Microbiome composition of larval Zeugodacus cucurbitae
Three out of 27 phyla dominated the larval microbiomes: Proteobacteria,
Firmicutes and Bacteroidetes (S7). In total, 122 orders, 205 families
and 527 genera were identified in the larval microbiomes. The most
abundant genera were Enterobacter (25%), Leuconostoc(9%), Acinetobacter (7.21%), Lactococcus (6.6%),Gluconobacter (6.15%) and Empedobacter (5.9%).
Across all host plants we identified seven core genera :Acinetobacter, Enterobacter, Klebsiella, Paenibacillus,
Pseudomonas, Stenotrophomonas and Sphingobacterium .
Larval microbiomes showed a high ASV richness (mean = 145.36, SD =
75.56) but low phylogenetic diversity (mean = 13.51, SD = 4.58). Despite
their high ASV richness, larval microbiomes were dominated by very few
ASV as the ISI had a mean of 7.97 (SD = 6.21). The log transformed ISI
did not show consistent patterns across sites or host plants (S6- S7).
There was a significant interaction between host plant and site in the
fourth root transformed ACE, the log transformed ISI and the fourth root
transformed FPD (Table 1). The α diversity indices did not show
consistent patterns across sites and host plants. ASV richness as
estimated by ACE and FPD showed significantly higher values for Co in M,
but not in BP. Conversely evenness showed significantly higher values
for So in BP, but not in M (SI6-7).
PERMANOVA of the Generalized and Unweighted Unifrac distances revealed a
significant interaction for β diversity between host plant and site
(Table 2). However, pairwise comparisons did not provide additional
information as all were significant (S8).
The PCoA on presence/absence data (Unweighted Unifrac distances)
explains 46.15% of variation and suggests similarity between larvae
feeding on different host plants (Fig. 1), however this pattern is not
recovered in the PCoA on weighted abundancies (Generalized Unifrac
distances, 59.03% of variation).
Several microbial genera showed a significant differential abundance
between larvae raised on different host plants (S9 and Fig. 2-3).Ketogulonicigenium , Devosia, Salmonella, Allorhizobium and
three possible unidentified genera from the family Enterobacteriaceae
genera were more abundant on Co than on Cu and So. Most of the genera
with higher abundance in So larvae belonged to the Order
Enterobacterales, with Rahnella being detected in So larvae only.
Compared to Cu larvae, larvae on So also showed enrichment in a few
genera belonging to the Acetobacteriaceae, the class Bacilli and in the
genus Pseudomonas . Larvae raised on Cu showed higher abundance
for genera belonging to the bacterial families Clostridia and
Negativicutes from the phylum Firmicutes and for the genusLeucobacter . An additional three genera of the Clostridia were
more abundant in Cu than in Co larvae. In contrast, taxa belonging to
Acetobacteraceae and to genus Pseudomonas were less abundant in
larvae raised on Cu compared to larvae from other host plants.
Between sites, only the genus Comamonas showed a pattern of
differentiation, as its relative abundance was 8.5x lower in larvae from
M than in larvae from BP. Within hosts, more bacterial genera showed
differential abundance between M and BP (S12).
Predictions from Tax4Fun2 suggested that bacteria might be involved in a
variety of KEGG level 2 pathways important for host-symbiont
interactions. The most abundant KEGG pathways include carbohydrate
metabolism (18.8% of pathways), amino acid metabolism (11.8%),
membrane transport (14.9%), cellular community (8.3%), cofactor and
vitamin metabolism (6.1%), energy metabolism (6.3%), lipid metabolism
(4.3%), nucleotide metabolism (4%) and xenobiotics degradation and
metabolism (3.8%). Together these pathways involved over 76% of the
predicted metabolic pathways (Fig. 4). Tax4Fun2 also identified some
pathways associated with plant-pathogen interactions at low abundance
(0.14%) and antibiotic resistance (2.5%).
Based upon the predictions of Tax4Fun2, there appear to be functional
differences between larval and adult microbiomes. Fig. 4 shows that,
overall, adults are more enriched in pathways related to carbohydrate
metabolism and membrane transport. In contrast, amino acid metabolism
appears to be more enriched in larvae than in adults.
There was considerable variation between pooled larval samples in the
fraction of ASV’s and sequences that could be used by Tax4Fun2 to make
these predictions. Between 51.3% and 95.5% of all ASV’s and between
63% and 99.9% of sequences in samples were used in metagenomic
predictions.