Large-scale RNAi screen of G protein-coupled receptors involved in larval growth, molting and metamorphosis in the red flour beetle

Abstract

Background: The G protein-coupled receptors (GPCRs) belong to the largest superfamily of integral cell membrane proteins and play crucial roles in physiological processes including behavior, development and reproduction. Because of their broad and diverse roles in cellular signaling, GPCRs are the therapeutic targets for many prescription drugs. However, there is no commercial pesticide targeting insect GPCRs. In this study, we employed functional genomics methods and used the red flour beetle, Tribolium castaneum, as a model system to study the physiological roles of GPCRs during the larval growth, molting and metamorphosis.

Results: A total of 111 non-sensory GPCRs were identified in the T. castaneum genome. Thirty-nine of them were not reported previously. Large-scale RNA interference (RNAi) screen was used to study the function of all these GPCRs during immature stages. Double-stranded RNA (dsRNA)-mediated knockdown in the expression of genes coding for eight GPCRs caused severe developmental arrest and ecdysis failure (with more than 90% mortality after dsRNA injection). These GPCRs include dopamine-2 like receptor (TC007490/D2R) and latrophilin receptor (TC001872/Cirl). The majority of larvae injected with TC007490/D2R dsRNA died during larval stage prior to entering pupal stage, suggesting that this GPCR is essential for larval growth and development.

Conclusions: The results from our study revealed the physiological roles of some GPCRs in T. castaneum. These findings could help in development of novel pesticides targeting these GPCRs.

Figure 1

Comparison of larva and pupa mortality caused by GPCR RNAi in T. castaneum. Stage-specific mortality pattern among different GPCR RNAi insects was compared. RNAi for five GPCRs results in most lethality occurred during the larval stage (Figure. 1A), while RNAi for 12 GPCRs caused relatively more pupal mortality than the larval mortality (Figure. 1B). dsRNA for malE (control) and selected T. castaneum GPCR were injected into one-day old final instar larvae. The percent mortality caused by dsRNA injection during the larval and pupal stages is shown. Mean ± SE of two independent experiments are shown.

Figure 2

Phenotypes observed after the injection of dsRNA for select GPCR into T. castaneum. dsRNA for malE (control) and selected T. castaneum GPCR were injected into one-day old final instar larvae. Phenotypes observed in control insects at three days after ecdysis to the final instar larval stage (A); quiescent stage, a non-feeding prepupal stage (B); three days after ecdysis to the pupal stage (C); three days after adult eclosion (D) are shown. (E). Phenotypes of TC007490/D2R RNAi insects. (F). Phenotypes of TC001872/Cirl RNAi insects. Accumulation of the ommatidia (black arrow) at quiescent stage is shown at the upper-left panel at higher magnification. (G). Phenotypes of TC012521/stan RNAi insects with wings attached to the ventral side of the abdomen (black arrow). (H). Phenotypes of TC014055/fz RNAi insects. The black arrow points to the split in the dorsal thoracic region. (I). Phenotypes of TC005545/smo RNAi insects with unextended pupal wings (white arrow). (J). Phenotypes of TC009370/mthl RNAi insects with improperly folded wings (white arrow) and unshed exuviae (white arrow head). Scale bar: 1.0 mm.

Figure 3

Stage-specific gene expression of six GPCRs in the whole body determined by qRT-PCR. Total RNA was extracted from pools of five larvae, pupae, or adult (three day-old males and females) for each time point. The Y-axis denotes expression levels normalized using Tcrp49 mRNA levels as an internal control. Mean ± SE of three replications are shown. Means with the same letter are not significantly different (α = 0.05; ANOVA).