Bingzhong Ren

Systematic comparison of constitutive promoters and the doxycycline-inducible promoter.

Constitutive promoters are used routinely to drive ectopic gene expression. Here, we carried out a systematic comparison of eight commonly used constitutive promoters (SV40, CMV, UBC, EF1A, PGK and CAGG for mammalian systems, and COPIA and ACT5C for Drosophila systems). We also included in the comparison the TRE promoter, which can be activated by the rtTA transcriptional activator in a doxycycline-inducible manner. To make our findings representative, we conducted the comparison in a variety of cell types derived from several species. We found that these promoters vary considerably from one another in their strength. Most promoters have fairly consistent strengths across different cell types, but the CMV promoter can vary considerably from cell type to cell type. At maximal induction, the TRE promoter is comparable to a strong constitutive promoter. These results should facilitate more rational choices of promoters in ectopic gene expression studies.

Identification and Comparison of Candidate Olfactory Genes in the Olfactory and Non-Olfactory Organs of Elm Pest Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) Based on Transcriptome Analysis

The leaf beetle Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) is a predominant forest pest that causes substantial damage to the lumber industry and city management. However, no effective and environmentally friendly chemical method has been discovered to control this pest. Until recently, the molecular basis of the olfactory system in A. quadriimpressum was completely unknown. In this study, antennae and leg transcriptomes were analyzed and compared using deep sequencing data to identify the olfactory genes in A. quadriimpressum. Moreover, the expression profiles of both male and female candidate olfactory genes were analyzed and validated by bioinformatics, motif analysis, homology analysis, semi-quantitative RT-PCR and RT-qPCR experiments in antennal and non-olfactory organs to explore the candidate olfactory genes that might play key roles in the life cycle of A. quadriimpressum. As a result, approximately 102.9 million and 97.3 million clean reads were obtained from the libraries created from the antennas and legs, respectively. Annotation led to 34344 Unigenes, which were matched to known proteins. Annotation data revealed that the number of genes in antenna with binding functions and receptor activity was greater than that of legs. Furthermore, many pathway genes were differentially expressed in the two organs. Sixteen candidate odorant binding proteins (OBPs), 10 chemosensory proteins (CSPs), 34 odorant receptors (ORs), 20 inotropic receptors [1] and 2 sensory neuron membrane proteins (SNMPs) and their isoforms were identified. Additionally, 15 OBPs, 9 CSPs, 18 ORs, 6 IRs and 2 SNMPs were predicted to be complete ORFs. Using RT-PCR, RT-qPCR and homology analysis, AquaOBP1/2/4/7/C1/C6, AquaCSP3/9, AquaOR8/9/10/14/15/18/20/26/29/33, AquaIR8a/13/25a showed olfactory-specific expression, indicating that these genes might play a key role in olfaction-related behaviors in A. quadriimpressum such as foraging and seeking. AquaOBP4/C5, AquaOBP4/C5, AquaCSP7/9/10, AquaOR17/24/32 and AquaIR4 were highly expressed in the antenna of males, suggesting that these genes were related to sex-specific behaviors, and expression trends that were male specific were observed for most candidate olfactory genes, which supported the existence of a female-produced sex pheromone in A. quadriimpressum. All of these results could provide valuable information and guidance for future functional studies on these genes and provide better molecular knowledge regarding the olfactory system in A. quadriimpressum.

Drone and Worker Brood Microclimates Are Regulated Differentially in Honey Bees, Apis mellifera.

Background Honey bee (Apis mellifera) drones and workers show differences in morphology, physiology, and behavior. Because the functions of drones are more related to colony reproduction, and those of workers relate to both survival and reproduction, we hypothesize that the microclimate for worker brood is more precisely regulated than that of drone brood. Methodology/Principal Findings We assessed temperature and relative humidity (RH) inside honey bee colonies for both drone and worker brood throughout the three-stage development period, using digital HOBO® Data Loggers. The major findings of this study are that 1) both drone and worker castes show the highest temperature for eggs, followed by larvae and then pupae; 2) temperature in drones are maintained at higher precision (smaller variance) in drone eggs and larvae, but at a lower precision in pupae than the corresponding stages of workers; 3) RH regulation showed higher variance in drone than workers across all brood stages; and 4) RH regulation seems largely due to regulation by workers, as the contribution from empty honey combs are much smaller compared to that from adult workers. Conclusions/Significance We conclude that honey bee colonies maintain both temperature and humidity actively; that the microclimate for sealed drone brood is less precisely regulated than worker brood; and that combs with honey contribute very little to the increase of RH in honey bee colonies. These findings increase our understanding of microclimate regulation in honey bees and may have implications for beekeeping practices.

Phylogeny and acoustic signal evolution of a pure tone song katydid Pseudophyllus titan (Orthoptera: Tettigoniidae) based on the complete mitogenome

Abstract Ensifera is an extremely diverse group in the order Orthoptera, and it has been the subject of considerable research, especially in terms of phylogeny and bioacoustics. Katydids are known for using high-frequency broadband signals whereas most cricket songs are pure tones with a frequency range of 2–8 kHz. Pseudophyllus titan is a special katydid emits low-frequency pure tone songs similar to that of crickets. Acoustic communication of Ensifera were widely studied, but song evolution could deserve new examination taking into account the newly available phylogenies. To examine the song evolution of P. titan in the Ensifera, the mitogenomes of P. titan and three other ensiferan species (Sphagniana ussuriana, Oecanthus sinensis, Truljalia hibinonis) were obtained by high-throughput sequencing. In addition, the phylogeny of Ensifera was reconstructed including 63 in-group taxa, and the divergence time was further estimated for major ensiferan lineages. Lastly, the evolutions of song frequency were evaluated based on an ancestral character state reconstruction (ACSR). Moreover, we have studied the calling songs of 78 katydids and performed ACSR based on five genes. All phylogeny analyses confirmed that the superfamily Schizodactyloidea was placed basally to the non-grylloid clade. P. titan was in the Pseudophyllinae clade, and this clade had a distant relationship form other lineages in Phaneropteridae. The ACSR showed that P. titan evolved low-frequency pure tone songs independently after ancestors of katydids evolved high-frequency broadband noise. Referring to the geologic time scale, we suggest that the song frequency evolution in the Ensifera to some extent caused by adaptations to the changing habitat under conditions of climate change and plant evolution.

Identification and evolution of olfactory genes in the small poplar longhorn beetle Saperda populnea.

Saperda populnea is a serious pest of poplar and willow trees in the Palaearctic region, causing extensive damage to forests and the lumber industry. Until recently, there is no safe and effective chemical method to control this pest due to the lack of sufficient knowledge on the molecular basis of its olfactory genes, moreover, the evolutionary history of the olfactory gene family in subfamily Lamiinae is still fully unknown. Our RNA sequencing of the antennae of S. populnea identified 43 odorant binding proteins (OBPs), 15 chemosensory proteins (CSPs), 56 odorant receptors (ORs) and 24 inotropic receptors (IRs) in S. populnea. The RT-PCR results showed several genes were expressed in a sex specific manner, suggesting that these genes might play key role in their olfactory-sensing and sex-related behaviors. Further evolutionary studies were performed on these olfactory genes, overall comparison of the Ka/Ks values of orthologous genes in S. populnea and two other Lamiinae species showed three main conclusions: 1. olfactory genes have evolved more rapidly than the non-olfactory genes in the tested long horn beetles; 2. the IR gene family are under a strong purifying selection; 3. the OBPs of Monochamus alternatus evolved more rapidly than the other two species, which is speculated to be correlated with differentiation of selective pressure in different geographic origins. Detailed evolutionary studies on each olfactory genes showed that several OBPs and ORs are under significantly purifying/relaxed selective pressure, and several positive selection sites were also detected, modeling of SpopOR14 and SpopOBP4/5 showed that most of the positive selection sites were distributed at the N-terminus of SpopOR14, while the positive selection sites in SpopOBP4/5 were located in H-bond donors, results suggest that these sites are more likely to be linked with the selectivity of modeled olfactory genes. The research provided a better understanding of the molecular basis and evolutionary history of the olfactory genes in Lamiinae, through elaborating the mechanism whereby amino structural evolution affects specific variants in OBPs and ORs.

Selectivity and ligand-based molecular modeling of an odorant-binding protein from the leaf beetle Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) in relation to habitat-related volatiles

In this study, the most abundant and antenna-specific odorant-binding protein (OBP) of the elm pest A. quadriimpressum, AquaOBP4, was expressed and purified. The selectivity of AquaOBP4 was investigated by screening against a panel of 40 habitat-relevant compounds. Based on the obtained results, a homologous model of AquaOBP4 was established. This model indicated that AquaOBP4 is highly homologous to DmelOBP LUSH and includes two main binding sites. A docking analysis showed that four of five active ligands bound at Site 1, whereas the other ligand was situated at Site 2. Furthermore, new ligands were docked in the model, and the results of fluorescence-based binding assays of these compounds were highly consistent with the binding conformation and binding affinity predicted by our model. Additionally, three binding odorants derived from elm leaves elicited a strong electroantennogram response and exerted a significant attractive effect on adult A. quadriimpressum. All of the results showed that AquaOBP4 is likely linked to the foraging behavior of A. quadriimpressum. This study provides a new reliable tool for future large-scale compound screenings and revealed several functional chemicals that might aid the development of a better pest management approach for A. quadriimpressum.

Review and key to all known species of the genus Epacromius Uvarov, 1942 (Orthoptera: Acrididae)

This paper provides a review and key to all species of the genus Epacromius Uvarov, 1942. The first description of the male of the species Epacromius fallax Wang, 2007 is given.

Micromorphological differentiation of left and right stridulatory apparatus in crickets (Orthoptera: Gryllidae)

The present study compared micromorphological differentiation of stridulatory apparatus between the functional right tegmen and non-functional left tegmen, analyzed under scanning electron microscope (SEM), among eight Gryllidae species. The results showed that the main differences were found on the length and shape of files and teeth. The length of stridulatory file and teeth number were lower on the left stridulatory apparatus than that on right stridulatory apparatus in all species. However, the ratio between the length of stridulatory teeth and the interval of stridulatory teeth was significantly higher on the left stridulatory apparatus than that on right stridulatory apparatus in Dianemobius fascipes, Polionemobius taprobanensis, Pteronemobius gifuensis, Teleogryllus occipitalis and Oecanthus longicauda. In addition, the length of stridulatory teeth was positively related to number of stridulatory teeth, however, the interval of stridulatory teeth was negatively related to the ratio between the length of stridulatory teeth and the interval of stridulatory teeth for left and right stridulatory apparatus. Our result illustrated that the length of left and right stridulatory file and teeth length could be an effective character to distinguish species. Left stridulatory apparatus was not entirely degraded than right stridulatory apparatus.

The Complete Mitochondrial Genome and Song Evolution of the Monotypic Genus U. Tarbinsky, 1932 (Orthoptera: Tettigoniidae)

Abstract The insect Uvarovites inflatus Uvarov is highly appreciated in China. It is known for its distinctive songs and horn-like forewings and is raised commercially for insect lovers. U. inflatus was previously categorized as part of the monotypic genus Uvarovites; however, there was little molecular evidence to support this taxonomic classification. This study obtained and investigated the mitogenome of U. inflatus, and its songs were characterized and compared with other Ensifera species whose mitogenomes are available. By performing the mitochondrial analysis, we were able to assess the phylogenetic relationships between these species and discuss the evolution of Ensifera calling songs. The mitogenome of U. inflatus is 15,956 bp in length and contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region. The organization and orientation of the U. inflatus mitogenome are similar to those of other Tettigonioidea species. Phylogenetic analysis based on 13 protein-coding genes showed that the superfamily Tettigonioidea is monophyletic, as are the other six tested subfamilies from Tettigonioidea. Our results also indicated that Grylloidea is monophyletic. A Bayesian relaxed clock analysis showed that the differentiation of U. inflatus and Gampsocleis gratiosa Brunner occurred in the middle Miocene, suggesting that their speciation occurred over a long evolutionary period. The results provide significant support for the establishment of the monotypic genus Uvarovites. Calling song analysis showed that at least two discrete steps of independent evolution occurred during the change from pure tone to broadband noise, and that the ancestor of existing Ensifera was more likely to have emitted pure-tone songs than broadband signals. Together, the mitogenome, molecular clock, and acoustic data allowed us to clarify the taxonomic state of U. inflatus and propose a timeline for the evolution of Ensifera songs.

Acoustic, genetic and morphological variations within the katydid Gampsocleis sedakovii (Orthoptera, Tettigonioidea)

Abstract In an attempt to explain the variation within this species and clarify the subspecies classification, an analysis of the genetic, calling songs, and morphological variations within the species Gampsocleis sedakovii is presented from Inner Mongolia, China. Recordings were compared of the male calling songs and analysis performed of selected acoustic variables. This analysis is combined with sequencing of mtDNA - COI and examination of morphological traits to perform cluster analyses. The trees constructed from different datasets were structurally similar, bisecting the six geographical populations studied. Based on two large branches in the analysis, the species Gampsocleis sedakovii was partitioned into two subspecies, Gampsocleis sedakovii sedakovii (Fischer von Waldheim, 1846) and Gampsocleis sedakovii obscura (Walker, 1869). Comparing all the traits, the individual of Elunchun (ELC) was the intermediate type in this species according to the acoustic, genetic, and morphological characteristics. This study provides evidence for insect acoustic signal divergence and the process of subspeciation.