赤拟谷盗Tribolium castaneum与锈赤扁谷盗Cryptolestes ferrugineus种间竞争的研究

在30℃、75%RH条件下研究赤拟谷盗Tribolium castaneum与锈赤扁谷盗Cryptolestes ferrugineus的种间竞争,并采用Votka-Volterra种间竞争模型进行拟合。结果表明,赤拟谷盗与锈赤扁谷盗混合饲养时的种群数量均小于单独饲养时的种群数量,两物种之间存在着显著的种间竞争关系。属于资源利用型竞争;单独饲养和混合饲养条件下赤拟谷盗的种群数量均大于锈赤扁谷盗的种群数量;混合饲养条件下两个物种可以共存,并形成稳定的平衡状态,在这个竞争系统中,赤拟谷盗为优势种。

Genome-wide mapping of conserved microRNAs and their host transcripts in Tribolium castaneum

MicroRNAs （miRNAs） are endogenous 22-nt RNAs, which play important regulatory roles by post-transcriptional gene silencing. A computational strategy has been developed for the identification of conserved miRNAs based on features of known metazoan miRNAs in red flour beetle （Tribolium castaneum）, which is regarded as one of the major laboratory models of arthropods. Among 118 putative miRNAs, 47% and 53% of the predicted miRNAs from the red flour beetle are harbored by known protein-coding genes （intronic） and genes located outside （intergenic miRNA）, respectively. There are 31 intronic miRNAs in the same transcriptional orientation as the host genes, which may share RNA polymerase II and spliceosomal machinery with their host genes for their biogenesis. A hypothetical feed-back model has been proposed based on the analysis of the relationship between intronic miRNAs and their host genes in the development of red flour beetle.

Identification,Characterization, and Expression of P450 Gene Encoding CYP6BQ13v2 from the Red Flour Beetle,Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae)

An allele of CYP6BQI3, named CYP6BQ 13v2 （GenBank accession no. FJ209361）, was isolated from the red flour beetle, Tribolium castaneum （Herbst） （Coleoptera： Tenebrionidae） by RT-PCR. The cDNA sequence of CYP6BQ13v2, 1 563 bp in length, contains an open reading frame of 1 554 nucleotides encoding a putative protein of 518 amino acid residues with a predicted molecular weight of 59.92 kDa and a theoretical pl of 7.60. The putative protein contains the classic hemebinding sequence motif F××G×××C×G （residues 456-465） conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s. It shares 98% identity with the previously published sequence of CYP6BQ13 （GenBank accession no. XP967146） from the T. castaneum genome project. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated that there was closer phylogenetic relationship of CYP6BQ 13v2 with CYP302A1 and CYP307A1 mediating synthesis of the insect molting hormone, distant relationship with CYP6B1 metabolizing plant allelochemicals, CYP6D 1 linking to pyrethroid resistance and other members of CYP6 family. Expression test of the gene in the adults and immature stages of T. castaneum by quantitative real-time PCR revealed that CYP6BQ13v2 is expressed in all life stages investigated. The mRNA expression level in 1st instar larvae was 14.9- and 3.86-fold higher than those in pupae and adults, respectively. The CYP6BQ13v2 expression levels appeared in the order of 1st instar larvae, followed by 4th instar larvae, 7th instar larvae, adult, and pupae from high to low. The more bioinformation of CYP6BQ 13v2 was also analyzed.

Transcriptome analysis of hsp18.3 functions and regulatory systems using RNA-sequencing in the red flour beetle,Tribolium castaneum

The red flour beetle, Tribolium castaneum, is a major agriculture pest of stored grain, cereal products and peanuts for human consumption. It is reported that heat shock protein 18.3 of T. castaneum（Tchsp18.3） plays a significant role in stress resistance, development and reproduction. However, the regulatory systems of Tchsp18.3 remain unknown. Therefore, we compared the global transcriptome profiles of RNA interference（RNAi）-treated larvae（ds-Tchsp18.3） and control larvae of T. castaneum using RNA sequencing. Overall, we obtained 14 154 435 sequence reads aligned with 13 299 genes. Additionally, 569 differentially expressed genes（DEGs） were identified from the ds-Tchsp18.3 and control groups, of which 246 DEGs were annotated in the 47 Gene Ontology（GO） functional groups and 282 DEGs were assigned to 147 Kyoto Encyclopedia of Genes and Genomes（KEGG） biological signaling pathways. The DEGs encoding viperin, dorsal, Hdd11, PGRP2, defensin1 and defensin2 were simultaneously related to immunity and stress responses, which suggests that cross-talk might exist between the immunity and stress responses of T. castaneum. The knockdown of Tchsp18.3 gene expression suppressed the antioxidant activity process, which most likely modulated the effects of Tchsp18.3 on development and reproduction. Furthermore, the DEGs, including Blimp-1, Gld, Drm, Kinesin-14, Pthr2, Delta（11）-like and EGF-like domain protein 2, were also associated with the development and reproduction of ds-Tchsp18.3 insects. Additionally, knockdown of Tchsp18.3 amplified the serine protease（SP） signaling pathway to further regulate stress responses and innate immunity as well as development and reproduction of the red flour beetles. These results provide valuable insight into the molecular regulatory mechanism of Tchsp18.3 involved in insect physiology and further facilitate the research of suitable and sustainable management for pest control.

Prevalence of Wolbachia in 10 Tenebrionidae stored-product insects and spatiotemporal infection dynamics in Tribolium confusum(Jaquelin Du Val)(Coleoptera:Tenebrionidae)

Insect symbionts Wolbachia used for pest population control has focused on vector pest species and agricultural insects while rare reports in Chinese stored-product insect samples.In this paper,we surveyed the prevalence of Wolbachia using a PCR detection method in ten Tenebrionidae stored-product insects.Subsequently,the spatiotemporal Wolbachia infection dynamics in Tribolium confusum and Wolbachia elimination patterns using tetracycline treatment were investigated in detail by TaqMan®probe real-time quantitative PCR,and host reproductive fitness parameters were compared.T.confusum was the only Wolbachia infected species in all the surveyed species.Wolbachia infection density consistently increased with the development of T.confusumand plateaued at 3.7×107 wsp copies per individual insect at the young adult stage.Wolbachia densities in females were higher than that in males with a significant difference at the pupae stage and varied among different tissues and organs.Uninfected female beetles were completely incapable of producing mature progenies when crossed with Wolbachia infected males.Embryogenesis and egg hatch rate were specifically inhibited after Wolbachia elimination,while other traits,including the number of eggs,pupation rate and sex ratio,remained unaffected by tetracycline treatment.Our results showthat the TaqMan®probe qPCR is a reliable detection method for quantifying the density of Wolbachia as compared to qualitative detection of wsp gene by PCR and relatively quantified by real-time qPCR.The fitness results indicated that Wolbachia infection was not an obligate symbiont and benefited the host confused flour beetle.

Resistance of Tribolium castaneum to Phosphine and the Relationship between Complete Lethal Concentration and Exposure Time of Phosphine in Hainan Province

[Objeetive] The paper was to investigate the resistance of Tribolium castaneum to phosphine in Hainan Province. [Method] Using the measurement method of phosphine resistance of stored grain pests recommended by FAO, the phosphine resistance of eight strains of T. castaneum adults in Hainan Province was determined. [Result] The resistance coefficients of different strains of ？7. castaneum against phosphine ranged from 612 to 1 045, and all strains reached extremely high resistance level （Rf〉 160）. When the concentration of phosphine was 200 mL/m^3, Rf612, Rf826.4 and Rfl 045 strains had complete lethal time of 8, 9 and 11 d, respectively. [Condusion] T. castarveum in Hainan Province had very strong resistance to phosphine, and the operation scheme of low concentration and long duration should be adopted in elimination of T. castaneum.

Rapid Molecular Identification of Tribolium destructor

In this study, a rapid molecular identification method of Tribolium destructor was established with PCR and PCR-RFLP （polymerase chain reaction-restriction fragment length polymorphism） technology. According to the results, （ 1 ） with PCR method, specific primers were designed based on CO1 gene of T. destructor for PCR amplification, and electrophoresis detection confirmed that PCR method could be used to rapidly and accurately identify T. destructor; （2） with PCR-RFLP method, two pairs of degenerate primers were used to amplify CO1 gene of Tribolium species, PCR products were digested with HindIII and detected by electrophoresis, results indicated that PCR-RFLP method could also be used for rapid identification of T. destructor in quarantine practice.

Mortality of Nile Crocodile (<i>Crocodylus niloticus</i>) Eggs Caused by the Flour Beetle (<i>Tribolium castaneum</i>)

It is estimated that quarter to half a million specimens of the Nile crocodile (Crocodylus niloticus Laurenti, 1768) live in liberty. Ranching crocodiles for their skins has been successfully implemented in several African countries but also in Israel. Recently, in one of these ranches, an increased mortality of crocodile eggs was observed and it was thought that insects were causing their death. Two crocodile eggs were kept together with 100 adults of Tribolium castaneum (Herbst, 1797), while the third egg was kept without beetles. No food for beetles was added to the boxes. The eggs were observed twice a month for any kind of changes on their surface. After two months, at least 39 holes were observed in one egg and 146 on the second egg, which was exposed to beetles, while no holes could be seen in the control egg. Some of the holes were deep enough to see the inner layers of the egg. There is enough evidence to believe that adults of T. castaneum are able to damage the eggs of the Nile crocodile and in some cases lead to their death;measures should be taken to protect the eggs from this and similar species of beetles.

The influence of different heating rates on mortality of Tribolium castaneum(Herbst)(Coleoptera: Tenebrionidae)

In order to elucidate the ability of stored product insects to resist high temperature at varying temperature conditions,the influence of different heating rates on the mortality of all life stages of Tribolium castaneum(Herbst)(Coleoptera:Tenebrionidae)was investigated.After acclimation from 28 to 42,44 and 46℃ at different heating rates(0,1.25,2.50,5.00,and 10.00℃/h),eggs,larvae,pupae,and adults of T.castaneum were exposed at 50℃ for 0(as a control),10,15,20,25 and 30 min,respectively.The corrected mortality of T.castaneum was determined after 24 h.The results showed that different exposure times and heating rates had significant effects on the mortality of T.castaneum.The mortality of all life stages of T.castaneum with the same acclimation experience increased significantly with the increasing exposure time,and acclimation improved the heat tolerance of T.castaneum.The mortalities exposed at 50℃ for 30 min of T.castaneum eggs(acclimated to 46℃ at 5.00℃/h),larvae(acclimated to 44℃ at 2.50℃/h),pupae(acclimated to 46℃ at 5.00℃/h),and adults(acclimated to 46℃ at 2.50℃/h)were 62.50%,54.44%,58.89%and 53.33%,respectively,significantly lower than that of corresponding life stages of T.castaneum acclimated at 1.25 and 10.00℃/h.The present results are in favor of developing an effective heat treatment protocol for pest control.

Bioefficacy of Ethanolic Leaves Extract of Azadirachta indica Against Stored Product Insect Pest, Tribolium castaneum (Herbst.)

Tribolium castaneum is a species of beetle in the family tenebrionidae,the darkling beetle.Conventional insecticides used for controling stored product pests are expensive and are proven to have side effects hence the need to have an alternative biopesticides from plants which are proven to be effective against many insects.Azadrachta indica has been used for the control many insects,literature review show no report of its effects on Tribolium castaneum.This study is aimed at evaluating the bioinsecticidal activity of ethanolic leaves extract of Azadirachta indica against stored Tribolium castaneum.Different concentrations(60,70,80,90,and 100%)of ethanolic leaves extract of A.indica was applied on the filter paper and were allowed to dry.Control was done by applying water only on the filter paper.For the determination of the percentage mortality,15 adults of the insects was taken into the Petri dishes embedded with the filter paper covered with a lid.The A.indica extracts at different concentrations tested showed significant insecticidal activity against T.castaneum.Secondary metabolites screening indicated that the A.indica extract have secondary metabolites that are related to insecticidal activity.The study has shown that the ethanolic leaves extract A.indica is effective in managing stored product pest.

A pattern recognition receptor C-type lectin TcCTL14 contributes to immune response and development in the red flour beetle, Tribolium castaneum

Evidence is accumulating that pattern recognition receptor(PRR)C-type lectins(CTL)play essential roles in recognition of pathogens.TcCTL14(accession no.TC00871)contains the most domains among all CTL of Tribolium castaneum.Yet the biological function of TcCTL14 remains unclear.In this study,TcCTL14 exhibiting typical motif and domain of CTL was cloned from T.castaneum.The expression pattern analysis showed that TcCTL14 was highly expressed in late pupae and central nervous system,and was upregulated after treatment with Escherichia coli and Staphylococcus aureus,respectively.Analysis of binding affinity revealed that recombinant TcCTL14 not only could bind to lipopolysaccharide and peptidoglycan in a dose-dependent fashion,but possibly could bind to and agglutinate different bacteria in a Ca^(2+)-dependent fashion.Knockdown of TcCTL14 before injection with bacteria led to the downregulation of nuclear factor-κB transcription factors of Toll/IMD and 4 antimicrobial peptides.Knockdown of TcCTL14 also caused suppressed metamorphosis,reduced fecundity,and delayed embryogenesis of T.castaneum.Further observation discovered that knockdown of TcCTL14 inhibited the development of ovaries and embryos.The detection of signaling pathways revealed that TcCTL14 may be involved in metamorphosis and fecundity by impacting 20-hydroxyecdysone and vitellogenin,respectively.Overall,these results indicate that TcCTL14 may contribute to immune response by agglutination or regulating the expression of antimicrobial peptides by the Toll/IMD pathway,and is required for T.castaneum development including metamorphosis,fecundity,and embryogenesis.These findings will improve the functional cognition of PRR CTL in insects and provide the new strategy for pest control.

肉桂醛对赤拟谷盗的熏蒸作用及对成虫保护酶活性的影响

为明确肉桂醛对赤拟谷盗Tribolium castaneum各发育阶段的杀虫活性及对成虫几种保护酶活性的影响,本试验采用三角瓶密闭熏蒸法测定了肉桂醛对赤拟谷盗低龄幼虫、高龄幼虫、蛹和成虫的熏蒸作用及24 h致死中浓度熏蒸后成虫体内超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性。结果表明,肉桂醛的熏蒸效果与其浓度和赤拟谷盗发育阶段有关,肉桂醛2.5μL/L熏蒸72 h对赤拟谷盗成虫的熏蒸效果最好,幼虫次之。在24 h致死中浓度处理下,赤拟谷盗成虫体内SOD、POD、CAT活性分别在48、12 h和36 h达到最高,其活性分别为对照的2.70、1.03倍和1.96倍。随着熏蒸时间的延长,成虫POD活性在处理36 h后低于对照,SOD、CAT活性分别在处理36~60 h和24~48 h显著高于对照。综上所述,经肉桂醛熏蒸处理后,赤拟谷盗成虫体内动态平衡受到破坏,进而产生毒害作用。因此,肉桂醛对赤拟谷盗的生物防治有较高的应用价值。

2种植物提取物对杂拟谷盗的生物活性及几种酶活性的影响

杂拟谷盗(Tribolium confusum)是一种重要的储粮害虫,熏蒸是目前主要的防治方法。相比化学熏蒸,植物源杀虫剂作用方式多样且对环境和粮食安全。因此本论文研究了2种植物[瑞香狼毒(Stellera chamaejasme L.)根石油醚提取物和百里香(Thymus mongolicus Ronn.)甲醇提取物]对杂拟谷盗的熏蒸、拒食和驱避活性,以及对杂拟谷盗2种靶标酶和3种解毒酶活性的影响。结果表明,瑞香狼毒根石油醚提取物和百里香甲醇提取物熏蒸杂拟谷盗24 h后,致死中浓度(Lethal concentration,LC 50)分别为88.50和119.57 mg·L^(-1);拒食处理杂拟谷盗5 d后的拒食中浓度(Antifeedant concentration,AFC 50)分别为23.00和14.78 mg·L^(-1);4 h的驱避率分别为63.49%和71.90%。2种植物提取物对杂拟谷盗乙酰胆碱酯酶和三磷酸腺苷酶活性均有抑制作用。2种植物提取物处理杂拟谷盗后,细胞色素P450和谷胱甘肽-S-转移酶活性被诱导升高,而羧酸酯酶活性降低。本试验结果为未来进一步开发利用这2种植物源农药防治储粮害虫提供了一定的理论基础。

Expression of the Tribolium castaneum (Coleoptera: Tenebrionidae) hsp83 gene and its relation to oogenesis during ovarian maturation

Heat shock proteins （HSP） can protect organisms and cells from thermal damage. In this study, we cloned the full length cDNA encoding the HSP83 protein （the homologue of HSP90） of Tribolium castaneum（red flour beetle）. The isolated cDNA contains the full coding sequence, a partial 5＇ untranslated region of 55 bp and the complete 3＇ untranslated region. We found the hsp83 gene is located on chromosome 5 of the T. castaneum genome. The predicted HSP83 protein sequence has a high similarity （on average 86.77%） with that of other insect species. The expression of the hsp83 gene in the whole body and in the ovary could be induced with heat stress （40℃ for 1 h） in newly hatched （within 3 h post emergence） and mature （10 days post emergence） beetles. Under normal conditions, the hsp83 expression in the ovary is about 3-fold higher than in the whole body at both stages. No significant difference in hsp83 expression was observed between the two ovarian developmental stages regardless if the beetles were treated with heat shock or not. The expression of the HSP83 protein in the whole body could also be induced with heat stress in newly hatched and mature beetles. However, in the ovary, HSP83 was only expressed in the follicle cells of mature beetles and not in newly hatched beetles, regardless if the beetles were treated with heat shock or not. Furthermore, the females were not able to produce mature oocytes after knock-down of the hsp83 expression by injecting dsRNA. These results suggest that the HSP83 protein is involved in protection against heat stress and could be involved in oogenesis during ovarian maturation of T. castaneum.

大黑粉盗与赤拟谷盗和锈赤扁谷盗对磷化氢的耐受力比较研究

潜在危害的储粮害虫大黑粉盗Cynaeus angustus研究缺乏,掌握其磷化氢耐受力有助于科学治理。采用快速击倒和FAO推荐方法测定了磷化氢对大黑粉盗的KT_(50)值和毒力方程,并与赤拟谷盗Tribolium castaneum和锈赤扁谷盗Cryptolestes ferrugineus进行了比较,测定了100、200、300、400、500 mL/m^(3)磷化氢体积分数下3种害虫卵、幼虫、蛹和成虫不同时间的死亡率。磷化氢对大黑粉盗、赤拟谷盗和锈赤扁谷盗的KT 50值分别为7、127、3736 min,相应毒力方程斜率值(b)为3.67、8.28和9.94,相应LC 50值为0.008、1.34和5.88 mg/L。不同害虫虫态在100~500 mL/m^(3)下的半数致死时间LT 50值于大黑粉盗卵为4~2 h、幼虫4~1 h、蛹4~1 h、成虫3~1 h,于赤拟谷盗为卵12~5 d、幼虫11~5 d、蛹13~7 d、成虫10~4 d,于锈赤扁谷盗为卵28~13 d、幼虫为18~9 d,蛹26~11 d,成虫17~9 d。相应的完全致死时间(LT_(100))于大黑粉盗卵为21~6 h、幼虫8~3 h、蛹9~6 h、成虫5~3 h,于赤拟谷盗卵为21~18 d、幼虫21~15 d、蛹27~18 d、成虫21~12 d,于锈赤扁谷盗卵为54~30 d、幼虫42~30 d、蛹48~30 d、成虫36~25 d。所测大黑粉盗为磷化氢敏感品系,其各虫态对磷化氢的耐受力为卵>蛹>幼虫>成虫,其耐受力远小于赤拟谷盗和锈赤扁谷盗磷化氢抗性品系。

Characterization and functional analysis of hsp18.3 gene in the red flour beetle, Tribolium castaneum

Small heat shock proteins (sHSPs) are diverse and mainly function as molecular chaperones to protect organisms and cells from various stresses. In this study, hsp 18.3, one Tribolium castaneum species-specific shsp、has been identified. Quantitative real-time polymerase chain reaction illustrated that Tchsp 18.3 is expressed in all developmental stages, and is highly expressed at early pupal and late adult stages, while it is highly expressed in ovary and fat body at the adult period. Moreover, it was up-regulated 4532 ± 396-fold in response to enhanced heat stress but not to cold stress;meanwhile the lifespan of adults in ds-Tchsp 18.3 group reduced by 15.8% from control group under starvation. Laval RNA interference (RNAi) of Tchsp 18.3 caused 86.1%±4.5% arrested pupal eclosion and revealed that Tchsp 18.3 played an important role in insect development. In addition, parental RNAi of Tchsp 18.3 reduced the oviposition amount by 94.7%. These results suggest that Tchsp 18.3 is not only essential for the resistance to heat and starvation stress, but also is critical for normal development and reproduction in T. castaneum.

Genome-wide identification and characterization of long non-coding RNAs in Tribolium castaneum

Long non-coding RNAs(lncRNAs)are poorly understood in insects.In this study,we performed genome-wide analysis of lncRNAs in Tribolium castaneum by RNA-seq.In total,4516 lncRNA transcripts corresponding to 3917 genes were identified from late embryos,early larvae,late larvae,early pupae,late pupae and early adults of T.castaneum,including 3152 novel lncRNAs and 1364 known lncRNAs.These lncRNAs have few exons and transcripts,and are short in length.During development,they exhibited nine different expression patterns.Functionally,they can act either by targeting messenger RNAs(1813 lncRNAs)and lncRNAs(45 lncRNAs)or as micro RNA(miRNA)precursors(46 lncRNAs).LncRNAs were observed to target the metabolic enzymes of glycolysis,TCA cycle and amino acids,demonstrating that lncRNAs control metabolism by regulating metabolic enzymes.Moreover,lncRNAs were shown to participate in cell differentiation and development via their targets.As miRNA precursors,lncRNAs could participate in the ecdysone signaling pathway.This study provides comprehensive information for lncRNAs of T.castaneum,and will promote functional analysis and target identification of lncRNAs in the insect.

赤拟谷盗与杂拟谷盗侵染对小麦粉挥发性物质的GC-IMS分析

目的本研究旨在探究赤拟谷盗(Tribolium castaneum)与杂拟谷盗(Tribolium confusum)侵染对小麦粉中挥发性物质的种类、含量和成分的影响。方法采用气相色谱-离子迁移谱技术(Gas Chroma-tography-Ion Mobility Spectrometry,GC-IMS),对正常小麦粉、受赤拟谷盗与杂拟谷盗侵染的小麦粉进行定性定量测定。结果在3种小麦粉中共检测到了50个峰,鉴定出了41种挥发性物质。赤拟谷盗侵染后,小麦粉中含有高浓度的己醛、2-乙基己醇、壬醛、2,6-双甲基吡啶、(E)-2-庚烯醛、1-辛烯-3-酮、二甲基三硫化合物、庚醛、糠醛。而杂拟谷侵染后小麦粉中则含有高浓度的1-戊烯-3-醇、己醇、2-庚酮、戊醇、3-甲基丁醇。与对照组相比,受两试虫侵染后,小麦粉中的醛类与醇类含量显著增加。结论通过对小麦粉中挥发性物质的分析,并结合气相色谱-离子迁移谱、指纹图谱和主成分分析揭示了正常小麦粉与经过赤拟谷盗与杂拟谷盗侵染的小麦粉中挥发性成分的差异,该研究结果为预测小麦粉储存过程中早期害虫发生提供了参考依据。