25 cycles), followed by 72°C for 5 min. PCR products were cleaned with AMPure XP beads. A second-stage PCR (PCR2) was carried out using 5 μl PCR1 purified to attach dual indices and Illumina sequencing adapters using the Nextera XT Index Kit. Different combinations of index (i5 and i7) were used for each sample. The PCR2 was executed under the same conditions of PCR1 but with eight PCR cycles. A clean-up of the PCR2 products with AMPure XP beads was performed before quantification. PCR2 products were quantified and normalized at 7 ng/μl using PicoGreen dsDNA Quantitation Assay and were generated an equimolar pool (7 ng/μl). Before proceeding to high-throughput sequencing (HTS), the final pool was quantified by qPCR (kit KAPA SYBR FAST qPCR ABI Prism readymix KK4604) and 7 pM of denatured final pool was loaded on MiSeq reagent kits v3 (600 cycles). All the processes were carried out by the GIGA-Research Center of the University of Liège (ULiège, Belgium) using Illumina MiSeq Technology for paired-end sequencing (2 × 300 bp reads).

Data analysis

The data analysis was achieved as previously described [40]. The 16S rRNA Illumina Miseq sequencing reads were analyzed using UPARSE [60] bioinformatics pipeline. For each aphid sample, forward and reverse sequences from paired-end reads were assembled and the resulting consensus sequences were filtered based on their respective quality (expected number of errors <1.0 and a length >450 nt). Sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs). Chimera were removed using a reference-based filtering with UCHIME and the gold database of the corresponding software. A second level of quality-filtering was carried out in order to discard OTUs with a number of sequences <0.005% of the total number of sequences, as recommended previously [61]. As previously recommended [62,63], an additional filtering was performed by analyzing the negative controls in order to remove OTUs corresponding to potential contaminations. Bacterial taxonomic assignments of each OTU were obtained using the dada2 R package [64] and the Greengenes (v.13.8) database [65]. Finally, to improve the taxonomic assignment, the representative OTUs were compared to the sequences in the GenBank using BLASTn. To compare samples, the number of sequences was standardized or rarefied to 50,000 per sample. After rarefaction, the OTU table was analyzed (See S2 Table. Commands and options used to build the OUT table). All bacterial sequences found in A. corni are given in S3 and S4 Tables (Supporting information). Raw data were deposited European Nucleotide Archive (ENA) as a file under accession number PRJEB35700.

Phylogenetic relationships of bacteria associated with A. corni and representative endosymbionts of other aphids were established using SeaView v4.6.1 to align 16S rRNA sequences [66] and GBlocks v0.91b [67] to remove poorly aligned positions and divergent regions of DNA alignments. We selected the best fit evolutionary models using PartitionFinder v1.1.0 [68]. The phylogenetic tree was reconstructed using the neighbor joining method with SeaView v4.6.1 and bootstrap values were computed for each branch node (N = 1000).

Results

Library basic statistics

On average, NGS sequencing produced 187,592 bacterial 16S rRNA reads per sample (Table 1).

Table 1

Summary of sequencing data.

Raw data
Average size (Mb) per sample	3
Raw number of sequence per sample	187,592
After assembly of paired sequences
Average size (Mb) per sample	169.4
Average number of sequence per sample	173,117
Average of the median sequence length	466
After quality filtering
Average size (Mb) per sample	72.7
Average number of sequence per sample	150,605
Sequence length (min; median; max)	(450; 465; 584)

The assembly of paired sequences resulted in consensus sequences with an average length of 466 bp. The quality-based filtering of the consensus sequences resulted in an average number of high-quality sequences per sample of 150,605.

OTU clustering and taxonomic assignment

Initially, high-quality reads were clustered using >97% sequence similarity into 23 OTUs. Based on the analysis of the negative controls, 13 OTUs that count for 0.23% of the total number of reads were identified as contaminants and removed (S4 Table). Reads were therefore clustered into 10 biologically relevant OTUs (Table 2 and S3 Table).

Table 2

Taxonomic assignment of OTUs by Greengenes and GenBank, including the three top BLAST hits, GenBank accession numbers and % identity.

OTU no.	PC reads.	Greengenes identification	Id%	GenBank identification	Accession	Id%
		Taxon		Three closest GenBank matches
OTU_01	58.77	Buchnera	98.28	Buchnera aphidicola/Geoica urticularia	AJ296751.1	98.28
				Buchnera aphidicola/Myzus persicae	CP002703.1	96.34
				Buchnera aphidicola/Myzus persicae	CP002701.1	96.34
OTU_02	20.08	Buchnera	96.15	Buchnera aphidicola/Pemphigus matsumurai	KF311221.1	96.15
				Buchnera aphidicola/Pemphigus sinobursarius	KF311219.1	96.15
				Buchnera aphidicola/Pemphigus yunnanensis	HQ792326.1	96.15
OTU_03	17.34	Buchnera	91.24	Buchnera aphidicola/Anoecia oenotherae	CP033012.1	97.85
				Buchnera aphidicola/Anoecia fulviabdominalis	JX998094.1	97.2
				Buchnera aphidicola/Eulachnus mediterraneus	LT600356.1	94.22
OTU_04	2.62	Serratia	99.14	Serratia symbiotica/Aphis fabae	KT176010.1	99.35
				Serratia symbiotica/soil	MG287131.1	99.14
				Serratia symbiotica/soil	KX900450.1	99.14
OTU_05	0.89	-	90.15	Serratia symbiotica/Prociphilus longianus	MG831336.1	99.35
				Serratia symbiotica/Prociphilus longianus	MG835393.1	98.92
				Serratia symbiotica/Prociphilus longianus	MG835392.1	98.92
OTU_06	0.04	Wolbachia	96.64	Wolbachia pipientis/Pentalonia nigronervosa	KJ786950.1	96.64
				Wolbachia pipientis/Pentalonia nigronervosa	KJ786949.1	96.64
				Wolbachia pipientis/Pentalonia nigronervosa	KC522606.1	96.64
OTU_7	0.01	Buchnera	98.21	Buchnera aphidicola/Geoica urticularia	AJ296751.1	98.21
				Buchnera aphidicola/Myzus persicae	CP002703.1	95.92
				Buchnera aphidicola/Myzus persicae	CP002701.1	95.92
OTU_8	0.01	Candidatus Phlomobacter	95.05	Arsenophonus/Aleurodicus dispersus	AY264664.1	95.91
				Arsenophonus/Macrosteles sexnotatus	AB795344.1	95.27
				Arsenophonus/Stomaphis takahashii	FJ655541.1	95.27
OTU_9	0.01	Uliginosibacterium	98.92	Dactylopiibacterium carminicum/Dactylopius opuntiae	GQ853370.1	98.92
				Dactylopiibacterium carminicum/Dactylopius opuntiae	GQ853369.1	98.92
				Sphingomonas/soil	JX944513.2	96.34
OTU_10	0.01	Buchnera	98.06	Buchnera aphidicola/Geoica urticularia	AJ296751.1	98.06
				Buchnera aphidicola/Myzus persicae	CP002703.1	96.13
				Buchnera aphidicola/Myzus persicae	CP002701.1	96.13
OTUs 11–23	0.23	Contaminants identified from negative control analysis

PC reads, cluster size in percent; Id, identity %.

All OTUs correspond to the group of Proteobacteria and include three bacterial orders: Enterobacteriales, Rhodocyclales and Rickettsiales. Our results indicate that the microbial profile of A. corni is dominated by the order of Enterobacteriales, which includes the obligate symbiont B. aphidicola and the facultative symbionts S. symbiotica and Arsenophonus.

B. aphidicola was detected in all the samples (100%) and was represented by 5 OTUs (OTUs 1, 2, 3, 7 and 10) that account for 96.23% of all reads. OTUs 1, 7 and 10 differed by only 3 to 4 bp whereas OTUs 2 and 3 differed from these OTUs by 20 to 38 bp. Different B. aphidicola haplotypes are present in an aphid colony with the dominance of a single haplotype (Fig 2). The vast majority of the reads clustered into a single OTU for most aphid species (OTUs 1, 2 or 3) and many minor OTUs (OTUs 7 and 10) were detected in all samples. In aphid colonies from the Casablanca-Settat region, The OTU1 (with related minor OTUs 2, 3, 7 and 10) was detected and matched a sequence of B. aphidicola previously reported on Geoica urticularia. OTUs 2 and 3 were common to all samples from Béni Mellal-Kénifra region and OTU3 was detected on the Anoecia genus.

Fig 2

Relative abundance of bacterial taxa from Illumina sequencing of 16S rRNA amplicons, represented as a heat map based on the log-transformed values.

The warm colors indicating higher and cold colors indicating lower abundance. Each color bar corresponding to one sampled colony.

The next most abundant OTUs were presented by the facultative symbiont S. symbiotica (OTUs 4 and 5) that account for 3.52% of all reads. Taxonomic identification of bacterial OTUs resulted in three additional taxa including Wolbachia (OTU 6), Arsenophonus (OTU 8) and D. carminicum (OTU 9). Phylogenetic analyses including the symbionts associated with A. corni (i.e. B. aphidicola, S. symbiotica, Arsenophonus and Wolbachia) and representative symbionts of other species of aphids and insects are shown in S1–S4 Figs (Supporting information).

Diversity of bacterial communities in the samples

The bacterial communities of the samples were mainly composed of the obligate symbiont B. aphidicola and mostly complemented by the facultative symbionts S. symbiotica (with a high abundance of reads from three samples) and Wolbachia (with reads detected from four samples) (Fig 2).

The facultative symbiont Arsenophonus was detected in most of the samples (13/16), but with an extremely low number of reads. D. carminicum was also found in most of the samples with an extremely low number of reads.

Discussion

Our approach based on 16S rRNA enabled us to identify five bacterial genera in the dogwood aphid A. corni, a species that spends most of its life cycle on Poaceae roots. Due to its subterranean lifestyle, we expected to find in this species a bacterial diversity that includes environmental bacteria such as gut bacteria, plant associates, pathogens and environmental contaminants. Xylem vessels are the primary entry routes for many soil-borne bacteria infecting plants [56]. Although primarily phloem-feeders, aphids are also capable of ingestion from the xylem vessels, a strategy displayed for maintaining water balance [69,70]. OTUs that were identified correspond to five bacterial genera, mostly related to symbionts already found associated with aphids: Buchnera, Serratia, Wolbachia, Arsenophonus and Dactylopiibacterium. All these genera have been previously described as symbiotic partners of insects. We did not find any bacterial partners that can be considered as environmental-related (e.g. Pseudomonas spp., Erwinia spp., etc.) as in the case of other aphid species, including those that feed on cereal crops [40,42].

The nutritional obligate symbiont Buchnera was found in all samples with distinctive 16S haplotypes in a single aphid colony. These results might be a consequence of Buchnera polyploidy, as evidenced by the 16S rRNA copy-number variation. Alternatively, a clone from a single colony may contain Buchnera strains with different haplotypes. Co-infection with multiple B. aphidicola strains was reported in several aphid genera [71,72].

The secondary endosymbiont S. symbiotica is one of the most common symbiont species in aphid populations [73] and was identified in three of the eight colonies surveyed. This symbiont includes a wide variety of strains ranging from co-obligate nutritional partners, that are mainly found in the Lachninae and the Chaitophorinae subfamilies [12,74], to facultative strains whose reported associated effects in the pea aphid Acyrthosiphon pisum are heat stress resistance and protection against parasitoids [75-77]. Strains detected in this study are probably of facultative nature, as S. symbiotica was not found in all colonies.

Interestingly, Wolbachia was detected in four colonies. This symbiont, an α-proteobacterium, is commonly found in insects and studies suggest that Wolbachia is present in at least 65% of arthropod species [78]. It is known to manipulate the reproduction of their host [79], promote the oogenesis in certain wasp species [80], display a nutritional function in certain bedbug and whiteflies species by producing B vitamins [81,82], and is associated with antiviral protection by influencing the vector competence of several species of mosquitoes for viruses [83,84]. Although some studies have reported the presence of Wolbachia in aphid populations, it is considered rare in these insects [85-90]. The biological significance of Wolbachia in aphids is still unknown. It has been hypothesized that the symbiont play a role in the proliferation of asexual lineages [87], and its role in the production of B vitamins in the banana aphid Pentalonia nigronervosa is currently debated [86,91,92]. Wolbachia infections in aphids could also be acquired by horizontal transmission from other insects such as parasitoid wasps, known to be infected by this symbiont [93,94]. To our knowledge, the stability of Wolbachia infections in aphids has never be tested and A. corni could be a suitable candidate to elucidate the biological significance of this symbiont in aphids.

The genera Arsenophonus and Dactylopiibacterium were also detected in most of the sampled aphids, but with a much lower read abundance than for S. symbiotica and Wolbachia. Arsenophonus is a bacteria found in many insect species including aphids, scale insects, leafhoppers, whiteflies and wasps [8,82,95-97]. Despite the fact that the prevalence of Arsenophonus can reach up to 70% in species of the Aphis genus [98], the phenotypes associated with this symbiont remain unclear in aphids. Bacteriophages required for protective symbiosis were found in various strains of the symbiont [99], but no defensive properties were found in Aphis glycines infected by Arsenophonus [98]. Recent studies suggest that Arsenophonus may be involved in host nutrition, probably by mediating host plant range [35,100-102]. In whiteflies, comparative genomics suggests that Arsenophonus is a source of B vitamins [82]. However, no genome of strains associated with aphids has yet been sequenced. While the presence of a symbiont in specialized host cells such as bacteriocytes and sheath cells are important clues for determining the mutualistic and heritable nature of a symbiont [4], no such information are available for this particular symbiont.

One OTU was assigned to D. carminicum (β-proteobacteria, family Rhodocyclaceae). So far, this bacterial species has only been reported in the scale insect species Dactylopius coccus (Hemiptera: Coccoidea: Dactylopiidae), where it has been described as a nitrogen-fixing symbiont [103,104]. Dactylopius coccus is now well established in Morocco where it ravages the plants of Opuntia ficus-indica. Although D. carminicum is considered a symbiont capable of passing through the reproductive organs in scale insects, this species remains largely undocumented. It cannot be excluded at present that this newly discovered species resides in the soil, in the host plant or lives in other insects.

In recent years, several studies have characterized overall all the bacteria present in aphids by deep sequencing of 16S rRNA [40,42,45]. A common point throughout these studies is the reduced abundance of environmental bacteria relative to the primary and secondary endosymbionts. Besides theses heritable symbionts and in contrast to our results, many environmental bacteria have been reported in R. padi, e.g. the phytopathogenic members of the Pseudomonas genus and some saprophytes of plant and soil (Acinetobacter and Staphyloccocus genera). Moreover, gut symbiotic bacteria of aphids were also found, i.e. Pantoea and Erwinia genera [40,42].

In conclusion, although A. corni lives in the rhizosphere, an environment that is very rich in bacteria and other microorganisms, the number of bacterial taxa detected in this species is surprisingly low [105]. Despite its subterranean lifestyle, A. corni shelter a bacterial diversity mainly limited to known bacterial endosymbionts. Few species of facultative endosymbionts have been detected in the context of this study. It is now recognized that some species of aphids are more likely to harbor facultative symbionts than others [4,73,106]. It should be noted that our sampling covers only a small part of Morocco, and the diversity of symbionts of aphid populations can change dramatically in response to various environmental conditions [4]. However, our study provides a snapshot of the bacterial community associated with a poorly studied aphid species, and identified bacterial taxa that may play a role in the biology of A. corni, in particular the facultative symbionts Arsenophonus and Wolbachia whose associated phenotypes in aphids are still elusive. A. corni could represent a suitable species to investigate the role of these symbiotic bacteria in aphids. Finally, insect-associated bacterial communities, and in particular heritable symbionts, have received much attention in recent decades, somewhat overshadowing the diversity of other types of microorganisms that can associate with insects, such as fungi. For example, in some species of cicadas, grasshoppers, and aphids, certain fungi species have become obligate symbionts by replacing ancestral bacterial symbionts [107-111], suggesting that fungi may establish more or less long-lasting relationships with insects and become an established part of their microbiota. Although fungal diversity was not explored in our study, it would likely deserve more attention in future work, and insects with a subterranean lifestyle are likely interesting candidates for such an investigation.

Phylogenetic analysis of Buchnera associated with A. corni and its placement compared to strains associated with other aphid species, based on a sequence from the V3-V4 region of bacterial 16S rRNA.

The evolutionary history was inferred using the Neighbor Joining (NJ) methods, with a J-C model. The percentage of replicate trees was verified with bootstrap of 1000 replicates. Designations in bold are strains sequenced in this study. Host names are followed by the GenBank accession number of each bacterial sequence. Geneious version 6.1 created by Biomatters.

(TIF)

Click here for additional data file.

Phylogenetic analysis of Serratia symbiotica associated with Anoecia corni and their placement compared to strains associated with other aphid species, based on a sequence from the V3-V4 region of bacterial 16S rRNA.

The evolutionary history was inferred using the Neighbor Joining (NJ) methods, with a J-C model. The percentage of replicate trees was verified with bootstrap of 1000 replicates. Designation in bold are is strains sequenced in this study. Host names are followed by the GenBank accession number of each bacterial sequence. Geneious version 6.1 created by Biomatters.

(TIF)

Click here for additional data file.

Phylogenetic analysis of Wolbachia associated with Anoecia corni and its placement compared to strains associated with other insect species, based on a sequence from the V3-V4 region of bacterial 16S rRNA.

The evolutionary history was inferred using the Neighbor Joining (NJ) methods, with a HKY model. The percentage of replicate trees was verified with bootstrap of 1000 replicates. Designation in bold is strain sequenced in this study. Host names are followed by the GenBank accession number of each bacterial sequence. Geneious version 6.1 created by Biomatters.

(TIF)

Click here for additional data file.

Phylogenetic analysis of Arsenophonus associated with Anoecia corni and its placement compared to strains associated with other insect species, based on a sequence from the V3-V4 region of bacterial 16S rRNA.

The evolutionary history was inferred using the Neighbor Joining (NJ) methods, with a HKY model. The percentage of replicate trees was verified with bootstrap of 1000 replicates. Designation in bold is strain sequenced in this study. Host names are followed by the GenBank accession number of each bacterial sequence. Geneious version 6.1 created by Biomatters.

(TIF)

Click here for additional data file.

Summary of collection details and 16S rRNA gene sequencing results for aphid samples.

(DOCX)

Click here for additional data file.

Data analysis: Commands and options used to build the OUT table.

(DOCX)

Click here for additional data file.

Bacterial representative sequences (operational taxonomic units) found in A. corni.

(DOCX)

Click here for additional data file.

Contaminant OTUs identified in this study.

(DOCX)

Click here for additional data file.

13 May 2021

PONE-D-20-40214

Insight into the Microbiome of the Subterranean Aphid Anoecia corni

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

**********

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Reviewer #3: Yes

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Reviewer #1: The manuscript by Fakhour and colleagues describes the bacterial microbiome associated with 16 samples of the aphid Anoecia corni collected on wheat roots in two regions of Morocco.

The manuscript is well written and presented in general, but it has limited interest even to those who specifically study insect microbiomes. The bacterial species identified in this work were already known to be associated with aphids, so the manuscript adds little to the current understanding about the aphid microbiome. As a general comment, the in silico study of microorganisms other than bacteria (e.g. fungi, which are key components of the plant rhizosphere) would have increased the novelty and interest of the paper. Insect microbiomes, in fact, include also fungi, viruses, and protozoa. As A. corni lives in close association with soil and wheat roots, at least the fungal component of the insect microbiome should have been explored.

In addition, environmental parameters associated to each sampling site (e.g. precipitation, temperature, etc…) are not provided. Such data, for example, might have shed light to the possible impact of the environment on the biodiversity indexes, which, by the way, are totally missing.

Minor issues

The title: as the Authors analyzed only the bacterial component of the aphid microbiome, I suggest they modify the title accordingly.

Abstract line 28: the Authors claimed that the bacterial microbiome of aphids is “poorly diversified”, but later in the Introduction they stated that aphids are associated “with a wide range of bacterial symbionts”. Please explain this contradiction.

Abstract line 42: In this study the Authors took into consideration only the bacterial endosymbionts, so it is obvious that the bacterial diversity found in A. corni samples is limited to this part of the insect microbiome. Please rephrase.

Table 2: What does “PC reads.” stands for?

Introduction page 3: “B. aphidicola” and “A. corni” should be written without abbreviations as it is the first time they are cited in the manuscript. Check the spelling of “Hamiltonella defensa”

Page 4 line 90: the correct spelling is “dioecious”

Page 6 and Materials and Methods in general: if the procedures are already described in another paper, I suggest to cite the paper and keep the description short, providing only relevant modifications from the previous protocols/approaches, if there are any.

Page 11 line 214: OTU_6 (and not OTU 7) corresponds to Wolbachia (Table 2) and I assume that OTU 18 is actually OTU 8 and OTU 19 is OTU 9, is it correct?

Figure 1 improve resolution and associate the library codes to the sampling locations.

Supplementary figures: phylogenetic trees should first report bacterial names and then their host names in brackets, otherwise they seem to represent insect phylogeny, instead of that of their symbionts. In some cases there are bacterial names and insect names on different branches of the same tree.

Reviewer #2: Overall comments :

In the present paper, the authors use 16S rRNA sequencing to investigate for the first time the microbial communities associated to the subterranean aphid Anocia comi.

They identified a total of 23 OTUs, 10 of them being associated to 5 putative symbiont genera, including the obligatory symbiont Buchnera aphipicola and 4 known facultative symbionts. Among them was Wolbachia, which is considered rare in aphids.

Overall, I am pleased by the results presented in the paper. However, some elements are unclear and need to be reworked to make the paper easier to understand and reproduce for other researchers. In particular, figures need to be reworked, and it seems (to me at least) that there is some confusion in sample naming and OTU numbering.

Hereafter are my comments. Most of them are typos or minor comment but some of them are more important for the comprehension of the paper.

Abstract :

37 : I'd remove "hosting"

42 : I don't understand this sentence. What is expected apart from bacterial endosymbionts?

Data analysis : for the sake of reproducibility, the commands and options used for the different software should be supplied.

161 : missing citation for dada2

167 : There is no correpondance given between the identifiers you used for your samples and the ENA identifiers. Could you please add the accessions in sup Table 1 for instance?

169 : missing citation for SeaView

Table 1 : Maybe add median sequence length after filtering?

More detailled statstics (min-median-max) would also be useful

186 : 0.23% is not much but is massive compared to the abundance of Wolbachia for instance. I would at least remove the "only".

Also, the complete abundance table for the 23 OTUs should be available as supplementary.

188 : The contaminant OTUs are also "genuine". I would use "biologically relevant"

Table 2 :

OTU identifiers do not match with the figures.

I'd appreciate a comment on the discrepancies between the Greengenes identification (from dada2?) and the Blast based

Fig 2 :

- The naming of samples is very unclear. I understand (from the supplementary) that the number indicates the locality. But this naming convention gives the impression that samples are paired. There is also a H12 sample which is puzzling to me.

- Could also indicate on Fig2 the geographic origin of samples?

- Text should be bigger (also for other figures)

- Apart from the presence absence of Serratia, the Figure is hard to read. Maybe use a log scale? And it's also quite redundant with Fig. 3

Fig 3 :

Legend : How is that "relative abundance"? It seems to me that these are absolute counts. The Figure with relative abundances would be Fig. 2.

I would rephrase "Each color bar corresponding" by "Each column represents"

You mention log counts, but the counts on the scale do not seem log transformed. I believe only the scale is logarithmic.

Maybe a heatmap made only of secondary symbions in addition to this one would be useful to understand where they are (ideally with read counts or relative abundances written on the heatmap)

217 - Fig S1-S4 : Overall the trees are poorly supported and not discussed in the paper.

234 : "Extremely low number of reads" : Please give numbers, in absolute (read counts) and relative (%).

The count table for all OTUs and all samples should be available as supplementary.

You make no comment on the fact that the different Buchnera OTUs are not evenly distributed across samples.

249 : Could it be because you used a different extraction protocol?

Reviewer #3: PLOS ONE

Review: “Insight into the Microbiome of the Subterranean Aphid Anoecia corni”

Authors: Samir Fakhour, François Renoz, Jérôme Ambroise, Inès Pons, Christine Noël, Jean- Luc Gala, Thierry Hance

General Comments

The study has an interesting premise to investigate the microbiota of ground dwelling, root feeding aphids. The paper provides a strong hypothesis as to why these aphids might harbor more microbes, and sets out to examine this using High Throughput Sequencing. The incorporation of phylogenetic relatedness of microbial organisms to support whether they are aphid, soil, or plant associated is good, although this concept should be mentioned in the introduction.

Overall this manuscript provides a good discussion of the 5 main bacterial OTUs found in A. corni. An overview of the functions these microbes in other organisms was provided along with the bacteria’s potential role in this aphid. The conclusion provided a nice summary of additional lines of investigation for the role of Arsenophonus and Wolbachia in A. corni.

Major Comments

Flow and organization can be enhanced

-Some sentences in the discussion have repetitive content and could be enhanced by restructuring or merging two sentences into one. Reminders throughout the text of the main ideas are helpful, however the sentence repetition does not help drive the manuscript content forward.

-Incorporate relevance into the introduction

-The introduction should mention the importance of phylogenetic relatedness in determining microbial associations, to prime the reader for the Neighbor Joining tree analyses in the materials and methods.

Clarify Materials and methods

-Based upon the sample collection information, it is not entirely clear the process for obtaining aphids and then raising the colonies. Were apterous adult aphids collected on wheat roots, then placed into colonies and then used to generate clones? Then were these aphid clones used to assess microbial diversity? It is unclear.

-Revise discussion for clarity and flow

-The manuscript makes a bold claim that no environmental bacteria were found associated with aphids, however only the aphids were sampled and not the plant or xylem fluids. The way that this is written should be framed in the context that bacterial symbionts were not more abundant in ground dwelling aphids despite being in contact with soil microbes and having access to xylem. An average or assessment of microbes identified in other aphids beyond the typical symbionts should be provided if this comparison is to be made.

-While after reading L239 to L234 it is clearer to the reader that the phylogenetic assessment using Neighbor Joining (NJ) trees were intended to clarify the relationship of microbial organisms to aphid hosts, the relevance of phylogenetic relationships among microbes and their environment/host association should be mentioned in the introduction to prep the reader for this in the materials and methods and then discussion.

Incorporate Citations

-Lacks some citations for aphid studies in the past 5 years that used HTS to examine microbiota composition and/or phylogenetic relationships of microbes. While the citations used are fine, the predominant use of older/foundational references is noticeable.

Minor Comments

L62: Update italicization from genus and species being italicized to Candidatus being italicized and genus and species being unitalicized

L81: Suggested update “microbiota associate” to “microbiota associated”

L91: Suggested update to “many whose ecological and taxonomic position remain largely unknown”

L101: change “microbiome” to “microbiota”

L126: change “step” to “steps”

L186: suggested to add the 3 OTUs that were removed as negative controls. “were identified as contaminants and removed (e.g. contaminant 1, contaminant 2, contaminant 3).”

L247: Dactylopiibacterium symbionts are noted to be found in scale insects (Vera-Ponce de León et al. Genome Biol Evol. 2017 and Bustamante-Brito et al. Life. 2019.). Where is the citation to support that this is a known associate of aphids? This sentence should have some citations provided.

L249: Revise to something along the lines of “We did not find any bacterial partners that can be considered as environmental-related (e.g. Pseudomonas spp., Erwinia spp., etc.) as in the case of other aphid species, including those that feed on cereal crops [29, 31].”

L252 – L253: Recommendation to combine both sentences into one: “S. symbiotica is one of the most common symbiont species in aphid populations [55] and was identified in three of the eight colonies surveyed.”

L259 – L261: Suggested reduction and clarification of sentences into one: “This symbiont, an α-proteobacterium, is commonly found in insects and studies suggest that Wolbachia is present in at least 65% of arthropod species [59].”

L300: Change “Quite few” to “Few”

References

Update references so that the first letter of the title is capitalized and the remaining portion of the title is in lower case. Also make sure to italicize scientific names of microbial organisms.

L354, L357, L470, L472, L475, L477, L485, L491, L498, L503, L508: Italicize Wolbachia

L371: Italicize Buchnera

L379: Remove all caps from manuscript title

L520, L526, L532: Italicize Arsenophonus

L543: Update italicization from genus and species being italicized to Candidatus being italicized and genus and species being unitalicized

Table 2

In Id% columns change the formatting from a “,” to a “.”. For example “98,28” would be formatted as “98.28”.

Figure 1

The map figure needs greater resolution as it is currently blurry. For someone unfamiliar with this area, the map is not very useful. It would be nice to have each point labeled with the colony name and then have this linked to the supplementary Table S1. GPS coordinates.

Figure 2

This figure is also blurry and needs greater resolution. Not all the colors labeled that correspond to different microbes are show in the graph. It would be helpful to clearly label the X and Y axis with what they represent. Also it is highly recommended to change the color of either Serratia or Wolbachia, as those with red-green colorblindness will be unable to distinguish between the two colors selected.

Figure 3

While the samples are labeled in figure 3, they are in the opposite orientation as those in Figure 2. This should be updated so that the sample text faces the same direction for both Fig 2 and 3. Also the figure resolution should be increased.

Table S2

Update font size in this supplementary document so that it is consistent for all OTUs, rather than size 10 font in some places and size 12 font in others.

Supplementary figures 1-4

-Greater resolution on figures needed.

-Neighbor joining trees have very low branch support values. In the context of this study, could be interpreted as distantly related bacterial strains still being affiliated with other insects. However, when it comes to Wolbachia from A. corni the low support (75%) from the neighbor joining tree and separation from all other strains of Wolbachia could be that this is a closely related bacteria masquerading as Wolbachia.

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26 Jul 2021

Dear Academic Editor,

Please find attached the revised version of our manuscript "Insight into the Microbiome of the Subterranean Aphid Anoecia corni" (Manuscript [PONE-D-20-40214] - [EMID:8d559adb517b70dd]). We thank you and the reviewers for their relevant comments and encouragement. This reinforces the idea of intensifying our research efforts in using next generation sequencing approaches to decipher how bacterial communities (and beyond) are shaped in insect populations. All of the reviewers' comments have been addressed and the manuscript has been revised accordingly. Our responses are in red in this letter as well as in the new version of the manuscript. We hope that the changes and additions have sufficiently improved the manuscript to meet PLOS ONE standards.

We would like to point out that, following the reviewers' comments, we have changed the title to "Insight into the bacterial communities of the Subterranean Aphid Anoecia corni".

We look forward to your response

Sincerely,

Samir Fakhour and co-authors

Dear Dr. Fakhour,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I am really sorry for the long time that all this review process took; your manuscript has been evaluated by three independent reviewer and although one has recommended rejection, the other two say it has great value and should be published after revisions. So I kindly ask you to reply to all the reviewers and try to integrate their comments and suggestions in your revised manuscript

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Reply: Based on your suggestion, the correction has been made. The map has been changed to another figure with better resolution and the image of the map is not under copyright. The aphid colony code has been reported in this figure as shown in Table S1.

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Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

________________________________________

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript by Fakhour and colleagues describes the bacterial microbiome associated with 16 samples of the aphid Anoecia corni collected on wheat roots in two regions of Morocco.

The manuscript is well written and presented in general, but it has limited interest even to those who specifically study insect microbiomes. The bacterial species identified in this work were already known to be associated with aphids, so the manuscript adds little to the current understanding about the aphid microbiome. As a general comment, the in silico study of microorganisms other than bacteria (e.g. fungi, which are key components of the plant rhizosphere) would have increased the novelty and interest of the paper. Insect microbiomes, in fact, include also fungi, viruses, and protozoa. As A. corni lives in close association with soil and wheat roots, at least the fungal component of the insect microbiome should have been explored.

In addition, environmental parameters associated to each sampling site (e.g. precipitation, temperature, etc…) are not provided. Such data, for example, might have shed light to the possible impact of the environment on the biodiversity indexes, which, by the way, are totally missing.

We agree with these remarks and the limitations pointed out by the reviewer. This study was initially based on a large sampling campaign covering the cereal growing areas of Morocco and targeting cereal aphids and their associated bacterial communities. With this exploratory study, we had the ambition to report the bacterial diversity associated with an aphid receiving little attention and living in close contact with the soil microbial flora, with the hypothesis that they are potentially associated with a diverse bacterial community given their lifestyle. Obviously, this is not the case and the contrary would certainly have made the study more interesting (but the results are as they are...). The comment about identifying other types of microorganisms is also relevant, especially about fungal diversity, which was not tested here. It is true that this is an aspect that has been little explored in aphids and beyond, whereas recent studies have shown that in some species, the fungal diversity encountered could even be at the origin of new endosymbioses where bacteria are replaced by fungi. This is the case in some cicadas, grasshoppers, and aphids where fungi of the genus Ophicordyceps have become obligate symbionts by replacing ancestral bacterial symbionts (Suh, Noda and Blackwell 2001; Vogel and Moran 2013; Xue et al. 2014; Blackwell 2017; Matsuura et al. 2018). Insect-associated bacterial communities have received much attention over the past two decades, somewhat eliding fungal diversity that would certainly deserve more attention, and aphids having a subterranean lifestyle might be good models to begin such an exploration. Although this could not be done in our study, this perspective is now mentioned in the manuscript.

L308-316: We added perspectives on the importance of assessing fungal diversity in insects.

Minor issues

The title: as the Authors analyzed only the bacterial component of the aphid microbiome, I suggest they modify the title accordingly.

Reply: we changed the title which is now: “Insight into the bacterial communities of the Subterranean Aphid Anoecia corni” (L1).

Abstract line 28: the Authors claimed that the bacterial microbiome of aphids is “poorly diversified”, but later in the Introduction they stated that aphids are associated “with a wide range of bacterial symbionts”. Please explain this contradiction.

Reply: Although there is ambiguity, there is no contradiction. Indeed, aphids are known to be associated with low bacterial diversity compared to other insects such as fruit flies, ants, termites, etc. which may be associated with a high diversity of environmental bacteria residing in their digestive tract. In aphids, this low bacterial diversity in the digestive tract stems from the fact that the phloem sap on which they feed is known to be virtually devoid of microorganisms (Grenier, Nardon and Rahbé 1994). Thus, aphid-associated bacterial communities are largely dominated by a few symbiont taxa (there are about a dozen, but an aphid rarely harbors more than three) as in other sap-feeding insects (Colman, Toolson and Takacs-Vesbach 2012; Jing et al. 2014). Thus, aphids are characterized as being associated with a low diversity bacterial community, which essentially revolves around a dozen species of bacterial symbionts. If the bacterial community associated with the aphid is low, on the other hand, the symbiont diversity is rather broad since most insects do not harbor heritable bacteria (at the exception of Wolbachia) but do harbor a large diversity of transient bacteria.

L53: We added the term "heritable" which specifies more precisely what we mean by symbiont (there is a durable and heritable character in the relationship maintained with the insect).

Abstract line 42: In this study the Authors took into consideration only the bacterial endosymbionts, so it is obvious that the bacterial diversity found in A. corni samples is limited to this part of the insect microbiome. Please rephrase.

Reply: We do not understand this comment. In this study, the 16S rRNA amplicon Illumina sequencing approach was used to assess the composition of the overall bacterial communities associated with our sampled aphids. It is true that the presence of other bacteria, for example living in the digestive tract, is deemed to be rare in aphids, as the phloem sap is considered an almost sterile environment (Grenier, Nardon and Rahbé 1994). Nevertheless, some studies have revealed the sporadic presence of extracellular bacteria have been in the digestive tract of aphids. These include beneficial gut symbionts, pathogens, plant pathogens, and environmental contaminants (Harada et al. 1997; Sevim, Çelebi and Sevim 2012; Gauthier et al. 2015). In a previous study on five species of cereal aphids, and in addition to the bacterial endosymbionts, bacteria from the genera Pseudomonas, Acinetobacter, Pantoea, Erwinia and Staphyloccocus were detected (Fakhour et al. 2018). Extracellular strains of the symbiont Serratia symbiotica could also be detected in the digestive tract (Pons et al. 2021). These observations thus suggest that the aphid microbiome is not necessarily limited to intracellular endosymbiotic bacteria alone. Therefore, our approach focused on all bacteria present in the sampled aphids. We did not consider only heritable symbionts, but we found almost only that.

However, to be more accurate, we have replaced in the abstract "symbiotic diversity" by "bacterial diversity" (L30).

Table 2: What does “PC reads.” stands for?

Reply: PC reads corresponds to cluster size in percent. We have added a caption to line 204.

Introduction page 3: “B. aphidicola” and “A. corni” should be written without abbreviations as it is the first time they are cited in the manuscript. Check the spelling of “Hamiltonella defensa”

Reply: Based on your suggestion, the correction has been made (L56 & L62).

Page 4 line 90: the correct spelling is “dioecious”

Reply: The correction has been made (L90).

Page 6 and Materials and Methods in general: if the procedures are already described in another paper, I suggest to cite the paper and keep the description short, providing only relevant modifications from the previous protocols/approaches, if there are any.

Reply: We have shortened some procedures already mentioned in one of our previous studies (Fakhour et al. 2018). By rereading the manuscript, we confirm that there is nothing to change on this side.

Page 11 line 214: OTU_6 (and not OTU 7) corresponds to Wolbachia (Table 2) and I assume that OTU 18 is actually OTU 8 and OTU 19 is OTU 9, is it correct?

Reply: The correction has been made (L221 & L222).

Figure 1 improve resolution and associate the library codes to the sampling locations.

Reply: Based on your suggestion, the correction has been made.

Supplementary figures: phylogenetic trees should first report bacterial names and then their host names in brackets, otherwise they seem to represent insect phylogeny, instead of that of their symbionts. In some cases there are bacterial names and insect names on different branches of the same tree.

Reply: We agree with this comment. Phylogenetic trees have been modified according to your suggestion (Lines 668 to 698 and Fig S1, S2, S3 and S4).

Reviewer #2: Overall comments:

In the present paper, the authors use 16S rRNA sequencing to investigate for the first time the microbial communities associated to the subterranean aphid Anocia comi.

They identified a total of 23 OTUs, 10 of them being associated to 5 putative symbiont genera, including the obligatory symbiont Buchnera aphipicola and 4 known facultative symbionts. Among them was Wolbachia, which is considered rare in aphids.

Overall, I am pleased by the results presented in the paper. However, some elements are unclear and need to be reworked to make the paper easier to understand and reproduce for other researchers. In particular, figures need to be reworked, and it seems (to me at least) that there is some confusion in sample naming and OTU numbering.

Hereafter are my comments. Most of them are typos or minor comment but some of them are more important for the comprehension of the paper.

Abstract:

37 : I'd remove "hosting"

Reply: Based on your suggestion, the correction has been made (L37).

42 : I don't understand this sentence. What is expected apart from bacterial endosymbionts?

Reply: Bacteria residing in the digestive tract, pathogens, plant pathogens and environmental contaminants. Please, see comments above (reviewer 1) and the manuscript L67-71 for example.

Data analysis: for the sake of reproducibility, the commands and options used for the different software should be supplied.

Reply: Based on your suggestion. We added the commands and options used for the different software in supporting information (S2 table. Data analysis: Commands and options used to build the OUT table).

161 : missing citation for dada2

Reply: The appropriate citation has been added (Callahan et al. 2016) (L163).

167 : There is no correpondance given between the identifiers you used for your samples and the ENA identifiers. Could you please add the accessions in sup Table 1 for instance?

Reply: Based on your suggestion, the the ENA identifiers (sample accession ENA) has been added for each sample in S1.Table (second column of the table).

169 : missing citation for SeaView

Reply: The appropriate citation has been added (Gouy, Guindon and Gascuel 2010) (L172).

Table 1 : Maybe add median sequence length after filtering?

More detailled statstics (min-median-max) would also be useful

Reply: Based on your suggestion, we added the Sequence length (min; median; max) in the Table 1 (page 8).

186 : 0.23% is not much but is massive compared to the abundance of Wolbachia for instance. I would at least remove the "only".

Also, the complete abundance table for the 23 OTUs should be available as supplementary.

Reply: We add a S4 Table. Contaminant OTUs identified in this study in the Supplementary files. That presents the 13 contaminant OTUs identified from negative control analysis with their size in percent (OTU 11 to 23).

188 : The contaminant OTUs are also "genuine". I would use "biologically relevant"

Reply: Based on your suggestion, the correction was made (L190).

Table 2 :

OTU identifiers do not match with the figures.

I'd appreciate a comment on the discrepancies between the Greengenes identification (from dada2?) and the Blast based

Reply: Greengenes databse is discontinued and not been updated since many years, also, there are several mistakes in the taxonomy (> 20%), so for that we have somes discrepancies.

Fig 2 :

- The naming of samples is very unclear. I understand (from the supplementary) that the number indicates the locality. But this naming convention gives the impression that samples are paired. There is also a H12 sample which is puzzling to me.

- Could also indicate on Fig2 the geographic origin of samples?

- Text should be bigger (also for other figures)

- Apart from the presence absence of Serratia, the Figure is hard to read. Maybe use a log scale? And it's also quite redundant with Fig. 3

Fig 3 : Legend : How is that "relative abundance"? It seems to me that these are absolute counts. The Figure with relative abundances would be Fig. 2.

I would rephrase "Each color bar corresponding" by "Each column represents"

You mention log counts, but the counts on the scale do not seem log transformed. I believe only the scale is logarithmic.

Maybe a heatmap made only of secondary symbions in addition to this one would be useful to understand where they are (ideally with read counts or relative abundances written on the heatmap)

Reply: Based on your suggestion, the correction was made. The Figure 2 has been removed to avoid redundancy. Figure 3 then becomes Fig. 2 and it has been improved according to your instructions.

217 - Fig S1-S4 : Overall the trees are poorly supported and not discussed in the paper.

Reply: We hope it's better now. However, we keep them as an indication. It is true that we do not develop these analyses and results. In our opinion, these trees are informative and should be left in the data supplements: they have been built on the basis of short sequences (unavoidable here with short 16S sequences of 450 bp) and are therefore not extremely robust (L222 to L224).

234 : "Extremely low number of reads" : Please give numbers, in absolute (read counts) and relative (%). The count table for all OTUs and all samples should be available as supplementary.

Reply: Based on your suggestion, the correction was made. The tables S3 and S4 in the supplementary files have been improved and now present the numbers in absolute (read counts) and relative (%) for each OUT (representative and contaminants).

You make no comment on the fact that the different Buchnera OTUs are not evenly distributed across samples.

Reply: we have added details on the different haplotypes associated with aphids from the two regions of Morocco in results and discussion sections (Lines 209 to 218 and lines 258 to 262, respectively).

249 : Could it be because you used a different extraction protocol?

Reply: On the contrary, we used the same extraction protocol as the one used in the previous study on cereal aphids (Fakhour et al. 2018).

Reviewer #3: PLOS ONE

Review: “Insight into the Microbiome of the Subterranean Aphid Anoecia corni”

Authors: Samir Fakhour, François Renoz, Jérôme Ambroise, Inès Pons, Christine Noêl, Jean- Luc Gala, Thierry Hance

General Comments

The study has an interesting premise to investigate the microbiota of ground dwelling, root feeding aphids. The paper provides a strong hypothesis as to why these aphids might harbor more microbes, and sets out to examine this using High Throughput Sequencing. The incorporation of phylogenetic relatedness of microbial organisms to support whether they are aphid, soil, or plant associated is good, although this concept should be mentioned in the introduction.

Overall this manuscript provides a good discussion of the 5 main bacterial OTUs found in A. corni. An overview of the functions these microbes in other organisms was provided along with the bacteria’s potential role in this aphid. The conclusion provided a nice summary of additional lines of investigation for the role of Arsenophonus and Wolbachia in A. corni.

Major Comments

Flow and organization can be enhanced

-Some sentences in the discussion have repetitive content and could be enhanced by restructuring or merging two sentences into one. Reminders throughout the text of the main ideas are helpful, however the sentence repetition does not help drive the manuscript content forward.

Reply: We have reread the manuscript several times, but without more details from you, it is difficult to know exactly which part to change. The introduction is quite direct, short, without redundancy. Same thing for the discussion which is quite short. The only slight redundancy is in the discussion to remind the interest of these aphids (subterranean lifestyle) in the light of the overall results. So, if you think some syntax adjustments are necessary, please give us more details.

-Incorporate relevance into the introduction

Reply: Again: what do you mean? Could you clarify this?

-The introduction should mention the importance of phylogenetic relatedness in determining microbial associations, to prime the reader for the Neighbor Joining tree analyses in the materials and methods.

Reply: With all due respect, we prefer to leave things as they are. Indeed, reviewer 2 is not very convinced of the approach, which we understand. Phylogenetic trees are based on the alignment of rather short 16S DNA sequences (about 450 bp). This does not provide good robustness (probably at least 1500 bp and preferably several concatenated genes). The trees are indicative, but given their lack of robustness (despite appreciable node values with the new analyses) we think it is more appropriate to leave them in the data supplements section to show that one is cautious and not to start speculating on these results (probably with longer reads and more genes the topologies would have been different).

Clarify Materials and methods

-Based upon the sample collection information, it is not entirely clear the process for obtaining aphids and then raising the colonies. Were apterous adult aphids collected on wheat roots, then placed into colonies and then used to generate clones? Then were these aphid clones used to assess microbial diversity? It is unclear.

Reply: In fact, we do not understand where this is not clear. We went to the field and directly collected the insects which were directly placed in tubes filled with ethanol. We did not generate clones in the laboratory. What we did was pretty standard (Fakhour et al. 2018).

-Revise discussion for clarity and flow

Reply: Once again, we have re-read the discussion. We feel that it is short, fluid and the sentences are short. If sections of the discussion are not well written or obscure, please give us more information so we can do the best job possible.

-The manuscript makes a bold claim that no environmental bacteria were found associated with aphids, however only the aphids were sampled and not the plant or xylem fluids. The way that this is written should be framed in the context that bacterial symbionts were not more abundant in ground dwelling aphids despite being in contact with soil microbes and having access to xylem. An average or assessment of microbes identified in other aphids beyond the typical symbionts should be provided if this comparison is to be made.

Reply: We hope it's better now (L307-314).

-While after reading L239 to L234 it is clearer to the reader that the phylogenetic assessment using Neighbor Joining (NJ) trees were intended to clarify the relationship of microbial organisms to aphid hosts, the relevance of phylogenetic relationships among microbes and their environment/host association should be mentioned in the introduction to prep the reader for this in the materials and methods and then discussion.

Reply: We hope it's better now (L102-103).

Incorporate Citations

-Lacks some citations for aphid studies in the past 5 years that used HTS to examine microbiota composition and/or phylogenetic relationships of microbes. While the citations used are fine, the predominant use of older/foundational references is noticeable.

Reply: We have updated some citations for aphid studies in the past 5 years.

- We add : Tian et al., 2019 (L61) ; Skaljac et al., 2018 (L62) ; Kaech and Vorburger, 2020 (L62) ; Polin et al., 2015 (L62) ; Romanov et al., 2020 (L63) ;

- The Jamin et al, 2019 and Parker et al., 2021 citations have replaced Scarborough et al., 2005 (L61)

- The Moreira et al., 2019 and Ren et al., 2020 citations have replaced Augustinos et al., 2011 and Gomez-Valero et al., 2004 (L63)

- The Mathé-Hubert et al., 2019 and Guidolin et al., 2018 citations have replaced Fukatsu et al., 2001(L64)

- The Leclair et al., 2021; Chevignon et al., 2018 ; Oliver and Higashi, 2018, Heyworth and Ferrari, 2015 and Frago et al., 2017 citations have replaced Oliver et al., 2003 and Scarborough et al., 2005 (L65)

- The Tsuchida, 2016 and Nikoh et al., 2018 citations have replaced Tsuchidaet al., Tsuchida et al., 2004 (L65)

- The Heyworth et al., 2020 citation replaced Burke et al., 2010 (L66)

- The Lenhart & White, 2020 and Wagner et al., 2015 citations have replaced Tsuchida et al., 2004and Koga et al., 2003 (L66)

Minor Comments

L62: Update italicization from genus and species being italicized to Candidatus being italicized and genus and species being unitalicized

Reply: Based on your suggestion, the correction has been made (L62).

L81: Suggested update “microbiota associate” to “microbiota associated”

Reply: Based on your suggestion, the correction has been made (L81).

L91: Suggested update to “many whose ecological and taxonomic position remain largely unknown”

Reply: Based on your suggestion, the correction has been made (L91-92).

L101: change “microbiome” to “microbiota”

Reply: Based on your suggestion, the correction has been made (L101).

L126: change “step” to “steps”

Reply: Based on your suggestion, the correction has been made (L128).

L186: suggested to add the 3 OTUs that were removed as negative controls. “were identified as contaminants and removed (e.g. contaminant 1, contaminant 2, contaminant 3).”

Reply: Based on your suggestion, we added some information’s concerning de 13 (not 3) OTU removed as negative controls in the tables S4 Table. Contaminant OTUs identified in this study (see supplementary files).

L247: Dactylopiibacterium symbionts are noted to be found in scale insects (Vera-Ponce de León et al. Genome Biol Evol. 2017 and Bustamante-Brito et al. Life. 2019.). Where is the citation to support that this is a known associate of aphids? This sentence should have some citations provided.

Reply: At present, this bacterium has only been identified in these insects. It is thus for the first time (and it is a bit curious) that this species is reported in aphids. We cannot therefore include references related to aphids for this bacterium...

L249: Revise to something along the lines of “We did not find any bacterial partners that can be considered as environmental-related (e.g. Pseudomonas spp., Erwinia spp., etc.) as in the case of other aphid species, including those that feed on cereal crops [29, 31].”

Reply: Based on your suggestion, the correction has been made (L255-257).

L252 – L253: Recommendation to combine both sentences into one: “S. symbiotica is one of the most common symbiont species in aphid populations [55] and was identified in three of the eight colonies surveyed.”

Reply: Based on your suggestion, the correction has been made (L263-264).

L259 – L261: Suggested reduction and clarification of sentences into one: “This symbiont, an α-proteobacterium, is commonly found in insects and studies suggest that Wolbachia is present in at least 65% of arthropod species [59].”

Reply: Based on your suggestion, the correction has been made (L270-272).

L300: Change “Quite few” to “Few”

Reply: Based on your suggestion, the correction has been made (L318).

References

Update references so that the first letter of the title is capitalized and the remaining portion of the title is in lower case. Also make sure to italicize scientific names of microbial organisms.

L354, L357, L470, L472, L475, L477, L485, L491, L498, L503, L508: Italicize Wolbachia

L371: Italicize Buchnera

L379: Remove all caps from manuscript title

L520, L526, L532: Italicize Arsenophonus

Reply: Based on your suggestions, all requested changes have been made.

L543: Update italicization from genus and species being italicized to Candidatus being italicized and genus and species being unitalicized

Reply: Based on your suggestion, the correction has been made.

Table 2

In Id% columns change the formatting from a “,” to a “.”. For example “98,28” would be formatted as “98.28”.

Reply: These changes have been made.

Figure 1

The map figure needs greater resolution as it is currently blurry. For someone unfamiliar with this area, the map is not very useful. It would be nice to have each point labeled with the colony name and then have this linked to the supplementary Table S1. GPS coordinates.

Reply: We have made the requested changes. We hope that the new map is more suitable.

Figure 2

This figure is also blurry and needs greater resolution. Not all the colors labeled that correspond to different microbes are show in the graph. It would be helpful to clearly label the X and Y axis with what they represent. Also it is highly recommended to change the color of either Serratia or Wolbachia, as those with red-green colorblindness will be unable to distinguish between the two colors selected.

Figure 3

While the samples are labeled in figure 3, they are in the opposite orientation as those in Figure 2. This should be updated so that the sample text faces the same direction for both Fig 2 and 3. Also the figure resolution should be increased.

Reply: Based on your suggestions of those the reviewer 2, the figure 2 has been removed to avoid redundancy. Figure 3 then becomes Fig. 2 and it has been improved according to your instructions.

Table S2

Update font size in this supplementary document so that it is consistent for all OTUs, rather than size 10 font in some places and size 12 font in others.

Reply: Based on your suggestion, the correction has been made.

Supplementary figures 1-4

-Greater resolution on figures needed.

Reply: Based on your suggestion, the correction has been made.

-Neighbor joining trees have very low branch support values. In the context of this study, could be interpreted as distantly related bacterial strains still being affiliated with other insects. However, when it comes to Wolbachia from A. corni the low support (75%) from the neighbor joining tree and separation from all other strains of Wolbachia could be that this is a closely related bacteria masquerading as Wolbachia.

Reply: We have improved the branch support values of the phylogenetic trees (Figure S1-4). However, the analyzes were performed on the basis of short sequences (16S sequences of 450bp), which may explain these weak supports.

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29 Jul 2021

Insight into the bacterial communities of the Subterranean Aphid Anoecia corni

PONE-D-20-40214R1

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Reviewers' comments:

2 Aug 2021

PONE-D-20-40214R1

Insight into the bacterial communities of the Subterranean Aphid Anoecia corni

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