Fig 1.
Kratom field located in the southern part of Thailand from which insect and non-insect leaves were sampled.
The map of Thailand was generated using R statistical software [42] with the raster colors indicating the mean elevation above sea level. The map was generated using publicly available data and open-source R packages (suitable for use under the CC BY 4.0 license). The world administrative boundaries shapefile was obtained from https://public.opendatasoft.com/explore/dataset/world-administrative-boundaries/export/ and imported into R software using the st_read function from the sf package. The digital elevation map (DEM) for Thailand was accessed using getData function of the raster package and cropped to the country of Thailand using the crop function of the terra package, while the final visualization was done using the ggplot2 package.
Fig 2.
Visual observations of insect attack on kratom leaves in the plantation in Nampu subdistrict, Ban Na San district, Surat Thani province.
Table 1.
Gradient condition program for the mobile phase to separate mitragynine.
Table 2.
Summary of genomic DNA analysis of the potential insect samples, including specimen ID, collecting date, stage, DNA concentration, COI barcode sequencing, and GenBank match results for insects samples collected from the kratom field in Surat Thani province.
Fig 3.
Insects identified in the kratom plantation;
(a) a larva from the lepidopterous insect family, with the same morphology as LEP12, (b) a larva of Parotis marinata (LEP17 sample), (c) a insect beetle of Miridiba sp., with the same morphology as COL06, (d) a insect beetle of Apogonia sp., and (e) kratom stem damaged by beetle attack.
Table 3.
Morphological and physiological characteristics of insect-infested kratom leaves and healthy leaves in Nampu Subdistrict, Ban Na San District, Surat Thani Province. The statistical difference (P-value) is obtained from the t-test statistical test, where a value greater than 0.05 indicates no statistically significant difference between the two groups, while *** indicates a significant difference. The superscripted letters indicate any differences in mean comparison.
Table 4.
Summary of the number of SMs, with those significantly loading the first two principle axes (PC) of PCA also indicated.
Fig 4.
Mitragynine content found in kratom leaves collected from undamaged (Non-Insect) and insect-damaged (Insect) leaves.
The P-value represents the result of a statistical test using the t-test at a 95% confidence level.
Fig 5.
PCA score plots as obtained from the LC-MS data for M. speciosa or kratom leaves characterized by insect and non-insect groups for the positive ion mode.
Fig 6.
The PCA scores plot characterizing the separation of insect (green) and non-insect (orange) samples based on the first two principal components, PC1 and PC2.
The variance explained by the first two principle components is indicated in the axis labels.
Fig 7.
The PLS-DA plot characterizing the separation between insect (green) and non-insect (orange) samples using two latent variables, Component 1 and Component 2.
Fig 8.
The VIP scores identifying SMs that significantly contributed to differentiating between the insect and non-insect samples as determined by the PLS-DA model. Chemical names are listed in supplementary Table S3.
Fig 9.
Cross-validated scores plot using OPLS-DA scatter plot (top panel) to determine the class separation between the insect and non-insect groups.
The points are labeled based on class membership into “In” and “No”, with the x and y-axes representing the predictive (tp) and orthogonal (to) components and the ellipse representing the 99% confidence intervals as determined by the Hotelling’s T² distribution. The scatterplot between the covariance and correlation or the S-plot is shown in the bottom panel, highlighting key metabolites contributing to group discrimination, based on P-value < 0.1 and VIP > 1.
Fig 10.
Box plots comparing the relative abundance of three metabolites between “Insect” (green boxes) and “Non-insect” (orange boxes) groups, with the metabolite indicated on the y-axis.
Statistical significance is indicated by * (P-value < 0.0) and ** (P-value < 0.01).