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1.
J Agric Food Chem ; 67(40): 11035-11043, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517486

RESUMO

Ca2+-binding proteins (CaBPs) are widely distributed as Ca2+ sensor relay proteins that regulate various cellular processes, including Ca2+ homeostasis. Diamide insecticides such as cyantraniliprole kill insects by disrupting the Ca2+ homeostasis in muscle cells. However, less attention has been paid to the roles of CaBPs in response to insecticides. In this study, two CaBP genes (BtCaBP1 and BtCaBP2) were identified in the whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and their functions in response to cyantraniliprole were investigated. After expression of BtCaBP1 and BtCaBP2 in vitro, the results of Ca2+ imaging and cytotoxicity assay revealed that the overexpression of each of the BtCaBPs stabilized Ca2+ concentration in the cytoplasm after exposure to cyantraniliprole and decreased the toxicity of cyantraniliprole against Sf9 cells. However, the knockdown of BtCaBP1 or BtCaBP2 in vivo significantly increased the toxicity of cyantraniliprole to B. tabaci. Taken together, these results provide evidence that BtCaBP1 and BtCaBP2 play a role in response to cyantraniliprole exposure through stabilization of Ca2+ concentration in whiteflies.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Hemípteros/efeitos dos fármacos , Hemípteros/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Pirazóis/farmacologia , ortoaminobenzoatos/farmacologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Hemípteros/classificação , Hemípteros/metabolismo , Proteínas de Insetos/genética , Filogenia
2.
J Agric Food Chem ; 67(36): 9979-9988, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31411878

RESUMO

A delta class glutathione S-transferase gene (BoGSTd2) is identified from Bradysia odoriphaga for the first time. Developmental expression analysis showed that expression of BoGSTd2 is significantly higher in the fourth instar larval stage and the adult stage. Tissue-specific expression analysis found that BoGSTd2 was expressed predominantly in the midgut and Malpighian tubules in the fourth instar larvae and the abdomen of adults. Expression of BoGSTd2 was significantly upregulated following exposure to chlorpyrifos and clothianidin. In vitro inhibition and metabolic assays indicated that recombinant BoGSTd2 could not directly metabolize chlorpyrifos and clothianidin. Nevertheless, disk diffusion assays indicated that BoGSTd2 plays an important role in protection against oxidative stress. RNAi assays showed that BoGSTd2 participates in the elimination of reactive oxygen species induced by chlorpyrifos and clothianidin. These results strongly suggest that BoGSTd2 plays an important role in chlorpyrifos and clothianidin detoxification in B. odoriphaga by protecting tissues from oxidative stress induced by these insecticides.


Assuntos
Dípteros/enzimologia , Glutationa Transferase/metabolismo , Proteínas de Insetos/metabolismo , Inseticidas/metabolismo , Animais , Clorpirifos/metabolismo , Dípteros/genética , Dípteros/crescimento & desenvolvimento , Dípteros/metabolismo , Glutationa Transferase/genética , Guanidinas/metabolismo , Inativação Metabólica , Proteínas de Insetos/genética , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Neonicotinoides/metabolismo , Estresse Oxidativo , Tiazóis/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-31454682

RESUMO

Heortia vitessoides Moore is a notorious defoliator of Aquilaria sinensis (Lour.) Gilg trees. Chitin deacetylases (CDAs) catalyze the N-deacetylation of chitin, which is a crucial process for chitin modification. Here, we identified and characterized HvCDA1 and HvCDA2 from H. vitessoides. HvCDA1 and HvCDA2 possess typical domain structures of CDAs and belong to the Group I CDAs. HvCDA1 and HvCDA2 were highly expressed before and after the larval-larval molt. In addition, both exhibited relatively high mRNA expression levels during the larval-pupal molt, the pupal stage, and the pupal-adult molt. HvCDA1 and HvCDA2 transcript expression levels were highest in the body wall and relatively high in the larval head. Significant increases in the HvCDA1 and HvCDA2 transcript expression levels were observed in the larvae upon exposure to 20-hydroxyecdysone. RNA interference-mediated HvCDA1 and HvCDA2 silencing significantly inhibited HvCDA1 and HvCDA2 expression, with abnormal or nonviable phenotypes being observed. Post injection survival rates of the larvae injected with dsHvCDA1 and dsHvCDA2 were 66.7% and 46.7% (larval-pupal) during development and 23.0% and 6.7% (pupal-adult), respectively. These rates were significantly lower than those of the control group insects. Our results suggest that HvCDA1 and HvCDA2 play important roles in the larval-pupal and pupal-adult transitions and represent potential targets for the management of H. vitessoides.


Assuntos
Amidoidrolases/metabolismo , Pupa/enzimologia , Amidoidrolases/genética , Animais , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/enzimologia , Muda/genética , Muda/fisiologia , Mariposas/enzimologia , Pupa/crescimento & desenvolvimento , Interferência de RNA
4.
Pestic Biochem Physiol ; 159: 107-117, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400772

RESUMO

Latrophilin (LPH) is an adhesion G protein-coupled receptor (aGPCR) that participates in multiple essential physiological processes. Our previous studies have shown that lph is not only indispensable for the development and reproduction of red flour beetles (Tribolium castaneum), but also for their resistance against dichlorvos or carbofuran insecticides. However, the regulatory mechanism of lph-mediated insecticide susceptibility remains unclear. Here, we revealed that knockdown of lph in beetles resulted in opposing changes in two chemoreception genes, chemosensory protein 10 (CSP10) and odorant-binding protein C01 (OBPC01), in which the expression of TcCSP10 was downregulated, whereas the expression of TcOBPC01 was upregulated. TcCSP10 and TcOBPC01 were expressed at the highest levels in early pupal and late larval stages, respectively. High levels of expression of both these genes were observed in the heads (without antennae) of adults. TcCSP10 and TcOBPC01 were significantly induced by dichlorvos or carbofuran between 12 and 72 h (hrs) after exposure, suggesting that they are likely associated with increasing the binding affinity of insecticides, leading to a decrease in sensitivity to the insecticides. Moreover, once these two genes were knocked down, the susceptibility of the beetles to dichlorvos or carbofuran was enhanced. Additionally, RNA interference (RNAi) targeting of lph followed by exposure to dichlorvos or carbofuran also caused the opposing expression levels of TcCSP10 and TcOBPC01 compared to the expression levels of wild-type larvae treated with insecticides alone. All these results indicate that lph is involved in insecticide susceptibility through positively regulating TcCSP10; and the susceptibility could also further partially compensated for through the negative regulation of TcOBPC01 when lph was knockdown in the red flour beetle. Our studies shed new light on the molecular regulatory mechanisms of lph related to insecticide susceptibility.


Assuntos
Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Receptores de Peptídeos/metabolismo , Tribolium/efeitos dos fármacos , Tribolium/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Insetos/genética
5.
Pestic Biochem Physiol ; 159: 154-162, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400777

RESUMO

The migratory locust, Locusta migartoria, is a major agricultural insect pest and its resistance to insecticides is becoming more prevalent. Cytochrome P450 monooxygenases (CYPs) are important enzymes for biotransformations of various endogenous and xenobiotic substances. These enzymes play a major role in developing insecticide resistance in many insect species. In this study, we heterologously co-expressed a CYP enzyme (CYP6FD1) and cytochrome P450 reductase (CPR) from L. migartoria in Sf9 insect cells. The recombinant enzymes were assayed for metabolic activity towards six selected model substrates (luciferin-H, luciferin-Me, luciferin-Be, luciferin-PFBE, luciferin-CEE and 7-ethoxycoumarin), and four selected insecticides (deltamethrin, chlorpyrifos, carbaryl and methoprene). Recombinant CYP6FD1 showed activity towards 7-ethoxycoumarin and luciferin-Me, but no detectable activity towards the other luciferin derivatives. Furthermore, the enzyme efficiently oxidized deltamethrin to hydroxydeltamethrin through an aromatic hydroxylation in a time-dependent manner. However, the enzyme did not show any detectable activity towards the other three insecticides. Our results provide direct evidence that CYP6FD1 is capable of metabolizing deltamethrin. This work is a step towards a more complete characterization of the catalytic capabilities of CYP6FD1 and other xenobiotic metabolizing CYP enzymes in L. migratoria.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Família 6 do Citocromo P450/metabolismo , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Locusta migratoria/efeitos dos fármacos , Locusta migratoria/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Família 6 do Citocromo P450/genética , Proteínas de Insetos/genética
6.
Pestic Biochem Physiol ; 159: 27-33, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400781

RESUMO

Imidacloprid has been used to control one of most serious pests, Bemisia tabaci. However, B. tabaci has developed imidacloprid resistance mainly by over-expressing CYP6CM1. It was reported that imidacloprid-resistant B. tabaci showed no or low level of cross-resistance against dinotefuran. Here, we expressed CYP6CM1 variants using Sf9/baculovirus and/or Drosophila S2 cells and showed that CYP6CM1 variants metabolized imidacloprid but not dinotefuran. In addition, we demonstrated that imidacloprid and pymetrozine competed for a CYP6CM1 variant more efficiently than dinotefuran, using a luminescent substrate competition assay. These results suggest that lack of metabolic activity of CYP6CM1 variants against dinotefuran caused no or low level of cross-resistance.


Assuntos
Guanidinas/metabolismo , Guanidinas/farmacologia , Hemípteros/efeitos dos fármacos , Hemípteros/metabolismo , Inseticidas/metabolismo , Inseticidas/farmacologia , Neonicotinoides/metabolismo , Neonicotinoides/farmacologia , Nitrocompostos/metabolismo , Nitrocompostos/farmacologia , Animais , Hemípteros/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Triazinas/metabolismo , Triazinas/farmacologia
7.
Pestic Biochem Physiol ; 158: 40-46, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378359

RESUMO

Aphis gossypii Glover is an economically important pest of numerous crops throughout the world. Some field populations of A.gossypii in China have developed moderate level of resistance to sulfoxaflor, a newly released sulfoximine insecticide for management of sap-feeding pests. To evaluate the effect of sulfoxaflor resistance on the fitness cost of A. gossypii, the life history traits of sulfoxaflor-resistant strain (SulR) and an isogenic susceptible strain (SS) were compared using the age-stage, two-sex life table approach. The results showed that the resistant strain had a reduction in fitness (relative fitness = 0.917), along with significantly decreases in longevity, fecundity, net reproductive (R0), mean generation time (T) and gross reproductive rate (GRR). Compared to the susceptible strain, SulR strain showing a shorter developmental duration of each nymph instar stage. Moreover, the adult pre-oviposition period (APOP) and total preoviposition period (TPOP) of SulR strain were also significantly shorter than that of the susceptible strain. Investigation of six development and reproduction related genes indicated that EcR, USP and JHBP were overexpressed in the SulR strain, while the mRNA transcript level of Vg was decreased significantly compared to the susceptible strain. These results suggest that there is a fitness cost associated with sulfoxaflor resistance in A. gossypii and the different expression of EcR, USP, JHBP, and Vg may play very important role in this trade-off.


Assuntos
Afídeos/efeitos dos fármacos , Inseticidas/farmacologia , Piridinas/farmacologia , Compostos de Enxofre/farmacologia , Animais , Afídeos/genética , Afídeos/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Ninfa/efeitos dos fármacos , Ninfa/genética , Ninfa/metabolismo
8.
Pestic Biochem Physiol ; 158: 47-53, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378360

RESUMO

Buprofezin is a chitin synthesis inhibitor that is very effective against Homopteran pests, such as the white-backed planthopper (WBPH), S. furcifera (Horvath). In the present study, resistance selection, cross-resistance and mechanisms of buprofezin resistance were investigated in this planthopper species. However, the mechanism associated with resistance to growth regulator insecticides (IGRs) remains largely unknown. A resistant strain (Bup-R) with a resistance level (22-fold) to buprofezin was developed through continuous selection for 47 generations from a laboratory susceptible strain (Bup-S). The results showed that the Bup-R exhibited no cross-resistance to other tested insecticides. Synergism tests showed that piperonyl butoxide (PBO) (SR = 3.9-fold) and diethyl maleate (DEM) (SR = 1.8-fold) had synergistic effects on buprofezin toxicity in the resistant strain (F47). Enzyme activity results revealed an approximate 5.7-fold difference in cytochrome P450 monooxygenase and a 2-fold difference in glutathione S-transferase (GST) between the resistant and susceptible strains, suggesting that the increased activity of these two enzymes is likely the main detoxification mechanism involved in resistance to buprofezin in this species. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) and GST genes by quantitative real-time PCR results indicated that sixteen P450 and one GST gene were significantly overexpressed in the Bup-R strain, among which thirteen P450 genes and one GST gene were >2-fold higher than in the Bup-S strain. The present study increases our knowledge of the buprofezin resistance mechanism in S. furcifera and provides a useful reference for integrated pest management (IPM) strategies.


Assuntos
Hemípteros/efeitos dos fármacos , Inseticidas/farmacologia , Tiadiazinas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hemípteros/metabolismo , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Maleatos/metabolismo , Butóxido de Piperonila/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
9.
Pestic Biochem Physiol ; 158: 69-76, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378363

RESUMO

Sex pheromones are crucial for communication between females and males in moths, and pheromone receptors (PRs) play a key role in peripheral coding of sex pheromones. During the last decade, many PR candidates have been identified based on transcriptome sequencing and bioinformatic analysis, but their detailed functions remain mostly unknown. Here, focusing on four PR candidates of Athetis dissimilis (AdisOR1, AdisOR6, AdisOR11 and AdisOR14) identified in a previous study, we first cloned the full-length cDNAs and determined the tissue expression profiles by quantitative real-time PCR (qPCR). The results revealed that expression of three of these genes were male antennae-specific, while AdisOR11 was similar in expression between male and female antennae. Furthermore, the expression level of AdisOR1 was much higher than those of the other three genes. Then, functional analysis was conducted using Xenopus oocyte system. AdisOR1 responded strongly to the sex pheromone component Z9-14:OH and the potential pheromone component Z9,E12-14:OH, suggesting its important role in the sex pheromone perception; AdisOR14 showed specificity for Z9,E12-14:OH; while AdisOR6 and AdisOR11 did not respond to any of the pheromone components and analogs tested. Taken together, this study contributes to elucidate the molecular mechanism of sex pheromone reception and provides potential targets for development of OR based pest control techniques in A. dissimilis.


Assuntos
Proteínas de Insetos/metabolismo , Lepidópteros/metabolismo , Receptores de Feromonas/metabolismo , Animais , Feminino , Proteínas de Insetos/genética , Lepidópteros/genética , Masculino , Feromônios/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Feromonas/genética
10.
J Agric Food Chem ; 67(32): 8896-8904, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31339308

RESUMO

The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans, including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). Bacillus thuringiensis (Bt) is widely used as a biopesticide, which produces Cry toxins for mosquito control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in response to Cry toxins. Our results showed by far-western blot and ELISA methods that the CTLTGA9 protein interacted with brush border membrane vesicles (BBMVs) of A. aegypti larvae and with ALP1, APN, and Cry11Aa proteins. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the larvicidal activity of Cry toxins. According to the results of molecular docking, CTLGA9 may compete with Cry11Aa for binding to ALP1 and APN receptors and thus decreases the mosquitocidal toxicity of Cry11Aa. Our results provide further insights into better understanding the mechanism of Cry toxins and help improve the Cry toxicity for mosquito control.


Assuntos
Aedes/efeitos dos fármacos , Aedes/metabolismo , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/metabolismo , Aedes/química , Aedes/genética , Animais , Proteínas de Bactérias/química , Sítios de Ligação , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos
11.
DNA Cell Biol ; 38(8): 773-785, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339741

RESUMO

Pierisin-5 protein (pie-5) belongs to a family of proteins possessing DNA-dependent ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous protein subunits for basic scientific research, in addition to the development and production of diagnostics and vaccines. In this study, a new method for the in vitro expression of the cytotoxic protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5 protein (rpie-5) was investigated in different human cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5 protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5 protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 µg/mL) was more sensitive when compared with HepG2 (IC50 1.9 µg/mL) and AGS (IC50 3.7 µg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of lactose dehydrogenase (LDH) enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating protein-X), Bad, APAF-1 (apoptotic protease activating factor-1), Cyt-c, and caspase-3/9 and the downregulation of Bcl-2 (B-cell lymphoma 2) in rpie-5-treated cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.


Assuntos
ADP Ribose Transferases/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Proteínas Recombinantes/farmacologia , ADP Ribose Transferases/genética , Animais , Apoptose/fisiologia , Baculoviridae/genética , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Proteínas de Insetos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/genética , Células Sf9 , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
12.
Arch Insect Biochem Physiol ; 102(1): e21592, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31276235

RESUMO

Ferritin, which is ubiquitous among all living organisms, plays a crucial role in maintaining iron homeostasis, immune response, and detoxification. In the present research, we identified an iron-binding protein, ferritin heavy chain subunit, from Papilio xuthus and named PxFerHCH. The complete complementary DNA of PxFerHCH was 1,252 bp encoding a sequence of 211 amino acids, which includes an iron-responsive element. Phylogenetic analysis showed that PxFerHCH is clustered with Manduca sexta and Galleria mellonella ferritin heavy chain subunits. Expression levels of PxFerHCH in various tissues were analyzed by reverse transcription quantitative polymerase chain reaction, and the results exhibited that PxFerHCH was expressed in all tissues with the highest expression in the fat body. The relative expression level of PxFerHCH in response to bacterial (Escherichia coli and Staphylococcus aureus) challenges sharply increased by about 12 hr postinfection (hpi) and then decreased at 24 hpi. In addition, the iron-binding capacity and antioxidation activity of recombinant PxFerHCH protein were also investigated. These results reveal that PxFerHCH might play an important role in defense against bacterial infection.


Assuntos
Apoferritinas/metabolismo , Borboletas/metabolismo , Ferro/metabolismo , Sequência de Aminoácidos , Animais , Apoferritinas/genética , Apoferritinas/isolamento & purificação , Sequência de Bases , Borboletas/genética , Borboletas/imunologia , Escherichia coli , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Staphylococcus aureus
13.
Virology ; 533: 137-144, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31247402

RESUMO

Angiotensin-converting enzyme (ACE) plays diverse roles in the animal kingdom. However, whether ACE plays an immune function against viral infection in vector insects is unclear. In this study, an ACE gene (LsACE) from the small brown planthopper was found to respond to Rice stripe virus (RSV) infection. The enzymatic activities of LsACE were characterized at different pH and temperature. Twenty planthopper proteins were found to interact with LsACE. RSV infection significantly upregulated LsACE expression in the testicle and fat body. When the expression of LsACE in viruliferous planthoppers was inhibited, the RNA level of the RSV SP gene was upregulated 2-fold in planthoppers, and all RSV genes showed higher RNA levels in the rice plants consumed by these planthoppers, leading to a higher viral infection rate and disease rating index. These results indicate that LsACE plays a role in the immune response against RSV transmission by planthoppers.


Assuntos
Hemípteros/imunologia , Hemípteros/virologia , Proteínas de Insetos/imunologia , Insetos Vetores/imunologia , Insetos Vetores/virologia , Peptidil Dipeptidase A/imunologia , Tenuivirus/fisiologia , Sequência de Aminoácidos , Animais , Hemípteros/genética , Hemípteros/fisiologia , Proteínas de Insetos/genética , Insetos Vetores/genética , Insetos Vetores/fisiologia , Oryza/virologia , Peptidil Dipeptidase A/genética , Filogenia , Doenças das Plantas/virologia , Tenuivirus/classificação , Tenuivirus/genética , Tenuivirus/isolamento & purificação
14.
Pestic Biochem Physiol ; 157: 143-151, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153462

RESUMO

Autophagy is a cell adaptive response that involves the process of microbial infections. Our previous study has indicated that Bombyx mori nucleopolyhedrovirus (BmNPV) infection triggers the complete autophagic process in BmN-SWU1 cells, which is beneficial to the viral infection. Autophagy-related (ATG) protein ATG13, as part of the ULK complex (a serine-threonine kinase complex composed of ULK1, ULK2, ATG13, ATG101, and FIP200), is the most upstream component of the autophagy pathway, and how it affects virus infections will improve our understanding of the interaction between the virus and the host. This study has determined that the overexpression of the BmAtg13 gene promotes the expression of viral genes and increases viral production in BmN-SWU1 cells, whereas knocking down the BmAtg13 gene suppresses BmNPV replication. Moreover, the BmAtg13 overexpression transgenic line contributed to viral replication and increased mortality rate of BmNPV infection. In contrast, the BmAtg13 knockout transgenic line reduced viral replication 96 h post-infection. Furthermore, BmATG13 directly interacted with viral protein BRO-B, forming a protein complex. Taken together, the findings of this study suggest that BmATG13 may be utilized by the BRO-B protein to promote BmNPV replication and proliferation, which, in turn, provides important insights into the mechanism that autophagy influences viral infection.


Assuntos
Proteínas de Insetos/metabolismo , Nucleopolyhedrovirus/patogenicidade , Replicação Viral/fisiologia , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Insetos/genética , Ligação Proteica , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genética
15.
Pestic Biochem Physiol ; 157: 196-203, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153469

RESUMO

Overexpression of the cytochrome P450 monooxygenase CYP6A51 has been previously associated with pyrethroid resistance in the Mediterranean fruit fly (medfly) Ceratitis capitata, an important pest species worldwide; however, this association has not been functionally validated. We expressed CYP6A51 gene in Escherichia coli and produced a functional enzyme with preference for the chemiluminescent substrate Luciferin-ME EGE. In vitro metabolism assays revealed that CYP6A51 is capable of metabolizing two insecticides that share the same mode of action, λ-cyhalothrin and deltamethrin, whereas no metabolism or substrate depletion was observed in the presence of spinosad or malathion. We further expressed CYP6A51 in vivo via a GAL4/UAS system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. Toxicity bioassays indicated that CYP6A51 confers knock-down resistance to both λ-cyhalothrin and deltamethrin. Detection of CYP6A51 - associated pyrethroid resistance in field populations may be important for efficient Insecticide Resistance Management (IRM) strategies.


Assuntos
Ceratitis capitata/efeitos dos fármacos , Ceratitis capitata/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Piretrinas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Família 6 do Citocromo P450/genética , Família 6 do Citocromo P450/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Nitrilos/farmacologia
16.
Pestic Biochem Physiol ; 157: 26-32, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153474

RESUMO

Nitenpyram is very effective in controlling Nilaparvata lugens (brown planthopper, BPH), and its resistance has been reported in field populations; however, the resistance mechanism remains unclear. In the present study, cross-resistance and resistance mechanisms in nitenpyram-resistant BPH were investigated. A resistant strain (NR) with a high resistance level (164.18-fold) to nitenpyram was evolved through successive selection for 42 generations from a laboratory susceptible strain (NS). The bioassay results showed that the NR exhibited cross-resistance to imidacloprid (37.46-fold), thiamethoxam (71.66-fold), clothianidin (149.17-fold), dinotefuran (98.13-fold), sulfoxaflor (47.24-fold), cycloxaprid (9.33-fold), etofenprox (10.51-fold) and isoprocarb (9.97-fold) but not to triflumezopyrim, chlorpyrifos and buprofezin. The NR showed a 3.21-fold increase in cytochrome P450 monooxygenase (P450) activity compared to that in the NS, while resistance was also synergized (4.03-fold) with the inhibitor piperonyl butoxide (PBO), suggesting a role of P450. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) genes by quantitative real-time PCR results indicated that twelve P450 genes were significantly overexpressed in the NR strain, especially CYP6ER1 (203.22-fold). RNA interference (RNAi) suppression of CYP6ER1 through injection of dsCYP6ER1 led to significant susceptibility in the NR strain. The current study expands our understanding of the nitenpyram resistance mechanism in N. lugens, provides an important reference for integrated pest management (IPM), and enriches the theoretical system of insect toxicology.


Assuntos
Hemípteros/efeitos dos fármacos , Neonicotinoides/farmacologia , Animais , Carbamatos/farmacologia , Guanidinas/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Nitrocompostos/farmacologia , Piretrinas/farmacologia , Piridinas/farmacologia , Pirimidinonas/farmacologia , Interferência de RNA , Tiazóis/farmacologia
17.
J Insect Sci ; 19(2)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31222323

RESUMO

Pheromonal communication is important in insect mate finding and reproduction. Identifying components of pest insect pheromone system is a first step to disrupt pest insect reproduction. In this study, we identified and cloned the pheromone biosynthesis activating neuropeptide receptor (PBANR) from the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), which is one of the most damaging pests of corn and other crops in parts of Asia and Australia. The O. furnacalis PBANR (OstfuPBANR) gene has an ORF of 1,086 bp and encoded 362 amino acids with seven transmembrane domains and had a high sequence identity to known lepidopteran PBANRs. Expression analysis showed that OstfuPBANR was highly expressed in the pheromone glands compared with other tissues, consistent with other studies. Interestingly, OstfuPBANR was expressed higher in the larval stages compared to the pupal or adult stages, suggesting that OstfuPBANR may have broad functions in larva beyond adult pheromone synthesis.


Assuntos
Mariposas/metabolismo , Receptores de Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Expressão Gênica , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mariposas/genética , Feromônios/biossíntese , Receptores de Neuropeptídeos/genética , Análise de Sequência de DNA
18.
Parasit Vectors ; 12(1): 311, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234914

RESUMO

BACKGROUND: The cuticle is an indispensable structure that protects the mosquito against adverse environmental conditions and prevents pathogen entry. While most cuticles are hard and rigid, some parts of cuticle are soft and flexible to allow movement and blood-feeding. It has been reported that 3, 4-dihydroxyphenylacetaldehyde (DOPAL) synthase is associated with flexible cuticle formation in Aedes aegypti. However, the molecular function of DOPAL synthase in the ontogenesis of mosquito remains largely unknown. In this study, we characterized gene expression profiles of DOPAL synthase and investigated its functions in larvae and female adults of Aedes agypti by RNAi. RESULTS: Our results suggest that the expression of DOPAL synthase is different during development and the transcriptional level reached its peak at the female white pupal stage, and DOPAL synthase was more highly expressed in the cuticle and midgut than other tissues in the adult. The development process from larva to pupa was slowed down strikingly by feeding the first-instar larvae with chitosan/DOPAL synthase dsRNA nanoparticles. A qRT-PCR analysis confirmed that the dsRNA-mediated transcription of the DOPAL synthase was reduced > 50% in fourth-instar larvae. Meanwhile, larval molt was abnormal during development. Transmission electron microscopy results indicated that the formation of endocuticle and exocuticle was blocked. In addition, we detected that the dsDOPAL synthase RNA caused significant mortality when injected into the female adult mosquitoes. CONCLUSIONS: Our findings demonstrate that DOPAL synthase plays a critical role in mosquito larval development and adult survival and suggest that DOPAL synthase could be a good candidate gene in RNAi intervention strategies in mosquito control.


Assuntos
Aedes/enzimologia , Aedes/genética , Descarboxilases de Aminoácido-L-Aromático/genética , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimento , Interferência de RNA , Animais , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Controle de Mosquitos/métodos
19.
Parasit Vectors ; 12(1): 319, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238963

RESUMO

BACKGROUND: Bacillus thuringiensis israelensis (Bti) is a widely used mosquitocidal microbial pesticide due to its high toxicity. ATP-binding proteins (ABP) are prevalently detected in insects and are related to reaction against Bti toxins. However, the function of ABP in mosquito biocontrol is little known, especially in Aedes aegypti. Therefore, this study aimed to clarify the function of ABP in Ae. aegypti against Bti toxin. RESULTS: Aedes aegypti ABP (GenBank: XM_001661856.2) was cloned, expressed and purified in this study. Far-western blotting and ELISA were also carried out to confirm the interaction between ABP and Cry11Aa. A bioassay of Cry11Aa was performed both in the presence and absence of ABP, which showed that the mortality of Ae. aegypti is increased with an increase in ABP. CONCLUSIONS: Our results suggest that ABP in Ae. aegypti can modulate the toxicity of Cry11Aa toxin to mosquitoes by binding to Bti toxin. This could not only enrich the mechanism of Bt toxin, but also provide more data for the biocontrol of this transmission vector.


Assuntos
Aedes/genética , Bacillus thuringiensis/patogenicidade , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Aedes/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Bioensaio , Clonagem Molecular , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Mosquitos Vetores/microbiologia , Controle Biológico de Vetores , Ligação Proteica
20.
BMC Genomics ; 20(1): 412, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31117959

RESUMO

BACKGROUND: Parts of Europe and the United States have witnessed dramatic losses in commercially managed honey bees over the past decade to what is considered an unsustainable extent. The large-scale loss of bees has considerable implications for the agricultural economy because bees are one of the leading pollinators of numerous crops. Bee declines have been associated with several interactive factors. Recent studies suggest nutritional and pathogen stress can interactively contribute to bee physiological declines, but the molecular mechanisms underlying interactive effects remain unknown. In this study, we provide insight into this question by using RNA-sequencing to examine how monofloral diets and Israeli acute paralysis virus inoculation influence gene expression patterns in bees. RESULTS: We found a considerable nutritional response, with almost 2000 transcripts changing with diet quality. The majority of these genes were over-represented for nutrient signaling (insulin resistance) and immune response (Notch signaling and JaK-STAT pathways). In our experimental conditions, the transcriptomic response to viral infection was fairly limited. We only found 43 transcripts to be differentially expressed, some with known immune functions (argonaute-2), transcriptional regulation, and muscle contraction. We created contrasts to explore whether protective mechanisms of good diet were due to direct effects on immune function (resistance) or indirect effects on energy availability (tolerance). A similar number of resistance and tolerance candidate differentially expressed genes were found, suggesting both processes may play significant roles in dietary buffering from pathogen infection. CONCLUSIONS: Through transcriptional contrasts and functional enrichment analysis, we contribute to our understanding of the mechanisms underlying feedbacks between nutrition and disease in bees. We also show that comparing results derived from combined analyses across multiple RNA-seq studies may allow researchers to identify transcriptomic patterns in bees that are concurrently less artificial and less noisy. This work underlines the merits of using data visualization techniques and multiple datasets to interpret RNA-sequencing studies.


Assuntos
Abelhas/genética , Dicistroviridae/patogenicidade , Dieta , Proteínas de Insetos/genética , Estado Nutricional , Transcriptoma , Viroses/virologia , Animais , Abelhas/fisiologia , Abelhas/virologia , Regulação da Expressão Gênica , Marcadores Genéticos , Polinização
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