三叶斑潜蝇热激蛋白Hsp90基因的克隆及其在高温胁迫下的表达分析

采用RT-PCR及RACE技术克隆了三叶斑潜蝇Hsp90基因全长cDNA序列,并用实时定量RT-PCR的方法检测其在不同发育阶段受到高温胁迫后的表达水平。该基因的cDNA序列全长2 408 bp,开放阅读框为2 145 bp,编码714个氨基酸;5′非编码区为151 bp,3′非编码区为112 bp。该基因推导的氨基酸序列与其他昆虫同源序列比较有很高的相似性（80%~99%）。聚类分析结果显示三叶斑潜蝇与美洲斑潜蝇和南美斑潜蝇的亲缘关系最近。实时荧光定量PCR检测结果表明三叶斑潜蝇Hsp90基因的表达受到热胁迫的诱导,诱导3龄幼虫最大表达量的温度比诱导其他发育阶段的温度低,在43 ℃时预蛹和蛹的表达量在整个生命周期中最高,在检测的高温胁迫条件下,雄虫比雌虫的表达量更高。该结果为阐明三叶斑潜蝇胁迫耐受能力及其对其他潜蝇种群的取代机制奠定了分子基础。     

温度胁迫下沙葱萤叶甲小热激蛋白基因GdHsp20.6的克隆及表达分析

为明确小热激蛋白在沙葱萤叶甲Galeruca daurica应对温度胁迫中的作用,采用PCR方法克隆沙葱萤叶甲小热激蛋白基因Hsp20（GdHsp20.6）完整的开放阅读框（open reading frame,ORF）序列,应用在线软件对GdHsp20.6基因进行生物信息学分析,通过原核表达技术诱导表达及纯化其编码蛋白,并通过实时荧光定量PCR（real-time quantitative PCR,RT-qPCR）技术分析不同温度胁迫下GdHsp20.6基因的表达量。结果显示,GdHsp20.6基因ORF序列长度为543 bp,编码180个氨基酸,预测分子量为20.6 kD,无跨膜区和信号肽。GdHsp20.6氨基酸序列有高度保守的α-结构域。GdHsp20.6氨基酸序列与其它鞘翅目昆虫的Hsp20氨基酸序列有较高的一致性,其中与花绒寄甲Dastarcus helophoroides的Hsp20.99氨基酸序列的一致性最高,为63%。GdHsp20.6基因在大肠杆菌Escherichia coli BL21（DE3）细胞系中成功表达,经异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-β-d-thiogalactoside,IPTG）诱导后GdHsp20.6蛋白成功表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）和Western-blot分析表明融合蛋白大小与预测大小一致,并纯化获得了纯度较高的目的蛋白GdHsp20.6。低温（-10~5℃）和高温（35~40℃）处理1 h以及处理后25℃恢复30 min均能诱导GdHsp20.6基因表达上调,并且0℃处理30~120 min也能诱导GdHsp20.6基因表达上调。表明GdHsp20.6基因在沙葱萤叶甲应对低温和高温胁迫中可能起着重要作用。     

三叶草斑潜蝇hsp70的克隆及其表达量在高低温胁迫下的变化

为了明确高低温胁迫对三叶草斑潜蝇hsp70表达量的影响,采用RT-PCR和RACE技术获得了1条三叶草斑潜蝇诱导型热激蛋白基因hsp70,命名为Lthsp70-1,并利用实时荧光定量PCR技术检测其在温度胁迫后的表达量.该基因的开放阅读框为1923 bp,编码640个氨基酸.氨基酸序列中含有HSP70家族的签名序列IFDLGGGTFDVSIL和IVLVGGSTRIPK、DnaK特征基序IDLGTT(Y)S(C)V、非细胞器基序RARFEEL,以及C末端的保守序列EEVD.实时荧光定量PCR结果显示:成虫在31～33℃范围内,其hsp70表达量随温度升高而上升,33℃时达到最高峰;35～39℃时,hsp70表达量迅速下降;蛹经0℃胁迫0.5～2.0 h,其hsp70表达量随时间延长呈上升趋势.由此可见,高低温胁迫均能诱导三叶草斑潜蝇hsp70的表达.     

沙葱萤叶甲表皮蛋白基因GdAbd的克隆及对温度胁迫的响应

为揭示沙葱萤叶甲Galeruca daurica表皮蛋白基因GdAbd在其应对温度胁迫中的作用,采用RACE技术克隆表皮蛋白基因GdAbd的cDNA全长序列,并进行生物信息学分析;利用实时荧光定量(qPCR)技术比较GdAbd经不同温度处理1 h及25℃恢复30 min后在沙葱萤叶甲2龄幼虫体内的表达水平。结果表明,GdAbd基因全长708 bp(GenBank登录号:MG874710),开放阅读框为477 bp,编码158个氨基酸;蛋白预测分子量为16.98 kD,等电点为4.26;编码蛋白具有典型的表皮蛋白RR保守结构域,属于RR-2亚族;具有1个跨膜结构和1个信号肽。同源序列比对和系统发育分析表明,GdAbd与马铃薯甲虫Leptinotarsa decemlineata SgAbd-4的同源性最高,氨基酸序列一致性达50.63%。qPCR检测结果表明,与25℃对照相比,GdAbd基因表达水平在-10～5℃低温胁迫时未发生显著变化,而在35℃高温胁迫时发生显著上调;-10、-5和0℃低温胁迫后25℃恢复30 min可诱导GdAbd显著上调表达,但5℃低温和35℃高温胁迫处理与对照差异不显著。说明短时低温胁迫不能显著影响GdAbd表达,但胁迫后回温可诱导其上调表达。     

小麦条锈菌hsp70基因的克隆及热胁迫下的表达特征分析

为明确热激蛋白70在小麦条锈菌对高温适应性中的作用,采用RACE和PCR技术扩增得到小麦条锈菌hsp70的基因全长,并利用实时荧光定量PCR技术对热胁迫下不同温度敏感类型小麦条锈菌中hsp70的表达特征进行分析。克隆得到的小麦条锈菌hsp70基因组序列全长为2 817bp,包含7个内含子,cDNA序列全长为2 267bp,其中开放阅读框为1 917bp,编码639个氨基酸,分子量为66.7ku,等电点为5.15。编码蛋白含3个保守的HSP70氨基酸序列。qRT-PCR试验结果表明,28℃热激后,hsp70的转录水平随热激时间的延长而略有增加,2h达到最高值。不同温度敏感类型菌株在热激2h后hsp70的相对表达量具有显著差异。低敏感类型菌株hsp70平均表达量为未经处理对照的7.12倍,而高敏感类型菌株hsp70平均表达量仅为对照的1.63倍。     

小麦小GTP结合蛋白基因TaRop3克隆及其表达分析

利用同源克隆法从小麦抗条锈病基因Yr5近等基因系（Taichung29*6/Yr5）克隆到编码Rop蛋白基因的全长cDNA序列,并将基因命名为TaRop3（Triticum aestivum Rop3）,聚类分析其所在Rop蛋白家族中的亚组,并利用半定量RT-PCR方法分析其组织表达特异性和不同诱导条件下表达动态。序列分析表明,TaRop3开放阅读框为639 bp,预测编码含213个氨基酸残基Ⅱ型Rop蛋白,C末端具有膜定位基序,与大麦HvRop4和水稻OsRac4聚类在同一亚组。TaRop3基因在小麦幼苗和成株根、茎、叶片和茎节、柱头及花药中均有表达,其中在幼苗及成株茎部表达水平较高。非亲和条锈菌小种CYR17侵染和水杨酸处理诱导TaRop3表达增强,而干旱、高温胁迫以及脱落酸、乙烯利和茉莉酸甲酯处理均降低该基因表达水平。     

向日葵3个谷胱甘肽-S-转移酶GST基因的克隆及表达模式分析

从向日葵转录组测序结果中获得了3个对盐胁迫有响应的谷胱甘肽-S-转移酶（GST,EC2.5.1.18）GST基因,构建了系统进化树并进行分析,得知这3个基因属于Tau型GST,并将它们命名为HaGSTU26（HanXRQChr04g0127901）、HaGSTU8（HanXRQChr06g0177581）、HaGSTU27（HanXRQChr10g0316331）,然后以向日葵Sk02R为试验材料,克隆这3个基因,并进行了不同组织和不同胁迫条件下的表达分析。序列分析表明：HaGSTU26基因组为1674bp,CDS（编码蛋白序列）长666bp,编码221个氨基酸,由2个外显子和1个内含子组成;HaGSTU8基因组为2271bp,CDS长654bp,编码217个氨基酸,由2个外显子和1个内含子组成; HaGSTU27基因组为663bp,CDS长663bp,编码220个氨基酸,由1个外显子组成。实时荧光定量PCR分析表明：向日葵HaGSTU26、HaGSTU8、HaGSTU27基因在不同组织（根、幼叶、成熟叶、茎、幼茎、苞叶）中表达量不同,其中,HaGSTU26基因和HaGSTU27基因在根中表达量最高,而HaGSTU8基因在苞叶中表达量最高,但这3个基因均在成熟茎中的表达量最低。在不同胁迫条件下,测定这3个基因在向日葵幼苗中的表达量,结果表明在盐及ABA胁迫下,基因表达量均随着处理时间的增加而呈现先增加后下降的趋势。其中,在盐胁迫下,HaGSTU26基因在12 h后相对表达量最高,HaGSTU8及HaGSTU27基因表达量在3h后相对表达量最高。在ABA胁迫后,HaGSTU26及HaGSTU27基因表达量在12 h后相对表达量最高,HaGSTU8在24 h后相对表达量最高。在冷胁迫下,HaGSTU26和HaGSTU27基因上调表达,它们分别在3、24 h后相对表达量最高,HaGSTU8基因下调表达,其相对表达量随处理时间的延长呈现逐渐减少的趋势。在热胁迫条件下,这3个基因的相对表达量随着胁迫时间的延长而增加,均在24 h后表达量最高。以上结果说明这3个基因对不同非生物胁迫（盐、ABA、冷、热胁迫）均有响应。     

苹果蠹蛾瞬时感受器离子通道基因的克隆及温度胁迫表达分析

瞬时感受器离子通道(transient receptor potential,TRP)是影响昆虫温度感知系统的关键组成。为探讨苹果蠹蛾温度感知和温度适应的机理,本研究以苹果蠹蛾Cydia pomonella为研究对象,通过分子克隆获得TRPA家族中的CpPainless和CpWater_witch基因,进行生物信息学分析,并利用实时荧光定量PCR技术分析靶基因在高低温胁迫后的表达。结果表明,CpPainless基因编码938个氨基酸,N端有8个锚蛋白重复序列。CpWater_witch基因编码980个氨基酸,N端有10个锚蛋白重复序列,二者编码产物均有6个跨膜结构,具瞬时感受器离子通道家族成员结构典型特征。表达分析结果显示,和对照26℃相比,高低温胁迫1 h后,CpPainless在5龄幼虫雌虫体内的表达量显著下调,而5龄幼虫雄虫经低温胁迫1 h后CpPainless表达量显著上调,但高温胁迫1 h后表达差异不显著;CpWater_witch在雌雄虫体内均没有显著变化。研究结果为研究瞬时感受器离子通道在苹果蠹蛾温度感知中的作用奠定基础。     

烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析

为探讨UV-B胁迫对烟蚜Myzus persicae热激蛋白Hsp90基因表达量的影响,采用RT-PCR与RACE技术克隆了烟蚜热激蛋白Hsp90基因的全长,并对其进行生物信息学分析,利用实时荧光定量PCR技术研究了烟蚜Hsp90基因在不同时长UV-B胁迫下的表达量变化。结果表明,烟蚜Hsp90基因的cDNA全长为2 670 bp,编码728个氨基酸,编码蛋白质的相对分子量为82.6 kD,等电点为4.95,获得的氨基酸序列具有Hsp90蛋白家族的1个签名序列及C末端MEEVD基序,推测其属于胞质型热激蛋白。系统进化树结果显示,烟蚜Hsp90与其它昆虫Hsp90具有很高的相似性。实时荧光定量PCR结果表明,不同时长UV-B胁迫下烟蚜Hsp90均有表达,随着照射时间延长,Hsp90表达量表现为先上升后下降的趋势;与对照相比,照射时间为15、30、60、90和120 min时,Hsp90表达量均显著升高,且在60 min时Hsp90表达量达最大,是对照组的2.05倍。表明Hsp90基因在不同时长UV-B胁迫下差异表达,在烟蚜适应紫外胁迫的分子机制中具有重要作用。     

加州新小绥螨对朱砂叶螨的控制作用

为明确加州新小绥螨Neoseiulus californicus（McGregor）国内种群对朱砂叶螨Tetranychus cinnabarinus（Boisduval）的控制潜力,应用功能反应、数值反应及实验种群生命表参数评价了该捕食螨对朱砂叶螨的控制能力。加州新小绥螨对朱砂叶螨的功能反应能很好地拟合Holling Ⅱ方程。在25℃,加州新小绥螨雌成螨对朱砂叶螨卵、幼螨、若螨和成螨的功能反应参数a/T_h值分别为42.4195、81.6275、54.3044和17.9399,对朱砂叶螨幼螨、若螨和卵的控制力高于成螨。在19~31℃,加州新小绥螨对朱砂叶螨雌成螨日均捕食量和对猎物的控制能力a/T_h随温度升高而增大,在31℃时达最大值,分别为10.80头和29.5364。在28℃时,加州新小绥螨的净增殖率和内禀增长率分别为26.1522和0.2213。表明加州新小绥螨国内种群表现出重要的生物防治潜能。     

Biotransformation of clomazone in rice (Oryza sativa) and early watergrass (Echinochloa oryzoides)

Rice (Oryza sativa), a relatively tolerant species, and early watergrass (Echinochloa oryzoides; EWG), a relatively susceptible species, were exposed to ¹⁴C-labeled clomazone to determine accumulation, biotransformation, and mass balance. On a total mass basis, rice absorbed more clomazone than EWG (p < 0.05), but on a nmol/g basis, there was no significant difference between the two species (p > 0.05). Rice contained more extractable ¹⁴C residues (7.7 ± 0.5 vs. 4.8 ± 0.5 nmol in rice vs. EWG, respectively; p < 0.5), but the concentration in EWG was significantly higher (4.2 ± 0.5 vs. 1.8 ± 0.1 nmol/g in EWG vs. rice, respectively; p < 0.01). More metabolized residue was measured in EWG compared to rice (84.1% vs. 67.9%; p < 0.01). Both species produced hydroxylated forms, β-d-glucoside conjugates, and several other unidentified polar metabolites, but EWG generally produced higher metabolite concentrations. The concentration of the suspected active metabolite, 5-ketoclomazone, was significantly higher in EWG vs. rice (21 ± 2 vs. 5.7 ± 0.5 pmol/g, respectively; p < 0.01). Differences in sensitivity to clomazone between rice and EWG appear to be due to differential metabolism, but in this case the more susceptible EWG qualitatively and quantitatively metabolized more clomazone than the more tolerant rice. This is consistent with the action of a metabolically activated herbicide. This metabolic difference could be exploited to develop herbicide safeners for use with clomazone.     

20-(S)-喜树碱7-C-取代衍生物的合成及杀松材线虫活性研究

为研究喜树碱(CPT)类化合物的杀线虫活性,以喜树碱为原料,经烷基化、氧化、酯化等步骤合成了13个7-C-取代的20-(S)-喜树碱衍生物,其中化合物 14 未见文献报道,所有衍生物的结构经红外光谱(FT-IR)、核磁共振氢谱(1H NMR)和液-质联用(LC-MS)等分析手段进行了表征。采用浸渍法测定了化合物对松材线虫Bursaphelenchus xylophilus的毒杀活性。结果表明:与母体化合物喜树碱相比,7-C-取代的20-(S)-喜树碱衍生物具有更强的杀线虫活性,其中化合物7-苄基喜树碱、7-甲酰基喜树碱、7-苯甲酰氧甲基喜树碱在24 h的致死中浓度(LC50值)分别为2.28、2.21和1.37 mg/L,明显高于母体化合物喜树碱的LC50值12.18 mg /L。     

Green nano-emulsion intervention for water-soluble glyphosate isopropylamine (IPA) formulations in controlling Eleusine indica (E. indica)

This article describes the development of environmentally friendly nano-emulsion system for water-soluble herbicide application. Pseudoternary phase diagrams were established in the emulsion system of fatty acid methyl esters (FAMEs)/alkylpolyglucosides (APG) and/or 3-(3-hydroxypropyl)-heptamethyltrisiloxane (organosilicone)/water encompassed with 41% (w/w) glyphosate isopropylamine (IPA) as herbicide active. Pre-formulations were selected from isotropic (L) region in the phase diagrams and their emulsion system characteristics were determined. The microemulsion systems were chosen and then dispersed into water using low-energy stirring method (200 rpm for 5 min). Oil-in-water (O/W) nano-emulsions were formed with particle sizes of diameter less than 200 nm. The nano-emulsion systems showed significantly lower surface tension than a commercial formulation (Roundup®). In the biological application study, treatments of nano-emulsion formulations and Roundup® were applied on narrow-leaved weed Eleusine indica. Multiple doses of glyphosate IPA of the treatments were applied for the construction of dose-response curves for determination of effective dose (ED₅₀). The nano-emulsion formulation showed lower ED₅₀ was 0.40 kg a.e./ha in controlling the weed than Roundup® was 0.48 kg a.e./ha. This finding suggested that the possibility of using nano-emulsion system to increase penetration and uptake of glyphosate IPA.     

Transformation a mutant Monochoria vaginalis acetolactate synthase (ALS) gene renders Arabidopsis thaliana resistant to ALS inhibitors

Acetolactate synthase (ALS) genes from Monochoria vaginalis resistant (R) and susceptible (S) biotypes against ALS inhibitors found in Korea revealed a single amino acid substitution of Proline (CCT), at 169th position based on the M. vaginalis ALS sequence numbering, to serine (TCT) in conserved domain A of the gene (equal to the proline 197 in Arabidopsis thaliana ALS gene sequence). A. thaliana plants transformed with the single mutated (Pro₁₆₉ to Ser) M. vaginalis ALS gene (including transit signal peptide) showed cross-resistance patterns to ALS-inhibiting herbicides, like as sulfonylurea-herbicide bensulfuron methyl (R/S factor of 9.5), imidazolinone-herbicide imazapyr (R/S factor of 5.1), and triazolopyrimidine-herbicide flumetsulam (R/S factor of 17.6) when measuring hypocotyls’ length of A. thaliana. The ALS activity from the transgenic A. thaliana plants confirmed the cross-resistance pattern to these herbicides like as R/S factor of 8.3 to bensulfuron methyl, 2.3 to imazapyr, and 13.2 to flumetsulam.     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

Prevention of progeny formation in Drosophila melanogaster by 1-arylimidazole-2(3H)-thiones

Effects of 1-arylimidazole-2(3H)-thiones (AITs) and 1-(substituted benzyl)imidazole-2(3H)-thiones (BITs) were tested on progeny formation in Drosophila melanogaster. Some AITs showed inhibitory activities at laying eggs and delayed eclosion by 1 day. The inhibitory activity was nullified by adding octopamine (OA) or noradrenaline (NA) to the medium for progeny formation in D. melanogaster. The effect of AIT on the contents of OA and NA was analyzed in adults of D. melanogaster by high-performance liquid chromatography with electrochemical detection. Flies fed with AIT decreased OA and NA levels and increased TA content. Taken together, the inhibitory activity of AIT could be due to inhibition of tyramine β-hydroxylase and dopamine β-hydroxylase.     

二化螟盘绒茧蜂寄生对寄主二化螟幼虫免疫反应的影响

为了探明二化螟盘绒茧蜂Cotesia chilonis (Matsumura)寄生对寄主二化螟Chilo suppressalis (Walker)幼虫血细胞和体液免疫反应的影响,观察并测定了二化螟盘绒茧蜂寄生引起二化螟幼虫血细胞数量、延展、存活、吞噬和包囊作用以及血淋巴酚氧化酶活性等的变化。结果显示,二化螟幼虫血细胞总数在寄生后1天即显著高于对照。其中,颗粒血细胞和浆血细胞在寄生后的数量变化均与血细胞总数变化相似,颗粒血细胞在寄生后3天、浆血细胞在寄生后0.5天起即分别与其对照差异显著,但两种血细胞在血细胞总数中各自所占的比例与对照多无显著差异。二化螟幼虫血细胞延展能力在寄生后0.5天受到显著抑制,此后与对照无显著差异;同时寄生可增加寄主血细胞的死亡率。寄主幼虫血细胞的吞噬作用在寄生后2天起显著降低;包囊作用则在寄生后1天起显著降低。二化螟幼虫被二化螟盘绒茧蜂寄生后,血淋巴酚氧化酶活性显著升高,但随后呈逐渐下降趋势。研究结果说明,二化螟盘绒茧蜂寄生使寄主二化螟幼虫的免疫反应呈现一定的规律变化。     

Methoxyfenozide and pyriproxifen alter the cellular immune reactions of Eurygaster integriceps Puton (Hemiptera: Scutelleridae) against Beauveria bassiana

Effects of the two insect growth regulators (IGRs) methoxyfenozide, 20-hydroxyecdysone (20E) agonist, and pyriproxifen, Juvenile hormone (JH) agonist, were examined on the cellular immune responses of the Sunn pest, Eurygaster integriceps versus the entomopathogenic fungus Beauveria bassiana. The simultaneous treatment with the IGRs and the fungal spores altered haemocyte count (total and differentiate), nodulation response and phenoloxidase (PO) activity in a dose- and time-dependent manner. It was observed that different concentrations of methoxyfenozide increased total and differentiate haemocyte numbers as well as B. bassiana-induced nodulation response. In contrast with the JH agonist, pyriproxifen significantly decreased total and differentiate haemocyte numbers and inhibited nodule formation in E. integriceps adults. The 20E agonist displayed major effects when injected at the doses 2.79 and 5.59 μg/mg adult. In contrast, injecting adults by pyriproxifen significantly impaired their ability to raise an efficacious response against the fungal spores. The ability of the two IGR analogues to interfere with activity of the PO system in haemolymph of E. integriceps adults was also investigated 6 h after injection by fungal spores. Methoxyfenozide had an excitatory effect on PO activity when the 5.59 μg/mg concentration was used against adults. Conversely, pyriproxifen had an inhibitory effect on PO activity when used at 1.49 μg/mg adult concentration. These findings demonstrate that pyriproxifen may interfere with cell-mediated immunity of E. integriceps. So, pyriproxifen could be a good candidate for the integrated control of the Sunn pest.     

亚洲玉米螟6-磷酸葡萄糖异构酶基因(Pgi)全序测定及分析

为进一步从核酸水平上研究亚洲玉米螟Pgi基因相关特性,采用反转录PCR及RACE等技术对该基因编码序列及DNA全序列进行了测定,与Gen Bank中其它昆虫Pgi基因相关信息进行比较分析,并构建了系统发育树。结果表明:亚洲玉米螟Pgi基因编码区序列长为1 671 bp,共编码556个氨基酸;其DNA序列全长为10 078 bp(短序列为9 311 bp),由12个外显子与11个内含子镶嵌而成;各外显子长度与鳞翅目大部分昆虫相同,介于95~188 bp之间,内含子序列总长度为8 407 bp(短序列为7 640 bp),各内含子长度介于418~1 547 bp之间,且在内含子3、4、5、11上均发现杂合现象。系统发育结果显示,大部分昆虫Pgi基因c DNA严格按物种聚类,除了双翅目的家蝇Musca domestica与黑森瘿蚊Mayetiola destructor出现一定的交叉现象外,其余没有出现交叉现象。