Detection of temporal changes in insect body reflectance in response to killing agents

Abstract

Computer vision and reflectance-based analyses are becoming increasingly important methods to quantify and characterize phenotypic responses by whole organisms to environmental factors. Here, we present the first study of how a non-destructive and completely non-invasive method, body reflectance profiling, can be used to detect and time stress responses in adult beetles. Based on high-resolution hyperspectral imaging, we acquired time series of average reflectance profiles (70 spectral bands from 434-876 nm) from adults in two beetle species, maize weevils (Sitophilus zeamais) and larger black flour beetles (Cynaus angustus). For each species, we acquired reflectance data from untreated controls and from individuals exposed continuously to killing agents (an insecticidal plant extract applied to maize kernels or entomopathogenic nematodes applied to soil applied at levels leading to ≈100% mortality). In maize weevils (exposed to hexanic plant extract), there was no significant effect of the on reflectance profiles acquired from adult beetles after 0 and 12 hours of exposure, but a significant treatment response in spectral bands from 434 to 550 nm was detected after 36 to 144 hours of exposure. In larger black flour beetles, there was no significant effect of exposure to entomopathogenic nematodes after 0 to 26 hours of exposure, but a significant response in spectral bands from 434-480 nm was detected after 45 and 69 hours of exposure. Spectral bands were used to develop reflectance-based classification models for each species, and independent validation of classification algorithms showed sensitivity (ability to positively detect terminal stress in beetles) and specificity (ability to positively detect healthy beetles) of about 90%. Significant changes in body reflectance occurred at exposure times, which coincided with published exposure times and known physiological responses to each killing agent. The results from this study underscore the potential of hyperspectral imaging as an approach to non-destructively and non-invasively quantify stress detection in insects and other animals.

Fig 1

Average body reflectance profiles acquired from maize weevils on maize kernels with/without hexanic plant extract across all exposure times or for exposure times of 60 and 144 hours (a). Open dots represent spectral bands selected in linear discriminant classification of healthy and terminally stressed weevils. 95% confidence intervals as percentage of average reflectance in each spectral band (b). Relative effect (treated / untreated) of hexanic plant extract treatment on body reflectance profiles from maize weevils for all five exposure times (0–144 hours) (c).

Fig 2

Average body reflectance profiles acquired from larger black flour beetles exposed to soil with/without entomopathogenic nematodes (a). Open dots represent spectral bands selected in linear discriminant classification of healthy and terminally stressed weevils. 95% confidence intervals as percentage of average reflectance in each spectral band (b). Relative effect (treated / untreated) of hexanic plant extract treatment on body reflectance profiles from larger black flour beetles for all seven exposure times (0–69 hours) (c).

Fig 3

Results (F-values) from analyses of variance in 70 spectral bands from 434–876 nm of reflectance data from maize weevils on maize kernels with/without hexanic plant extract (a). Separate analyses were conducted for all combinations of spectral bands and exposure times. Arrow indicates position of spectral band at 448 nm. Average reflectance at 448 nm from maize weevils exposed to maize kernels with/without hexanic plant extract (b). Selection of time intervals for treatment responses according to results in b (selected) or one time point earlier (early) or later (late) (c). “*” denotes difference at the 0.05-level, “**” denotes difference at the 0.01-level, and “***” denotes difference at the 0.001-level.

Fig 4

Results (F-values) from analyses of variance in 70 spectral bands from 434–876 nm of reflectance data from larger black flour beetles on soil with/without entomopathogenic nematodes (a). Separate analyses were conducted for all combinations of spectral bands and exposure times. Arrow indicates position of spectral band at 448 nm. Average reflectance at 448 nm from larger black flour beetles exposed to soils with/without entomopathogenic nematodes (b). Selection of time intervals for treatment responses according to results in b (selected) or one time point earlier (early) or later (late) (c). “*” denotes difference at the 0.05-level, “**” denotes difference at the 0.01-level, and “***” denotes difference at the 0.001-level.