Proteomics/qPCR approach on estimating physical ages of wild male oriental fruit flies

Authors: Chang, Chiou

Submitted to: Journal of Asia Pacific Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/23/2016
Publication Date: 3/31/2016
Citation: Chang, C.L. 2016. Proteomics/qPCR approach on estimating physical ages of wild male oriental fruit flies. Journal of Asia Pacific Entomology. 19:347-351.

Interpretive Summary: Information on the age of individual wild-caught insects is important to diverse research areas such as applied pest management, behavioral and evolutionary ecology. Knowledge of ages has been crucial to fruit fly control programs, especially for monitoring population structures in Sterile Insect Technique programs. Data on the ages of wild-caught male fruit flies in field attractant studies is essential. Many techniques to estimate wild-caught insect physical ages have been introduced. All these methods may achieve some degree of success, but none are ideal. Oriental fruit flies are becoming model research organisms, frequently reared in research institutions. Research specimens domesticated in a lab-reared setting may have different behavioral phenotypes compared to their wild counterparts. Additionally, the absence of “key” stimuli in the physical environment of captive animals may result in altered biological patterns such as the length of life cycle. This study was to conduct a regression analysis of the ratio of odorant binding protein 99b expression from 4 to 12-d-old males, relative to 1-d-old males to estimate the physical ages of wild male fruit flies. Our findings indicated that despite strong behavioral differences between flies from laboratory stock and wild-caught, the physical ages of both groups are identical. We suggest proteomic and qPCR analysis of selected genes and the proteins they encode may be developed onto reliable tools for determining the ages of wild-caught animals, including oriental fruit flies.

Technical Abstract: Male fruit flies reared in the laboratory in DKI-PBARC rearing facility in Hilo, Hawaii, were collected and whole insects were run through standard proteomic analysis. An odorant binding protein 99b (OBP) (Bdor0907381) located at molecular weight between 9226 dalton and PI 4.56 was identified throughout 1-12 days old. Two regression lines based on proteomics and qPCR protocols were established to assess the wild fly ages: Y= -0.7768+ 0.7205X, R2= 0.89 (for proteomics) and Y= -0.6478+0.344X, R2 =0.64 (for qPCR). Two data validation protocols were used: proteomics approach and qPCR approach. Data for validation were including known ages of males from irradiated lab pupae with 100 gys and 30 gys (Chang et al. 2015), unknown ages of wild strain attracted by methyl eugenol (ME), laboratory strain attracted to methyl eugenol with known ages (8, 9, and 12 d old) (Chang et al., 2013), males from lufenuron treatment, control, and recovery, virgin males (24 d old), virgin males (13, 14, 15 days old), and mated males (13, 14, 15, 16 days old) (Chang et al., 2012; 2014). Three biological samples were analyzed by 2D-gel electrophoresis and Delta 2D software to validate these data using regression analysis created from 4-12 days old lab strain. All known ages samples were validated first, then further to validate the regression with samples from wild strain.