UBE2I stimulates female gonadal differentiation in chicken (Gallus gallus) embryos

Without known analogous sex-determining factors like SRY (sex determining region Y) in mammals, the chicken (Gallus gallus) sex determination mechanism still remains unclear, which highly restricts the biological research on chicken development and poultry single-sex reproduction. Here we not only characterized a new female-biased gene UBE2I and identified the expression pattern by qRT-PCR, but also described the functional role of UBE2I in the gonadal development of chicken embryos. Results showed that UBE2I exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads and robust expression in ovaries of newborn chickens. Most importantly, we successfully developed an effective method to interfere or overexpress UBE2I in chicken embryos through the intravascular injection. The qRT-PCR analysis showed that the sex-related genes (FOXL2, CYP19A1 and HINTW) in females were upregulated (P<0.05) under the overexpression of UBE2I and the sex-related genes (SOX9, DMRT1 and WT1) in females were downregulated (P<0.05) after interfering UBE2I. Furthermore, the change of UBE2I expression was associated with the level of estradiol and its receptors (AR and ESR), which suggests that UBE2I is necessary to initiate the female-specific development in chickens. In conclusion, this work demonstrates that UBE2I is a crucial sex differentiation-related gene in the embryonic development of chickens, which provides insights for further understanding the mechanism of sex determination in chickens.     

中国梨木虱冬型成虫性信息素成分的提取和GC-MS鉴定

通过鉴定中国梨木虱性信息素的成分,为进一步开发其性引诱剂提供基础资料.本试验利用正己烷分别提取梨木虱冬型雌、雄成虫体表浸提物,用Y型嗅觉仪测试了浸提物对中国梨木虱冬型雌和雄成虫行为反应的影响,并运用气相色谱-质谱联用仪对浸提物进行了分析和鉴定.雌虫浸提物对雄性梨木虱有较强的引诱作用,雄虫浸提物对雄木虱,雌、雄浸提物对雌木虱均无引诱作用,说明雌虫浸提物中含有雌木虱释放的性信息素.气相色谱-质谱联用仪(GC-MS)分析结果表明,雌虫浸提物中13-甲基二十七烷和2-甲基二十七烷的含量多于雄虫浸提物中的含量.在保留时间为23.136 min时,雌虫浸提物出现色谱峰,而雄虫浸提物没有出峰.这3种化合物可能是中国梨木虱冬型雌成虫的性信息素成分.     

Accurate recognition of the reproductive development status and prediction of oviposition fecundity in Spodoptera frugiperda (Lepidoptera: Noctuidae) based on computer vision

Spodoptera frugiperda (Lepidoptera: Noctuidae) is an important migratory agricultural pest worldwide, which has invaded many countries in the Old World since 2016 and now poses a serious threat to world food security. The present monitoring and early warning strategies for the fall army worm (FAW) mainly focus on adult population density, but lack an information technology platform for precisely forecasting the reproductive dynamics of the adults. In this study, to identify the developmental status of the adults, we first utilized female ovarian images to extract and screen five features combined with the support vector machine (SVM) classifier and employed male testes images to obtain the testis circular features. Then, we established models for the relationship between oviposition dynamics and the developmental time of adult reproductive organs using laboratory tests. The results show that the accuracy of female ovary development stage determination reached 91%. The mean standard error (MSE) between the actual and predicted values of the ovarian developmental time was 0.2431, and the mean error rate between the actual and predicted values of the daily oviposition quantity was 12.38%. The error rate for the recognition of testis diameter was 3.25%, and the predicted and actual values of the testis developmental time in males had an MSE of 0.7734. A WeChat applet for identifying the reproductive developmental state and predicting reproduction of S. frugiperda was developed by integrating the above research results, and it is now available for use by anyone involved in plant protection. This study developed an automated method for accurately forecasting the reproductive dynamics of S. frugiperda populations, which can be helpful for the construction of a population monitoring and early warning system for use by both professional experts and local people at the county level.     

Development of a high-efficiency sex pheromone formula to control Euproctis pseudoconspersa

The tea tussock moth (Euproctis pseudoconspersa) is one of the most destructive chewing pests in tea plantations and causes a serious allergic reaction on the skin of tea plantation workers. The sex pheromone components of its Japanese population were first identified as 10,14-dimethylpentadecyl isobutyrate (10Me14Me-15:iBu) and 14-methylpentadecyl isobutyrate (14Me-15:iBu), with a stereogenic center. Only 10Me14Me-15:iBu has been identified in the Chinese E. pseudoconspersa population. However, field tests have shown that 10Me14Me-15:iBu cannot meet the demand of effective pest control in China. To develop a high-efficiency E. pseudoconspersa sex pheromone formula, electroantennogram (EAG) recordings of (S)- and (R)-enantiomers of 10Me14Me-15:iBu and 14Me-15:iBu were obtained in the present study. The results demonstrated that the EAG responses of male antennae to (R)-enantiomers were significantly higher than responses to the (S)-enantiomers, and 14Me-15:iBu also elicited EAG activity. Field tests showed that the catch numbers of male moths by (R)-enantiomers were significantly higher (P<0.05) than those of (S)-enantiomers. Addition of 14Me-15:iBu significantly increased the catch numbers of both the (S)- and (R)-enantiomers. The efficient pheromone formula containing 0.75 mg (R)-10Me14Me-15:iBu and 0.1 mg 14Me-15:iBu showed significantly higher attractiveness than commercial pheromone products. Our study demonstrated that (R)-10Me14Me-15:iBu was the major sex pheromone component of E. pseudoconspersa, and 14Me-15:iBu might be the minor sex pheromone component. Furthermore, a high-efficiency sex pheromone formula for E. pseudoconspersa control was defined in this study.     

Volatiles from Sophora japonica flowers attract Harmonia axyridis adults (Coleoptera: Coccinellidae)

The multicolored Asian lady beetle, Harmonia axyridis(Coleoptera: Coccinellidae), is a common generalist predator in China and is occasionally found gathering on the Chinese pagoda tree, Sophora japonica, in summer. In a field investigation, we found that H. axyridis adults preferred S. japonica during its flowering period even though their optimal prey(aphid) is absent at this time. In addition, male and female adults were attracted to S. japonica flowers to a similar extent in a Y-tube olfactometer assay. Using coupled gas chromatography-electroantennogram detection(GC-EAD), we identified a flower odor component(nonanal) that elicited a significant electrophysiological response in H. axyridis. Electroantennogram(EAG) dose-dependent responses revealed that the amplitude of the adult beetle's EAG response increased with increasing concentration of nonanal, peaking at 10 mg mL~(–1). In Y-tube olfactometer behavioral tests, H. axyridis adults preferred a 10 mg mL~(–1) nonanal source over a 100 mg mL~(–1) diluent. Under field conditions, the adults were significantly attracted to both concentrations(10 and 100 mg mL~(–1)), and high concentrations generally had greater attraction. All these results suggest that nonanal, a volatile compound of S. japonica flowers, greatly attracts H. axyridis adults. This study provides a basis for the development of synthetic attractants of H. axyridis, with the potential to promote biocontrol services of this generalist predator in the native area(e.g., China) and to suppress its population by mass trapping in its invasive areas.     

Estimation of the potential geographical distribution of a new potato pest (Schrankia costaestrigalis) in China under climate change

Global food security is threatened by the impacts of the spread of crop pests and changes in the complex interactions between crops and pests under climate change. Schrankia costaestrigalis is a newly-reported potato pest in southern China. Early-warning monitoring of this insect pest could protect domestic agriculture as it has already caused regional yield reduction and/or quality decline in potato production. Our research aimed to confirm the potential geographical distributions (PGDs) of S. costaestrigalis in China under different climate scenarios using an optimal MaxEnt model, and to provide baseline data for preventing agricultural damage by S. costaestrigalis. Our findings indicated that the accuracy of the optimal MaxEnt model was better than the default-setting model, and the minimum temperature of the coldest month, precipitation of the driest month, precipitation of the coldest quarter, and the human influence index were the variables significantly affecting the PGDs of S. costaestrigalis. The highly- and moderately-suitable habitats of S. costaestrigalis were mainly located in eastern and southern China. The PGDs of S. costaestrigalis in China will decrease under climate change. The conversion of the highly- to moderately-suitable habitat will also be significant under climate change. The centroid of the suitable habitat area of S. costaestrigalis under the current climate showed a general tendency to move northeast and to the middle-high latitudes in the 2030s. The agricultural practice of plastic film mulching in potato fields will provide a favorable microclimate for S. costaestrigalis in the suitable areas. More attention should be paid to the early warning and monitoring of S. costaestrigalis in order to prevent its further spread in the main areas in China’s winter potato planting regions.     

Diagnosis and characterization of the ribosomal DNA-ITS of potato rot nematode (Ditylenchus destructor) populations from Chinese medicinal herbs

The potato rot nematode (Ditylenchus destructor) is a very economically important nematode in agronomic and horticultural plants worldwide. In this study, 43 populations of D. destructor were collected from different hosts across China, including 37 populations from Chinese herbal medicine plants. Obtained sequences of ITS-rDNA and D2–D3 of 28S-rDNA genes of D. destructor were compared and analyzed. Nine types of significant length variations in ITS sequences were observed among all populations. The differences in ITS1 length were mainly caused by the presence of repetitive elements with substantial base substitutions. Reconstructions of ITS1 secondary structures showed that the minisatellites formed a stem structure. Ten haplotypes were observed in all populations based on mutations and variations of helix H9. Among them, 3 known haplotypes (A–C) were found in 7 populations isolated from potato, sweet potato, and Codonopsis pilosula, and 7 unique haplotypes were found in other 36 populations collected from C. pilosula and Angelica sinensis compared with 7 haplotypes (A–G) according to Subbotin’ system. These unique haplotypes were different from haplotypes A–G, and we named them as haplotypes H–N. The present results showed that a total of 14 haplotypes (A–N) of ITS-rDNA have been found in D. destructor. Phylogenetic analyses of ITS-rDNA and D2–D3 showed that all populations of D. destructor were clustered into two major clades: one clade only containing haplotype A from sweet potato and the other containing haplotypes B–N from other plants. For further verification, PCR-ITS-RFLP profiles were conducted on 7 new haplotypes. Collectively, our study suggests that D. destructor populations on Chinese medicinal materials are very different from those on other hosts and this work provides a paradigm for relevant researches.     

Effects of rearing system(floor vs. cage) and sex on performance,meat quality and enteric microorganism of yellow feather broilers

This study investigated the effects of floor and cage rearing systems on performance, meat quality, and enteric microorganisms of male and female yellow feather broilers. Two hundred Xueshan chickens(42 days old; 100 of each sex) were divided into four groups, according to sex and rearing system(stainless-steel cage or litter floor). Male and cage-reared broilers exhibited better(P0.001) performance and higher(P≤0.001) eviscerated percentage than female and floor-reared broilers. The interaction between rearing system and sex had an effect(P0.05) on the percentage of breast and thigh muscles. Female floor-reared broilers presented the highest(P0.02) percentage of breast and thigh muscles among all the groups. In both sexes, the abdominal fat content in cage-reared broilers was higher(P0.01) than that in floor-reared broilers. Female cage-reared broilers exhibited better(P0.05) meat quality of breast muscle than other groups. An analysis of welfare observations indicated that the feather quality of floor-reared broilers was better(P0.01) than that of cage-reared broilers, whereas the gait scores were not significantly affected(P0.05) by rearing system. The enteric microbiota assessment by 16 S rRNA amplicon-based sequencing showed that Firmicutes, Proteobacteria, and Bacteroidetes were the most prevalent phyla in both rearing systems, and that the cecum was relatively stable in terms of microbiome composition. Floor-reared broilers exhibited a richer diversity of bacteria in the intestinal tract than cage-reared broilers, while alpha diversity was not significantly different(P0.05) among the groups. The ratio of Bacteroidetes to Firmicutes and the abundance of Helicobacter and Romboutsia could potentially affect the production of broilers. These findings indicate that cage rearing improved the production of male Xueshan broilers, which may be due to the difference in enteric microbiota between cage and floor rearing systems.     

中西印度洋的黄鳍金枪鱼繁殖生物学研究

根据我国国家金枪鱼渔业观察员2012年10月-2013年1月和2013年10月-2014年2月两个阶段在印度洋中西部采集1233尾黄鳍金枪鱼的样本,对其进行了繁殖生物学的研究,主要分析黄鳍金枪鱼纯重、叉长、性比、性成熟率和性腺指数等繁殖生物学参数。结果表明:(1)雌性雄性叉长-纯重关系式分别是W=7×10-6L3.244和W=6×10-6L3.197;(2)雌雄之比为0.74∶1,达到性成熟的雌雄个体性别比为0.69∶1;(3)雌性性腺指数为0.39~5.92,在雌性个体中性腺指数大于2的占52.03%;雄性性腺指数为0.44~3.37,在雄性个体中性腺指数大于2的占31.43%。(4)雌、雄性成熟叉长的Logistic曲线分别是P=1/[1+e0.08276×(122.991-FLm)]和P=1/[1+e0.03767×(118.618-FLm)];(5)采样期间(10月至翌年2月)雌、雄性个体分别在12月和1月的性成熟率达到最高,在2月份性成熟率最低;(6)根据体长分布推算,叉长为115 cm可以作为黄鳍金枪鱼个体分为大小两个群体的基准,雌雄样本中大个体所占比例分别是63.13%和75.40%,两个群体中,雌雄个体大小存在显著性差异(P0.05)。本文的研究结果有助于更好地了解印度洋西部黄鳍金枪鱼的生物学特征,为我国进一步开发该种渔业资源提供基础资料。     

Mutagenesis of odorant coreceptor Orco reveals the distinct role of olfaction between sexes in Spodoptera frugiperda

Odorant receptor (OR) is crucial for insects to detect and recognize external chemical cues closely related to their survival. The insect OR forms a heteromeric complex composed of a ligand-specific receptor and a ubiquitously odorant receptor coreceptor (Orco). This study used the CRISPR/Cas9 technique to knock out (KO) Orco and reveal its essential role in acting on OR-meditated olfactory behavior in a critical invasive agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda. Electroantennogram (EAG) results suggested that the Orco mutants of both male and female moths severely reduced their electrophysiological responses to the eight tested plant volatiles and two sex pheromones. However, the Orco gene played distinct roles in mating behavior between sexes: the mating behavior was fully disrupted in mutated males but not in mutated females. The oviposition result indicated that the Orco KO females displayed reduced egg laying by 24.1% compared with the mated wild type (WT) females. Overall, these results strongly suggest that Orco is an excellent target for disrupting FAW’s normal behavior and provides a feasible pest control approach.     

笔管榕榕小蜂的群落结构与物种多样性

笔管榕为桑科榕属落叶乔木,雌雄同株,为福建地区的常见树种。笔管榕榕果内生活着多种榕小蜂,它们与笔管榕共同构成了榕树-榕小蜂共生体系。通过全年(2011年7月至2012年7月间)对福州地区不同样地的30株笔管榕进行的定时、定点、定株观察与采集,在15批次830个榕果中,共收集到50817头榕小蜂。笔管榕榕果内有7种小蜂,隶属于小蜂总科中的6个科(亚科)7个属,其中榕小蜂科的Platyscapa ishiiana为传粉小蜂,其余6种为非传粉小蜂,分别属于金小蜂科、广肩小蜂科、姬小蜂科及隐针榕小蜂亚科。非传粉小蜂中的Philotrypesis sp.、Acophila mikii、Otitesella ako、Sycophila sp.和Sycobiomorpha sp.小蜂几乎在全年的每个月都可采集到,属常见种。Aprostocetus sp.全年仅收集到2头,为偶见种。笔管榕榕果内的榕小蜂种类在不同月份、不同季节没有明显变化,但不同种类榕小蜂的数量变化明显。在笔管榕榕小蜂群落结构中,全年有9个月均是传粉小蜂占优势,但在1月、9月和11月的采集批次中,非传粉小蜂占优势。非传粉小蜂(Acophila mikii、Sycophila sp.、Philotrypesis sp.和Otitesella ako)常呈爆发性发生,每批次中往往是1-2种的非传粉小蜂数量较多,但不同批次,数量较多的非传粉小蜂种类往往不同,因此在小蜂群落结构中未见某种非传粉小蜂占明显优势。福州地区笔管榕榕果内小蜂群落的的多样性指数为0.72,均匀度指数为0.37,丰富度指数为0.55,优势集中性指数为0.68。分析了6种小蜂的性比,结果表明:传粉小蜂的性比极度偏雌,非传粉小蜂虽然也偏性,但均高于传粉小蜂,推测榕小蜂性比与雄蜂的翅型和交配场所有关。非传粉小蜂的数量和传粉小蜂总数、雌蜂数和雄蜂数均呈极显著负相关,和性比呈极显著正相关关系,说明非传粉小蜂对传粉小蜂的雌、雄蜂都有负面影响,且传粉小蜂雌蜂减少的幅度大于传粉小蜂雄蜂,从而导致其性比升高。非传粉小蜂中Acophila mikii、Sycobiomorpha sp.、Otitesella ako Ishii为造瘿类群,Philotrypesis sp.、Sycophila sp.属于寄居者或寄生者类群,非传粉小蜂中的寄生或寄居类群与造瘿类群相比,对传粉小蜂的负面影响更大。研究结果为榕-蜂协同进化研究,以及城市绿化和生物多样性保护提供科学依据。     

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.     

Selection,effective dominance,and completeness of Cry1A.105/Cry2Ab2 dual-protein resistance in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)

In the U.S., Helicoverpa zea (Boddie) is a major pest targeted by both transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins. Resistance of insect to Bt maize and cotton containing cry1A and cry2A genes has widely occurred in the U.S. In this study, two trials were performed to investigate larval survival and development of a Cry1A.105/Cry2Ab2 dual-protein resistant (VT2P-RR), a susceptible, and an F₁ heterozygous (VT2P-RS) populations of H. zea on ears of nine Bt and three non-Bt maize hybrids. The Bt maize hybrids evaluated represent five common pyramided traits expressing two or three of the Cry1A.105, Cry1Ab, Cry1F, Cry2Ab2, and Vip3Aa20 proteins. In the laboratory, neonates of the three H. zea populations were inoculated on silks of ears collected from maize at R1–R2 plant stages; and larval survivorship was checked 10 d after neonate release. All three insect populations survived normally on non-Bt maize ears. Varied numbers of VT2P-RR and VT2P-RS survived on ears of Cry1A.105/Cry2Ab2 maize, while all larvae of the three populations died or could not develop on ears of Vip3Aa20-expressing maize. The results demonstrated that the dual-protein resistant H. zea was not cross-resistant to Vip3Aa20-expressing maize, and thus traits with vip3Aa20 gene should be effective to manage Cry1A.105/Cry2Ab2-resistant H. zea. The resistance in VT2P-RR was determined to be incomplete on Cry1A.105/Cry2Ab2 maize. The effective dominance levels varied greatly, from recessive to incompletely dominant, depending on maize hybrids and trials, suggesting that proper selection of maize hybrids could be important for mitigating the Cry1A.105/Cry2Ab2 resistance. The data generated should aid in modeling multiple-protein Bt resistance in H. zea.     

寄主大小及寄生顺序对蝇蛹佣小蜂寄生策略的影响

寄主大小模型认为寄生蜂后代性比与寄主大小相关,寄生蜂倾向于在大寄主上产出更多雌性后代,在小寄主上产出更多雄性后代。探讨了以家蝇蛹为寄主时,蝇蛹佣小蜂后代产量和性比变化;单次寄生情况下,寄主大小及寄生顺序对寄生蜂后代性比等影响。结果表明,蝇蛹佣小蜂的产卵期为(8.93±3.34) d,单头雌蜂能产雌性后代(34.11±16.34)头和雄性后代(11.04±8.87)头,且雄性百分比为0.24±0.11。随成蜂日龄的增大,寄生蜂产生雄性后代的比率显著增加。蝇蛹佣小蜂在寄生家蝇蛹时,会优先选择寄生个体较大的蛹;在单次寄生的情况下,蝇蛹佣小蜂倾向于在较大的家蝇蛹内产出更多的雌性后代。     

甘蔗转化酶抑制子(SoInvInh1)基因克隆和表达分析

采用cDNA末端快速克隆(Rapid amplification of cDNA ends,RACE)技术,以甘蔗未成熟茎cDNA为模板克隆转化酶抑制子,命名为SoInvInh1,GenBank登录号KF575175。SoInvInh1基因的cDNA序列全长为678bp,3′-UTR长146bp。开放阅读框长531bp,编码176个氨基酸,预测分子质量和等电点分别为18.09ku和8.52。SoInvInh1不含内含子序列。预测该蛋白N端具有1个信号肽和跨膜结构,19—172位氨基酸是PMEI蛋白的保守结构域。不同物种间InvInh蛋白氨基酸序列同源性较低,但都具有4个保守的Cys位点。荧光定量PCR分析表明在甘蔗茎、叶、花序和花序轴中都能检测到SoInvInh1基因表达。在甘蔗生长的不同时期,SoInvInh1在叶中表达无明显规律。而在工艺成熟期和生理成熟前期SoInvInh1整体上表现为幼茎中基因表达量高,而老茎中基因表达量低,表明茎中该基因可能与酸性转化酶活性相关。     

Molecular diagnosis and direct quantification of cereal cyst nematode (Heterodera filipjevi) from field soil using TaqMan real-time PCR

Heterodera filipjevi continues to be a major threat to wheat production worldwide. Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease. In the present study, a TaqMan-minor groove binder (TaqMan-MGB) probe-based fluorescence quantitative real-time PCR (qPCR) was successfully developed and used for quantifying H. filipjevi from DNA extracts of soil. The primers and probe designed from the obtained RAPD-SCAR marker fragments of H. filipjevi showed high specificity to H. filipjevi using DNA from isolates-confirmed species of 23 Heterodera spp., 1 Globodera spp. and 3 Pratylenchus spp. The qPCR assay is highly sensitive and provides improved H. filipjevi detection sensitivity of as low as 4^–3 single second-stage juvenile (J2) DNAs, 10^–3 female DNAs, and 0.01 μg μL^–1 genomic DNAs. A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H. filipjevi in naturally infested field soils. There was a high correlation between the H. filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay. qPCR potentially provides a useful platform for the efficient detection and quantification of H. filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.