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2.
Front Physiol ; 10: 1256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31611820

RESUMO

Spodoptera exigua and S. litura are two sympatric species in China and many other countries. Both moths employ a multiple component sex pheromone blend, including a common component Z9,E12-14:OAc, and two specific components Z9-14:OH and Z11-16:OAc for S. exigua, and one specific component Z9,E11-14:OAc for S. litura. For the two species, it has been well documented that males are able to recognize and behaviorally attracted by their species-specific sex pheromone, which functions as a means of reproductive isolation, but whether males could mutually recognize pheromone components of its sympatric species is unknown. In the present study, the electroantennogram (EAG) and field evaluation were conducted to address this topic. The EAG recordings revealed that males of each species could significantly respond to specific components of its sympatric species, although the response values were lower than that to its own major component. In field tests, the specific components Z9-14:OH and Z11-16:OAc of S. exigua strongly inhibited the male catches of S. litura to its conspecific sex pheromone, while specific component Z9,E11-14:OAc of S. litura significantly reduced the male catches of S. exigua to its sex pheromone. Furthermore, the combined lure of the two species completely inhibited male catches of S. litura, and significantly decreased the male catches of S. exigua, compared to the species-specific lure alone. The results demonstrated that males of the two sibling species could perceive the specific components of its counterpart, suggesting that mutual recognition of pheromone components may function to strengthen the behavioral isolation between the two species. Our study has added new knowledge to the reproductive isolation via sex pheromone communication system in sympatric moth species, and provided a base for designing of mating disruption tactics targeting multispecies by using insect sex pheromones.

3.
Insect Biochem Mol Biol ; 115: 103244, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31560967

RESUMO

Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.


Assuntos
Comunicação Animal , Antenas de Artrópodes/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Insetos/fisiologia , Percepção Olfatória , Spodoptera/fisiologia , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Feminino , Masculino , Mutação , Atrativos Sexuais
4.
Ying Yong Sheng Tai Xue Bao ; 30(7): 2447-2456, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31418248

RESUMO

The static chamber-gas chromatography method was applied to observe CO2 fluxes across the water-air interface from different aquaculture systems, including four groups of mesocosms, i.e., blank control system (CK), monoculture system of Sebastes schlegelii (F), monoculture system of Chlamys farreri (B) and polyculture system of S. schlegelii and C. farreri (FB). Meanwhile, the physical, chemical and biological indices of the water were measured. The results showed that the group CK was a stable CO2 source, with the mean flux of 12.42 mg·m-2·h-1. The group B was a CO2 source during the early and middle periods of the experiment and a CO2 sink at the end of the experiment, with the mean flux during the experiment being 10.95 mg·m-2·h-1. The change trends of CO2 flux in the group F and FB were generally consistent, acting as CO2 sources in the early period and as CO2 sinks subsequently. The mean fluxes of those two groups during the expe-riment were -3.53 and -10.49 mg·m-2·h-1, respectively. Water pH was a good predictor for CO2 flux across the water-air interface according to the result of regression analysis, with pH value of 8.25 as the critical threshold between efflux and influx. The net primary production of water column was the main factor influencing the CO2 flux, indicating that phytoplankton might be the main internal force regulating the CO2 flux across the water-air interface. Our results indicated that C. farreri at present stocking density in the polyculture system could promote phytoplankton biomass as well as primary production and therefore enhance the CO2 sink function.


Assuntos
Poluentes Atmosféricos/análise , Aquicultura , Dióxido de Carbono/análise , Pectinidae , Animais , Metano , Água
5.
Insects ; 10(9)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31454982

RESUMO

Gustatory receptors (GRs) are crucial in the peripheral coding of the non-volatile compounds in insects, and thus play important roles in multiple behaviors including feeding, mating, and oviposition. However, little research has been done on GRs in lepidopteran pests. In the current work with Spodoptera litura, an important worldwide crop's pest, a candidate fructose GR gene (SlitGR8) was cloned in full length, and its spatial and temporal expression profiles were determined by quantitative real-time PCR (qPCR). It revealed that SlitGR8 was highly expressed in antennae of both male and female adults, as well as in larva of first, fifth and sixth instar. Functional analyses were further conducted using the Xenopus oocyte system. SlitGR8 responded specifically to D-fructose among 12 tested sugar compounds. In addition, the behavioral assay demonstrated that both female and male moths could respond with proboscis extension behavior to D-fructose applied onto the antenna, but females showed higher sensitivity than males. The results provide an important base for further elucidation of molecular mechanisms of gustation, and a potential target for development of feeding interfering technique in S. litura.

6.
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
7.
Pestic Biochem Physiol ; 156: 152-159, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31027575

RESUMO

Sex pheromone biosynthesis in moths relies on the activity of multiple enzymes, including Δ9 desaturase, which plays an important role in catalyzing desaturation at the Δ9 position of the carbon chain. However, the physiological function of moth Δ9 desaturase has not been elucidated in vivo. In this study, we used the CRISPR/Cas9 system to knockout the Δ9 desaturase gene (SlitDes11) of Spodoptera litura to analyze its role in sex pheromone biosynthesis. First, through the direct injection of SlitDes11-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, gene editing was induced in around 30% of eggs 24 h after injection and was induced in 20.8% of the resulting adult moths. Second, using a sibling-crossing strategy, insects with mutant SlitDes11 (bearing a premature stop codon) were selected, and homozygous mutants were obtained in the G5 generation. Third, pheromone gland extracts of adult female homozygous SlitDes11 mutants were analyzed using Gas chromatography (GC). The results showed that titers of all three ester sex pheromone components; Z9, E11-14:Ac, Z9,E12-14:Ac, and Z9-14:Ac; were reduced by 62.40%, 78.50%, and 72.50%, respectively. This study provides the first direct evidence for the role of SlitDes11 in sex pheromone biosynthesis in S. litura, and indicates the gene could be as potential target to disrupt sexual communication in S. litura for developing a new pollution-free insecticide.


Assuntos
Proteínas de Insetos/metabolismo , Atrativos Sexuais/metabolismo , Spodoptera/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Mutação em Linhagem Germinativa , Proteínas de Insetos/química , Proteínas de Insetos/genética , Mariposas/metabolismo , Mutação/genética , RNA Mensageiro , Alinhamento de Sequência
8.
Mar Pollut Bull ; 141: 387-397, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955748

RESUMO

To investigate the effects of mariculture on the sources, distribution and preservation of sedimentary organic carbon (SOC), sediments from an mariculture area in Ailian Bay, China, and a control area were analyzed for grain size composition, total organic carbon (TOC), total nitrogen (TN), carbon/nitrogen (C/N) ratio, and stable carbon and nitrogen isotopic composition (δ13C and δ15N). The sedimentary type of sediments in study area was clay silt. TOC, TN, C/N, δ13C, and δ15N ranged from 0.58 to 1.21%, 0.06-0.17%, 6.29-9.82, -23.20 to -18.50‰, and 6.17-7.38‰, respectively, and followed similar spatial patterns. TOC, TN and δ13C were higher in mariculture area than in control area. Biodeposit and kelp OC contributions greater of SOC in mariculture area than the control area (biodeposits: 20.10 ±â€¯4.84 to 6.2 ±â€¯1.3%; kelp: 15.3 ±â€¯6.63 to 5.2 ±â€¯0.84%). Overall, mariculture activities significantly influence the sources, distribution and preservation of SOC.


Assuntos
Aquicultura/métodos , Monitoramento Ambiental/métodos , Gastrópodes/crescimento & desenvolvimento , Sedimentos Geológicos/química , Kelp/crescimento & desenvolvimento , Poluentes Químicos da Água/análise , Animais , Baías/química , Carbono/análise , Ciclo do Carbono , Isótopos de Carbono/análise , China , Nitrogênio/análise , Isótopos de Nitrogênio/análise
9.
Artigo em Inglês | MEDLINE | ID: mdl-30861459

RESUMO

Acute heat stress is common in aquaculture and can affect diverse physiological processes in fish; however, different species of fish have various mechanisms for heat stress adaptation. In this study, we profiled the transcriptome responses of the Atlantic salmon (Salmo salar) to heat stress at 23 °C for 6 or 24 h, compared with that of fish at a normal temperature of 13 °C. The liver was selected as the target tissue for this analysis. A total of 243 and 88 genes were differentially expressed after 6 and 24 h of heat stress, respectively. Of these, only 22 were common to both time points, and most of these common genes were molecular chaperones such as heat shock cognate 71 kDa protein and heat shock protein 90-alpha. Genes such as activating transcription factor 6, calreticulin, protein disulfide isomerase A3, and protein kinase R-like endoplasmic reticulum kinase-eukaryotic initiation factor 2-alpha were only up-regulated after 6 h of heat stress; most of these genes are involved in the endoplasmic reticulum stress pathway. Indeed, endoplasmic reticulum stress was identified at 6 h but not at 24 h, suggesting that stress response plays an important role in the adaptation of Atlantic salmon to acute heat stress. Other up-regulated genes at 6 h were related to the insulin and nucleotide oligomerization domain-like receptor signaling pathways, which directly eliminate misfolded proteins and sustain sugar and lipid homeostasis. At 24 h, heat stress influenced the expression of steroid and terpenoid backbone biosynthesis, which may influence the sexual development and differentiation of Atlantic salmon. Overall, our results elucidate the transcriptome mechanisms that contribute to short-term heat tolerance in the liver of Atlantic salmon.


Assuntos
Resposta ao Choque Térmico , Salmo salar/genética , Transcriptoma , Adaptação Fisiológica , Animais , Aquicultura , Proteínas de Peixes/genética , Salmo salar/fisiologia
10.
Insect Sci ; 26(3): 388-399, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29058383

RESUMO

Pheromone-binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target-gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1-sgRNA injected eggs and 19.5% for PBP3-sgRNA injected eggs. Second, by an in-crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA-injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.


Assuntos
Antenas de Artrópodes/fisiologia , Proteínas de Insetos/fisiologia , Mariposas/fisiologia , Atrativos Sexuais/metabolismo , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Feminino , Homozigoto , Masculino , Mutação
11.
Ying Yong Sheng Tai Xue Bao ; 29(9): 3102-3110, 2018 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-30411588

RESUMO

Rainbow trout (Oncorhynchus mykiss) is a cold-water fish species with optimal temperature of 12-18 ℃. Temperature stress is a common issue in commercial culture of the Salmonid fish in summer. Both maple trout and steelhead trout are rainbow trout breeding species with good quality. The purpose of this study was to explore the impact of acute temperature stress on antioxidant responses of two kinds of rainbow trout. The juvenile maple trout was diploid, and the juvenile steelhead trout was triploid. The fishes were exposed to four different temperature treatments: 13, 17, 21, and 25 ℃, and hepatic samples were collected at 0, 1, 6, and 12 h. After acute heat stress, the fish were transferred to the tank with optimal temperature (13 ℃) and hepatic samples were collected after 1, 12, 24, and 48 h. The biomarkers, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), lipid peroxidation (LPO), malondialdehyde (MDA), and heat shock protein 70 kDa (HSP70) were measured. Results showed that the activities of SOD, CAT, and GPx in both maple trout and steelhead trout in 17 ℃ treatment had no significant increase after acute temperature stress. The SOD activities of maple trout and steelhead trout in 21 ℃ treatment significantly increased in response to acute temperature stress, but the SOD activity of maple trout returned to normal during the recovery process. The activities of SOD, CAT, and GPx for both species in 25 ℃ treatment were significantly increased after acute temperature stress, which returned to normal for 24 h recovery. The amount of HSP70 produced by maple trout at 17, 21, and 25 ℃ treatments was significantly higher than that at 13 ℃ treatment, while that of steelhead trout was significantly higher for the 21 and 25 ℃ treatments than that at 13 ℃ treatment. A variety of antioxidant parameters were analyzed by integrated biomarker response (IBR) values. Results showed that the antioxidant response of maple trout was significantly higher than that of steelhead trout in 17 ℃ and 21 ℃ treatments, but significantly lower than steelhead trout in 25 ℃ treatment. Such a result indicated that the antioxidant responses of different ploidy rainbow trouts were different under acute stress at different temperatures.


Assuntos
Antioxidantes/metabolismo , Oncorhynchus mykiss/fisiologia , Estresse Fisiológico/fisiologia , Animais , Melhoramento Vegetal , Ploidias , Temperatura Ambiente
12.
J Insect Physiol ; 111: 25-31, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30336148

RESUMO

Chrysoperla sinica is an important natural predator of many notorious agricultural pests. Understanding its olfactory mechanism can help enhance the effectiveness of C. sinica in biological control. In the present study, we investigated the tissue expression patterns of 12 odorant-binding protein (OBP) genes from C. sinica (CsinOBPs). The results of quantitative real-time polymerase chain reaction (qPCR) showed that CsinOBP1, CsinOBP2, CsinOBP3, CsinOBP4, CsinOBP6, CsinOBP7, CsinOBP9, CsinOBP10, and CsinOBP12 were predominantly expressed in the antennae of both sexes, indicating their roles in olfaction. Additionally, the qPCR analysis revealed that the 12 CsinOBP genes had distinct expression patterns, while the motif-pattern investigation suggested that the OBPs had different ligands. The ligand-binding assay showed that CsinOBP1 and CsinOBP10 had broader binding spectra than did the other OBPs. Thus, CsinOBP1 was able to bind not only plant volatiles (such as farnesol, cis-3-hexenyl hexanoate, geranylacetone, ß-ionone, 2-tridecanone, and trans-nerolidol) but also the aphid alarm pheromone (E)-ß-farnesene. On the other hand, CsinOBP2 and CsinOBP6 exhibited relatively narrow binding spectra, only binding ethyl benzoate. The study also identified several compounds that can potentially be used to develop slow-release agents attracting C. sinica and to improve search strategies for insect pest control.


Assuntos
Proteínas de Insetos/genética , Insetos/genética , Feromônios/metabolismo , Receptores Odorantes/genética , Compostos Orgânicos Voláteis/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Masculino , Comportamento Predatório , Ligação Proteica , Receptores Odorantes/metabolismo
13.
J Insect Sci ; 18(5)2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30260453

RESUMO

The high sensitivity of the olfactory system is essential for feeding and oviposition in moth insects, and some chemosensory proteins (CSPs) are thought to play roles in this system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. In this study, to identify the olfactory CSPs from a repertoire of 21 CSP members in the notorious rice pest Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), tissue expression patterns were firstly examined by quantitative real-time polymerase chain reaction (qPCR). It showed that CSP2 was antennae specific and seven more CSPs (CSP1, 3, 4, 6, 15, 16, and 17) were antennae biased in expression, suggesting their olfactory roles; while other CSPs were multiple-tissue expressed and non-antennae biased, suggesting other functions for these genes. To further determine the ligand binding specificity, three putative olfactory genes (CSP1-3) were expressed in Escherichia coli cells, and binding affinity of these three recombinant CSP proteins were measured for 35 plant volatiles by the ligand binding assays. CSP1 and CSP2 exhibited high binding affinities (Ki ≤ 10.00 µM) for four (2-tridecanone, benzaldehyde, laurinaldehyde and 2-pentadecanone) and two (2-heptanol and (+)-cedrol) host plant volatiles, respectively; the three CSPs also showed moderate binding affinity (Ki = 10.01-20.00 µM) for 16 plant volatiles. Our study suggests that the three CSPs play essential roles in the perception of host plant volatiles, providing bases for the elucidation of olfactory mechanisms in this important pyralid pest.


Assuntos
Antenas de Artrópodes/fisiologia , Proteínas de Insetos/metabolismo , Mariposas/fisiologia , Percepção Olfatória , Compostos Orgânicos Voláteis/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Mariposas/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores Sexuais
14.
J Chem Ecol ; 44(10): 886-893, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30094705

RESUMO

Orthaga achatina (Lepidoptera: Pyralidae) is the most serious pest in south China of camphor trees, Cinnamomum camphora (L.) Presl, an important urban tree species. Gas chromatography-electroantennographic detection (GC-EAD) of the sex pheromone of O. achatina showed three EAD-active components. Coupled gas chromatography/mass spectrometry analyses identified these as (Z)-11-hexadecenol (Z11-16:OH), (Z)-11-hexadecenyl acetate (Z11-16:OAc), and (3Z,6Z,9Z,12Z,15Z)-tricosapentaene (Z3,Z6,Z9,Z12,Z15-23:H). In field tests using different combinations of the three compounds, male moths were attracted to a mixture of Z11-16:OAc and Z3,Z6,Z9,Z12,Z15-23:H, but less attracted to other blends. Further field tests with different ratios of the two compounds determined the optimal ratio of the binary blend as 500:250. The addition of Z11-16:OH to Z11-16:OAc, or to the binary mixture of Z11-16: OAc and the pentaene did not yield higher catches. This shows that O. achatina uses a mixture of Type I and Type II sex pheromone components. Orthaga achatina is the third Pyraloidea species found to utilize Z3,Z6,Z9,Z12,Z15-23:H as a sex pheromone component.


Assuntos
Lepidópteros/química , Lepidópteros/efeitos dos fármacos , Atrativos Sexuais/química , Atrativos Sexuais/farmacologia , Animais , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Comportamento Sexual Animal/efeitos dos fármacos
15.
Insect Biochem Mol Biol ; 99: 37-53, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29800678

RESUMO

The functions of the Ionotropic Receptor (IR) family have been well studied in Drosophila melanogaster, but only limited information is available in Lepidoptera. Here, we conducted a large-scale genome-wide analysis of the IR gene repertoire in 13 moths and 16 butterflies. Combining a homology-based approach and manual efforts, totally 996 IR candidates are identified including 31 pseudogenes and 825 full-length sequences, representing the most current comprehensive annotation in lepidopteran species. The phylogeny, expression and sequence characteristics classify Lepidoptera IRs into three sub-families: antennal IRs (A-IRs), divergent IRs (D-IRs) and Lepidoptera-specific IRs (LS-IRs), which is distinct from the case of Drosophila IRs. In comparison to LS-IRs and D-IRs, A-IRs members share a higher degree of protein identity and are distinguished into 16 orthologous groups in the phylogeny, showing conservation of gene structure. Analysis of selective forces on 27 orthologous groups reveals that these lepidopteran IRs have evolved under strong purifying selection (dN/dS≪1). Most notably, lineage-specific gene duplications that contribute primarily to gene number variations across Lepidoptera not only exist in D-IRs, but are present in the two other sub-families including members of IR41a, 76b, 87a, 100a and 100b. Expression profiling analysis reveals that over 80% (21/26) of Helicoverpa armigera A-IRs are expressed more highly in antennae of adults or larvae than other tissues, consistent with its proposed function in olfaction. However, some are also detected in taste organs like proboscises and legs. These results suggest that some A-IRs in H. armigera likely bear a dual function with their involvement in olfaction and gustation. Results from mating experiments show that two HarmIRs (IR1.2 and IR75d) expression is significantly up-regulated in antennae of mated female moths. However, no expression difference is observed between unmated female and male adults, suggesting an association with female host-searching behaviors. Our current study has greatly extended the IR gene repertoire resource in Lepidoptera, and more importantly, identifies potential IR candidates for olfactory, gustatory and oviposition behaviors in the cotton bollworm.


Assuntos
Regulação da Expressão Gênica/fisiologia , Genoma de Inseto/fisiologia , Estudo de Associação Genômica Ampla , Proteínas de Insetos , Lepidópteros , Receptores Ionotrópicos de Glutamato , Animais , Drosophila melanogaster , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Lepidópteros/genética , Lepidópteros/metabolismo , Receptores Ionotrópicos de Glutamato/biossíntese , Receptores Ionotrópicos de Glutamato/genética
16.
J Insect Physiol ; 103: 29-35, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28927827

RESUMO

The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis of biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene in a noctuid pest Spodoptera litura. A fragment of SlitBLOS2 was identified by analyzing a S. litura transcriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy. To clarify the function of SlitBLOS2, CRISPR/Cas9 based target mutagenesis of SlitBLOS2 was achieved, displaying a mosaic translucent integument in 62.3-70.6% larvae of G0 generation. Further PCR-based genotype analysis demonstrated various mutations occurred at the SlitBLOS2 specific target site. A homozygote mutant individual was obtained in G1 generation, in which the yellow strips and white spots on the larval integument completely disappeared. Our study clearly demonstrates the function of SlitBLOS2 in the integument coloration, and thus provides a useful marker gene for genome editing based gene functional study and pest control strategy in S. litura as well as other lepidopteran pests.


Assuntos
Proteínas de Insetos/fisiologia , Pigmentação/genética , Spodoptera/fisiologia , Animais , Sistemas CRISPR-Cas , Feminino , Marcadores Genéticos , Larva , Estágios do Ciclo de Vida , Masculino
17.
Sci Rep ; 7(1): 8470, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28814748

RESUMO

Pheromone binding proteins (PBPs) are thought to play crucial roles in perception of the sex pheromones particularly in noctuid moths, but this is rarely in vivo evidenced due to lacking an effective technique. Here, we reported an in vivo functional study of PBP1 in the important lepidopteran pest Helicoverpa armigera (HarmPBP1), by using the CRISPR/Cas9 system. Efficient and heritable mutagenesis was achieved by egg injection of mixture of Cas9-mRNA and HarmPBP1-sgRNA. The TA cloning and sequencing revealed various insertion and/or deletion (indel) mutations at the target site. Among those, one mutation resulted in a premature stop codon at the target site, which led to a highly truncated protein with only 10 amino acids. The HarmPBP1 with this mutation would completely loss its function, and thus was used to select the homozygous mutant insects for functional analysis. The electroantennogram recording showed that the mutant male adults displayed severely impaired responses to all three sex pheromone components (Z11-16:Ald, Z9-16:Ald and Z9-14:Ald). Our study provides the first in vivo evidence that HarmPBP1 plays important role in perception of female sex pheromones, and also an effective methodology for using CRISPR/Cas9 system in functional genetic study in H. armigera as well as other insects.


Assuntos
Proteínas de Transporte/genética , Mariposas/genética , Sequência de Aminoácidos , Animais , Antenas de Artrópodes/fisiologia , Sistemas CRISPR-Cas , Fenômenos Eletrofisiológicos , Feminino , Mutação INDEL , Proteínas de Insetos/genética , Masculino , Atrativos Sexuais
18.
Artigo em Inglês | MEDLINE | ID: mdl-28807837

RESUMO

As the first biochemical step of olfactory reception and recognition, odorant binding proteins (OBPs) have been demonstrated to be essential. Considering functional diversities of OBPs within a single species, we here extended the characterization of two other OBPs from Spodoptera exigua, belonging to insect Classic OBPs. With a combination of transcriptome and Rapid Amplification of cDNA End (RACE) approaches, two OBP genes in S. exigua were identified, namely SexiOBP1 and OBP7. Expression pattern analysis revealed that both of them exhibited a distinct expression pattern, where OBP1 was broadly and highly expressed in several tissues including antennae of adults whereas OBP7 was abundant only in the antennae of both sexes, strongly indicative of olfactory roles. Further, binding assays showed that the two SexiOBPs shared a common odorant-response spectrum with considerable affinities to host odorants of acetophenone, farnesol and ß-ionone (Ki<20µM). Specially, OBP1 could strongly bind an insect attractant ß-caryophyllene (Ki=2.76µM) released by maize. Intriguingly, the major sex pheromone of S. exigua, Z9,E12-14:Ac, was the best ligand for OBP7 with Ki value of 7.58µM. Ligand structural analysis revealed that the two SexiOBPs were capable of accommodating different types of ligands in shape and size, possibly implying the plasticity of binding pockets. Ultimately, comparison of binding properties among 10 SexiOBPs including the two OBPs in this study implied a cross-talk in functions, i.e. different OBPs are also suitable to accept some common odorants except for unique ligands. Taken together, this study has provided evidence for their involvements in seeking and orientation of host plants, and meanwhile indicates functional conservation and differences between OBP1 and OBP7 from S. xigua.


Assuntos
Receptores Odorantes/metabolismo , Spodoptera/metabolismo , Animais , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Receptores Odorantes/genética
19.
PLoS One ; 12(1): e0170072, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28081263

RESUMO

The American cockroach, Periplaneta americana, is a vector of many pathogenic organisms associated with human diseases. Olfaction plays a crucial role in guiding cockroach behaviors and contributes to their ability to transmit pathogens. Odorant binding proteins (OBPs), abundant in the insect olfactory sensilla, are important for insect olfaction. In this study, three OBP genes, PameOBP1, 2 and 3, were cloned from P. americana. Sequence alignment and phylogenetic analysis revealed that PameOBP1, 2 and 3 belong to the Minus-C OBP, Classic OBP, and Plus-C OBP subfamilies, respectively. Expression pattern and ligand-binding analysis showed that PameOBP1 and 2 were specifically expressed in antennae, and exhibited high binding affinities (Ki < 2 µM) to farnesene, farnesol, 2-tridecanone, and tetradecane, suggesting roles in volatile perception. Conversely, PameOBP3 was ubiquitously expressed in most of the tissues examined at high levels and displayed very weak binding affinities (Ki > 40 µM) for all 87 ligands tested. Our study provides insights into the functional diversity of PameOBP genes and provides some volatiles that can potentially be used in behavioral interference of P. americana.


Assuntos
Proteínas de Insetos/metabolismo , Periplaneta/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Sequência de Aminoácidos , Animais , Antenas de Artrópodes/metabolismo , Antenas de Artrópodes/patologia , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Cetonas/química , Cetonas/metabolismo , Ligantes , Masculino , Filogenia , Ligação Proteica , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Receptores Odorantes/química , Receptores Odorantes/classificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Espectrometria de Fluorescência
20.
Ying Yong Sheng Tai Xue Bao ; 28(11): 3784-3788, 2017 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-29692123

RESUMO

Environmental factors affect the insect sex pheromone communication, and subsequently the attractiveness of sex pheromone lures used in pest control. In order to provide the guidance for the sex pheromone use in control of Chilo suppressalis, effects of temperature on the sex pheromone production in female moths and electro-physiological response of male moths to sex pheromones, were explored in our present study. The insects were treated with different temperatures (15, 20, 25, 30 and 35 ℃) at pupa and adult stages, then the contents and relative proportion of each of three pheromone components were determined by a gas chromatography (GC), and the electro-physiological response to sex pheromones were detected by an electroantennograph (EAG). Results showed that the contents of all three sex pheromone components (Z9-16:Ald, Z11-16:Ald and Z13-18:Ald) were highest in 25 ℃ treated females, significantly higher than those in other temperature treated females, and the relative proportion of Z13-18:Ald was significantly reduced in 25 ℃ treated females. On the male part, EAG responses to each of the three sex pheromone components and the tertiary blend showed no significant difference at temperatures between 15-25 ℃, but EAG va-lues decreased significantly as the temperature increased to 30 ℃ and further to 35 ℃ for component Z9-16:Ald, Z11-16:Ald and the blend, and to 35 ℃ for Z13-18:Ald. Taken together, the optimum temperature for the sex pheromone communication was 20-25 ℃ in C. suppressalis, and too higher or lower temperature will affect the normal communication. The study provided an important reference for the use of sex pheromone in pest control, and for prediction of the development of pest population at extremely low or high temperature.


Assuntos
Comunicação Animal , Lepidópteros , Atrativos Sexuais , Animais , Feminino , Masculino , Mariposas , Feromônios , Pupa , Temperatura Ambiente
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