Structural changes in the bacterial communities
The number of valid reads varied among different samples (Table 2). A total of 6,274,405
high quality reads with an average length of 422 bp were obtained. Rarefaction analysis indicated that the number of species increased rapidly before reaching a plateau (Fig. S1).
Bacterial diversity among all the larvae group fed with different host plants were highly significant as demonstrated by three parameters (observed species richness, ACE and Chao1 (Table 1, Fig.1, Fig.S2, Fig. S3). Shannon index showed no significant difference among all the larvae group from the different host plants, although significant differences existed between larvae samples fed with celocia (L3) and cucumber (R1) with p value = 0.0207 by Shannon wilcox test. Simpson index also showed no significant differences among all the larvae groups fed with the various host plants.
Principal component analysis (PCA) by clustering showed that the bacterial communities of all the larvae groups were not totally different although some groups like M1 and R1 groups were distinct from other larvae groups (Fig. S4). This indicates that host plants had a significant effect on the larvae of FAW gut bacterial communities. Principal coordinate analysis (PCoA) was calculated based on weighted and unweighted UniFrac, which considers both community membership and relatedness of community members. These distances were visualized by PCoA plots which displayed similarity between communities (Fig. 2). The unweighted pair group method with arithmetic means (UPGMA) clustering analyses also indicated that the entire bacterial communities of bacteria group were clearly distinct from each other (Fig.3). Analysis of molecular variance (AMOVA) revealed that the bacterial communities were significantly different among the various larvae groups (p=0.002).
Comparison of Bacterial communities of FAW larvae from different host plants
Nine phyla (Firmicutes. Proteobacteria. Cyanobacteria. Actinobacteria. Bacteroidetes Acidobacteria Patescibacteria Chloroflexi and Verrucomicrobia) were present in all the samples at varied proportions (Fig.4a). Firmicutes was the most abundant phylum and accounted for 44.1 % of the total bacteria with 15.1% and 14.2 % from larvae fed with celocia (L3) and sweet potato (R3) plants respectively (Table S1). Proteobacteria with 28.5% of the total bacteria followed in abundance and was dominated in the larvae group fed on green amaranth (L2) and sugar cane (M1) with 18.5% and 14.3% respectively. Cyanobacteria constituted 11.4% of the total phyla and was mostly enriched by larvae groups fed with cabbage (L1) and cucumber (R1) with 20.0% and 17.9% respectively. Actinobacteria recorded 9.4% bacteria phyla representation and was mostly enriched in M3 and R2 with 24.3% and 20.9% respectively. Phylum Bacteroidetes was only 5.4% and was mostly enriched by R1 and L2 with 36.2% and 22.8% respectively. Acidobacteria recorded 0.35% of the total bacteria phyla and was enriched by M1and R1 with 40.7%and 38.3% respectively. Chloroflexi constituted 0.09% of the total bacteria phyla in the gut of S. frugiperda and was mostly contributed by R1 and M1 with 40.8% and 35.4% respectively. Phylum Rokubacteria constituted only 0.05% of total bacteria and was found in three groups; M1, R1 and M2 with 47.9%, 34.1% and 17.9 % respectively (Table S1). Unweighted Hierarchical clustering tree of all gut bacterial groups based on taxon distribution revealed gemmatimodetes and Chloroflexi to be among the top ten bacteria phyla in the gut of S. frugiperda and was only recorded in three samples (R1, M1 and M2) (Fig.3).
Eight bacteria classes were prevalent across all the larvae groups; Gammaproteobacteria, Bacilli, Erysipelotrichia, Oxyphotobacteria, Actinobacteria, Bacteroidia, Alphaproteobacteria, and Clostridia. Bacilli was the most dominant class representing 28.8% of the total classes followed by Gammaproteobacteria with 23.2%. Class Bacilli was mostly enriched by L3 and M1 with 21.3% and 15.8% respectively while Gammaproteobacteria was mostly enriched by L2 and M1 with 20.4 and 14.9% respectively (Fig.4b). Erysipelotrichia was 13.9% and was mostly enriched by M3 and L2 groups with 19.5% and 19.5% representation respectively (Table S2). The number and relative abundance of bacterial phylum to species levels varied among the different groups fed with different host plants, although there were no significant differences among them (Table 3). However, M1 sample recorded highest number of bacteria order and families with 196 and 328 respectively while R1 recorded highest number of bacterial genus and species with 578 and 416 respectively. Order Lactobacillales was the most dominant order observed in all the samples, followed by Erysipelotrichales (Fig. S5). They constituted 28.4% and 13.9% of all the bacteria order sequenced respectively. The order Lactobacillales and Erysipelotrichales were mostly enhanced in L3 and M3 larvae samples with 21.6% and 19.5% respectively (Table S3).
The resulting heatmap shows the relative abundances of the top 35 OTUs at the family level facilitating the identification of species that vary in abundance in each sample (Fig.5). Enterococcaceae was the predominant bacterial family in all the larvae samples constituting 28.3 % of all the bacteria families recorded (Fig. S6). Erysipelotrichaceae with 13.9 % and Moraxellaceae (13.3%) followed Enterococcaceae in occurrence. The family Enterococcaceae was mostly enhanced by L3 and M1 with 21.59% and 15.8% respectively while Erysipelotrichaceae and Moraxellaceae were mostly enriched by M3(20.5%) and L2(30.3%) respectively (Table S4). The relative abundance and number of bacterial genera varied in different larvae samples. The predominant genus Enterococcus (28.2%) was mostly enhanced in L3 followed by M1 with 21.7% and 15.7% (Fig. S7). Erysipelatoclostridium (13.5%) and Acinetobacter (13.33%) followed Enterococcus in relative abundance among the top 10 bacteria genera recorded. Erysipelatoclostridium and Acinetobacter were mostly enriched in L2 sample with 20.0% and 30.3% respectively (Table S5). The genus Klebsiella was mostly supplied by M1 (58.7%) while Corynebacterium_1 was dominant in M3 sample. Other minor bacteria genera apart from the top ten found in the different larvae samples was about 34.4%. Larvae samples fed with R1 was mostly dominated by genus Weeksella (64.0%).
The number and relative abundance of bacterial species also differed in larvae from different host plants ranging from 205 to 416. R1 sample with 416 number of bacterial species has higher number of bacteria species followed by M1 (415). The least bacterial species was recorded in L2 sample (Table 3). Uncultured_Clostridium_sp with 13.48 % of all the bacteria species was the most dominant species and was enriched in L2 and M3 with 20.0% and 19.2% respectively (Fig.S8). Acinetobacter_calcoaceticus and Klebsiella_variicola were mostly enhanced by L2 and M1 with 41,86% and 58,72% representation respectively. Pectobacterium_carotovorum_subsp._carotovorum and uncultured_ Flavobacterium_sp were mostly enhanced in M1 (50.2 %) and R1 (64.1%) respectively (Table S6). In total, 38724 OTUs were identified in all the larvae groups from different host plants. The number of OTUs significantly varied among the groups. The number of OTUs ranged among the larvae groups in the order of M1>R1>M2>R3>R2>L2>L1>L3>M3 (Table 3). M1 had the highest OTU numbers while M3 was the least among all the groups in the number of OTUs.
LEfSe analyses revealed that Phylum Cyanobacteria of class oxyphotobacteria, order chloroplast species Sphingobacterium_multivorum were enriched in larvae fed by L1 host plant (Fig. S9). Phylum Firmicutes of class Bacilli, order lactobacillales and family Enterococcaceae, genus Enterococcus and species Acinetobacter_baylyi were enriched in L3 fed larvae. Phylum protobacteria of class Gammaproteobacteria, order Pseudomonadales and family Moraxellaceae, genus Acinetobacter, species Acinetobacter_calcoaceticus as well as order Cytophagales of family Spirosomaceae and Leadbetterella were enriched in L2 host plant as well as order Flavobacteriales of families Flavobacteriaceae and Weeksellaceae, order _Corynebacteriales of family Tsukamurellaceae, Micrococcaceae and order Rhodobacterales of family Rhodobacteraceae were enriched in R1 host samples
M1 host plants were also enriched in Class bacilli of the order Lactobacillales, the family Enterococcaceae and genus Enterococcus. M3 enhanced predominance of the class_Actinobacteria of the order Corynebacteriales within the family Corynebacteriaceae, genus Corynebacterium_ and species Corynebacterium_variabile (Fig. S10). A significant variation was found between the R1 and R3 groups. We found that 6 taxa were overrepresented and 10 were underrepresented, e.g. order Flavobacteriales (Fig. 11).