Minus-C odorant binding protein TcasOBP7G contributes to reproduction and defense against phytochemical in the red flour beetle, Tribolium castaneum

Gao Shanshan,Zhang Xinyi,Zhou Can,Zhang Guangyan,Zhang Yong-lei,Li Rui-min,Zhang Kun-peng 한국응용곤충학회 2023 Journal of Asia-Pacific Entomology Vol.26 No.4

Insect odorant binding proteins (OBPs) paly an important role in the recognition and transmission signals. One odorant binding protein gene, TcasOBP7G, was significantly up-regulated after exposure to eucalyptol. Never theless, the causal role of TcasOBP7G in Tribolium castaneum is still unclear. Here, TcasOBP7G was cloned and characterized from T. castaneum, aligning with the typical characteristic of Minus-C OBPs. The analysis of gene expression profile showed that TcasOBP7G is highly expressed at late larval and adult stage, with particularly high expression levels in ovary and testis of adults, suggesting that TcasOBP7G might be associated with reproduction of T. castaneum. Furthermore, RNAi of TcasOBP7G led to a decrease in spawning, while the development of gonads was normal, suggesting that TcasOBP7G may medicate the reproduction of T. castaneum. Additionally, the mRNA level of TcasOBP7G was significantly increased after exposure to eucalyptol, and knockdown of TcasOBP7G resulted in an increased susceptibility of T. castaneum to eucalyptol, indicating that TcasOBP7G is associated with the tolerance of T. castaneum to eucalyptol. Altogether, these results indicate that TcasOBP7G is required for both reproduction and defense against phytochemical in T. castaneum. This study will provide a theoretical basis for the development of novel control strategies based on the olfaction of insects.

A sigma class glutathione S-transferase gene regulated by the CncC pathway is required for phytochemical tolerance in the red flour beetle, Tribolium castaneum

Gao Shan-shan,Li Dong-yu,Huo Zhuang-kun,Zhang Yong-lei,Cao Yi-zhuo,Tan Yue-yao,Guo Xin-long,Zhang Jia-hao,Zhang Kun-peng,Li Rui-min 한국응용곤충학회 2022 Journal of Asia-Pacific Entomology Vol.25 No.4

Insect glutathione S-transferases (GSTs) play a crucial role in the detoxification of exogenous compounds, especially insecticides and plant allelochemicals. A sigma class GST gene, TcGSTS7, mediates the response to eugenol in Tribolium castaneum. However, the mechanism underlying this effect remains largely unknown. In this study, TcGSTS7, which exhibits a structural motif and domain organization characteristic of GSTs, was cloned from the T. castaneum genome. Spatiotemporal expression analysis revealed that TcGSTS7 was most highly expressed at the late larva stage and was mainly expressed in the fat body and epidermis of larvae and adults, suggesting that TcGSTS7 may play a potential role in the protection against toxic xenobiotics in T. castaneum. Furthermore, the expression of TcGSTS7 was significantly induced after exposure to eugenol, while RNA inter ference (RNAi) targeting TcGSTS7 enhanced the sensitivity of the beetle to eugenol, indicating that TcGSTS7 is involved in the tolerance of T. castaneum to this insecticide. Interestingly, the depletion of TcCncC, which encodes a transcription factor of the CncC pathway that has been associated with the regulation of detoxification-related genes in insects, led to a reduction in the TcGSTS7 transcript level following exposure to eugenol, which suggests that TcGSTS7 acts downstream of the CncC pathway. Combined, these results indicated that TcGSTS7 partici pates in the tolerance of T. castaneum to phytochemicals in a CncC pathway-dependent manner. These findings have implications for the development of novel drugs for use in pest control.

Activation of immune-associated phospholipase A₂ is functionally linked to Toll/Imd signal pathways in the red flour beetle, Tribolium castaneum

Shrestha, S.,Kim, Y. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2010 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.34 No.5

Bacterial challenge enhances phospholipase A<SUB>2</SUB> (PLA<SUB>2</SUB>) activity, which in turn induces biosynthesis of various eicosanoids that mediate non-self recognition signal to immune effectors in insects. But, there is little information on how PLA<SUB>2</SUB> activity is controlled after the non-self recognition. A recent genome analysis of the red flour beetle, Tribolium castaneum, has annotated both Toll and Imd signal pathways that are presumably considered to specifically respond to different microbial infections to express specific antimicrobial peptides (AMPs). This study set up a hypothesis that PLA<SUB>2</SUB> activation is linked to Toll and Imd pathways in T. castaneum. Bacterial challenge to the larvae of T. castaneum induced expressions of Toll and Imd genes. Different AMP genes were induced in larvae infected with Gram-positive or -negative bacteria. RNA interference using double-stranded RNAs (dsRNAs) specific to different Toll and Imd genes showed differential inhibition of these AMP expressions, indicating that the Toll and Imd pathways play critical roles in the production of AMPs by specifically responding to various bacterial challenges in T. castaneum. These Toll and Imd immune signals are also linked to the activation of PLA<SUB>2</SUB> in T. castaneum. Activation of PLA<SUB>2</SUB> was significantly induced in response to bacterial challenge, but was inhibited by dsRNAs specific to different Toll and Imd genes. The activation of PLA<SUB>2</SUB> via Toll and Imd pathways could be explained by induction of PLA<SUB>2</SUB> gene expression because the dsRNA treatments of Toll and Imd genes suppressed the gene expression of PLA<SUB>2</SUB> in response to bacterial challenge. The functional links were further supported by an immunofluorescence assay of PLA<SUB>2</SUB> intracellular translocation. Upon bacterial challenge, hemocytes from control larvae showed intracellular translocation of their PLA<SUB>2</SUB>s near to cell membrane, but hemocytes from larvae treated with dsRNAs of the Toll and Imd genes did not show the translocation, at which the PLA<SUB>2</SUB>s appeared to be evenly spread in the cytoplasm. These results suggest that recognition of bacterial challenge initiates Toll and Imd pathways in T. castaneum, which subsequently induces the activation of immune-associated PLA<SUB>2</SUB>s as well as gene expression of various AMPs.

Isolation and identification of fungal strains on red flour beetle, Tribolium castaneum collected from RPC

Sook-Young Park,Tae-Sung Yun,Jihyun Yu,Yujin Hwang,Ki-Jeong Hong 한국응용곤충학회 2017 한국응용곤충학회 학술대회논문집 Vol.2017 No.10

The red flour beetle, Tribolium castaneum, is one of economically important pests and can transmit several fungalspecies with their movement in stored grains. We collected T. castaneum samples from 34 rice processing complexes(RPC) nationwide during 2016-2017 and identified the contamination of fungal species on the surface of T. castaneum.Using sequence analysis of ITS region, 24 fungal genera were found. Among them, the genus Aspergillus spp. (28.8%)was the most frequently presented, followed by Cladosporium spp. (12.3%), Hyphopichia spp. (8.9%), Penicillium spp.(8.2%), and so on. In addition, as a known mycotoxin-producing fungal species, Aspergillus flavus (16 isolates) and Fusariumspp. (3 isolates) were also identified. Our study indicates that T. castaneum would play an important role in the contaminationof fungi and contribute potential production of mycotoxin in storage rice.

Contact toxicity and transcriptomic analysis of terpinen-4-ol exposure in Tribolium castaneum

Shan-shan Gao,Yong-lei Zhang,Kun-peng Zhang,Wang Xing-yun,Qing-bo Tang,Yuan-chen Zhang 한국응용곤충학회 2022 Journal of Asia-Pacific Entomology Vol.25 No.3

The terpene, terpinen-4-ol (T4ol), exhibits contact toxicity in Tribolium castaneum. However, the molecular mechanisms underlying this toxicity have not been elucidated. This study examined changes in the expression of four classic enzymes after exposure of T. castaneum to T4ol. Acetylcholinesterase and glutathione S-transferase activities were markedly inhibited after exposure to T4ol, while that of the detoxifying enzyme cytochrome oxidase P450 increased markedly. Carboxylesterase activity did not show significant changes. Furthermore, RNA sequencing revealed 260 differentially expressed genes (DEG) between the T4ol-treated and control samples, and qRT-PCR was used to validate the RNA-Seq data. The Gene Ontology analysis classified the DEGs into 36 functional groups, including the immune system processes, response to stimulus, and developmental processes. T4ol altered the response to stimulus and the immune system process of beetles by inducing the expression of the genes Stabilin-1, Attacin 1, and Defensin 1. Furthermore, the DEGs receptor tyrosine kinase Torso-like protein (RTKTsl), Frizzled 4 (Fz4), Protein Wnt-5b, Ecdysone-induced protein 78C (E78), Zinc finger protein GLIS1 (ZFPGLIS1) were classified as participating in beetle development, and Fz4 and Protein Wnt-5b also mapped to the Wnt signaling pathway. This indicated that pathways associated with development are inhibited after exposure to T4ol. T4ol also induced CYP9Z6/GSTs7 overexpression, and RNAi targeting these genes significantly increased larvae mortality on T4ol exposure, supporting the participation of CYP9Z6/GSTs7 in the response to T4ol in T. castaneum. The results of this study will facilitate understanding of the toxic mechanisms of T4ol and provide a basis for controlling the pests of stored products.

Loss of function of the yellow-e gene causes dehydration-induced mortality of adult Tribolium castaneum

Noh, M.Y.,Kramer, K.J.,Muthukrishnan, S.,Beeman, R.W.,Kanost, M.R.,Arakane, Y. Academic Press 2015 Developmental Biology Vol.399 No.2

Yellow protein (dopachrome conversion enzyme, DCE) is involved in the melanin biosynthetic pathway that significantly accelerates pigmentation reactions in insects. Recent studies have suggested that the insect yellow genes represent a rapidly evolving gene family generating functionally diverse paralogs, but the exact physiological functions of several yellow genes are still not understood. To study the function(s) of one of the yellow genes, yellow-e (TcY-e), in the red flour beetle, Tribolium castaneum, we performed real-time PCR to analyze its developmental and tissue-specific expression, and utilized immunohistochemistry to identify the localization of the TcY-e protein in adult cuticle. Injection of double-stranded RNA for TcY-e (dsTcY-e) into late instar larvae had no effect on larval-pupal molting or pupal development. The pupal cuticle, including that lining the setae, gin traps and urogomphi, underwent normal tanning. Adult cuticle tanning including that of the head, mandibles and legs viewed through the translucent pupal cuticle was initiated on schedule (pupal days 4-5), indicating that TcY-e is not required for pupal or pharate adult cuticle pigmentation in T. castaneum. The subsequent pupal-adult molt, however, was adversely affected. Although pupal cuticle apolysis and slippage were evident, some of the adults (~25%) were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults rapidly became highly desiccated. Interestingly, both the failure of the pupal-adult molt and desiccation-induced mortality were prevented by maintaining the dsTcY-e-treated insects at 100% relative humidity (rh). However, when the high humidity-rescued adults were removed from 100% rh and transferred to 50% rh, they rapidly dehydrated and died, whereas untreated beetles thrived throughout development at 50% rh. We also observed that the body color of the high humidity-rescued dsTcY-e-adults was slightly darker than that of control animals. These results support the hypothesis that TcY-e has a role not only in normal body pigmentation in T. castaneum adults but also has a vital waterproofing function.

Cloning of phospholipase A2 gene and its role in mediating cellular immune response in red flour beetle, Tribolium castaneum

Sony Shrestha,Yoonseong Park,Yonggyun Kim 한국응용곤충학회 2008 한국응용곤충학회 학술대회논문집 Vol.2008 No.05

Phospholipase A2 (PLA2) is the committed catalytic step of eicosanoid biosynthesis, which has been a common molecular target of several entomopathogens to induce insect immunosuppression. Despite critical importance of PLA2 in insect immunity, its gene structure was not known. This study identified insect PLA2 gene associated with immune reactions in the red flour beetle, Tribolium castaneum. Based on a previous study that an immune-associated PLA2 in insect is secretory type of PLA2 (sPLA2), five highly matched cDNA sequences were obtained from T. castaneum genome database using an sPLA2 sequence probe encoded in Drosophila melanogaster. The expressions of these five putative PLA2 were confirmed by reverse transcriptase-polymerase chain reaction. Out of five genes, one PLA2 gene called TcPLA2B was chosen because it showed specific expression in hemocyte and fat body. TcPLA2B was cloned and expressed in Escherichia coli and its protein was purified. The purified TcPLA2B showed PLA2enzyme activity, which was specifically inhibited by bromophenacyl bromide (a specific sPLA2inhibitor) and dithiothreitol (reducing agent of disulfide bond). It was sensitive to pH (optimum at pH 6.0) and reaction temperature (optimum at 10-30°C), and calcium dependency. An immunofluorescence assay indicated that TcPLA2B was localized near to cellular membrane of the cytosol in the hemocytes of T. castaneum at immune chanlenge. Double-stranded RNA (dsRNA) of TcPLA2B-treated larvae showed knockdown of its mRNA expression and did not form hemocyte nodule formation, while control larvae could exhibit time- and bacterial dose-dependent nodule formation in response to bacterial challenge. Addition of arachidonic acid (the catalytic product of PLA2) to the dsRNA-treated larvae rescued the inhibition of nodule formation. These results suggest that TcPLA2B gene is associated with insect immune reaction.

Isolation and Identification of Fungal Species from the Insect Pest Tribolium castaneum in Rice Processing Complexes in Korea

Yun, Tae-Seong,Park, Sook-Young,Yu, Jihyun,Hwang, Yujin,Hong, Ki-Jeong The Korean Society of Plant Pathology 2018 Plant Pathology Journal Vol.34 No.5

The red flour beetle, Tribolium castaneum, is one of the most common and economically important pests of stored cereal products worldwide. Furthermore, these beetles can act as vectors for several fungal post-harvest diseases. In this study, we collected T. castaneum from 49 rice processing complexes (RPCs) nationwide during 2016-2017 and identified contaminating fungal species on the surface of the beetles. Five beetles from each region were placed on potato dextrose agar media or Fusarium selection media after wet processing with 100% relative humidity at $27^{\circ}C$ for one week. A total of 142 fungal isolates were thus collected. By sequence analysis of the internal transcribed spacer region, 23 fungal genera including one unidentified taxon were found to be associated with T. castaneum. The genus Aspergillus spp. (28.9%) was the most frequently present, followed by Cladosporium spp. (12.0%), Hyphopichia burtonii (9.2%), Penicillium spp. (8.5%), Mucor spp. (6.3%), Rhizopus spp. (5.6%), Cephaliophora spp. (3.5%), Alternaria alternata (2.8%) and Monascus sp. (2.8%). Less commonly identified were genera Fusarium, Nigrospora, Beauveria, Chaetomium, Coprinellus, Irpex, Lichtheimia, Trichoderma, Byssochlamys, Cochliobolus, Cunninghamella, Mortierella, Polyporales, Rhizomucor and Talaromyces. Among the isolates, two known mycotoxin-producing fungi, Aspergillus flavus and Fusarium spp. were also identified. This result is consistent with previous studies that surveyed fungal and mycotoxin contamination in rice from RPCs. Our study indicates that the storage pest, T. castaneum, would play an important role in spreading fungal contaminants and consequently increasing mycotoxin contamination in stored rice.

Isolation and Identification of Fungal Species from the Insect Pest Tribolium castaneum in Rice Processing Complexes in Korea

Tae-Seong Yun,Sook-Young Park,Jihyun Yu,Yujin Hwang,Ki-Jeong Hong 한국식물병리학회 2018 Plant Pathology Journal Vol.34 No.5

The red flour beetle, Tribolium castaneum, is one of the most common and economically important pests of stored cereal products worldwide. Furthermore, these beetles can act as vectors for several fungal post-harvest diseases. In this study, we collected T. castaneum from 49 rice processing complexes (RPCs) nationwide during 2016-2017 and identified contaminating fungal species on the surface of the beetles. Five beetles from each region were placed on potato dextrose agar media or Fusarium selection media after wet processing with 100% relative humidity at 27°C for one week. A total of 142 fungal isolates were thus collected. By sequence analysis of the internal transcribed spacer region, 23 fungal genera including one unidentified taxon were found to be associated with T. castaneum. The genus Aspergillus spp. (28.9%) was the most frequently present, followed by Cladosporium spp. (12.0%), Hyphopichia burtonii (9.2%), Penicillium spp. (8.5%), Mucor spp. (6.3%), Rhizopus spp. (5.6%), Cephaliophora spp. (3.5%), Alternaria alternata (2.8%) and Monascus sp. (2.8%). Less commonly identified were genera Fusarium, Nigrospora, Beauveria, Chaetomium, Coprinellus, Irpex, Lichtheimia, Trichoderma, Byssochlamys, Cochliobolus, Cunninghamella, Mortierella, Polyporales, Rhizomucor and Talaromyces. Among the isolates, two known mycotoxin- producing fungi, Aspergillus flavus and Fusarium spp. were also identified. This result is consistent with previous studies that surveyed fungal and mycotoxin contamination in rice from RPCs. Our study indicates that the storage pest, T. castaneum, would play an important role in spreading fungal contaminants and consequently increasing mycotoxin contamination in stored rice.

RNAi‐mediated knockdown of juvenile hormone esterase causes mortality and malformation in Tribolium castaneum

Xu Zhanyi,Yan Ru,Qian Jiali,Chen Dongping,Guo Yirong,Zhu Guonian,Wu Huiming,Chen Mengli 한국곤충학회 2022 Entomological Research Vol.52 No.11

RNA interference is an efficient approach for gene function identification and a potential novel strategy for selectively controlling pests. The red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae), is a major global storage pest, which causes great economic loss. Juvenile hormone esterase (JHE), a carboxylesterase, is responsible for the degradation of juvenile hormone. However, the knockdown effect of jhe on metamorphosis of pupae or adult in T. castaneum is unknown. In this study, we analyzed the expression profiles of Tc j h e in the larval stage, we found that Tc j h e was expressed throughout the whole larval instars and the expression levels were relatively high right after molting. Furthermore, we knocked down the expression of Tcjhe by injecting dsTc j h e , which significantly increased the mortality of adults and decreased the eclosion rate. In addition, abnormal developmental phenotypes, including wing deformitity, pupal–adult monsters and shrunken adults, were observed. Our finding indicates that JHE plays an important role in the metamorphosis and development in T. castaneum, suggesting that Tc j h e could be used as a potential target for the development of RNAi-based control strategies in T. castaneum.