Effects of Various Degrees of Antennal Ablation on Mating and Oviposition Preferences of the Diamondback Moth,Plutella xylostella L.

Using scanning electron microscopy, we investigated the distribution of the trichoid, basiconic, and coeloconic sensilla on the antennae of the diamondback moth （DBM;Plutella xylostella）. The trichoid sensilla were the most abundant sensory organ, and the male moth antennae host signiifcantly more trichoid sensilla than female moth antennae. Conversely, basiconic and coeloconic sensilla were found more frequently on female than on male antennae. We performed experiments with various degrees of antennal ablation and demonstrated that DBM antennae played a key role in the control of mating and oviposition. We found that neither oviposition preference nor mating behaviors changed signiifcantly when less than 1/4 of both antennae were removed. However, there was a signiifcant behavioral change when the antennae were ablated by more than half. As the length of the antenna was shortened, the successful mating rate decreased and mating peak was delayed. An otherwise consistent host preference for oviposition was eliminated when both antennae were completely removed. Furthermore, we found that the number of trichoid sensilla was positively correlated with mating rate and oviposition preference. However, the numbers of basiconic and coeloconic sensilla were not correlated with mating rate and mating peak, but highly correlated with oviposition preference. Taken together, our results indicate that antennal sensory information plays a critical role in the mating and oviposition behaviors of this economically important pest.     

Analysis of sex pheromone production and field trapping of the Asian corn borer(Ostrinia furnacalis Guenée) in Xinjiang,China

Identifying the sex pheromone systems of local pest populations facilitates their management, especially for moth species that show significant geographic variation in sex pheromone communication. We investigated the pheromone production and behavioral responses of the Asian corn borer(Ostrinia furnacalis Guenée; ACB) in Xinjiang, China. The ACB produces three compounds:(Z)-12-tetradecenyl acetate(Z12-14:Ac) and(E)-12-tetradecenyl acetate(E12-14:Ac)which are two sex pheromone compounds, and n-te...     

A larval specific OBP able to bind the major female sex pheromone component in Spodoptera exigua(Hübner)

Odorant binding proteins(OBPs) in insects are postulated to solubilize and transport the hydrophobic odorants across the hydrophilic antennal lymph to the olfactory receptors(ORs) located on the dendrite membrane of the sensory neurons. OBPs in adult insects have been intensively reported, but those in larvae are rarely addressed. In our study, a full-length OBP c DNA, namely Sexi OBP13, was cloned by RT-PCR and RACE strategy from the heads of Spodoptera exigua larvae. The quantitative real-time PCR(q PCR) measurement indicated that Sexi OBP13 was highly expressed in larval head, but very low in other parts of larva and was not detected in any tissues of adult. The binding affinities of Sexi OBP13 to plant volatiles and female sex pheromone components were measured by competitive binding assays. Interestingly, Sexi OBP13 displayed a high binding affinity(Ki=3.82 μmol L–1) to Z9,E12–14:Ac, the major sex pheromone component of S. exigua, while low affinities to the tested host plant volatiles(Ki27 μmol L–1). The behavioral tests further confirmed that Z9,E12–14:Ac was indeed active to elicit the behavioral activity of the third instar larvae of S. exigua. Taken together, our results suggest that Sexi OBP13 may play a role in reception of female sex pheromone in S. exigua larvae. The ecological significance of the larvae preference to the adult female sex pheromone was discussed.     

Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development

Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.     

Study on the Relationship Between Sex Differentiation and Ethylene in Ramie

To explore the role of ethylene in sexual determination in ramie, the ethylene release rates in ramie stem apex of different sex, bud of the same node in female and hermaphrodite ramie in the second crop, and single inflorescence of different sex in hermaphrodite ramie were measured by gas chromatography. Effects of two ethylene inhibitors on sex expression in ramie were investigated. The ethylene release rate of stem apex was higher in the second crop or female ramie than that in the third crop or hermaphrodite ramie during growth. Although ethylene release rates between lower nodes and higher nodes were little different, it was lower in the middle nodes in hermaphrodite ramie. The ethylene release rates were higher in the higher nodes of female ramie at the second crop. At the third crop, the ethylene release rates were higher in lower nodes, lower in middle nodes and then highest in higher nodes in female ramie. However, an opposite ethylene release pattern was observed in hermaphrodite ramie. The ethylene release rate was higher in female flower than male flower and mixed inflorescence in hermaphrodite ramie. The male flower could be distinctly induced by AVG （aminooethoxyvinylglycine）. The node of the first male flower, percentage of female flowers and ratio of female flowers to male in ramie were evidently depressed by AVG in contrast to water. The percentage of mixed male and female flowers was also increased and the percentage of female flower decreased by spraying AgNO3. There was a close relationship between sexual differentiation and ethylene release rate in ramie. The female ramie could be induced by high ethylene release rate. The female flower could be inhibited by AVG and AgNO3. AVG at a concentration of 300 mg L^-1 was most effective.     

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera （Hvbner）

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera(Hübner) was obtained from antennal cDNA libraries and expressed in Escherichia coli.The real time quantitative PCR(RT-qPCR) results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings.Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles.The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde,whereas methyl phenylacetate,2-decanone,1-pentanol,carvenol,isoborneol,nerolidol,2nonanone and ethyl heptanoate have relatively weak binding affinity.Moreover,the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38(α1),40-48(α2),62-72(α3),80-96(α4),98-108(α5),and 116-119(α6),two pairs of disulfide bridges Cys49-Cys55,Cys75-Cys78.The two amino acid residues,Ile94 and Trp101,may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.     

Cloning and Sequencing of a Gene Encoding GOBP2 in the Antenna of Spodoptera exigua

A pair of degenerate primers was designed, based on the comparison of five insects‘ GOBP2 gene sequences reported previously. A specific band (about 400bp in length) was amplified from cDNA of Spodoptera exigua antenna and another specific band (about 2kb in length) was amplified from genomic DNA.The two segments were cloned into T-easy vector, respectively. Results of sequencing and structural analysis showed that the full-length of GOBP2Sexi ORF is 426bp, 141 amino acid residues were encoded. The predicted MW and pI are 16.07ku and 5.09, respectively. There are six conservative Cys locus in the sequence, which is the typical characteristic of OBPs. GOBP2Sexi gene was inserted by two introns between amino acid residue 22 and 23 and between 82 and 83. The length of two introns is 160bp and 1403bp. Results of Northern blot showed that GOBP2 gene expressed specifically in the antenna of Spodoptera exigua, and the expression level is nearly equal in the antenna of male and female moths. The sequence was deposited in GenBank/EMBL and the accession number is AJ294809.     

斜纹夜蛾感受器超微形态特征

采用扫描电镜技术,系统研究了斜纹夜蛾触角、跗节和产卵器的感受器形态结构特征,发现触角感受器主要包括毛形、锥形、刺形、耳形和腔锥感受器.雌、雄虫触角锥形感受器形态特征区别明显,雄蛾锥形感受器长约10～15 μm,基部直径约4～5μm,显著比雌虫锥形感受器长、粗,雄蛾锥形感受器顶端有一小锥,雌蛾没有.雌成虫和雄成虫前足、中足、后足跗节均分布有毛形和刺形感受器,但数量明显少于触角同类感受器数量.雌成虫产卵器上主要为长毛形和短毛形感受器,这些感受器可能与其感受环境信息、产卵场所选择等行为有关.

Abstract：

The ultrastructures of the sensilla on the antennae, the tarsi and the ovipositor of Spodoptera litura were observed with scanning electron-microscopy. The antennae of both male and female moths were found to contain five kinds of antennal sensilla, namely, sensillum trichodeum, sensillum basiconicum,sensillum chaeticum, ear-shaped sensillum and sensillum coeloconicum. The characters of sensillums basiconicum on the antennae were different between the male and the female. The length was 10-15 μm, the diameter at the base was approximately 4-5 μm for the male antennae, which were significantly longer and bigger than the female antennae, and the male moths had onesmall bradawl at the bass of the antennae while the female moths did not. The tarsi of both male and female moths contained two kinds of sensilla:sensillum trichodeum and sensillum chaeticum; however, their numbers were significant less than those on the antennae. The long sensillun trichodeum and short sensillun trichodeum were mainly distributed on the ovipositor. These results provided basis for exploring the molecular mechanism of oviposition deterrent.     

Characterization of two novel heat shock protein 70s and their transcriptional expression patterns in response to thermal stress in adult of Frankliniella occidentalis (Thysanoptera: Thripidae)

Heat shock protein 70(HSP70) is one of the most important members in the heat shock protein family, and plays important roles in the thermotolerance of insect. To explore the molecular mechanism of thermotolerance of Frankliniella occidentalis adults, the difference in the expression of HSP70s in F. occidentalis male or female adults under the thermal stress was studied under the laboratory conditions. Two full length c DNAs of HSP70s gene(Fohsc704 and Fohsc705) were cloned from F. occidentalis by using RT-PCR and RACE. The genomic sequence was demonstrated by genomic validation, and the position and size of the intron were analyzed by sequence analysis of c DNA. Real-time PCR was used to analyze the HSP70 expression patterns. The c DNA of Fohsc704 and Fohsc705 possessed 2 073 and 1 476 bp which encoded 690 and 491 amino acids(aa) with a calculated molecular weight of 75 and 54 k Da, respectively. Four introns in Fohsc704 and six introns in Fohsc705 protein were found. However, the HSP70 protein sequences in our study were ended with EKKN and GIFL, which were different from the reported Fo HSP70s. Various expression patterns of Fohsc704 and Fohsc705 were found in both genders of F. occidentalis under thermal stress. The expression of Fohsc704 and Fohsc705 reached to the highest level at –12 and –8°C in male adults, respectively, and Fohsc705 expressed the highest level at 33°C in female adults. In conclusion, HSP70s of F. occidentalis in our study are novel heat shock proteins. There were difference in expression patterns of the two hsc70s in genders of F. occidentalis, and the two HSP70s play important roles in the thermotolerance of F. occidentalis.     

Gene Cloning and Tissue-Specific Microplitis mediator （Hymenoptera： Expression of G Protein β Subunit in Braconidae）

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator （Hymenoptera： Braconidae）. The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head （without antennae）, thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

Optimization of the sex pheromone-based method for trapping field populations of Phthorimaea operculella(Zeller) in South China

Despite the identification of the potato tuber moth Phthorimaea operculella(Zeller) sex pheromone, no effective application based on this pheromone has yet been developed and evaluated. This study investigated the effect of pheromone lures, trap densities, heights of trap deployment, and pheromone doses in Yunnan, China, for the purpose of increasing the control efficiency of P. operculella and improving the application of pheromone technology in the field. The results showed that lures made of corn oil and red PVC pipes attracted the highest number of moths(11.73±1.90 per trap per day). Sex pheromone loading of 100 μg was optimal for trapping moths, but higher doses of pheromone inhibited attraction. The density of traps did not affect capture rates; therefore, the optimum trap density was 30–40 traps ha~(–1). The optimum height of trap deployment was not above the height of the plant canopy. This study provides technical details necessary for the monitoring and control of potato tuber moth using sex pheromones.     

Gene Cloning and Tissue-Specific Expression of G Protein β Subunit in Microplitis mediator (Hymenoptera: Braconidae)

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator (Hymenoptera: Braconidae). The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head (without antennae), thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

RNA Interference-Mediated Downregulation of sAC Gene Inhibits Sperm Hyperactivation in Male Rats （Rattus norvegicus）

Hyperactivation is one of the most critical parts for fertilization, cAMP generated by soluble adenylyl cyclase （sAC） is necessary to activate sperm and is a prerequisite for sperm hyperactivation. The aim of this study is to investigate the function of sAC in hyperactivation in male rats. Four siRNAs of sAC gene were designed and separately transformed into rat sperm using electrotransformation method. Cultured for 12 and 24 h, physiological and biochemical indexes of these sperm were analyzed, and the expressions of some hyperactivation-related genes were detected using real-time PCR. We demonstrated 26.3-30.8% and 49.1-50.5% reduction in sAC at the protein by Western blot and mRNA levels by real-time PCR, respectively. The results showed that two siRNAs, Actb-717 and Actb-4205, were the best RNAi sites for silencing sAC. The VCL （curvilinear velocity） and ALH （amplitude of lateral head displacement） of RNA interference （RNAi）-transfected sperm were reduced, cAMP and protein phosphorylation in RNAi transfected sperm were also decreased. The hyperactivation-related genes, such as CatSper2, LDHC and PKA, were downregulated in the sperm, which sAC was knockdown. These findings demonstrated that sAC might play a critical role in cAMP signaling in the rat sperm hyperactivation, and downregulated sAC gene might prevent the expression of these hyperactivation-ralated genes resulting in sperm dysfunction. These findings suggest that these hyperactivation-ralated genes and sAC are functionally related in sperm hyperactivation and sAC falls into an expanding group of sperm proteins that appear to be promising targets for the development of male contraceptives.     

RNA Interference-Mediated Downregulation of sAC Gene Inhibits Sperm Hyperactivation in Male Rats(Rattus norvegicus)

Hyperactivation is one of the most critical parts for fertilization.cAMP generated by soluble adenylyl cyclase(sAC)is necessary to activate sperm and is a prerequisite for sperm hyperactivation.The aim of this study is to investigate the function of sAC in hyperactivation in male rats.Four siRNAs of sAC gene were designed and separately transformed into rat sperm using electrotransformation method.Cultured for 12 and 24 h,physiological and biochemical indexes of these sperm were analyzed,and the expressions of some hyperactivation-related genes were detected using real-time PCR.We demonstrated26.3-30.8%and 49.1-50.5%reduction in sAC at the protein by Western blot and mRNA levels by real-time PCR,respectively.The results showed that two siRNAs,Actb-717 and Actb-4205,were the best RNAi sites for silencing sAC.The VCL(curvilinear velocity)and ALH(amplitude of lateral head displacement)of RNA interference(RNAi)-transfected sperm were reduced.cAMP and protein phosphorylation in RNAi transfected sperm were also decreased.The hyperactivation-related genes,such as CatSper2,LDHC and PKA,were downregulated in the sperm,which sAC was knockdown.These findings demonstrated that sAC might play a critical role in cAMP signaling in the rat sperm hyperactivation,and downregulated sAC gene might prevent the expression of these hyperactivation-ralated genes resulting in sperm dysfunction.These findings suggest that these hyperactivation-ralated genes and sAC are functionally related in sperm hyperactivation and sAC falls into an expanding group of sperm proteins that appear to be promising targets for the development of male contraceptives.     

Differentiation of expression profi les of two calcineurin subunit genes in chicken skeletal muscles during early postnatal growth depending on anatomical location of muscles and breed

Calcineurin(Cn or CaN) is implicated in the control of skeletal muscle fiber phenotype and hypertrophy. However, little information is available concerning the expression of Cn in chickens. In the present study, the expression of two Cn subunit genes(Cn Aα and Cn B1) was quantified by q PCR in the lateral gastrocnemius(LG, mainly composing of red fast-twitch myofibers), the soleus(mainly composing of red slow-twitch myofibers) and the extensor digitorum longus(EDL, mainly composing of white fast-twitch myofibers) from Qingyuan partridge chickens(QY, slow-growing chicken breed) and Recessive White chickens(RW, fast-growing chicken breed) on different days(1, 8, 22, 36, 50 and 64 days post-hatching). Although Cn Aα and Cn B1 gene expressions were variable with different trends in different skeletal muscles in the two chicken breeds during postnatal growth, it is highly muscle phenotype and breed specific. In general, the levels of Cn Aα and Cn B1 gene expressions of the soleus were lower than those of EDL and LG in both chicken breeds at the same stages. Compared between the two chicken breeds, the levels of Cn Aα gene expression of the three skeletal muscles in QY chickens were higher than those in RW chickens on days 1 and 22. However, on day 64, the levels of both Cn Aα and Cn B1 gene expressions of the three skeletal muscles were lower in QY chickens than those in RW chickens. Correlation analysis of the levels of Cn Aα and Cn B1 gene expressions of the same skeletal muscle showed that there were positive correlations for all three skeletal muscle tissues in two chicken breeds. These results provide some valuable clues to understand the role of Cn in the development of chicken skeletal muscles, with a function that may be related to meat quality.     

Genome-Wide Expression Profile of Maize Root Response to Phosphorus Deficiency Revealed by Deep Sequencing

Phosphorus （P） is one of the three primary macronutrients that are required in large amounts for plant growth and development. To better understand molecular mechanism of maize and identify relevant genes in response to phosphorus deficiency, we used Solexa/Illumina＇s digital gene expression （DGE） technology to investigate six genome-wide expression profiles of seedling roots of the low-P tolerant maize inbred line 178. DGE studies were conducted at 6, 24 and 72 h under both phosphorus deficient and sufficient conditions. Approximately 3.93 million raw reads for each sample were sequenced and 6 816 genes exhibited significant levels of differential expressions in at least one of three time points in response to P starvation. The number of genes with increased expression increased over time from 6 to 24 h, whereas genes with decreased expression were more abundant at 72 h, suggesting a gradual response process for P deficiency at different stages. Gene annotations illustrated that most of differentially expressed genes （DEGs） are involved in different cellular and molecular processes such as environmental adaptation and carbohydrate metabolism. The expression of some known genes identified in other plants, such as those involved in root architecture, P metabolism and transport were found to be altered at least two folds, indicating that the mechanisms of molecular and morphological adaptation to P starvation are conserved in plants. This study provides insight into the general molecular mechanisms underlying plant adaptation to low-P stress and thus may facilitate molecular breeding for improving P utilization in maize.     

Gene Expression and Fungal Morphology in Submerged Culture Using Whole Barley of Aspergillus kawachii

The authors described a novel submerged batch culture system that produced high levels of amylase by Aspergillus kawachii using whole barley （WB）, the surface of which is covered by its husk. In this study, detailed analyses determining the amylase activities, residual sugars, fungal morphology and expression levels of genes were performed in a submerged culture using WB to address the mechanism underlying high amylase productivity in A. kawachii. High levels of glucoamylase and acid-stable u-amylase were produced in this culture, and expression levels of amylases, as well as glucose-repressive genes including high-affinity glucose transporter and peroxidase/catalase were also high. On the other hand, the morphology of mycelia was altered, with swollen, bulbous, multi-septum hyphae and conidiophores that normally form in a solid culture being partially generated. Furthermore, cell cycle and post-translational modification-related gene expression levels were altered, and were similar to those in the solid culture. These findings suggest that high amylase productivity in the submerged culture using WB is accompanied by both the up-regulation of amylase genes and activation of post-translational modifications due to fungal morphological changes being brought closer to those in the solid culture.     

Identification and Bioinformatics Analysis of SnRK2 and CIPK Family Genes in Sorghum

Through bioinformatic data mining, 10 SnRK2 and 31 CIPK genes were identified from sorghum genome. They are unevenly distributed in the sorghum chromosomes. Most SnRK2 genes have 8 introns, while the CIPK genes have a few （no intron or less than 3 introns） or more than I0 introns. Phylogenetic analysis revealed that SnRK2 genes belong to one cluster and CIPK genes form the other independent cluster. The sorghum SnRK2s are subgrouped into three parts, and CIPK into five parts. More than half SnRK2 and CIPK genes present in homologous pairs, suggesting gene duplication may be due to the amplification of SnRK family genes. The kinase domains of SnRK2 family are highly conserved with 88.40% identity, but those of the CIPK family are less conserved with 63.72% identity. And the identity of sorghum CBLinteracting NAF domains of CIPKs is 61.66%. What＇s more, regarding to the sorghum SnRK2 and CIPK kinases, they are characterized with distinct motifs and their subcellular localization is not necessarily the same, which suggests they may be divergent in functions. Due to less conserved sequences, complex subcellular localization, and more family members, sorghum CIPK genes may play more flexible and multiple biological functions. According to the phylogenetic analysis of SnRK genes and SnRK functional studies in other plants, it is speculated that sorghum SnRK2 and CIPK genes may play important roles in stress response, growth and development.