Fig 1.
Workflow of DNA extraction, PCR amplification, and Next-generation sequencing used for microbiome profiling of Phereoeca sp. larvae.
The process includes sample preparation, DNA extraction, DNA quality control, amplicon PCR, two-stage PCR amplification, PCR clean-up steps, library quality control, library normalization and pooling, followed by Illumina MiSeq sequencing.
Fig 2.
Representative larva of the household casebearer Phereoeca sp. collected from indoor environments in Peninsular Malaysia.
The larva is enclosed within a portable case constructed from silk, dust particles, and environmental debris. Scale bar = 0.5 mm.
Table 1.
List of Phereoeca sp. larval samples collected from three localities (Nilai, Benut, Bangi), including sample codes, species identification, and type of molecular analysis performed (COI barcoding or 16S rRNA metagenomics).
Table 2.
COI sequences used in phylogenetic analysis of Phereoeca spp., including reference sequences retrieved from GenBank and sequences generated in the present study.
Fig 3.
Agarose gel electrophoresis of PCR amplification products obtained from Phereoeca sp. larval samples.
(a) Amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene used for DNA barcoding. (b) Amplification of the bacterial 16S rRNA gene (V3-V4 region) used for microbiome analysis. Sample codes correspond to specimens collected from three localities: Nilai (1A-3A), Benut (1B–3B), and Bangi (1C-3C).
Fig 4.
Phylogenetic relationships of Phereoeca sp. inferred from mitochondrial COI sequences.
(a) Neighbor-Joining (NJ) tree constructed using Kimura 2-parameter genetic distances. (b) Maximum Parsimony (MP) tree reconstructed from the same dataset. Bootstrap support values are indicated at the nodes. Malaysian Phereoeca specimens form a distinct clade separate from reference sequences retrieved from GenBank.
Table 3.
Pairwise genetic distance matrix of COI sequences between Phereoeca sp. and related Lepidoptera species based on Kimura 2-parameter model.
Table 4.
Continuation of Table 4 showing additional genetic distance values among Phereoeca sp. specimens from different locations and reference sequences.
Fig 5.
Rarefaction curves showing sequencing depth and bacterial diversity recovered from Phereoeca sp. larvae.
The curves represent the number of observed amplicon sequence variants (ASVs) as a function of sequencing reads across samples from Johor, Negeri Sembilan (Nilai), and Selangor (Bangi). Curves approaching a plateau indicate adequate sequencing coverage.
Table 5.
Summary of sequencing output and microbial alpha diversity indices (Shannon, Simpson, Chao1, Observed ASVs) for Phereoeca sp. samples from Johor, Negeri Sembilan, and Selangor, Malaysia.
Fig 6.
Principal Coordinate Analysis (PCoA) plot illustrating differences in bacterial community composition associated with Phereoeca sp. larvae collected from three locations: Johor, Nilai (Negeri Sembilan), and Bangi (Selangor).
Samples cluster according to similarities in microbial community structure.
Fig 7.
Comparison of alpha diversity indices for bacterial communities associated with Phereoeca sp. larvae across sampling localities.
Diversity metrics include ACE, Chao1, Inverse Simpson, Observed ASVs, Shannon index, and Simpson index.
Table 6.
Top 25 most abundant bacterial classes identified in Phereoeca sp. samples across three locations, based on relative abundance.
Fig 8.
Venn diagram showing the number of shared and unique amplicon sequence variants (ASVs) among Phereoeca sp. samples from Johor, Nilai (Negeri Sembilan), and Bangi (Selangor).
Fig 9.
Relative abundance of bacterial phyla detected in Phereoeca sp. larvae from different sampling locations.
Each bar represents an individual sample, and colors indicate the proportion of major bacterial phyla identified through 16S rRNA gene sequencing.
Fig 10.
Relative abundance of dominant bacterial families associated with Phereoeca sp. larvae across sampling localities.
Each bar represents the microbial composition of individual samples.
Fig 11.
Relative abundance of bacterial genera detected in the microbiome of Phereoeca sp. larvae.
Bars represent individual samples, showing the distribution of dominant genera across different sampling sites.
Table 7.
Top 25 most abundant bacterial species (classified and unclassified) detected in Phereoeca sp. larvae, with corresponding relative abundance percentages.