Using near-infrared spectroscopy to resolve the species, gender, age, and the presence of Wolbachia infection in laboratory-reared Drosophila

Abstract

The aim of the study was to determine the accuracy of near-infrared spectroscopy (NIRS) in determining species, gender, age, and the presence of the common endosymbiont Wolbachia in laboratory-reared Drosophila. NIRS measures the absorption of light by organic molecules. Initially, a calibration model was developed for each study. An independent set with flies not involved in initial cross-validation was then used to validate the accuracy of each calibration model. Flies from the independent sets were correctly classified into Drosophila melanogaster and Drosophila simulans with 94% and 82% accuracy, respectively, whereas flies were successfully classified by gender with accuracy greater than 90%. In the age grading test, correlation plots of the actual and predicted age for males and females of D. melanogaster and D. simulans were shown to be overlapping between the adjacent age groups. It is, however, possible to predict the age of flies as less than 9 days of age with 62-88% accuracy and flies that are equal to or older than 9 days of age with 91-98% accuracy. Finally, we used NIRS to detect the presence of Wolbachia in flies. Flies from the independent sets were successfully identified as infected or not infected with Wolbachia with approximately 90% accuracy. These results suggest that NIRS has the potential to quantify the species, gender, and presence of Wolbachia in fly populations. However, additional optimization of the protocol may be necessary before the technique can reliably estimate fly age.

Keywords: Arthropods; parasite; population age structure; sex; species identification.

Figure 1

Mean spectra of all D. melanogaster and all D. simulans samples.

Figure 2

Regression coefficient plot for classifying D. melanogaster and D. simulans in the calibration model. The plot was generated with a 25-period moving average. (A) 1225 nm, (B) 1390 nm, (C) 1450 nm, (D) 1540 nm, (E) 1570 nm, (F) 1620 nm.

Figure 3

Near-infrared spectroscopy species prediction for D. melanogaster (D. mel) and D. simulans (D. sim). Plot shows actual species against predicted species. Dotted line indicates cut-off point for delimiting species.

Figure 4

NIRS gender classification of Drosophila species. (A) Actual sex against predicted sex for male (♂) and female (♀) D. melanogaster. (B) Actual sex against predicted sex for male (♂) and female (♀) D. simulans. Dotted line indicates cut-off point for classification of sex.

Figure 5

NIRS age grading of Drosophila species. Dotted lines refer to age classification of flies as either young (<9 days), middle-aged (9−18 days), or old (>18 days). Filled and nonfilled symbols represent values in the calibration model and independent set, respectively. (A) Actual vs. predicted age of male D. melanogaster Alst and Dah. Alst predicted age = 1.03 + (0.92 × actual age) (r² = 0.88) (B) Actual vs. predicted age of female D. melanogaster Alst and Dah. Alst predicted age = 1.22 + (0.90 × actual age) (r² = 0.86). (C) Actual vs. predicted age of male D. simulans Hw and Ky. Hw predicted age = 1.20 + (0.91 × actual age) (r² = 0.87) (D) Actual vs. predicted age of female D. simulans Hw and Ky. Hw predicted age = 1.28 + (0.90 × actual age) (r² = 0.87).

Figure 6

NIRS Wolbachia identification for Drosophila. Plot shows actual infection against predicted infection. Dotted line indicates cut off-point for delimiting infected and noninfected flies.