| G protein-coupled receptors (GPCRs) play vital roles in multiple physiological processes of eukaryotes, including growth, development, reproduction and so on. However, family-B of GPCRs has been poorly illustrated in insects. To largely understand the evolution and function of them, we have performed comparative genomic analysis in six insects. By using family-B GPCRs from Drosophila melanogaster, we have totally identified64family-B members in six insects,20in Tribolium castaneum,9in Apis mellifera,11in Bombyx mori,9in Acyrthosiphon pisum and14in Anopheles gambiae, and defined1novel methuselah-like (mthl) gene in D. melanogaster.Phylogenetic analysis revealed that there are three primary evolutionary patterns during the evolution of insect family-B GPCRs. The first category genes exhibit orthologous evolutionary relationships in the insect, embracing PDF receptor, Latrophilin and two Mthl receptors, which possibly arose before the insect differentiation. The genes with species-specific expansion belong to the second pattern, including human epididymis6(HE6) receptor-like gene and mthl. The former has largely duplicated in T. castaneum but lost in hemimetabolous and hymenopteran insects, which is likely to relate to its dry habitat; the latter has expanded in D. melanogaster, and is assumed to enhance the ability to deal with all kinds of environmental pressure. The third pattern genes just mildly duplicated or lost in certain insects, such as calcitonin receptor, CRF receptor and one Mthl receptor. As the annotation of family-B genes in T. castaneum and nondrosophiline insects has been completed, it will promote the function, deorphanization and signal transduction of these GPCRs.A striking discovery in this study is the finding of parathyroid hormone (PTH) receptor like (PTHRL) gene in T. castaneum and A. mellifera, which is firstly reported from invertebrates. Phylogenetic analysis indicated that PTHRL gene has lost at least two times in insects but duplicated in the vertebrates before the divergence of vertebrates, suggesting that PTHRL independently evolved and diverged in the function between insects and vertebrates.Based on gene structure and evolutionary relationship, Mth/Mthl was classified into four subgroups. The mthl genes in three of them have been identified in all insects, while the other subgroup has8mthl and1mth of D. melanogaster. Mth has been reported to be associated with lifespan, fertility and stress response, but our result suggested it lost in other insects. However, it is unkown whether its functions were replaced by other mthl genes or divergent in the functions of them. To answer the key problem, we have taken functional analysis of mthll from T. castaneum (Tcmthll), which show the highest similarity with mth. The results by experiments confirmed that Tcmthll with6exons in its coding region has the highest expression from late pupa to early adult. The result of RNA interferance (RNAi) showed that knocking down this gene had significantly lethal effect on the development of larva and pupa, and severely affected the adult eclosion in T. castaneum. Moreover, the treated adult beetles have significantly decreased the output and survival rate of eggs, which were confirmed to be induced by the treated female individual through further analysis. In contrast to the regulation of mth on stress response, the inhibition on expression of Tcmthll has largely decreased the resistance to starvation and high temperature in T. castaneum. These results all suggested that mth and mthl is divergent in the function in the insect. Additionally, the preliminary analysis indicates that Tcmthll could suppress the expression of JNK, Akt, ASK1and PI3K in T. castaneum, which will play fundamental roles in clarifying the mechanism of Mth/Mthl signal transduction in the future. |
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