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  • Monthly, Founded in 1950
    Supervisor:Chinese Academy of Sciences
    Sponsor:Institute of Zoology,Chinese Academy of Sciences
    The Entomological Society of China
    Domestic postal code: 2-153
    Foreign issuance code: Q61
    ISSN 0454-6296
    CN 11-1832/Q
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Sperm ultrastructure of Euricania clara Kato (Hemiptera: Fulgoroidea: Ricaniidae)
JIANG Zhen, Qin-Dao-Zheng
null    2018, 61 (2): 246-254.   DOI: 10.16380/j.kcxb.2018.02.012
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Abstract: 【Aim】 Sperm ultrastructure has great importance for species recognition and phylogenetics of insects. Studying sperm ultrastructure in the Fulgoroidea can provide more evidence towards phylogenetic analysis which, so far, remains controversial. 【Methods】Both the spermatozoon morphology and the ultrastructure of Euricania clara Kato were observed by ultramicrotomy in combination with light and transmission electron microscopy.【Results】 Mature spermatozoa of E. clara form a sperm bundle called spermatodesm, and each individual spermatozoon has no polymorphism and consists of a head, neck and long flagellum. The head includes the apical bilayered acrosome and nucleus. The neck region is comprised of a centriole and centriolar adjunct. The long flagellum mainly consists of a pair of symmetrical D-shaped mitochondrial derivatives, a pair of fishhook-shaped accessory bodies, and axoneme with the typical 9+9+2 microtubule pattern. 【Conclusion】The characteristics of fish-hook shaped accessory bodies of E. clara are generally consistent with those of other fulgoroids known to date, but are significantly different from other auchenorrhynchans. Furthermore, the number, size, and cross-section morphology and composition of spermatozoic mitochondrial derivatives of E. clara differ significantly from those of other insects. Although there is some consistency within the suborder Auchenorrhyncha, there are obvious differences. This study provides scientific data for phylogenetic analysis of the Fulgoroidea.
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Comparison of volatiles released from the host Juglans mandshurica in different damaged states and the GC-EAD and behavioral responses of Apriona germari (Coleoptera: Cerambycidae) to these volatiles
MA Yan, SHI Li-Yang, ZHAO Yi, XU Hua-Chao
null    2018, 61 (5): 574-584.   DOI: 10.16380/j.kcxb.2018.05.007
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【Aim】 This study aims to explore the volatiles from the host plant Juglans mandshurica, which affect the behavioral response of the brown mulberry longhorn beetle Apriona germari, and to provide a theoretical basis for screening plant-originated attractants or repellents of this insect. 【Methods】 Volatiles released from J. mandshurica in different damaged states [healthy (CK), and feeding, oviposition and boring damage by A. germari] were collected by using dynamic headspace adsorption, and those causing electrophysiological and behavioral responses in A. germari were identified by gas chromatography-mass spectrometry (GC-MS), gas chromatography-electrophysiological antennal detecting system (GC-EAD) and Y-tube olfactometer. 【Results】 In different damaged states, the main volatiles released from J. mandshurica included terpenes and aromatic compounds, and significant differences existed in the relative contents of several components of them. There were one or several unique volatile components in each damaged state. However, A. germari adults only had significant electrophysiological responses to six volatiles, including (1R)-(+)-alpha-pinene, cis-3-hexenyl acetate, nonanal, α-terpineol, 2-ethylhexyl acrylate and hexadecane, but had no responses to the other volatiles. Female adults had the strongest electrophysiological responses to nonanal, while male adults had the strongest electrophysiological responses to cis-3-hexenyl acetate. The olfactory reaction showed that 2-ethylhexyl acrylate had a significant attractivity to the female adults of A. germari (P<0.05), while cis-3-hexenyl acetate had an extremely significant attractivity to the male adults (P<0.01). And nonanal had a significant attractivity to both female and male adults (P<0.05), while α-terpineol had a significant repellent activity to them (P<0.05). 【Conclusion】 These results indicate that cis-3-hexenyl acetate, nonanal and 2-ethylhexyl acrylate show strong attractivity to A. germari adults, while α-terpineol shows good repellent activity.
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Effects of host plants on the feeding behavior and detoxification enzyme activities in Hyphantria cunea (Lepidoptera: Arctiidae) larvae
LI Lu-Sha, YUAN Yu-Fei, WU Lei, CHEN Min
null    2018, 61 (2): 232-239.   DOI: 10.16380/j.kcxb.2018.02.010
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【Aim】 The objective of this study is to quantify the feeding preference of Hyphantria cunea to different host plants and to verify its feeding strategy based on the contents of secondary metabolites in host plants and the activities of acetylcholinesterase (AchE) and detoxification enzymes in H. cunea larvae. 【Methods】Eight plants including Morus alba, Fraxinus pennsylvanica, Ailanthus altissima, Ulmus pumila, Populus nigra, Salix babylonica, Malus micromalus and Lonicera maackii in different damaged degrees in Beijing were selected as the host plants to raise H. cunea larvae. The feeding preference of the 4th instar larvae of H. cunea to these host plants was evaluated by measuring their feeding amount, selectivity and nutritional efficiency on leaves of different host plants. The contents of flavonoids, total phenols and tannin in host plants, and the activities of acetylcholinesterase (AchE), glutathione S transferases (GSTs), carboxylesterase (CarE) and cytochrome P450 (P450) in the 4th instar larvae of H. cunea fed on leaves of different host plants were assayed. 【Results】 The 4th instar larvae of H. cunea had different selectivity to host plants, and the preference order was F. pennsylvanica and A. altissima>M. alba>U. pumila>M. micromalus, S. babylonica and L. maackii>P. nigra. The flavonoid content in host plant leaves was negatively correlated with the feeding amount of H. cunea larvae (r=-0.657, P=0.017). The larvae feeding on different plants had different nutritional efficiency. H. cunea larvae fed on L. maackii had the highest approximate digestibility (AD) (0.97%±0.01%), relatively high relative consumption rate (RCR) (2.92±0.49 g/g·d), and the lowest efficiency of conversation of ingested food (ECI) (0.005±0.01%), efficiency of conversation of digested food (ECD) (0.005%±0.008%) and relative growth rate (RGR) (0.007±0.02 g/g·d). In addition, the larvae feeding on M. micromalus had the highest RGR (0.34±0.04 g/g·d) and relatively high levels of AD (0.72%±0.10%), ECI (0.16±0.01%), ECD (0.24%±0.06%) and RCR (2.19±0.38 g/g·d). The activities of AchE, GSTs, CarE and P450 in H. cunea larvae fed on different host plants were significantly different (P<0.01). 【Conclusion】 H. cunea larvae have different feeding preference and nutritional efficiency on different host plants. Their flexible feeding strategy and detoxification mechanism may be the main reasons for strong adaptability of this insect to different host plants.
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Analysis of the bacterial community structure and diversity in the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae) by 16S rRNA high-throughput sequencing
YANG Xiao-Qing, WANG Zheng-Liang, WANG Tian-Zhao, YU Xiao-Ping
null    2018, 61 (2): 200-208.   DOI: 10.16380/j.kcxb.2018.02.006
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【Aim】 To reveal the bacterial community structure and diversity in the small brown planthopper (SBPH), Laodelphax striatellus. 【Methods】 The Illumina MiSeq high-throughput sequencing platform was used to sequence the V3-V4 regions of the bacterial 16S rRNA gene in the newly emerged female and male adults (24 h after emergence) of L. striatellus. The numbers of sequences and operational taxonomic units (OTUs) and the abundance and diversity of the bacterial communities were analyzed using the related softwares and databases including USEARCH and Silva. 【Results】 A total of 29 333 valid tags and 55 OTUs were obtained from female adults of L. striatellus, while the numbers of valid tags and OTUs from male adults were 25 919 and 57, respectively. Among them, 20 OTUs were shared between the two samples of female and male adults, and the unique OTU numbers for female and male adults were 35 and 37, respectively. The total OTUs of the two samples were annotated into 7 phyla, 15 classes, 23 orders, 33 families, 56 genera, and 73 species. At the levels of phylum, class, order and family, the most dominant bacteria in both samples of female and male adults were from Proteobacteria (99.96% for female and 99.16% for male), Alphaproteobacteria (97.76% for female and 97.84% for male), Rhodospirillales (83.92% for female and 53.21% for male), and Acetobacteraceae (83.90% for female and 53.17% for male), respectively. At the genus level, the dominant bacteria with the highest abundance belonged to an undetermined genus in Acetobacteraceae (unclassified Acetobacteraceae). The second dominant genus was Wolbachia, accounting for 13.81% and 44.52% in female and male adults, respectively. At the species level, the numbers of specific bacterial species in male and female adults were 21 and 32, respectively. However, the abundance of the sex-specific species in the two samples was generally low (less than 0.5%). 【Conclusion】 The results indicate that the bacteria in L. striatellus adults are diverse and the bacterial community structure and diversity are different between female and male adults. This study lays the foundation for further studies on the excavation and utilization of microorganism resources for the biocontrol of L. striatellus.  
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Screening of host plants preferred for oviposition by female adults of Ostrinia furnacalis (Lepidoptera: Crambidae) and their electrophysiological responses to volatile components of Humulus scandens (Cannabaceae)
ZHANG Wen-Lu, WANG Wen-Qiang, BAI Shu-Xiong, ZHANG Tian-Tao, HE Kang-Lai, WANG Zhen-Ying
null    2018, 61 (2): 224-231.   DOI: 10.16380/j.kcxb.2018.02.009
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 【Aim】 To explore the chemical communication mechanism between the Asian corn borer Ostrinia furnacalis and host plants by identifying the components of volatiles from host plants used by female adults of this insect in searching for oviposition sites. 【Methods】 The oviposition preference of O. furnacalis to host plants including Polygonum lapathifolium, Abutilon theophrasti, Humulus scandens, Zea mays and Echinochloa crusgalli was screened, respectively, in field oviposition test and indoor wind tunnel behavioral test. Headspace volatiles of host plants were collected, and the components and contents of physiologically-active volatiles were identified by using two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS). The electroantennogram responses of O. furnacalis to the chemical components of the extract were tested by using coupled gas chromatography-electroantennographic detection (GC-EAD). 【Results】 The field oviposition test results showed that the number of eggs laid by female adults of O. furnacalis on different plants ranked as Z. mays>P. lapathifolium>H. scandens>E. crusgalli>A. theophrasti. In the wind tunnel bioassay, the adults showed an obvious oviposition preference to H. scandens. The GC×GC-TOFMS results showed that there were 45 volatile organic compounds from H. scandens, and the relative content of terpenoids was as high as 86.29% and that of aldehydes, ketones and esters in total was only 15.83%. Nine plant volatiles, i.e., linalool, α-caryophyllene, benzaldehyde, sabinene, myrcene, 2-pentylfuran, nonanal, α-farnesene, and trans-β-ocimene, caused significant GC-EAG reaction in female adults. 【Conclusion】 There are significant differences in oviposition preference of female adults of O. furnacalis to different hosts. The physical and chemical factors of host plants are the main factors affecting the host selection. O. furnacalis shows strong preference to the volatiles of H. scandens. Nine volatiles from H. scandens, including linalool, α-caryophyllene, benzaldehyde, sabinene, myrcene, 2-pentylfuran, nonanal, α-farnesene, and trans-β-ocimenemay, play important roles in oviposition host selection for the Asian corn borer.
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Effects of ectopic expression of Dichaete on leg development of Drosophila and its underlying mechanism
DONG Wei, KONG Yue, MA En-Bo, ZHANG Jian-Zhen, ZHANG Min, ZHANG Xu-Bo
null    2018, 61 (2): 149-155.   DOI: 10.16380/j.kcxb.2018.02.001
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【Aim】 The Drosophila Dichaete gene is a gene of Sox B subfamily, which is expressed throughout embryogenesis and plays an important role in early development. Dichaete also exists in the leg disc of larvae, but little is known about its function in legs. The aim of this study is to investigate the effect of ectopic expression of Dichaete on leg development of Drosophila and to explore the underlying mechanism. 【Methods】en-Gal4 and UAS-Dichaete of Drosophila melanogaster were crossed, and Dichaete was ectopicly expressed under the control of en-Gal4 in the progeny. Legs of the progeny were cut off and their structure was observed using binocular microscope. Immunohistochemistry was used to detect cell apoptosis and Distal-less (Dll) expression in leg discs. 【Results】 The ectopic expression of Dichaete resulted in distal leg defects of adults, enhanced apoptosis in the 3rd instar larvae, and increased expression of distal-less in D. melanogaster, but had no effect on Wg and Dpp signaling activities. Inhibition of apoptosis could not rescue the leg defects. 【Conclusion】 Ectopic expression of Dichaete interferes with maintenance of distal-less gene, leading to structural defects of adult legof D. melanogaster.
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Effects of larval non-sibling egg cannibalism on reproductive performance in Harmonia axyridis (Coleoptera: Coccinellidae)
SHEN Xin-Lan, SHA Xian-Lan, SHU Xiao-Han, LI Bao-Ping, MENG Ling
null    2018, 61 (2): 240-245.   DOI: 10.16380/j.kcxb.2018.02.011
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【Aim】 Egg cannibalism is common in predatory Coccinellidae. It is generally held that consumption of sibling eggs is critical to neonate larvae, but the nutritional value of non-sibling eggs cannibalized relative to prey aphids is still under debate. This study aims to examine the effect of feeding non-sibling eggs besides aphids in larval stage on the reproductive performance of female adults of Harmonia axyridis. 【Methods】Three diet treatments were set at larval stage of H. axyridis: the control (CK) with Rhopalosiphum padi aphids provided only, the dispersive egg consumption (DEC) treatment with non-sibling eggs provided in a small amount to ladybird larvae of each instar, and the aggregative egg consumption (AEC) treatment with non-sibling eggs provided in a large amount only to the final instar larvae as supplementary food together with R. padi aphids provided in the whole larval stage. The ladybird larvae with the above treatments were followed to adulthood, and the body volume of female adults, pre-oviposition period, 10-day fecundity, and egg hatching rate were surveyed. 【Results】 The diet treatment had no significant effect on the body volume of female adults of H. axyridis, which was 92.30, 89.73 and 93.98 mm3 in treatments CK, DEC and AEC, respectively. The diet treatment also had no significant effect on the pre-oviposition period, which was 7.50, 7.74 and 7.70 d in CK, DEC and AEC, respectively. The diet treatment, however, had a significant effect on 10-day fecundity. Female adults in AEC laid much fewer eggs (88) than in DEC (185) and CK (205) during the first 10 d after emergence, and no significant difference was found between DEC and CK. The diet treatment had no significant effect on egg hatching rate, which was 63.10%, 72.58% and 66.61% in CK, DEC and AEC, respectively. 【Conclusion】 These results suggest that consumption of a few non-sibling eggs across larval instars has negligible effects on reproductive performance of H. axyridis adults, but consuming a large number of non-sibling eggs at the final instar can considerably decrease its female adult fecundity.
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Diversity of gut bacterial communities in male adults of Odontolabis fallaciosa (Coleoptera: Scarabaeoidea: Lucanidae) with different mandibular forms
JIANG Yu, SUN Bing-Hua, CAO Yu-Yan, DI Yong-Ning, WAN Xia
null    2018, 61 (3): 322-330.   DOI: 10.16380/j.kcxb.2018.03.007
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【Aim】 To analyze and compare the community structure and diversity of the gut bacteria in adult males of Odontolabis fallaciosa with different mandibular forms. 【Methods】 The total DNA of guts from 31 male adults of O. fallaciosa with three mandibular forms (large, medium and small mandible forms) (including 15 adult males of large mandible form, 10 adult males of medium mandible form and 6 adult males of small mandible form) were extracted. The 16S rDNA gene fragments (V3-V4 region) of gut bacteria were sequenced by Illumina MiSeq, and the number of operational taxonomic units (OTUs), species composition, abundance, and alpha and beta diversity were analyzed. 【Results】 A total of 2 238 637 high quality sequences were obtained and clustered into 3 256 OTUs at a 97% similarity threshold. Analysis showed that they could be annotated into 42 phyla, 328 families and 542 genera. Among them, four phyla (Proteobacteria, Bacteroidetes, Firmicutes and Tenericutes) were prevalent in the gut bacteria communities of male adults, and four genera (Acinetobacter, Dysgonomonas, Bartonella and Chryseobacterium) were dominant. The alpha diversity analysis showed that the gut bacteria were quite abundant in these male samples of O. fallaciosa, and the beta diversity revealed that the composition of OTUs was different among the large, medium and small mandible males. 【Conclusion】 Using Illumina MiSeq sequencing technology, the structure and composition of the gut bacterial community in adult males of O. fallaciosa were analyzed. The microbial community composition in guts of adult males of O. fallaciosa has significant difference between the large and medium mandible adult males, whereas no significant difference exists between the large and small mandible adult males, or between the medium and small mandible adult males.
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Advances in the research of arginine kinase in insects
ZHENG Ya-Nan, LIU Pei-Xuan, SHI Yong, FAN Li-Chun
null    2018, 61 (3): 385-390.   DOI: 10.16380/j.kcxb.2018.03.013
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As one kind of phosphagen kinases, arginine kinase is widely distributed in various tissues of invertebrates. In the long-term evolutionary process, the spatial structures of arginine kinases of different insects have differentiated. However, their amino acid sequences are highly conserved. Functionally, arginine kinases are primarily involved in energy metabolism and maintain the normal life activity in insects and other invertebrates. In addition, with the development of modern scientific techniques, more and more studies show that arginine kinase also plays an important role in autoimmunity and paralysis of host insects. In this article, the distribution, structure and functions of arginine kinases in insects were reviewed.
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Effects of fluvalinate on the quality of reared queens of the western honey bee, Apis mellifera (Hymenoptera: Apidae)
LIAO Chun-Hua, ZHANG Bo, SHI Jing-Liang, WU Xiao-Bo
null    2018, 61 (2): 218-223.   DOI: 10.16380/j.kcxb.2018.02.008
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【Aim】 This study aims to determine the effects of three low doses of fluvalinate on the quality of reared queens of the western honey bee, Apis mellifera. 【Methods】 Queens were caged to lay eggs for 8 h. The larvae hatched from those eggs were transplanted into queen cells for queen rearing, and fed with 2 μL syrup containing different concentrations of fluvalinate (0, 0.05, 0.5 and 5 mg/kg) since the 2nd day of larval stage using a micro sampling syringe injector. The numbers of capped queen cells after feeding for three days and emerged queens in the four groups were recorded, and the birth weight and thorax width of newly emerged queens were also measured. The expression levels of three genes (Vg, hex110 and hex70b) in queen ovaries were also assayed by quantitative real-time PCR (qPCR). 【Results】 The results showed that the proportion of capped queen cells with larvae in the 5 mg/kg fluvalinate group was significantly lower than those in the 0.05 mg/kg fluvalinate group, 0.5 mg/kg fluvalinate group and the control group (0 mg/kg fluvalinate), while there was no significant difference among the latter three groups. The emergence rates of reared queens in the 0.5 mg/kg fluvalinate group and 5 mg/kg fluvalinate group were significantly lower than those in the 0.05 mg/kg fluvalinate group and the control group. There was no significant difference in the birth weight and thorax width of reared queens among the four experimental groups. The results of qPCR analysis indicated that the relative expression level of Vg and hex110 in the ovary of queens reared in the 5 mg/kg fluvalinate group were significantly lower than those reared in the 0.05 mg/kg fluvalinate group, 0.5 mg/kg fluvalinate group and the control group, while there was no significant difference among the latter three groups. The relative expression levels of hex70b gene in the ovary of queens reared in the 5 mg/kg fluvalinate group and 0.5 mg/kg fluvalinate group were significantly lower than that reared in the control group, while the relative expression level of hex70b in the ovary of queens reared in the 0.05 mg/kg fluvalinate group was not significantly different from those in the other three groups. 【Conclusion】 The results indicate that the quality of reared queens of A. mellifera will be affected when the residue of fluvalinate in their food exceeds 0.5 mg/kg.
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Differences in transcriptome and the expression of wing type related genes in nymphs and winged and apterous adults of Myzus persicae (Hemiptera: Aphididae)
ZHANG Chao, YANG Hong, YANG Mao-Fa, YU Hui-Ping, DAI Yuan-Feng
null    2018, 61 (2): 156-167.   DOI: 10.16380/j.kcxb.2018.02.002
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【Aim】 This study aims to understand the transcriptome features of Myzus persicae of different wing types (winged and apterous), to enrich its transcriptome data, and to reveal the molecular mechanisms of wing dimorphism of this insect. 【Methods】 The differences in transcriptome expression between nymphs, apterous adults, and winged adults of M. persicae were detected by high-throughput sequencing technology Illumina HiSeqTM2000. The expression patterns of wing type related genes were analyzed by real-time quantitative PCR (qPCR). 【Results】 The percentage of nucleotides with the quality score over 20 was not less than 94% (GenBank accession numbers: SRR6112438, SRR6112436 and SRR6112437). All reads were assembled into 128 065 unigenes with an average length of 607.08 bp. The resulting sequences were annotated to databases NR, NT, KOG, GO, KEGG, etc., and a total of 128 065 unigenes were annotated. Gene expression analysis showed that the genes of flight proteins, peptide hormones, receptor proteins, etc., which are related to wing formation during growth, were differentially expressed between winged adults and apterous adults or nymphs. qPCR analysis showed that there were significant differences in the expression levels of wing type differentiation related genes including Bursicon (bur- α and bur-β), flightin (Fln), Wntless (Wnt), Distal-less (Dll), Decapentaplegic (Dpp), juvenile hormone epoxide hydrolase gene (JHEH) and ecdysone receptor gene (EcR) between winged adults and apterous adults. 【Conclusion】 The expression levels of bur-α, bur-β , Fln, Wnt, Dll, Dpp, JHEH and EcR change during wing formation in M. persicae. Our results show the general expression characteristics of wing formation genes and provide a basis for further research of wing formation in M. persicae.
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Biological activities of Thymus vulgaris, Petroselinum sativum and Stachys lavandulifolia extracts against adult Callosobruchus maculatus (Coleoptera: Bruchidae) (In English)
Fahimeh HAMIDI, Ali MEHRVAR, Naser EIVAZIAN KARY, Hassan VALIZADEH
null    2018, 61 (3): 331-339.   DOI: 10.16380/j.kcxb.2018.03.008
Abstract454)      PDF(pc) (711KB)(198)       Save

【Aim】 Plants secondary metabolites are potentially considered as suitable alternatives for chemical pesticides if their extents of impacts were well studied earlier on different groups of pests. This study aims to evaluate the possible impacts of solvent polarity on the insecticidal activity of different plant extracts. 【Methods】 The extraction solvents were selected from a wide range of polarities, n-hexane, ethyl acetate and methanol. The lethal effects of Petroselinum sativum seed extracts as well as Thymus vulgaris and Stachys lavandulifolia shoot extracts on Callosobruchus maculatus adults at 28±2℃, 50%±5% RH and a photoperiod of 16L∶8D were evaluated, and their ovicidal and oviposition deterrence effects were also evaluated. 【Results】 The lowest LC50 value for contact toxicity to C. maculatus adults was associated with the n-hexane extract of T. vulgaris (0.05 g/mL) following with the ethyl acetate and methanol extracts, respectively. The ethyl acetate extract of T. vulgaris had the lowest LT50 value (10.04 h), with the highest relative speed of kill index (42.23%) among all the extracts. All of the extracts at LC20 and LC50 concentrations resulted in 100% ovicidal activity in contact toxicity to C. maculatus adults. In terms of oviposition deterrence, the methanol extract of S. lavandulifolia had the highest deterrence activity (97.54%). 【Conclusion】 Based on the results, it is concluded that T. vulgaris, P. sativum and S. lavandulifolia extracts show significant performances in terms of insecticidal and ovicidal toxicities as well as oviposition deterrence against C. maculatus adults. However, non-polar (n-hexane) extracts of the studied plants show better performances in comparison with other extracts.
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Analysis of the antennal transcriptome and olfaction-related genes of Callosobruchus chinensis (Coleoptera: Bruchuidae)
ZHENG Hai-Xia, ZHANG Yao-Wen, ZHANG Xian-Hong, YANG Mei-Hong
null    2018, 61 (2): 168-177.   DOI: 10.16380/j.kcxb.2018.02.003
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【Aim】 This study aims to establish the antennal transcriptome database of the azuki bean weevil, Callosobruchus chinensis, and to obtain the genetic data. 【Methods】The antennal transcriptome of C. chinensis adults was sequenced using an Illumina HiSeq 4000 platform and subjected to bioinformatics analysis. 【Results】 A total of 51.10 Gb clean data (NCBI SRA accession no.: SRP119884) were obtained, and the clean reads were assembled into 83 535 unigenes with an N50 length of 1 492 bp and 15 075 unigenes over 1 kb in length. A total of 22 148 unigenes in the antennal transcriptome of C. chinensis adults were annotated using BLASTX search against the public databases. Most unigenes (18 744 unigenes) were annotated to NR database, and the unigenes of C. chinensis had the highest similarity (39.57%) with those of Tribolium castaneum. All unigenes were divided into 54 branches of three categories (cellular components, molecular function and biological processes) using Gene Ontology (GO), and a lot of unigenes were annotated to be related to metabolic processes, binding activities and cellular processes. In KEGG database, 9 084 unigenes were assigned to 289 known metabolic pathways. Among these, most unigenes (516) are involved in metabolic pathways of ribosome. By further screening and identification, 140 olfaction-related genes of C. chinensis including 12 odorant binding protein (OBP) genes, 4 chemosensory protein (CSP) genes, 116 odorant receptor (OR) genes, 1 ionotropic receptor (IR) gene and 7 sensory neuron membrane protein (SNMP) genes were identified, and their expression levels were evaluated based on FPKM value. 【Conclusion】 This study obtained the antennal transcriptome data of C. chinensis. The results provide a foundation for the further study of gene functions and molecular mechanisms of olfactory reception of C. chinensis.
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Identification of cryptic species and detection of the susceptibility to cyantraniliprole and flupyradifurone of field populations of Bemisia tabaci (Hemiptera: Aleyrodidae) in Hainan, southern China
GUO Lei, HUANG Xin-Yi, LIANG Yan-Po, LIU Xiang-Xiang, CHU Dong
null    2018, 61 (2): 209-217.   DOI: 10.16380/j.kcxb.2018.02.007
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【Aim】 Determining the susceptibility of field populations of the whitefly, Bemisia tabaci in Hainan, China to two novel insecticides, cyantraniliprole and flupyradifurone, can help to provide the reference for the rational application of the insecticides and resistance monitoring of its field populations. 【Methods】 The cryptic species identity of field populations of B. tabaci collected from six localities in Hainan Province, southern China in January, 2017 was identified by sequencing of mitochondrial cytochrome oxidase I gene (mtCOI) and PCR-RFLP analysis of mtCOI. The LC50 values of cyantraniliprole and flupyradifurone against these populations were measured using leaf-dip method. 【Results】 Among the six populations, the population from Jiyang, Sanya city was identified as the cryptic species MEAM1, those from Yongfa, Chengmai county and Chongpo, Ledong county were identified as the mixed populations of the cryptic species MEAM1 (<10%) and MED (94.6% and 92.9%, respectively), and the other three were identified as the cryptic species MED. By comparison with the LC50 value of cyantraniliprole against the susceptible population, the population from Shisuo, Ledong county, had developed low-level resistance to cytraniliprole (RR: 5-10), while the other five were susceptible (RR<5). And by comparison with the LC50 values of flupyradifurone, all the six field populations were susceptible (RR<3). 【Conclusion】 Cyantraniliprole and flupyradifurone can be used as candidate insecticides in the control and resistance management of B. tabaci, and more attention should be paid to the resistance development of B. tabaci to cyantraniliprole in the field.
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Research and application of insect promoters
TONG Xiao-Ling, SHI Jin, GAI Ting-Ting, FANG Chun-Yan, DAI Fang-Yin
null    2018, 61 (3): 360-370.   DOI: 10.16380/j.kcxb.2018.03.011
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Promoter is an important component regulating gene expression. It binds to RNA polymerase to form a transcription initiation complex that controls the initiation time and the level of gene expression. Studies on promoters may contribute to elucidating the underlying mechanism of gene expression regulation so as to lay the foundation for the analysis of gene regulatory networks. In this article, we reviewed the research methods of insect promoters, mainly about the methods for the identification of insect promoters and their specific components and the application of insect promoters.
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Cloning and expression profiling of catalase gene in the oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae)
LI Hong-Bo, DAI Chang-Geng, ZHANG Chang-Rong, HE Yong-Fu, HU Yang
null    2018, 61 (2): 178-187.   DOI: 10.16380/j.kcxb.2018.02.004
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【Aim】 Catalase (CAT) plays an important role in decomposing hydrogen peroxide into water and oxygen which makes an organism avoiding the damage of oxidative stress. This study aims to explore the roles of CAT in anti-oxidative stress in the oriental armyworm, Mythimna separata. 【Methods】 The complete cDNA and genomic sequence of CAT in M.separata were cloned by RT-PCR and RACE. Bioinformatics programs were used to analyze the sequence characteristics of the gene and encoded protein. The expression levels of CAT in M. separata in different developmental stages (egg, 1st-6th instar larva, pupa and adult), tissues of the 5th instar larva (head, cuticle, foregut, midgut, hindgut and Malpighian tubules), and larvae under chlorpyrifos exposure, high temperature stress and larval crowding conditions were detected by quantitative real-time PCR (qPCR). 【Results】 The complete cDNA of CAT obtained from M. separata was named as MsCAT (GenBank accession no.: MF737386), which is 1 846 bp in length, with a 1 602 bp opening reading frame (ORF) encoding 533 amino acids. Sequence analysis indicated that MsCAT has three typical motifs of CAT family, including one proximal active site (88FDRERIPERVVHAKGAGA105), one NADPH binding site (216VTHQVLYLFGD226)and one proximal heme-ligand signature sequence (379RLFSYSDTH386). The DNA sequence of MsCAT contains a 612 bp intron that inserts into the location behind the 99th bp of the encoding region. Phylogenetic analysis indicated that CATs from Noctuidae moths could be assigned to one well-supported cluster. MsCAT was expressed in various developmental stages and tissues of the 5th instar larva of M. separata, and exhibited the highest expression level in the 6th instar larval stage and larval midgut, respectively. MsCAT was significantly up-regulated in larvae fed on corn leaves treated by low concentration (1 μg/mL) of chlorpyrifos for 24 h, but down-regulated in larvae fed on high concentrations of chlorpyrifos. The expression level of MsCAT was significantly up-regulated in larvae exposed to temperatures from 33 to 37℃, but declined in larvae exposed to 39℃ as compared with that of the control (24℃). The expression level of MsCAT in M. separata larvae was negatively related to their crowding degree. 【Conclusion】 The obtained full-length cDNA of MsCAT encoding CAT in M. separata is reliable, and MsCAT may play an important role in development and oxidative stress tolerance of M. separata.
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Contents of Vol. 61 Issue 3
null    2018, 61 (3): 391-391.  
Abstract395)      PDF(pc) (468KB)(206)       Save
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Molecular identification and phylogenetic analysis of 44 species of fleas in Qinghai Province, western China based on mtDNA COI gene sequence
MA Ying, Li-Hai-Long, HE Jian, ZHAO Yan-Mei, YANG Han-Qing, LU Liang, LIU Qi-Yong
null    2018, 61 (4): 488-497.   DOI: 10.16380/j.kcxb.2018.04.011
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【Aim】 DNA barcoding method was used to identify flea samples from Qinghai Province, western China in order to make up the deficiency of the traditional morphological classification method in this study. 【Methods】 The partial fragments (about 600 bp) of the mtDNA COI gene of 182 flea individuals that belong to 3 superfamilies, 6 families, 22 genera and 44 species were amplified by PCR, sequenced and aligned. The intra-and inter-species genetic distances were calculated with K2P model, and a phylogenetic tree was constructed with the neighbor-joining (NJ) method. 【Results】 Totally 182 COI gene sequences (GenBank accession no.: MG138154-MG138335) were sequenced successfully. The inter-species genetic distance (4%-12%) was significantly greater than the intra-species genetic distance (0.01%-2.90%). The NJ tree obtained showed that samples of the same species formed monophyletic groups with high support value, and inter-species branches were clear. 【Conclusion】 The results confirm that COI gene can be used in molecular identification of fleas.
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Evaluation of reproductive disturbance to Chinese bumblebees by the European bumblebee, Bombus terrestris (Hymenoptera: Apidae)
YUAN Xiao-Long, Muhammad NAEEM, ZHANG Hong, LIANG Cheng, HUANG Jia-Xing, AN Jian-Dong
null    2018, 61 (3): 348-359.   DOI: 10.16380/j.kcxb.2018.03.010
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【Aim】 Alien species may interfere with the normal reproduction of the closely related native species through mating competition. Although the European bumblebee, Bombus terrestris, is the most widely used pollinator in the world, it has caused biological invasion in many countries. This study aims to elucidate the possibility of reproductive disturbance to some Chinese bumblebees by B. terrestris. 【Methods】 The components of male cephalic labial gland secretions of B. terrestris and nine native bumblebee species in China were detected using gas chromatography-quadrupole time of flight mass spectrometry (GC-Q-TOF/MS), and the data were subjected to cluster analysis. The effects of male B. terrestris on the mating of the most important native bumblebee species under controlled environment were compared. 【Results】 The results showed that the GC-MS chromatograms of male cephalic labial gland secretions were unanimous within a bumblebee species, but significantly different between different bumblebee species. The components of male cephalic labial gland secretions of both B. lantschouensis and B. ignitus were similar to that of B. terrestris with 49.23% and 58.49% identity, respectively. The male of B. terrestris could mate with the queen of B. lantschouensis and significantly decreased the mating success of the latter (P<0.01). 【Conclusion】 It is concluded that the European bumblebee, B. terrestris, can interfere with the normal mating and has a high risk of reproductive disturbance to some local bumblebee species of China. In order to protect the local bumblebee resources and ecosystem balance from biological invasion, alien bumblebee species should be cautiously used in China.
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Molecular cloning, characterization and expression analysis of trehalase gene GdTre1 in Galeruca daurica (Coleoptera: Chrysomelidae)
CHEN Long, TAN Yao, ZHOU Xiao-Rong, PANG Bao-Ping, GE Xi-Ge-Du-Ren, SUN Zhi-Peng
null    2018, 61 (3): 271-281.   DOI: 10.16380/j.kcxb.2018.03.002
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 【Aim】 Trehalase (Tre), a key enzyme in trehalose metabolism of insects, plays important roles in the energy regulation and development of insects. This study aims to clone a trehalase gene from Galeruca daurica, and to analyze its expression patterns in order to investigate the functions of trehalase in the development of G. daleruca and summer diapause of its adults. 【Methods】 Based on the transcriptome data of G. daleruca, the full-length cDNA of the Tre gene was cloned using the rapid amplification of cDNA ends (RACE)-PCR and analyzed by bioinformatics. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression levels of the Tre gene in different developmental stages (egg, 1st-3rd instar larva, prepupa, pupa, and 3, 7, 10, 15, 25, 40, 60, 80 and 100 day-old adult) and various adult tissues (head, thorax and abdomen) of G. daleruca, and the 3 day-old adults under different stress temperature (15, 20, 25, 30, 35 and 40℃). The Tre activity in different day-old adults was assayed by the 3, 5-dinitrosalicylic acid colorimetry. 【Results】 A soluble trehalase gene was cloned from G. daleruca, and named GdTre1 (GenBank accession no.: KY697913). Its fulllength cDNA is 1 933 bp containing a 1 704 bp open reading frame (ORF) that encodes 567 amino acids with the predicted molecular weight of 66.56 kD and pI of 6.62. The coded protein contains typical functional domains in the trehalase superfamily with a signal peptide but without transmembrane domain. Homology and phylogenetic analyses showed that GdTre1 has the highest amino acid sequence identity (70.25%) with Leptinotarsa decemlineata Tre1b. RT-qPCR results revealed that GdTre1 was expressed throughout various developmental stages of G. daleruca, with higher expression levels in the egg and diapause adult, and lower expression levels in the larva, pre-pupa, pupa and pre-diapause adult. Tissue expression profiles revealed that the expression level of GdTre1 was the highest in the abdomen, second high in the thorax and the lowest in the head of adults. The expression level of GdTre1 in 3 day-old adults increased as the stress temperature increased, with the highest level at 30℃, and afterwards decreased a little with the temperature increasing. The expression levels of GdTre1 and the Tre activity were significantly different among different day-old adults and showed the same change trend. 【Conclusion】 Trehalase has a close relationship with the development and summer diapause in G. daleruca. The results provide a necessary foundation for further investigation on the molecular mechanisms of summer diapause in this insect.
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Foreground-background segmentation of lepidopteran specimen images based on fully convolutional networks
Zhu-Le-Qing, MA Meng-Yuan, ZHANG Zhen, MENG Zhao-Jun, WU Wei- Ren-Li-Li, GAO Cui-Qing, 南Xiao-Ning
null    2018, 61 (2): 255-262.   DOI: 10.16380/j.kcxb.2018.02.013
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 【Aim】 This study aims to realize the automatic foreground-background segmentation of lepidopteran specimen images by exploring the state-of-art computer vision technology. 【Methods】 First, the background is manually removed to form the ground truth of training set and testing set, and those images that are too large are resized to smaller ones. Then, the training set is enhanced by rotation, translation, scaling, etc., and their central areas are cropped as valid input and target images. Afterwards, the mean image of all the training samples is calculated and subtracted from all input images. Testing images are simply normalized but not enhanced. Fully convolutional networks (FCNs) are fine-tuned with training set until they converge. The parameter adjustment on later convolutional layers and de-convolutional layers is emphasized since their structures are different from those of original immigrated FCNs. When one given insect image is fed into the trained FCN after normalization, the segmentation result will be given. 【Results】 The proposed method was evaluated with the testing set including 823 samples, and the final mIoU (mean Intersection over Union) was as high s 94.96%. The visual effect of segmentation results given by FCN was much close to the manually produced results.【Conclusion】 The experimental results prove that the foregroundbackground of lepidopteran specimen images can be segmented efficiently by the trained FCN.
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Effects of mixtures of brood pheromone esters on the reared queen quality of the Chinese honeybee, Apis cerana cerana (Hymenoptera: Apidae)
HU Jing-Hua, ZHANG Li-Zhen, LIAO Chun-Hua, JIANG Wu-Jun, ZENG Zhi-Jiang, YAN Wei-Yu
null    2018, 61 (3): 300-307.   DOI: 10.16380/j.kcxb.2018.03.005
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 【Aim】 The objective of this study is to investigate the effects of mixtures of brood pheromone esters on the queen quality of the Chinese honeybee, Apis cerana cerana, reared by queenrearing technology without larvae-grafting, and to find a way to rear high-quality queen. 【Methods】 Queen-rearing technology without larvae-grafting was employed to rear queen of A. cerana cerana. Using a micro sampling syringe injector, 1 μL of mixture of brood pheromone esters with the concentration gradients of 0, 0.1%, 1.0% and 10.0% was added into queen cells, respectively, when the queen larvae were 60-64 h old. The body weight, the weight and width of thorax, the number of ovarioles in one ovary of the newly-emerged queens were tested. And the gene expression levels of vitellogenin (Vg), hexamerin70b (hex70b) and hexamerin110 (hex110) were quantified by qPCR. 【Results】 The results showed that the body weight of the newly-emerged queens of A. cerana cerana was increased significantly (P<0.05) in the treatments with the mixture of three brood pheromone esters (3E) (1.0% methyl linoleate, 16.0% methyl linolenate, and 35.0% methyl oleate) at all the three concentrations, while the width and weight of thorax of reared queens had no significant change as compared with the control (paraffin oil) (P>0.05). The number of ovarioles in one ovary was increased significantly (P<0.05) in 0.1% and 1.0% treatment groups. The expression levels of Vg and hex70b in all 3E treatment groups had no significant change, while that of hex110 in 1.0% and 10.0% 3E treatment groups increased significantly (P<0.05) as compared with the control. When the queens were reared with the mixture of four brood pheromone esters (4E) (4.5% methyl palmitate, 1.0% methyl linoleate, 16.0% methyl linolenate, and 35.0% methyl oleate), the body weight of the newly-emerged queens and the number of ovarioles in one ovary significantly increased in 10.0% 4E treatment group (P<0.05), while no significant difference of developmental indexes existed in 0.1% and 1.0% 4E treatment groups (P>005). The expression levels of Vg in ovaries in 0.1% and 1.0% 4E treatment groups, hex70b in 10.0% 4E treatment group and hex110 in 1.0% and 10.0% 4E treatment groups all significantly increased as compared with the control (P<0.05). When the queens were reared with the mixture of 10 brood pheromone esters (10E) (4.5% methyl palmitate, 2.5% methyl stearate, 35.0% methyl oleate, 1.0% methyl linoleate, 16.0% methyl linolenate, 3.5% ethyl palmitate, 1.5% ethyl stearate, 18.0% ethyl oleate, 0.5% ethyl linoleate, and 17.5% ethyl linolenate), the body weight of the newly-emerged queens, and the expression levels of Vg and hex110 in ovaries in all treatment groups significantly decreased as compared with the control (P<0.05), while the number of ovarioles in one ovary and the thorax indexes showed no significant change (P>0.05). The expression level of hex70b also reduced significantly in 10% 10E treatment group (P<0.05). 【Conclusion】 To a certain extent, the 1.0% 3E and 10.0% 4E added in queen-rearing process of A. cerana cerana could improve the queen quality.
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Expression profiling and functional analysis of RR-1 cuticular protein genes ApCP12 and ApCP23 in Antheraea pernyi (Lepidoptera: Saturniidae)
MA Yue-Yue, LIU Wei, WANG Yong, WANG De-Yi, LEI Piao, RU Yu-Tao, JIANG Yi-Ren, YANG Rui-Sheng, QIN Li
null    2018, 61 (2): 188-199.   DOI: 10.16380/j.kcxb.2018.02.005
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【Aim】 Cuticular proteins (CPs) are abundant in insects, playing important roles in insect growth and development. This study aims to enrich different types of CPs in Antheraea pernyi and to determine the expression patterns of CPs during different developmental stages. 【Methods】 Two CP genes were cloned from cuticle of A. pernyi larvae using PCR and 3′ RACE technology, and the phylogenetic relationship was analyzed by bioinformatics methods. The expression patterns of the two genes in different tissues of larvae and during embryonic development of A. pernyi were analyzed by semi-quantitative RT-PCR, and the relative expression levels of the two genes in A. pernyi at different developmental stages and in the 3rd instar larvae after RNAi treatment were measured using real-time quantitative PCR (qPCR) technology. 【Results】 Two CP genes were cloned from A. pernyi and named ApCP12 (GenBank accession number: MF318874) and ApCP23 (GenBank accession number: MF318875), respectively. Bioinformatic analysis indicated that ApCP12 is 690 bp in length with an open reading frame (ORF) of 336 bp, encoding a protein of 111 amino acid residues with the calculated molecular weight of 12 kD, while ApCP23 is 1 243 bp in length with an ORF of 594 bp, encoding a protein of 197 amino acid residues with the calculated molecular weight of 23 kD. Both proteins contain RR-1 consensus and are classified in different clusters with RR-2 cuticular proteins by phylogenetic analysis. Tissue-specific mRNA expression profiling showed  that ApCP12 was more widely distributed than ApCP23. In the progress of embryonic development, the expression level of ApCP12 increased gradually. During larval ecdysis stage, the expression level of ApCP12 increased by 3-fold, and that of ApCP23 increased by 13-fold in 3 d after ecdysis than in molting stage. During pupal melanization, the expression level of ApCP12 was higher than that of ApCP23. The expression levels of the two genes had no significant changes during the earlier stage of pupal eclosion, while on the last day before eclosion, the expression levels of ApCP12 and ApCP23 increased by 3.5- and 3-fold, respectively, as compared with that on the first day of 20E injection. After RNAi treatment, the expression levels of ApCP12 and ApCP23 in the 3rd instar larvae were downregulated 5- and 3-fold, respectively. 【Conclusion】 The results suggest that these two CPs of subtribe RR-1 participate in the cuticle building of larva, pupa and adult and have a close relationship with the whole life cycle of A. pernyi development.
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Analysis of genetic differentiation of different geographical populations of the melon fly, Bactrocera cucuribitae (Diptera: Tephritidae) in China based on microsatellite molecular markers
ZHANG Ya-Nan, NIU Li-Ming, ZHOU Shi-Hao, GONG Zhi, LI Lei, ZHANG Fang-Ping, HAN Dong-Yin, FU Yue-Guan
null    2018, 61 (5): 613-619.   DOI: 10.16380/j.kcxb.2018.05.011
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【Aim】 The melon fly, Bactrocera cucuribitae is an important invasive pest with wide host range, causing serious harm. This study aims to explore its population differentiation and genetic variation in China. 【Methods】 The genetic diversity of a total of 190 individuals collected from 21 regions of 7 provinces in China was analyzed using nine polymorphic microsatellite loci as the molecular markers. 【Results】 For the 21 geographical populations of B. cucuribitae, the average percentage of polymorphic loci was 97.08%, Shannon’s diversity index(I) was 0.8841, and the genetic differentiation coefficient among populations (FST) was 0.12806, suggesting that genetic differentiation occurs among the 21 geographical populations. The UPGMA cluster analysis showed that the populations of Hainan (excluding Sansha), Guangdong, Guangxi, Yunnan and Sansha of Hainan were clustered into one clade, while the populations of Fujian, Jiangxi and Sichuan clustered into separate clades, respectively. 【Conclusion】 A certain degree of genetic differentiation has occurred among the melon fly populations in China, but the accumulated variability is limited.
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Cloning, prokaryotic expression and expression profiling of Mfe2 from the Japanese sawyer beetle, Monochamus alternatus (Coleoptera: Cerambycidae)
LIU Xiao-Long, ZHANG Bin, TIAN Hao-Kai, NING Jing, WANG Hai-Xiang, ZHAO Li-Lin
null    2018, 61 (3): 282-291.   DOI: 10.16380/j.kcxb.2018.03.003
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【Aim】 Mfe2, peroxisomal multifunctional enzyme type 2 gene, plays a very important role in the β-oxidation of fatty acids. This study aims to clone and identify the Mfe2 gene from the Japanese sawyer beetle, Monochamus alternatus, to express its encoded protein in prokaryotic expression system, and to explore its expression patterns in different developmental stages and adult tissues of the beetle.【Methods】 The recombinant plasmid was constructed to clone the Mfe2 gene of M. alternatus using the fast cloning method through designing primers with overlapping region between the vector and target fragment, and the cloned sequence was analyzed with various bioinformatics tools. The expressed Mfe2 protein in prokaryotic expression system was inducted with IPTG, and the fusion protein was verified by Western blotting. The expression profiles of this gene in different developmental stages and adult tissues of M. alternatus were investigated using the RT-qPCR technology. 【Results】 Mfe2 was cloned from M. alternatus and named MaMfe2 (GenBank accession no.: MF944109). Its open reading frame is 2 148 bp, encoding a protein of 716 amino acids, with an estimated molecular mass of 77.56 kD. Bioinformatics analysis showed that MaMfe2 has an AICARFT_IMPCHas domain, with 91% amino acid sequence identity with peroxisomal multifunctional enzyme type 2 of Anoplophora glabripennis. The expressed protein in BL21 (DE3) strains of Escherichia coli bound specifically with its antibody in Western blot analysis. Developmental expression profiles showed that the expression level of MaMfe2 was the highest in the pupal stage, and lower in egg and larval stages, and tissue expression profiles revealed that the expression level of MaMfe2 was the highest in the fat body and the lowest in the protothorax of adults. 【Conclusion】 MaMfe2 encodes a peroxisomal multifunctional enzyme in M. alternatus, and its expression level is various in different developmental stages and adult tissues. This study provides the basis for further research on the function of this gene in M. alternatus.
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A life-history trait of the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Pyralidae)-positive correlation between body weight and temperature
HE Hai-Min, XIAO Liang, CHEN Qian-Wu, XUE Fang-Sen
null    2018, 61 (3): 340-347.   DOI: 10.16380/j.kcxb.2018.03.009
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【Aim】 Temperature is the most important environmental factor for ectotherms and affects all aspects of lifehistory traits. This study aims to understand how lifehistory traits vary with temperature in the Asian corn borer, Ostrinia furnacalis. 【Methods】 The developmental duration from egg hatching to pupation and from pupation to adult eclosion, and the pupal and adult weight were examined in the Nanchang population of O. furnacalis at 20, 22, 24, 26, 28, 30 and 32℃ under a photoperiod of 16L∶8D in the laboratory. 【Results】 The larval and pupal duration and total developmental duration of O. furnacalis were significantly decreased with the increasing of rearing temperature. The larval duration and total developmental duration were significantly shorter in males than in females, showing the protandry phenomenon. The growth rate was positively correlated with temperature. The growth rates of females were significantly lower than those of males at low temperatures but significantly higher than those of males at high temperatures. The relationship between body weight and rearing temperature in O. furnacalis did not follow the temperature-size rule, and both males and females gained heavier body weight at high temperature. Females were significantly larger in size than males at all temperatures, showing a female biased sex size dimorphism (SSD). Contrary to Rensch’s rule, the SSD index and body weight of O. furnacalis tended to increase with rising temperature. Male pupae lost significantly more weight at metamorphosis compared to females, resulting in higher SSD index in adults than in pupae. 【Conclusion】 High temperature not only significantly shortens developmental duration, but also results in heavier body weight at maturity in O. furnacalis. There are significant differences in life-history traits between females and males of O. furnacalis.
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Molecular cloning and functional characterization of N-β-alanyl-dopamine hydroxylase gene in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)
DU Xiao-Yan, FU Kai-Bin, XU Qing-Yu, Tursun AHMT, DING Xin-Hua, HE Jiang, GUO Wen-Chao
null    2018, 61 (6): 637-645.   DOI: 10.16380/j.kcxb.2018.06.002
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【Aim】 This study aims to clarify the function of N-β-alanyl-dopamine (NBAD) hydrolase gene important in melanin biosythesis in the Colorado potato beetle, Leptinotarsa decemlineata by RNA interference. 【Methods】 The NBAD hydrolase gene in L. decemlineata was characterized by data mining based on its transcriptome, its cDNA was cloned by RT-PCR, and the gene completeness and phylogeny were determined by multiple alignment and phylogenetic analysis, respectively. The expression levels of NBAD hydrolase gene in different developmental stages, tissues of the 4th instar larvae and male gonad and ovary of adults of L. decemlineata were detected by qPCR. The color change during larval growth was observed after RNAi, and the mechanism how the expression of NBAD hydrolase gene was influenced by juvenile hormone (JH) and molting hormone (MH) was assayed .【Results】 An NBAD hydroxylase gene was cloned from L. decemlineata and named Ldtan (GenBank accession no.: KY221866). Its encoded protein shows the highest amino acid sequence identity with the homologous proteins from Tribolium castaneum and Dendroctonus ponderosae and clustered into the same clade with them. The spatial expression profiles showed that Ldtan were highly expressed in ventral nerve cord, hindgut and cuticle of L. decemlineata, with the relative expression levels of 99.36±0.95, 17.79±3.11 and 9.21±0.12, respectively, while the temporal expression profiles showed that its expression level increased along with larval growth and reached the peak at the adult stage. Knockdown of Ldtan gene by feeding dsLdtan to the 2nd instar larvae not only led to tanned color, but also a degree of lethal effect. Knocking down the expression of JH synthesis and signal-related genes by RNAi downregulated the expression of Ldtan, while knocking down the expression of MH synthesis and signal-related genes by RNAi upregulated the expression of Ldtan. 【Conclusion】 The results suggest that Ldtan is involved in melanin synthesis in L. decemlineata, and JH and MH probably regulate its expression.
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cDNA cloning, prokaryotic expression and enzymatic characteristics of the glutathione S-transferase GmolGST6 in Grapholita molesta (Lepidoptera: Tortricidae)
LI Shuai, SU Li, LI Bo-Liao, LI Yi-Ping, LI Guang-Wei, WU Jun-Xiang
null    2018, 61 (4): 398-409.   DOI: 10.16380/j.kcxb.2018.04.002
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【Aim】This study aims to clone the glutathione S-transferase (GST) gene from the oriental fruit moth, Grapholita molesta, to analyze its structure properties, and to clarify the expression profiles and enzymatic characteristics of protein coded by this gene, so as to provide a theoretical basis for fundamental function research of this gene. 【Methods】 Based on the transcriptome data of female antennae from G. molesta, the complete coding sequence of GST gene was cloned using RT-PCR. The expression levels of this gene in antennae, head (without antennae), thorax, abdomen, leg and wing were measured by real-time fluorescence quantitative PCR (qPCR). The recombinant protein of GST was prokaryotically expressed, and then purified by Ni2+ affinity chromatography. In addition, the stability and kinetic parameters of the recombinant GST were analyzed. 【Results】 The complete coding region sequence of GmolGST6 (GenBank accession no.: MF503496) was obtained, and its ORF is 645 bp in length, encoding 214 amino acids with the predicted molecular mass of 24.21 kD and the theoretical isoelectric point of 5.10. The phylogenetic tree and the three-dimensional structure analysis indicated that GmolGST6 belonges to Delta subfamily of GSTs. qPCR results showed that GmolGST6 was primarily expressed in the antenna of male and female adults, and its expression level in the male antenna was significantly higher than that in the female antenna. The recombinant GmolGST6 showed catalytic activity towards the general substrate CDNB, and had the highest activity at pH 7.5 and 40℃. The Michaelis constant (Km) of the recombinant GmolGST6 was 0.21±0.06 mmol/L, and the maximum reaction rate (Vmax) was 14.02±1.40 μmol/min·mg. 【Conclusion】 Based on the expression profiles of GmolGST6 and the catalytic activities of the recombinant GmolGST6 towards CDNB, we speculate that GmolGST6 may be involved in the catabolism of exogenous substances and protecting the olfactory system from toxification of exogenous substances. 
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Feeding behaviour of Bactrocera minax (Diptera: Trypetidae) on male inflorescence of Castanea mollissima (Fagales: Fagaceae)
HE Zhang-Zhang, HUA Deng-Ke, DU Tian-Hua, WANG Fu-Lian, GUI Lian-You
null    2018, 61 (4): 458-467.   DOI: 10.16380/j.kcxb.2018.04.008
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【Aim】 This study aims to confirm and elucidate the feeding behaviour of adult Bactrocera minax on male inflorescence of Castanea mollissima in the wild, so as to provide references for prevention and control of this insect using non-chemical control methods in China. 【Methods】 The feeding behavior of adult B. minax on male inflorescence of C. mollissima was tracked with video technology and analyzed. 【Results】 The results showed that the feeding behaviour of adult B. minax was characterized by four phases, i. e., walking, feeding, grooming and inactivity, and the sequence of these four phases was not fixed. Except being unable to go from the inactivity phase to the feeding phase, adult B. minax could optionally transit from one phase to another among the four phases. The proportion of frequency of each behavior of adult B. minax in the order from high to low was as follows: walking>feeding>grooming>inactivity. The average time of the inactivity phase was significantly longer than that of the feeding, grooming and walking phase, and the difference in the average time was not obvious among the feeding, grooming and walking phases. There was no significant difference in the proportion of frequency and average time of each phase between females and males. 【Conclusion】 These results confirm the presence of feeding behavior of adult B. minax on male inflorescence of C. mollissima.
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Cloning, prokaryotic expression and tissue expression profiling of an odorant binding protein gene BminOBP25 from Bactrocera minax (Diptera: Tephritidae)
SI Pin-Fa, ZHOU Qiong, CUI Zhong-Yi
null    2018, 61 (5): 537-545.   DOI: 10.16380/j.kcxb.2018.05.003
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【Aim】 The odorant binding proteins (OBPs) of insects are closely related to olfactory recognition and play a key role in the successful delivery of fat-soluble odorant molecules in the lymph nodes of the antennal sensory receptors to the olfactory receptors. In order to understand the roles of OBPs in olfactory recognition and to lay foundation for studying the molecular mechanisms of olfactory transmission in Bactrocera minax, an OBP gene was cloned and its expression profiles were analyzed. 【Methods】 The full-length cDNA sequence of OBP gene was cloned from B. minax using RT-PCR and RACE techniques, and subjected to bioinformatics analysis. The recombinant expression vector pET28a(+)-BminOBP25 was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein was identified by SDS-PAGE and Western blotting. The expression profiles of OBP gene in different tissues of B. minax adults were detected by quantitative real-time PCR (qPCR). 【Results】 An OBP gene was cloned from B. minax and named BminOBP25 (GenBank accession no.: MH181875). Its ORF is 447 bp in length encoding 148 amino acids with a predicted molecular weight of 17.5 kD. The encoded protein has typical six conserved cysteines and six α-helices. The recombinant expression vector pET28a(+)-BminOBP25 was constructed and the target protein was stably expressed in host bacteria in the form of 6×His tag fusion protein after IPTG induction. qPCR analysis showed that BminOBP25 mRNA was expressed in antennae, head (without antennae), thorax, abdomen, leg, wing and ovipositor of adults, with higher expression levels in antenna, head (without antennae), leg and ovipositor.【Conclusion】 BminOBP25 has high transcriptional activity in antennae, heads, legs and ovipositors of B. minax adults, suggesting that BminOBP25 may also have physiological functions in non-olfactory tissues, and especially may play important roles during the selection of insect feeding and spawning sites. Its functions need further study. In this study, the prokaryotic expression of BminOBP25 was achieved, laying a foundation for further study of its functions.
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Cloning and expression analysis of olfactory receptors gene AcOr10 in the Chinese honeybee, Apis cerana cerana (Hymenoptera: Apidae)
YANG Le, JIANG Wu-Jun, HE Xu-Jiang, ZENG Zhi-Jiang
null    2018, 61 (3): 292-299.   DOI: 10.16380/j.kcxb.2018.03.004
Abstract281)      PDF(pc) (3643KB)(237)       Save
【Aim】 Olfactory receptor Or10 is one of the important receptor proteins for the perception of pheromone in the honeybee, Apis cerana cerana. The aim of this study is to clone the sequence of Or10 gene, and to analyze the expression levels of Or10 in drone antennae and brains across various physiological status, so as to provide the theoretical basis for further studies on the biological function of this gene. 【Methods】 The drone antennae of A. cerana cerana were collected, from which the total RNA was extracted to clone the sequence of Or10. The relative expression levels of Or10 in antennae and brains of drones, which were sexually immature at hive entrance, sexually mature at hive entrance, sexually immature at frames in hive and sexually mature at frames in hive, were detected using quantitative real-time PCR (qPCR). 【Results】 The cDNA sequence of Or10 gene (named as AcOr10) (GenBank accession no.: MF693365) of A. cerana cerana was obtained, with the length of 914 bp and a coding region of 738 bp, encoding 246 amino acids. The encoded protein has the molecular weight of 28.163 kD and isoelectric point of 5.50. The phylogenetic tree showed that AcOr10 clustered with Or10 of A. mellifera and Or4-like of A. florea. The relative expression levels of AcOr10 in antennae of sexually immature drones at hive entrance and sexually mature ones at frames in hive were significantly higher than those of sexually immature drones at hive entrance and sexually immature drones at frames in hive (P<0.05). However there was no significant difference in the relative expression level of AcOr10 in antennae between sexually mature drones at hive entrance and mature ones at frames in hive (P>0.05). There was also no significant difference in the relative expression level of AcOr10 in antennae between sexually immature drones at hive entrance and immature ones at frames in hive (P>0.05). The relative expression level of AcOr10 in brains of sexually mature drones at hive entrance was significantly higher than those of drones sexually immature at hive entrance, sexually immature at frames in hive and sexually mature at frames in hive (P<0.05), while there was no significant difference among the latter three groups (P>0.05). The relative expression levels of AcOr10 in antennae of drones sexually immature at hive entrance, sexually immature at frames in hive and sexually mature at frames in hive were all significantly higher than those in brains of drones with the same physiological status (P<0.05), but there was no significant difference in the expression level of AcOr10 between antennae and brains of drones sexually mature at hive entrance (P>0.05). 【Conclusion】 It is inferred that AcOr10 may be related with sexual maturity of A. cerana cerana drones, and flight activities have little influence on the expression of AcOr10 in antennae but cause the changes of brains, thereby affecting the mating behaviour of drones.
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cDNA cloning, prokaryotic expression and polyclonal antibody preparation of cathepsin L in Spodoptera frugiperda (Lepidoptera: Noctuidae)
CHENG Xing-An, LIN Xian-Wei, JIANG Xu-Hong, LIU Zhan-Mei, ZHONG Guo-Hua, Hu-Mei-Ying
null    2018, 61 (4): 410-422.   DOI: 10.16380/j.kcxb.2018.04.003
Abstract279)      PDF(pc) (5434KB)(126)       Save
 【Aim】 The objective of this study is to clone cathepsin L gene from Sf9 cells of Spodoptera frugiperda, to analyze its sequence features and to obtain the polyclonal antibody, so as to provide a theoretical basis for further studying the function of the gene in this insect. 【Methods】 Cathepsin L gene was cloned directly by using the specific primers which were designed based on the open reading frame (ORF) sequence of cathepsin L gene of S. exigua. The sequence characteristics of this gene were analyzed by using bioinformatics, and the deduced amino acid sequences of the gene were aligned using the ClustalX2 program. The three-dimensional structure of cathepsin L was modeled by homology modeling method. Finally, the anti-rabbit polyclonal antibody was produced by immunizing rabbit based on the prokaryotic expression of the gene. 【Results】 A cathepsin L gene named SfCatL (GenBank accession no.: HQ110065) was cloned from Sf9 cells of S. frugiperda, with the ORF of 1 035 bp in length, encoding 344 amino acids with the predicted N-terminal hydrophobic region of 16 amino acid residues displaying the characteristic features of a signal peptide. The predicted molecular weight (MW) and isoelectric point (pI) of SfCatL without signal peptide are 36.8 kD and 6.69, respectively. SfCatL has 53.7%-96.8% amino acid sequence identity with the cathepsin L proteins from other 13 species, showing the highest amino acid sequence identity (96.8%) with the cathepsin L protein from S. exigua. The three-dimensional structure of SfCatL showed that SfCatL folds into a shape of fortune cookie and contains three disulfide bonds, which are responsible for the stability of the structure. Hydrophilic amino acids are mainly coated on the surface of protein. After prokaryotic expression, the SfCatL protein was purified to produce the antiserum of SfCatL, whose titer reached 1∶40 000 by ELISA assay. Western blot assay indicated that the antiserum could specifically bind with the SfCatL protein from Sf9 cells. 【Conclusion】 The complete ORF of SfCatL was cloned, and its sequence features were analyzed. The SfCatL recombinant protein was prepared and purified, and finally the polyclonal antibody was acquired. This study provides a theoretical basis for further study of the gene function and the development of cathepsin inhibitor insecticides.
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Mating behavior of Sclerodermus guani (Hymenoptera: Bethylidae)
LUO Li-Lin, LI Li
null    2018, 61 (5): 604-612.   DOI: 10.16380/j.kcxb.2018.05.010
Abstract274)      PDF(pc) (2080KB)(171)       Save
 【Aim】 The semiectoparasitoid Sclerodermus guani tends to parasitize solitary wood-boring insects in concealed habitat (trunk or seed). Effective mating tactics are critical for population colonization and expansion of S. guani. This study aims to explore the mating behavior of S. guani and to unveil its mating tactics. 【Methods】 The mating process of both male and female adults of S. guani (♀∶♂=1∶1) was recorded with dissecting microscope, video camera and insect behavior tracker in the laboratory, and the differences in behavior performance, time allocation and speed of mating between female and male adults during the whole mating process were compared. 【Results】 The mating process of S. guani adults can be divided into three phases, i.e., pre-copulation, copulation and post-copulation, and the mating behavior shows a series of characteristic procedures including antennal drumming, mounting females, walking on the back of females, probing and copulation. In the pre-copulation phase, males walk around quickly, touch and drum females by antennae, and touch females by mouthpart. After mounting females, males walk on the back of females and still keep tapping females with antennae or touching with mouthpart. Females always keep a stereotypical posture of being stationary with head down. The total lasting time for the pre-copulation phase was about 653.617±54.160 s (mean±SE). During copulation, males insert their aedeagi, their forelegs incline to the front-end of abdomens of females at an angle of 45°, while their mid legs and hind legs hold the abdomens of females, and the mating duration was about 43.567±7.120 s. In the post-copulation phase, males signal the end of copulation by dismounting and moving away from the copulation site while females keep immobile for approximately 172 s. Both male and female adults exhibit multiple mating behaviors in the whole mating process. With the increasing of mating frequency, the mating duration increases at first and then decreases. In the whole mating process, the mating behavior of male and female adults is first decelerated and then accelerated. The mean mating velocities of S. guani in precopulation, copulation and post-copulation were 3.111, 0.595 and 1.016 cm/s for female adults and 2.754, 0.895 and 1.314 cm/s for male adults, respectively. 【Conclusion】 Male adults of S. guani behave actively during the mating process, suggesting that they play leading roles in spouse search, recognition and selection. The results of this study provide the theoretical basis for artificial reproduction, release and population rejuvenation of S. guani in the field.
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Comparative analysis of antioxidant enzyme activities in non-diapause,summer diapause and winter diapause pupae of Delia antiqua (Diptera: Anthomyiidae) (In English)
HAO You-Jin, GUO Qiang, CHEN Bin
null    2018, 61 (3): 263-270.   DOI: 10.16380/j.kcxb.2018.03.001
Abstract272)      PDF(pc) (1885KB)(337)       Save
【Aim】 In order to investigate the differences of antioxidant system in non-diapause (ND), summer diapause (SD) and winter diapause (WD) pupae of Delia antiqua, the variations of antioxidant enzyme activities and glutathione redox status were determined and compared. 【Methods】 Time-course variations of antioxidant enzyme activities of copper/zinc-SOD (Cu/Zn-SOD), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), and contents of reduced glutathione (GSH) and oxidized glutathione (GSSG) and their ratio were measured and compared between different stages of ND, SD and WD pupae of D. antiqua. Canonical discriminate analysis was also conducted to further explore the relationships between the major antioxidant components. 【Results】 The activities of all the five antioxidant enzymes dynamically changed during the whole pupal stage. Compared with ND and SD pupae, WD pupae at the pre-diapause phase had higher Cu/Zn-SOD activities. The mean Cu/Zn-SOD activity in SD or WD pupae at the diapause maintenance phase and post-diapause phase was much lower than that in ND pupae. In the whole pupal stage, ND pupae had higher MnSOD activities than SD and WD pupae, but no difference existed between SD and WD pupae. The average MnSOD activity was distinctly higher than that of Cu/Zn-SOD at the same developmental stage. The CAT activities in SD and WD pupae at the pre-diapause phase were statistically higher than that in ND pupae at the same developmental stage, however, the CAT activities in SD or WD pupae at the diapause maintenance phase and post-diapause phase were significantly lower than those in ND pupae. In either non-diapause or diapause pupae at the same developmental stage, the general GPx activities showed an opposite variation trend with GR activities. Canonical discriminant analysis revealed that antioxidant system in onion fly pupae showed diapause type specificity. 【Conclusion】 Redox status in ND pupae is significantly different from that in SD and WD pupae. Higher antioxidant enzyme activities and lower GSH/GSSG ratio in pupae at the pre-diapause phase and post-diapause phase suggest that the oxidative shift results from a higher respiration rate and development changes.
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Molecular cloning and characterization of transcription factor AP-1 gene in Apis cerana cerana (Hymenoptera: Apidae)
CHI Xue-Peng, WEI Wei, ZHANG Wei-Xing, WANG Hong-Fang, XU Bao-Hua
null    2018, 61 (4): 423-431.   DOI: 10.16380/j.kcxb.2018.04.004
Abstract265)      PDF(pc) (3561KB)(158)       Save
【Aim】 The objective of this study is to clone the cDNA sequence of transcription factor AP-1 gene (AccAP-1) from Apis cerana cerana and to explore its expression profiles in A. cerana cerana at different developmental stages, in different tissues and under different abiotic stress, so as to provide fundamental evidence for the future study of the physiological function of AccAP-1. 【Methods】 The full-length cDNA sequence of AP-1 gene was cloned from the entire tissues of A. cerana cerana by RT-PCR. Physiochemical properties and structure characteristics of the deduced amino acid sequence were analyzed by multiple bioinformatics methods. Phylogenetic tree between AP-1 from A. cerana cerana and its homologous AP-1 proteins from other hymenopteran insects was constructed using neighbor-joining method of MEGA5.2. The expression levels of AP-1 gene in A. cerana cerana at different developmental stages (1-6 d old larva, prepupa, white-eyed pupa, pink-eyed pupa, dark-eyed pupa, 1 d-old adult worker, and 15 d-old adult worker), in different tissues (head, muscle, epidermis, midgut, rectum and venom gland) of 15 d-old adult workers, and in 15 d-old adult workers under stress of extreme temperatures (4, 16 and 44℃) and fed with sucrose solutions containing pesticide (0.01 mL/L paraquat) and heavy metal (1 mg/mL CdCl2), respectively, were detected by real-time PCR. 【Results】 We obtained the full-length open reading frame (ORF) of AccAP-1 (GenBank accession no.: MF994311) from A. cerana cerana, which is 813 bp in length, encoding 270 amino acids with an estimated molecular weight of 30.24 kD and a predicted theoretical isoelectric point (pI) of 8.31. Conserved domain analysis indicated that AccAP-1 contains two highly conserved structures, i.e., Jun superfamily (AA 7-137) and bZIP_Jun (AA 195-255). The results of homologous sequence alignment and phylogenetic tree analysis indicated that A. cerana cerana was most closely related to Apis mellifera. The results of real-time PCR indicated that the expression levels of AccAP-1 at different developmental stages were significantly different (P<0.05) and the highest in 1 d-old larva and 15 d-old adult. AccAP-1 transcripts were expressed in all tissues tested, and the expression level was higher in muscles than in other tissues (P<0.05). The expression levels of AccAP-1 under different abiotic stress indicated that the low temperature 4℃ upregulated its expression (P<0.05), while the treatment at 16℃ for 0.5-2 h upregulated its expression (P<0.05), but extended treatment time at 16℃ (over 2 h) downregulated its expression. The expression level of AccAP-1 at 44℃ for 15 min was downregulated significantly (P<0.05), while those at 44℃ for the other treatment time had no significant difference (P>0.05). The expression of AccAP-1 was downregulated when the workers were fed with the sucrose solution containing paraquat (P<0.05); the expression of AccAP-1, however, was induced by CdCl2 (P<0.05). 【Conclusion】 The results suggest that AccAP-1 might play an important role in the response to abiotic stress in A. cerana cerana.
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Comparative transcriptome analysis of salivary glands of Southern rice black-streaked dwarf virus (SRBSDV)infected and uninfected adults of the whitebacked planthopper, Sogatella furcifera (Hemiptera: Delphacidae)
DENG Yao, LIU Yu-Di, WANG Xiang-Ping, Hou-Mao-Lin
null    2018, 61 (4): 449-457.   DOI: 10.16380/j.kcxb.2018.04.007
Abstract264)      PDF(pc) (3567KB)(165)       Save
【Aim】 The white-backed planthopper (WBPH), Sogatella furcifera, is a main vector of Southern rice black-streaked dwarf virus (SRBSDV). The salivary glands of the WBPH play an important role in feeding behaviour and virus transmission. This study aims to sequence the salivary gland transcriptomes of viruliferous (SRBSDV-infected) and nonviruliferous adults of the WBPH, to find the differentially expressed genes in salivary glands and to further infer the virus-related genes. 【Methods】 Salivary gland transcriptomes of viruliferous and nonviruliferous adults of S. furcifera were sequenced by using the Ion Proton II, PGM platform, and then were de novo assembled by SOAPdenovo software. Gene annotation was conducted by blastX, and GO term enrichment and KEGG metabolic pathway analysis were performed using Blast2go and Blastall software. The differentially expressed genes were calculated by RPKM values. 【Results】 A total of 52 062 unigenes from viruliferous salivary glands (VSGs) and 51 407 unigenes from nonviruliferous salivary glands (NVSGs) of S. furcifera with the mean length of 639 and 647 bp, respectively, were obtained, and their GenBank accession numbers are SRS851833 and SRS843978, respectively. A total of 18 431 unigenes have homologous sequences against the NR database, and the highest percentage of unigene sequences (16.23%) were matched to genes of Tribolium castaneum. All unigenes were enriched to 52 GO terms and 240 KEGG pathways. The results revealed that 89 unigenes had a significantly different expression level between VSGs and NVSGs. 【Conclusion】 Through the comparative analysis, the differentially expressed genes between viruliferous and nonviruliferous salivary glands of S. furcifera were found, and some of these genes might be involved in virus-vector interactions. The gene expression characteristics of salivary gland transcriptome provide useful information for the identification of genes involved in feeding and virus transmission and so lay the basis for investigating the interaction between SRBSDV and WBPH at the molecular level.
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Contents of Vol. 61 Issue 2
null    2018, 61 (2): 263-263.  
Abstract259)      PDF(pc) (478KB)(234)       Save
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Molecular cloning and function analysis of ATP synthase b subunit gene ATPSb in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)
FENG Ya-Lin, HAO Pei-Ying, YU Fei-Fei, LU Chao-Feng, ZHU Jia-Jun, YU Xiao-Ping
null    2018, 61 (5): 519-526.   DOI: 10.16380/j.kcxb.2018.05.001
Abstract258)      PDF(pc) (2578KB)(298)       Save
【Aim】 This study aims to investigate the functions of the ATP synthase b subunit gene in the rice brown planthopper (BPH), Nilaparvata lugens by RNAi and to explore the potential using the gene as a target to control this pest. 【Methods】 The full-length cDNA of ATP synthase b subunit gene was cloned from the BPH reared on rice variety TN1 by RACE according to the sequence information available in the transcriptome data previously obtained by our laboratory. The expression profiles of this gene in different developmental stages and different tissues of the 5th instar nymphs of N. lugens were detected with real-time quantitative PCR, and the RNAi experiments of the gene were carried out with the 2nd instar nymphs of N. lugens by using feeding method. 【Results】 The full-length cDNA of the ATP synthase b subunit gene of the BPH named ATPSb (GenBank accession number: MF973493) was successfully cloned. It contains an ORF of 843 bp, encoding a protein of 280 amino acid residues. Real-time quantitative PCR showed that the ATPSb gene was highly expressed in the 1st and 2nd instar nymphs, and its relative expression level decreased with the developmental stage. The mRNA level of ATPSb was higher in male adults than that in female adults. In the 5th instar nymphs, the mRNA level of ATPSb in the thorax was the highest among different tissues, while relatively low in the head, midgut, ovary and fat body. The RNAi results showed that the mRNA level of ATPSb was significantly decreased in the dsATPSb treatment group from the 6th day, and the RNAi treatment led to distinct mortality of the BPH nymphs. At 18 d after RNAi, the survival rate in the control group kept 80%, while no individual survived in the dsATPSb treatment group. 【Conclusion】 The results suggest that the ATPSb gene is essential to the survival of the BPH, and the RNAi of ATPSb shows an effective inhibition of the BPH. ATPSb may serve as a potential target gene for BPH control.
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Progress in targeted genome editing technology in the silkworm, Bombyx mori
LONG Ding-Pei, HAO Zhan-Zhang, XIANG Zhong-Huai, ZHAO Ai-Chun
null    2018, 61 (3): 371-384.   DOI: 10.16380/j.kcxb.2018.03.012
Abstract255)      PDF(pc) (2286KB)(262)       Save
The engineered endonuclease (EEN)-based genome editing technology is an important tool for genome function analysis and genetic improvement of biological varieties. The silkworm, Bombyx mori, is the first economic insect to be domesticated and bred by humans, and also an important model lepidopteran, which has been used for functional gene analysis in the post-genome era. In recent years, along with the rapid development of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR/Cas9 system-based novel genome editing technologies, the targeted genome editing technology in the silkworm also has had significant progress. In this review, the compositions and action principles of the three most commonly used EEN systems were introduced, and the establishment process of targeted genome editing technology in the silkworm and the application and research progress of this technology for point mutation of silkworm endogenous or exogenous genes, genome structure variation and integration of the exogenous genes into the silkworm genome were comprehensively and systematically summarized. This review also discussed the main problems and the corresponding strategies for the low-efficiency of the knockout of endogenous genes and integration of exogenous genes, and the off-target effects of the integration of exogenous genes in targeted genome editing techniques in the silkworm. In addition, the potential for multiplex gene editing and the development trend for the combined application and diversified development of different targeted genome editing systems of this technology in the silkworm were prospected. It is expected to provide insights and references for promoting the development of targeted genome editing technology in gene functional analysis, mutation model establishment and other research areas in insects.
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Identification of insect brain neuropils aided with the application of 3D printer technology
CHEN Qiu-Yan, CHANG Ya-Jun, GUO Qian-Qian, SU Ran-Ran, WANG Bo, HE Jing, XIE Gui-Ying, ZHAO Xin-Cheng
null    2018, 61 (4): 439-448.   DOI: 10.16380/j.kcxb.2018.04.006
Abstract252)      PDF(pc) (4506KB)(190)       Save
【Aim】 This study aims to identify the neuropil structure of the brain of male adult of Heliothis virescens, to reconstruct and print the three-dimensional brain models, and to use the established 3D printing protocol to print the brain models of Drosophila melanogaster, Apis mellifera and Schistocerca gregaria. 【Methods】 Immunohistochemical staining with a synaptic protein antibody was used to label the neuropil structures of H. virescens brain. The brain images were obtained by using a confocal laser scanning microscope, and the three-dimensional brain models were created by using imaging software and printed by using a 3D printer. 【Results】 The brain of male adult of H. virescens and its main neuropils including gnathal ganglion, antennal lobes, optic lobes, anterior optic tubercle, central body, and mushroom bodies were identified, and the digitalized three-dimensional brain models were created. For the first time, the three-dimensional brain models of male adult of H. virescens were printed by using a 3D printer and the digitalized models were transformed to the physical and solid models. Based on both digital and printed brain models of H.virescens and other three insects (D. melanogaster, A. mellifera and S. gregaria), the brain neuropils for the gustatory center, olfactory center, visual center, and the center for memory and learning were compared between these insect species. 【Conclusion】 The printed brain models offer a new form of brain visualization. The physical and solid insect brain models can be printed in desired size and be handled in hands for the visualization from any angles. The printed brain models facilitate the identification of neuropils and their spatial relationships, and the comparison for the equivalent structures in different insect species.
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Composition and diversity of microbes in the infrabuccal pocket of Camponotus japonicus (Hymenoptera: Formicidae)
ZHANG Kai-Xuan, WEI Cong, NAN Xiao-Ning , WANG Yun-Guo, HE Hong
null    2018, 61 (6): 686-697.   DOI: 10.16380/j.kcxb.2018.06.007
Abstract252)      PDF(pc) (3909KB)(164)       Save
【Aim】 This study aims to investigate the microbe composition (actinomycetes, bacteria and fungi) and the diversity of bacteria in the infrabuccal pocket of the Japanese carpenter ant, Camponotus japonicus, so as to explore their potential functions in food utilization and population immunity. 【Methods】 The culturedependent method and HiSeq high-througput sequencing technique were combined to analyze the microbes in the infrabuccal pocket, digestive tract and cuticle of C. japonicus workers collected in Yangling, Shaanxi, northwestern China. 【Results】 Ten actinobacterial strains were isolated from the infrabuccal pocket of C. japonicus workers, among which five strains belong to Streptomyce with the average isolation frequencies of 73.3%-96.7%. Seven bacterial strains were isolated and four of them were Bacillus, and the average isolation frequencies of the strains N-B1 and N-B4 were both above 70%. Three fungal strains were isolated, and the isolation frequency of the dominant strain P-F1 (Wickerhamiella) was up to 96.7%. All strains isolated from the crop, midgut and cuticle could be found in the infrabuccal pocket, and the species number of the strains and the number of microbial colonies isolated from the infrabuccal pocket were higher than those isolated from the cuticle, crop and midgut. The results of HiSeq high-throughput sequencing showed that the dominant bacterial groups in the infrabuccal pocket mainly belong to Proteobacteria, Firmicutes and Actinobacteria, but in the crop and midgut the dominant bacterial groups belong to Proteobacteria and Firmicutes. The dominant genera with high abundance were more in the infrabuccal pockets and crops than in the midgut, and the bacterial composition in the midgut was relatively simple. The dominant genera in infrabuccal pocket included Fructobacillus, Bacterium, Pseudomonas, Acinetobacter and Sphingtomonas. The microbial abundance and diversity in the infrabuccal pocket were higher than those in the crop and midgut. 【Conclusion】 Actinomycetes, Bacillus, yeasts and other predominant microbes generally exist in the infrabuccal pocket of C. japonicus, and their abundance and diversity levels are significantly higher than those in the digestive tract (crop and midgut). The potential roles of these microorganisms in food utilization, nutrient digestion and population immunity of ants need to be further studied.
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Determination of the promoter activity of diapause-associated sorbitol dehydrogenase genes in the silkworm, Bombyx mori
ZHU Juan, XIE Yu-Chen, CHEN Yan-Rong, TANG Shun-Ming, YI Yong-Zhu, SHEN Xing-Jia
null    2018, 61 (4): 391-397.   DOI: 10.16380/j.kcxb.2018.04.001
Abstract247)      PDF(pc) (1417KB)(279)       Save

【Aim】 To clarify the transcriptional characteristics of sorbitol dehydrogenase (BmSDH) genes (BmSDH-1, BmSDH-2a and BmSDH-2b) in the silkworm, Bombyx mori. 【Methods】 The transcription initiation sites of three BmSDH genes were determined by 5′RACE technique. The promoters of BmSDH about 1 kb in length and BmSDH-2a in different lengths were cloned by PCR. Then report plasmids with a fruit fly luciferase gene driven by BmSDH promoters in different lengths, pGL3-BmSDH-P-luc, were constructed. Using dual luciferase detection system, the promoter activity of BmSDH was detected by co-transfecting the BmN cells with pGL3-BmSDH-P-luc and pRL-CMV which contains a renin-luciferase reporter gene, and the effects of hormones on the promoter activity of BmSDH-2a was detected by adding juvenile hormone analogue (JHA), ecdysone hormone (20E) and diapause hormone (DH), respectively, to culture media in the gradient concentrations. 【Results】 The transcription initiation sites of BmSDH-1, BmSDH-2a and BmSDH-2b are located at 41, 41 and 40 bp upstream the translation initiation site, respectively. The promoter activity of BmSDH-2a was significantly higher than those of BmSDH-1 and BmSDH-2b. For the BmSDH-2a gene, the promoter activity of the 355 bp fragment was significantly higher than those of the 674 bp and 1 117 bp fragments. In BmN cells, the activity of 1 117 bp promoter of BmSDH-2a increased with the increase of DH concentration; however, when the DH concentration was higher than 100 ng/mL, the promoter activity was decreased to some degree but maintained at a high level. In JHA treated BmN cells, the promoter activity was decreased gradually as the hormone concentration increased. In 20E treated BmN cells, the promoter activity significantly increased when 0.1 ng/mL of 20E was applied, and then decreased gradually with the increase of hormone concentration. 【Conclusion】 The transcription initiation sites of the BmSDH genes were determined. The promoter activity of BmSDH-2a is significantly higher than those of BmSDH-1 and BmSDH-2b. A certain concentration of 20E can significantly enhance the promoter activity of BmSDH-2a. These results will contribute to clarifying the function of BmSDH genes in diapause process in B. mori.
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Research progress in bumblebee nutrition and feed
WANG Huan, XU Xi-Lian
null    2018, 61 (5): 620-626.   DOI: 10.16380/j.kcxb.2018.05.012
Abstract246)      PDF(pc) (855KB)(194)       Save
  Bumblebees are one group of the most important commercial pollinators. Domestication and utilization of bumblebee would make up for the decline of natural bee-pollinators and meet the demands of modern agriculture pollination. Nutrition and feed is one of the key issues to realize the domestication and industrial production of bumblebees. In this article, we reviewed how natural pollen, artificial diet with single or mixed pollen gathered by honeybee, feed with different ratios of protein and fat, feed with different amino acid levels, and feed with nutrient elements or royal jelly added affect the bumblebee development. The mixed pollen has promoting functions on larval weight as compared to the single pollen. The optimal ratio of protein to fat ingested by Bombus terrestris is 14∶1. High amino acid level can promote the oviposition of queens and maturity of larvae. Carbohydrates with nutrient elements added can promote the oviposition of queens and foundation of colonies. The royal jelly added in feed can enhance the survival and spawning rate of queens. We proposed that the future research should focus on the feed meeting the nutrient needs of different developmental stages of the local bumblebees and the intestinal symbiotic bacteria which may affect the metabolism and development of bumblebees.
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Dynamics of microfilaments and tubulin during spermiogenesis in the Chinese bush cricket, Gampsocleis gratiosa (Orthoptera: Tettigoniidae)
CHEN Jie, WANG Le, CHANG Yan-Lin
null    2018, 61 (3): 308-321.   DOI: 10.16380/j.kcxb.2018.03.006
Abstract235)      PDF(pc) (15001KB)(321)       Save
【Aim】 The objective of this research is to analyze the roles of tubulin and microfilaments in the Chinese bush cricket, Gampsocleis gratiosa, so as to build the foundation for exploring the mechanism of acrosomal complex formation and nucleus shaping during spermiogenesis in insects. 【Methods】 The development of sperms from testes, seminal vesicles and spermathecas in G. gratiosa adults including the localization of microfilaments and tubulin during spermiogenesis was observed by immunofluorescence, PAS-hematoxylin staining and transmission electron microscopy. 【Results】 In the early round spermatid testis, microfilaments gather to an area and tubulin is randomly distributed in cytoplasm during spermiogenesis. In the elongated spermatid, when the acrosomal complex is forming, microfilaments first emerge in subacrosomal space, become round-shaped and rodlet-shaped, and extend to two sides of the anterior region of the nucleus to form inverted ‘Y-shaped’ and then to form arrow-shaped. Tubulin exists around the acrosomal complex and the nucleus, and also in the flagellum. In sperm and spermatodesms of the seminal vesicle and the spermathecae from male and female adults, tubulin is only present in the flagellum, nevertheless, microfilaments and tubulin are absent in other areas. 【Conclusion】 The results suggest that microfilaments and microtubules as ‘scaffold’ participate in acrosomal complex formation and nucleus morphological reconstruction during spermiogenesis in G. gratiosa, and the ‘scaffold’ is removed in sperm maturing into spermatodesms.
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Distribution of 5-hydroxytryptamine in the optic lobes of the oriental armyworm, Mythimna separata (Lepidotpera: Noctuidae)
GUO Pei, MA Bai-Wei, ZHAO Xin-Cheng, WANG Gao-Ping, XIE Gui-Ying
null    2018, 61 (6): 668-675.   DOI: 10.16380/j.kcxb.2018.06.005
Abstract233)      PDF(pc) (3085KB)(182)       Save
【Aim】 This study aims to investigate the anatomy of the optic lobes of adults of the oriental armyworm, Mythimna separata (Lepidotpera: Noctuidae) and the distribution of 5-hydroxytryptamine (5-HT) in the neuroplis of the optic lobes. 【Methods】 The tissue embedding and section, immunohistochemical staining with synaptic protein antibody and anti-5-HT serum were used to label the neuropil structure of the optic lobe and 5-HT in M. separata adults, respectively. The digital images of the optic lobes were obtained by using laser scanning confocal microscopy. The identification of neuropils and the grouping and counting of 5-HT immunoreactive (5-HTi) cell bodies were performed using image software. 【Results】 The optic lobe of M. separata adults is composed of five neuropils, i.e., lamina, medulla, accessory medulla, lobula, and lobula plate. There are about 40 5-HTi cell bodies in each optic lobe, and all the five neuropil regions contain 5-HTi neural processes. 5-HTi neural processes in lamina are originated from the tangential neurons of the optic lobe, and the processes of medulla are from the tangential neurons, centrifugal neurons and amacrine neurons. Medulla is distributed into mainly three layers, in which the middle layer possesses the densest 5-HTi processes. A few of 5-HTi processes exist in accessory medulla. In the lobula and lobula plate, the 5-HTi processes are originated from the centrifugal neurons. At least two layers of 5-HTi processes in lobula and lobula plate were observed. A few of neural processes project to medulla and connect lobula, lobula plate and medula. 【Conclusion】 5-HT immunoreactive fibers are widely distributed in the optic lobe of M. separata. The results provide the basic knowledge of neural anatomy for further studying the role of 5-HT in the visual system of M. separata.
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Sequence analysis and expression profiling of pheromone binding protein genes in the litchi fruit borer, Conopomorpha sinensi (Lepidoptera: Gracillariidae)
LI Peng-Yan, LIU Yan-Ping, WANG Si-Wei, SUN Hai-Bin, BAI Jian-Shan, PENG Gang, GONG Xue-Hai
null    2018, 61 (5): 527-536.   DOI: 10.16380/j.kcxb.2018.05.002
Abstract227)      PDF(pc) (2596KB)(179)       Save
 【Aim】 This study aims to clone three pheromone binding protein (PBP) genes from the litchi fruit borer, Conopomorpha sinensis, and to analyze their sequences and expression characteristics, so as to provide essential basis for better use of sex pheromone for control. 【Methods】 The full-length cDNA of three PBP genes were cloned from the antennae of C. sinensis adults by transcriptome and RACE-PCR technique after extracting total RNA. The putative amino acid sequences were analyzed by bioinformatics software. The software I-TASSER was used to simulate 3D models, the software TM-align was used in protein homology modeling and the software COACH was used to speculate the binding sites. The expression profiles of the three genes in different developmental stages (larval and pupal stages), and different tissues of the 3 d-old female and male adults (antennae, head without antennae, thorax, abdomen, legs and wings) were analyzed by real-time PCR. 【Results】 Three PBP genes were cloned from the antennae of C. sinensis adults. They were named CsinPBP1, CsinPBP2 and CsinPBP3, and deposited under GenBank accession numbers MF093145, MF093146 and MF093147, respectively. Bioinformatic analysis revealed that the encoded proteins of the three genes have typical characteristics of odor binding proteins. Phylogenetic analysis revealed that CsinPBP1 has 72% amino acid sequence identity with PBP of Yponomeuta cagnagellus, CsinPBP2 has 55% amino acid sequence identity with PBP1 of Plutella xylostella, and CsinPBP3 has 39% amino acid sequence identity with PBP3 of Sesamia inferens. Software simulation analysis revealed that the 3D structures of CsinPBP1, CsinPBP2 and CsinPBP3 were the most similar with GOBP2 of Bombyx mori (PDB: 2wc6A), PBP1 of Amyetois transitetella (PDB: 4inxA) and PBP of B. mori (PDB: 2p70A), and were predicted to have 10, 7 and 8 binding sites, respectively. Expression profiling revealed that the three genes were only expressed in adult antennae, but neither in larval and pupal stages nor in other adult tissues including the head without antennae, thorax, abdomen, legs and wings. The relative expression levels of CsinPBP1, CsinPBP2 and CsinPBP3 in the antennae of male adults were 1.94, 28.19 and 32.94 times as high as those in the antennae of male adults, respectively. 【Conclusion】 Three PBP genes were cloned in C. sinensis. Their sequence and expression analysis suggest that they are related to sex pheromone sensing in males.
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cDNA cloning and expression profiling of small heat shock protein genes and their response to temperature stress in Monochamus alternatus (Coleoptera: Cerambycidae)
LI Hui, HE Xuan-Yu, TAO Rong, CHEN Hong-Jian, GONG Xin-Yue, LI Shou-Yin, HAO De-Jun
null    2018, 61 (7): 749-760.   DOI: 10.16380/j.kcxb.2018.07.001
Abstract225)      PDF(pc) (4018KB)(243)       Save
【Aim】 Monochamus alternatus, an important stem borer in pine forests in southern China, is a major vector of pine wilt disease. It has a wide range of distribution and strong resistance to temperature stress. The study aims to better understand the molecular mechanism of resistance to temperature stress in M. alternatus. 【Methods】 The full-length cDNA encoding small heat shock protein (sHSP) was cloned
from M. alternatus by RT-PCR and RACE techniques. The sequence characteristics of this sHSP were analyzed by bioinformatics methods. The expression patterns of this gene in different developmental stages, various tissues of the 4th instar larvae and the 4th instar larvae stressed under different low and high temperatures were detected by qPCR, and the stability of the reference genes at different temperatures was analyzed by geNorm, NormFinder and BestKeeper tools. 【Results】 The complete cDNA of the gene encoding sHSP was obtained from M. alternatus, and named MaltHSP21.20 (GenBank accession no.: MH091811). The complete cDNA (871 bp) encodes a protein of 187 amino acids, with a signal peptide of 18 amino acids in N-terminus, the molecular weight of 21.20 kD and the theoretical pI of 8.65. The domain prediction conforms to the characteristics of the sHSP family, containing 10 β-sheets and 7 β-sheets in the α-domain. Phylogenetic tree analysis showed that the sHSP sequence shares high homology with sHSP of Anoplophora glabripennis. The most stable reference gene was different identified by different methods, and assessed by three methods the most stable reference gene was RPL10. MaltHSP21.20 was expressed in all developmental stages of M. alternatus, with the highest expression level in diapause larvae, and moderate in egg and pupal stages. MaltHSP21.20 was expressed in different tissues of the 4th instar larvae, with the highest expression level in fat body. The expression of MaltHSP21.20 in the 4th instar larvae showed no response to low temperature, while was up-regulated significantly at 35℃, reached the maximum after exposure to 45℃ for 2 and 3 h, and down-regulated at 50℃. 【Conclusion】 RPL10 is a more stable reference gene under different temperature stress. Compared to other heat shock protein genes, MaltHSP21.20 is less sensitive to low temperature and presumably plays an important role in larval diapause during overwintering and the resistance to high temperature.
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Effects of regional agricultural landscape pattern on the community of ladybeetles in wheat fields
ZHANG Yong-Sheng, OUYANG Fang , MEN Xing-Yuan, GE Feng, YUAN Zhe-Ming
null    2018, 61 (4): 468-476.   DOI: 10.16380/j.kcxb.2018.04.009
Abstract218)      PDF(pc) (1109KB)(184)       Save
【Aim】 To clarify the effects of agricultural landscape pattern on ladybeetle community in wheat fields, so as to provide theoretical proofs for ecological regulation and management of insect pests. 【Methods】 Based on the remote sensing data, land cover classification and the survey data of ladybeetle abundance in wheat fields in 22 counties and cities in Shandong, the planting region of wheat was taken as a typical example, the landscape pattern metrics were calculated, and the effects of landscape pattern of farmland, non-crop habitat and regional agricultural landscape on ladybeetle abundance were analyzed using negative binomial generalized linear model. 【Results】 The ladybeetle abundance was positively correlated with mean patch area (AREA_MN) and area-weighted mean patch fractal dimension (FRAC_AM) of grassland and patch richness density (PRD) of regional landscape, and was negatively correlated with area-weighted mean Euclidean nearest neighbor distance (ENN_AM) of non-crop habitat. Grassland, clustered non-crop habitats and diverse regional landscapes benefited ladybeetle abundance. AREA_MN of grassland and ENN_AM of non-crop habitat could best predict ladybeetle occurrence. 【Conclusion】 Grassland, spatial distribution of non-crop habitats and regional landscape diversity are important landscape factors affecting the occurrence of ladybeetles in wheat fields.
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Electroantennogram and behavioral responses of Ectropis grisescens (Lepidoptera: Geometridae) to essential oils from three non-host plants
ZHANG Fang-Mei, QIAO Li, PAN Peng-Liang, WANG Guo-Jun, YIN Jian, CHEN Li-Jun
null    2018, 61 (5): 565-573.   DOI: 10.16380/j.kcxb.2018.05.006
Abstract215)      PDF(pc) (1911KB)(221)       Save
【Aim】 This study aims to screen the chemical attractants and repellents produced by non-host plants, which are potentially used for developing ‘push-pull’ strategy for controlling a common tea pest, Ectropis grisescens. 【Methods】 The behavioral and electroantennogram (EAG) responses of male and female adults of E. grisescens to essential oils from three non-host plants including Chenopodium ambrosioides, Mentha spicata and Artemisia annua were evaluated by using Y-tube olfactometer and EAG. 【Results】 Both sexes of E. grisescens adults showed EAG response to the essential oils from C. ambrosioides, M. spicata and A. annua in a dose-dependent manner, with the antennal response strengthening first and then decreasing with the concentration of essential oil, and reaching the maximum at the concentration of 1, 100 and 100 mg/L, respectively. Male adults showed slightly greater EAG response than females to the essential oils from C. ambrosioides and M. spicata, while their response to the essential oil from A. annua was opposite. The results of behavioral response tests showed that both sexes of E. grisescens adults displayed non-significant taxis to the essential oils from C. ambrosioides and A. annua and significantly negative taxis to the essential oil from M. spicata. 【Conclusion】 E. grisescens adults show significant EAG responses to the essential oils from the non-host plants C. ambrosioides, M. spicata and A. annua, and the essential oil from M. spicata has repellent activity against E. grisescens.
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Genetic differentiation and distribution of two sibling species of tea geometrids in tea-growing areas in Zhejiang, eastern China
BAI Jia-He, WANG Zhi-Bo, XIAO Qiang
null    2018, 61 (6): 741-748.   DOI: 10.16380/j.kcxb.2018.06.013
Abstract213)      PDF(pc) (1686KB)(117)       Save
【Aim】 To reveal the distribution of the sibling species of Ectropis grisescens and Ectropis obliqua in tea-growing areas in Zhejiang, eastern China. 【Methods】 A total of 533 specimens of tea geometrids collected from 17 counties in Zhejiang were identified by using mitochondrial cytochrome oxidase (mtCOI) gene as the molecular marker, and the geographical distribution of E. grisescens and E. obliqua in Zhejiang was also analyzed. 【Results】 The results showed that among the 17 surveyed localities, the tea geometrids from 11 localities in Zhejiang were all identified as E. grisescens, those from other three localities (Anji, Yuhang, Longwu) were all E. obliqua, and the rest three localities (Xihu, Lin′an and Fuyang districts of Hangzhou) were identified as the co-occurrence areas of the two sibling species. 【Conclusion】 E. grisescens is widely distributed in tea-growing areas in Zhejiang, the distribution of E. obliqua is narrower, and there exist co-occurrence areas of the two sibling species.
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