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.
Document Type
Article
Publication Date
8-1-2011
Digital Object Identifier (DOI)
http://dx.doi.org/10.1186/1471-2164-12-388
Repository Citation
Bai, Hua; Zhu, Fang; Shah, Kapil; and Palli, Subba R., "Large-scale RNAi Screen of G Protein-coupled Receptors Involved in Larval Growth, Molting and Metamorphosis in the Red Flour Beetle" (2011). Entomology Faculty Publications. 9.
https://uknowledge.uky.edu/entomology_facpub/9
Additional file 1
1471-2164-12-388-s2.pdf (22 kB)
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1471-2164-12-388-s5.pdf (9 kB)
Additional file 5
1471-2164-12-388-s6.xls (44 kB)
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Additional file 7
1471-2164-12-388-s8.pdf (392 kB)
Additional file 8
Notes/Citation Information
Published in BMC Genomics, v. 12, 388.
© 2011 Bai et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Additional files are listed at the end of this page.
Additional file 1:
List of identified Tribolium GPCR. 111 non-sensory Tribolium GPCRs and their closest Drosophila homologs are grouped into four classes. Official ID from BeetleBase http://beetlebase.org webcite is used for each Tribolium GPCR. Known or putative ligand for each Tribolium GPCR is based on previous publications. CG number, symbol and full name forDrosophila GPCRs are based on Flybase http://flybase.org/ webcite with modification.
Additional file 2:
Phylogenetic tree analysis of Class A Rhodopsin-like GPCRs from T. castaneum and D. melanogaster. The tree is rooted by D. melanogaster metabotropic glutamate receptor (CG11144). Tribolium and Drosophila GPCRs are indicated by official ID or CG number followed by putative ligands in parentheses.
Additional file 3:
Phylogenetic tree analysis of Class B Secretin receptor-like GPCRs from T. castaneum and D. melanogaster. The tree is rooted by D. melanogaster FMRFamide receptor (CG2114). Tribolium and Drosophila GPCRs are indicated by official ID or CG number followed by putative ligands in parentheses. Gene symbol of methuselah-like GPCRs, Cirl and stan are also shown.
Additional file 4:
Phylogenetic tree analysis of Class C Metabotropic glutamate receptor-like GPCRs from T. castaneum and D. melanogaster. The tree is rooted by D. melanogaster FMRFamide receptor (CG2114). Tribolium and Drosophila GPCRs are indicated by official ID or CG number followed by putative ligands in parentheses.
Additional file 5:
Phylogenetic tree analysis of Class D Atypical GPCRs from T. castaneum and D. melanogaster. The tree is rooted by D. melanogaster FMRFamide receptor (CG2114). Tribolium and Drosophila GPCRs are indicated by official ID or CG number followed by putative ligands in parentheses. Gene symbol of frizzled GPCRs are also shown.
Additional file 6:
Summary of mortality phenotype caused by RNAi targeting Tribolium GPCRs. Larva, pupa and adult mortality resulted from Tribolium GPCR RNAi are shown. E. coli malE gene is used as a negative control. Asterisk indicates the mortality is calculated as the average of two trials.
Additional file 7:
Knockdown efficiency of four selected GPCR RNAi during larval stage of T. castaneum. Total RNA was extracted from pools of five larvae (at quiescent stage) injected with malE or GPCR dsRNA. The Y-axis denotes relative expression levels normalized using Tcrp49 mRNA levels as an internal control. Mean ± SE of three replications are shown. Asterisk indicates a statistically significant difference between control and GPCR RNAi insects.
Additional file 8:
Selection of stable reference gene using BestKeeper. To determinate the stable housekeeping gene, the expression of five reference genes were examined across 72 cDNA samples collected from different developmental stages (Larva day 1 to day 5, Quiescent larva day 1 and day 2, Pupa day 0 to day 6, Adult day 0 to day 2). (A). Based on the two most important criteria for evaluating the stability of reference genes by Bestkeeper program [48], the stability (SD value) and the relation to the BestKeeper index (r and P-value), five reference genes, Actin, Elongation factor 1-α (Ef1α), Ribosomal protein 49 (Rp49), Ribosomal protein s3 (Rps3), and Tubulin alpha 6 (Tubulinα6) are all stable in different developmental stages of T. castaneum. From the analysis, rp49 was chosen as the reference gene to calculate relative expression levels because it showed the most stable expression among samples. (B). Stable expression of five reference genes, Actin, Ef1α, Rp49, Rps3, and Tubulinα6 are shown across 12 RNA samples isolated from different developmental stages (LD1: Larva day 1; Q1: Quiescent larva day 1; PD0: Pupa day 0; AD0: Adult day 0). Products obtained after 40 cycles of PCR amplification under conditions described in the Materials and Methods section were resolved on an agarose gel.