Cry2Ab蛋白对甜菜夜蛾和斜纹夜蛾低龄幼虫的抗性

【背景】在我国,由于Bt棉的种植,棉铃虫和红铃虫等靶标害虫得到了控制,但棉田其他鳞翅目害虫如甜菜夜蛾和斜纹夜蛾的危害仍较严重。美国商业化种植的双价棉BollgardⅡ所表达的Cry2Ab蛋白不仅对棉铃虫有较好的控制效果,而且对甜菜夜蛾和草地贪夜蛾有较好的控制作用。因此,该双价棉在我国被环境释放前,有必要研究其对棉田其他鳞翅目害虫的影响。【方法】在人工饲料中分别添加质量浓度为1．25、2．5、5．0、10．0和20．0μg·g^-1的Cry2Ab蛋白,采用生物测定的方法,在室内研究了其对甜菜夜蛾和斜纹夜蛾低龄幼虫存活率和体质量抑制率的影响。【结果】随着Cry2Ab蛋白浓度的增大,甜菜夜蛾初孵幼虫和1龄幼虫的存活率逐渐降低,2龄幼虫和3龄幼虫以及斜纹夜蛾各龄期幼虫的存活率在不同浓度处理下与对照差异均不显著。但与对照相比,高浓度处理对这2种害虫各龄期幼虫的体质量均有显著影响。【结论与意义】高浓度Cry2Ab蛋白（10．0和20．0μg·g^-1）对甜菜夜蛾低龄幼虫有较好的控制作用,但对斜纹夜蛾低龄幼虫的控制效果不太理想。这为该双价基因棉花在我国的推广提供了依据。     

草地贪夜蛾和其它五种重要鳞翅目害虫蛹的形态特征比较

【目的】田间草地贪夜蛾Spodopterafrugiperda(J.E.Smith)蛹与斜纹夜蛾Spodopteralitura Fabricius、甜菜夜蛾Spodoptera exigua(Hübner)、粘虫Mythimna separata(Walker)、棉铃虫Helicoverpa armigera(Hübner)和亚洲玉米螟Ostrinia furnacalis 5种重要鳞翅目害虫蛹容易混淆。明确草地贪夜蛾与其它5种重要的鳞翅目害虫蛹的形态差异,为田间鳞翅目害虫蛹的准确鉴别和田间调查提供依据。【方法】采用体式显微镜拍照观察并分析草地贪夜蛾蛹、斜纹夜蛾蛹、甜菜夜蛾蛹、粘虫蛹、棉铃虫蛹和亚洲玉米螟蛹的外部形态特征,根据相关特征差异编制了区分草地贪夜蛾与此5种鳞翅目害虫的检索表和快速区分表。【结果】草地贪夜蛾蛹主要特征为中足末端到达下颚末端之前,触角末端到达中足末端之前;中胸背板前缘略直;第4-7腹节背面前缘着生多行粗点刻;腹部末端臀棘上着生1对基部分开呈八字形的刺。草地贪夜蛾蛹腹部末端臀棘部位与其它5种害虫均不相同,辅以附肢位置、中胸背板前缘线和腹部点刻等特征可以准确将其与斜纹夜蛾、甜菜夜蛾、粘虫、棉铃虫和亚洲玉米螟的蛹区分。斜纹夜蛾中胸背板前缘线和臀棘上的刺与草地贪夜蛾有异;甜菜夜蛾下颚与中足位置及臀棘上的刺与草地贪夜蛾有异;粘虫腹部点刻和臀棘上的刺与草地贪夜蛾有异;棉铃虫触角末端位置和腹部末端的刺与草地贪夜蛾有异;亚洲玉米螟腹足遗迹和臀棘上的刺与草地贪夜蛾有异。【结论】根据蛹的结构特征,可以区分草地贪夜蛾与斜纹夜蛾、甜菜夜蛾、粘虫、棉铃虫和亚洲玉米螟等害虫的蛹。     

不同Bt蛋白对棉铃虫存活率和生长发育的影响

【背景】在我国Bt棉主要以Cry1Ab或Cry1Ac为主,其他新型Bt基因未被转入棉花中用来控制害虫,然而大面积种植单价Bt基因的棉花,将可能会大大增加靶标害虫对该类型Bt棉花抗性频率,因此研究其他新型Bt蛋白对靶标害虫的控制作用显得十分必要。【方法】采用蛋白混入人工饲料的生物测定方法,在室内测定了6种Bt蛋白对棉铃虫初孵幼虫的毒力,比较了浓度为1.0μg·g-1时不同Bt蛋白对棉铃虫幼虫生长发育的影响。【结果】毒力测定结果表明,不同Bt蛋白对棉铃虫初孵幼虫的毒力不同,LC50值由低到高依次为Cry1Ab 0.065μg·g-1、Cry1Ac 0.074μg·g-1、Cry2Ab 0.133μg·g-1、Cry2Aa 11.670μg·g-1、Cry1Ah 13.010μg·g-1和Cry1Ca20μg·g-1。生长发育测定结果表明,Cry1Ab和Cry1Ac对棉铃虫幼虫的生长发育影响最大,Cry2Ab次之;Cry1Ah和Cry2Aa对1龄幼虫的校正死亡率和体重抑制率差别不大,但对2龄幼虫的差异较大,Cry1Ah处理2龄幼虫后体重和生长发育参数与Cry2Ab接近,而Cry1Ca对棉铃虫幼虫生长发育几乎没影响。【结论与意义】Cry1Ah、Cry2Aa和Cry2Ab的毒力不如Cry1Ac和Cry1Ab,但仍可以作为控制棉铃虫幼虫的替代策略。     

不同Bt蛋白对棉铃虫存活率和生长发育的影响

在我国Bt棉主要以Cry1Ab或Cry1Ac为主,其他新型Bt基因未被转入棉花中用来控制害虫,然而大面积种植单价Bt基因的棉花,将可能会大大增加靶标害虫对该类型Bt棉花抗性频率,因此研究其他新型Bt蛋白对靶标害虫的控制作用显得十分必要。采用蛋白混入人工饲料的生物测定方法,在室内测定了6种Bt蛋白对棉铃虫初孵幼虫的毒力,比较了浓度为1．0μg· g-1时不同Bt蛋白对棉铃虫幼虫生长发育的影响。毒力测定结果表明,不同Bt蛋白对棉铃虫初孵幼虫的毒力不同,LC50值由低到高依次为Cry1Ab 0．065μg· g-1、Cry1Ac 0．074μg· g-1、Cry2Ab 0．133μg· g-1、Cry2Aa 11．670μg· g-1、Cry1Ah 13．010μg· g-1和Cry1Ca＞20μg· g-1。生长发育测定结果表明,Cry1Ab和Cry1Ac对棉铃虫幼虫的生长发育影响最大,Cry2Ab次之;Cry1Ah和Cry2Aa对1龄幼虫的校正死亡率和体重抑制率差别不大,但对2龄幼虫的差异较大,Cry1Ah处理2龄幼虫后体重和生长发育参数与Cry2Ab接近,而Cry1Ca对棉铃虫幼虫生长发育几乎没影响。Cry1Ah、Cry2Aa和Cry2Ab的毒力不如Cry1Ac和Cry1Ab,但仍可以作为控制棉铃虫幼虫的替代策略。     

棉铃虫ABC转运蛋白ABCG1的基因克隆、亚细胞定位及其与Cry1Ac毒力的关系

【目的】长期种植转Bacillus thuringiensis(Bt)基因作物使一些靶标害虫产生了Bt抗性,有研究表明小菜蛾Plutella xylostella的white基因表达下调使其产生了Cry1Ac抗性,由于Pxwhite蛋白与ABC转运蛋白ABCG1同属于ABCG亚家族,我们推测棉铃虫Helicoverpa armigera ABCG1基因(HaABCG1)的表达下调可能与棉铃虫对Cry1Ac毒素的抗性有关。【方法】克隆并分析HaABCG1的开放阅读框(open reading frame,ORF),通过构建HaABCG1基因的表达载体检测HaABCG1蛋白在TnH_5细胞中的亚细胞定位;通过细胞毒力实验验证HaABCG1蛋白与Cry1Ac毒素的关系;利用RNAi技术验证HaABCG1表达下调是否会降低棉铃虫对Cry1Ac毒素的敏感性。【结果】棉铃虫ABCG1基因的开放阅读框长1 896 bp,编码蛋白含631个氨基酸残基,分子质量估计为69.63 k D。离体昆虫细胞表达的HaABCG1蛋白主要定位在细胞的核膜和内质网。通过RNA干扰和生物测定实验发现HaABCG1下调表达不能使棉铃虫在Cry1Ac毒素浓度为0.05μg/m L的人工饲料上正常生长,3 d后处理组和对照组棉铃虫幼虫的体重变化无显著差异。经过细胞毒力实验证明HaABCG1蛋白不介导Cry1Ac毒素对TnH_5细胞的毒力,它既不是Cry1Ac毒素的受体,也不是其他3种Bt毒素Cry1Ca,Cry2Aa和Cry1Fa的受体。【结论】HaABCG1基因表达下调与棉铃虫对Cry1Ac的抗性不相关,HaABCG1不是Cry1Ac毒素的受体。这是首次报道ABCG1基因不参与棉铃虫的Cry1Ac抗性。     

四种鳞翅目害虫精氨酸激酶基因的表达谱及基于RNAi的功能分析

【目的】精氨酸激酶(arginine kinase, AK)(EC 2.7.3.3)是昆虫体内重要的磷酸原激酶(能量代谢调节因子),也是唯一能够形成有效ATP的磷酰基供体,起着与脊椎动物中肌酸激酶相同的作用。本研究旨在了解鳞翅目害虫AK基因的表达和功能。【方法】利用qRT-PCR方法测定AK基因在大螟Sesamia inferens、二化螟Chilo suppressalis、甜菜夜蛾Spodoptera exigua和斜纹夜蛾Spodoptera litura 这4种鳞翅目害虫不同发育阶段和3龄幼虫不同组织中的表达谱;通过终点法检测了这4种害虫不同发育阶段和幼虫不同组织中的AK酶活性;采用RNAi技术抑制该基因的表达并分析其功能。【结果】AK基因在大螟、二化螟、甜菜夜蛾和斜纹夜蛾这4种鳞翅目昆虫的不同发育阶段和3龄幼虫不同组织中均有表达,说明该基因的表达不具有发育时期和组织特异性。不同发育时期和3龄幼虫不同组织中AK酶活性与基因表达量变化趋势大体一致。注射以AK基因为靶标的dsRNA 6 d后,4种害虫体内AK基因的mRNA表达下降30%~50%,AK酶活性降低30%左右;14 d后幼虫的死亡率达50%左右,显著高于对照组幼虫的死亡率。【结论】AK基因在上述4种鳞翅目害虫中为组成型表达,RNAi抑制AK基因的表达可导致4种害虫的幼虫死亡,研究结果为开发以AK基因为靶标的鳞翅目害虫防治新技术提供了理论依据。     

草地贪夜蛾取食Cry1Ab和Cry1Fa蛋白后中肠转录组及ABC基因表达分析

【目的】为揭示草地贪夜蛾Spodoptera furgiperda幼虫取食Bt蛋白后与中肠上相关ATP结合盒转运子(ATP-binding cassette transporter, ABC)蛋白基因的表达量变化的关系。【方法】分别使用含活化晶体蛋白Cry1Ab (LC₇₀=240.2 μg/g)和Cry1Fa (LC₇₀=270.0 μg/g)蛋白的人工饲料饲喂草地贪夜蛾4龄幼虫48 h,利用高通量测序对中肠进行转录组测序并进行生物信息学分析,筛选处理后差异表达基因;利用RT-qPCR验证差异表达ABC基因的表达量。【结果】与饲喂正常人工饲料的对照相比,饲喂含240.2 μg/g Cry1Ab和270.0 μg/g Cry1Fa的人工饲料后草地贪夜蛾4龄幼虫中肠转录组中分别检测到1 305和1 202个差异表达基因。Cry1Ab和Cry1Fa处理组与对照组之间分别有994和912个差异表达基因被GO功能注释到生物学过程、分子功能和细胞组分三大类。在最终筛选到的9个差异表达的ABC家族基因中,Cry1Ab处理组与对照组之间有4个差异表达ABC基因,3个上调,1个下调; Cry1Fa处理组与对照组之间有5个差异表达ABC基因,2个上调,3个下调;Cry1Ab和Cry1Fa处理组与对照组之间有2个ABC基因(LOC118267200和LOC118267201)表达量均显著上调。RT-qPCR验证结果表明,与对照组相比,Cry1Ab处理组有3个ABC基因表达量极显著上调,2个ABC基因表达量下调;Cry1Fa处理组有5个ABC基因表达量上调,1个ABC基因表达量下调。【结论】Cry1Ab和Cry1Fa蛋白的摄入可以影响草地贪夜蛾幼虫中肠一些ABC家族基因的表达量变化,这些基因的表达量变化与昆虫抗性产生有关。经比对后发现,ABCC家族与ABCG8基因表达量变化显著。本研究为下一步明确草地贪夜蛾体内ABC转运蛋白在Bt蛋白杀虫机制中的作用,以及合理使用Bt蛋白防治草地贪夜蛾及延缓抗性提供了理论依据。     

甜菜夜蛾肽聚糖识别蛋白基因SePGRP-SA的克隆及功能鉴定

【目的】本研究旨在阐明甜菜夜蛾Spodoptera exigua幼虫体内肽聚糖识别蛋白(peptidoglycan recognition protein, PGRP)在响应苏云金芽胞杆菌Bacillus thuringiensis(Bt)感染过程中的功能。【方法】利用PCR方法扩增甜菜夜蛾幼虫肽聚糖识别蛋白基因SePGRP-SA全长cDNA;采用qRT-PCR分析SePGRP-SA在甜菜夜蛾不同发育阶段(卵、1-5龄幼虫、预蛹和蛹)及4龄幼虫不同组织(中肠、马氏管、围食膜、脂肪体、血淋巴和表皮)中的表达。通过RNAi技术沉默SePGRP-SA基因72 h后,qRT-PCR检测SePGRP-SA沉默效率及甜菜夜蛾4龄幼虫中肠抗菌肽相关基因(Ceropin, Attacin和Defensin)和细菌载量的变化。RNAi沉默SePGRP-SA 24 h后,以苏云金芽胞杆菌菌株Bt-GS57饲喂甜菜夜蛾4龄幼虫0, 24, 48, 72, 96和120 h,计算幼虫校正死亡率;饲喂甜菜夜蛾4龄幼虫Bt-GS57后0, 24, 48和72 h,利用qRT-PCR检测中肠SePGRP-SA, Ceropin, Attacin和Defensin的相对表达量。【结果】克隆获得甜菜夜蛾SePGRP-SA全长DNA(GenBank登录号：MW265930),开放阅读框长576 bp,编码191个氨基酸,其编码蛋白的预测分子量为21.59 kD。序列分析结果表明,SePGRP-SA具有典型的PGRP和Ami2保守结构域,信号肽为19个氨基酸,为分泌型蛋白;系统进化分析发现,SePGRP-SA与斜纹夜蛾Spodoptera litura的SlPGRP亲缘关系最近,氨基酸序列一致性达91.1%。发育表达谱结果表明SePGRP-SA在甜菜夜蛾4和5龄幼虫、预蛹和蛹中高表达;组织表达谱结果表明,SePGRP-SA在4龄幼虫各组织中均表达,其中以血淋巴中表达量最高。与注射dsEGFP(对照)相比,注射dsSePGRP-SA的甜菜夜蛾4龄幼虫在72 h时中肠SePGRP-SA基因表达量下调了95.26%,Cecropin, Attacin和Defensin表达量显著下调,中肠细菌载量显著升高。注射dsEGFP和dsSePGRP-SA的甜菜夜蛾4龄幼虫饲喂Bt-GS57,72 h时幼虫校正死亡率分别为50.00%和73.33%,表明幼虫对Bt-GS57的敏感性明显增加。甜菜夜蛾4龄幼虫取食Bt-GS57后,中肠SePGRP-SA, Cecropin, Attacin和Defensin表达量在48 h均显著增加,72 h时降低。【结论】Bt侵染能够引起甜菜夜蛾SePGRP SA基因激活抗菌肽相关基因Cecropin, Attacin和Defensin的表达。     

Cry1Ac和Cry2Ab杀虫蛋白对粘虫幼虫生长发育的影响

【目的】为探究Bt杀虫蛋白对次要靶标害虫粘虫Mythimna separata (Walker)(鳞翅目:夜蛾科)的杀虫活性及对其生长发育的影响。【方法】本文通过浸叶法饲喂初孵及2龄末粘虫不同剂量的Cry1Ac及Cry2Ab杀虫蛋白后,观察其死亡率,称量幼虫重,并统计了幼虫历期、化蛹率、蛹重、蛹期、蛹的羽化率、畸形率等指标。【结果】初孵幼虫取食浸泡含16、64、128μg/mLCry1Ac及Cry2Ab的玉米叶片后,随着时间的延长及浓度的增加,死亡率逐渐增加,且Cry1Ac杀虫蛋白对粘虫的生物活性高于Cry2Ab蛋白,在128μg/mL浓度下,取食Cry1Ac和Cry2Ab蛋白13d时的死亡率分别达到了65%及60%。取食两种蛋白后,初孵幼虫和2龄末幼虫重量均受到显著抑制,短期取食两种蛋白对幼虫历期、化蛹率、蛹重、蛹期、蛹的羽化率、畸形率没有影响。【结论】取食Cry1Ac和Cry2Ab杀虫蛋白后,对初孵幼虫有很好的杀虫活性,且Cry1Ac杀虫活性高于Cry2Ab杀虫蛋白;短期饲喂两种杀虫蛋白时,对2龄粘虫后期生长影响不大。本文结果为转Bt基因作物更好的应用于粘虫的防治提供了理论基础。     

棉铃虫对不同Bt蛋白的抗性及交互抗性研究

【目的】棉铃虫Helicoverpa armigera(Hübner)对Bt(Bacillus thuringiensis)的抗性问题是阻碍Bt抗虫棉持续发展的关键,探究棉铃虫对不同Bt蛋白的抗性演化趋势及交互抗性,可为选择合适的抗虫棉花备选基因提供参考。【方法】在室内通过连续多代筛选,获得了5个对Bt具有不同抗性的品系,利用饲料混合法测定了这些品系棉铃虫的交互抗性。【结果】经筛选后棉铃虫对Cry1Ac产生了超高水平的抗性、对Cry2Ab、Vip3Aa产生了超低抗性(耐性);Cry1Ac抗性棉铃虫停止筛选而换成Cry2Ab或Vip3Aa继续筛选后,棉铃虫对Cry1Ac抗性水平明显降低,对Cry2Ab或Vip3Aa产生低抗或超低抗性(耐性)。Cry1Ac抗性棉铃虫对Cry1Ab敏感性显著降低,而对Cry2Ab、Cry2Ah及Vip3Aa敏感性变化不显著;Cry2Ab抗性品系棉铃虫对Cry1Ac敏感性显著降低;Vip3Aa抗性品系棉铃虫对Cry1Ac敏感性变化不显著。说明Cry1Ac与Cry1Ab间存在明显交互抗性,与Cry2Ah、Vip3Aa没有交互抗性;而Cry2Ab与Cry1Ac间存在不对称的交互抗性。【结论】在筛选新杀虫基因或叠加基因时,应充分考虑抗性发展及交互抗性问题,Cry2Ah、Vip3Aa是治理Cry1Ac抗性棉铃虫或与Cry1Ac叠加最优的选择。     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Molecular identification of Taenia spp. in wolves (Canis lupus), brown bears (Ursus arctos) and cervids from North Europe and Alaska

Taenia tapeworms of Finnish and Swedish wolves (Canis lupus) and Finnish brown bears (Ursus arctos), and muscle cysticerci of Svalbard reindeer (Rangifer tarandus platyrhynchus), Alaskan Grant's caribou (Rangifer tarandus granti) and Alaskan moose (Alces americanus) were identified on the basis of the nucleotide sequence of a 396 bp region of the mitochondrial cytochrome c oxidase subunit 1 gene. Two species were found from wolves: Taenia hydatigena and Taenia krabbei. The cysticerci of reindeer, caribou and one moose also represented T. krabbei. Most of the cysticercal specimens from Alaskan moose, however, belonged to an unknown T. krabbei-like species, which had been reported previously from Eurasian elks (Alces alces) from Finland. Strobilate stages from two bears belonged to this species as well. The present results suggest that this novel Taenia sp. has a Holarctic distribution and uses Alces spp. as intermediate and ursids as final hosts.     

Tracking down the "head blob": comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless (wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.     

Phylogeny and Megasystematics of Phagotrophic Heterokonts (Kingdom Chromista)

Heterokonts are evolutionarily important as the most nutritionally diverse eukaryote supergroup and the most species-rich branch of the eukaryotic kingdom Chromista. Ancestrally photosynthetic/phagotrophic algae (mixotrophs), they include several ecologically important purely heterotrophic lineages, all grossly understudied phylogenetically and of uncertain relationships. We sequenced 18S rRNA genes from 14 phagotrophic non-photosynthetic heterokonts and a probable Ochromonas, performed phylogenetic analysis of 210–430 Heterokonta, and revised higher classification of Heterokonta and its three phyla: the predominantly photosynthetic Ochrophyta; the non-photosynthetic Pseudofungi; and Bigyra (now comprising subphyla Opalozoa, Bicoecia, Sagenista). The deepest heterokont divergence is apparently between Bigyra, as revised here, and Ochrophyta/Pseudofungi. We found a third universal heterokont signature sequence, and deduce three independent losses of ciliary hairs, several of 1-2 cilia, 10 of photosynthesis, but perhaps only two plastid losses. In Ochrophyta, heterotrophic Oikomonas is sister to the photosynthetic Chrysamoeba, whilst the abundant freshwater predator Spumella is biphyletic; neither clade is specifically related to Paraphysomonas, indicating four losses of photosynthesis by chrysomonads. Sister to Chrysomonadea (Chrysophyceae) is Picophagea cl. nov. (Picophagus, Chlamydomyxa). The diatom-parasite Pirsonia belongs in Pseudofungi. Heliozoan-like actinophryids (e.g. Actinosphaerium) are Opalozoa, not related to pedinellids within Hypogyristea cl. nov. of Ochrophyta as once thought. The zooflagellate class Bicoecea (perhaps the ancestral phenotype of Bigyra) is unexpectedly diverse and a major focus of our study. We describe four new biciliate bicoecean genera and five new species: Nerada mexicana, Labromonas fenchelii (=Pseudobodo tremulans sensu Fenchel), Boroka karpovii (=P. tremulans sensu Karpov), Anoeca atlantica and Cafeteria mylnikovii; several cultures were previously misidentified as Pseudobodo tremulans. Nerada and the uniciliate Paramonas are related to Siluania and Adriamonas; this clade (Pseudodendromonadales emend.) is probably sister to Bicosoeca. Genetically diverse Caecitellus is probably related to Anoeca, Symbiomonas and Cafeteria (collectively Anoecales emend.). Boroka is sister to Pseudodendromonadales/Bicoecales/Anoecales. Placidiales are probably divergent bicoeceans (the GenBank Placidia sequence is a basidiomycete/heterokont chimaera). Two GenBank ‘opalinid’ sequences are fungal; Pseudopirsonia is cercozoan; two previous GenBank ‘Caecitellus’ sequences are Adriamonas. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editior: Patnck J. Keeling]     

Removal of wastewater pathogen indicators in a constructed wetland in Leon, Spain

This paper studies the seasonal presence and removal of the pathogenous micro-organisms Escherichia coli, total coliforms (TC), Clostridium perfringens (Cp), faecal streptococci (FS), Giardia cysts, Cryptosporidium oocysts and helminth eggs, in a constructed wetland treatment system. The removal efficiency of this system with respect to the indicator micro-organisms achieved maximum values in spring and autumn at 99.9% for E. coli and TC, respectively, in winter at 97.0% for FS, in summer at 100% for Clostridium and throughout the year, also at 100%, in the case of Giardia cysts, Cryptosporidium oocysts and helminth eggs. In general, very low protozoan and helminth egg counts were found, and the system demonstrated efficient reduction of the wastewater indicator pathogens.     

A molecular phylogeny of Hebeloma species from Europe

In order to widen the scope of existing phylogenies of the ectomycorrhizal agaric genus Hebeloma a total of 53 new rDNA ITS sequences from that genus was generated, augmented by sequences retrieved from GenBank, and analysed using Bayesian, strict consensus and neighbour joining methods. The lignicolous Hebelomina neerlandica, Gymnopilus penetrans, and two species of Galerina served as outgroup taxa. Anamika indica, as well as representatives of the genera Hymenogaster and Naucoria, were included to test the monophyly of Hebeloma, which is confirmed by the results. Hebeloma, Naucoria, Hymenogaster and Anamika indica cluster in a strongly supported monophyletic hebelomatoid clade. All trees largely reflect the current infrageneric classification within Hebeloma, and divide the genus into mostly well-supported monophyletic groups surrounding H. crustuliniforme, H. velutipes, H. sacchariolens, H. sinapizans, and H. radicosum, with H. sarcophyllum being shown at an independent position; however this is not well supported. The section Indusiata divides with strong support into three groups, the position of the pleurocystidiate Hebeloma cistophilum suggests the possible existence of a third subsection within sect. Indusiata. Subsection Sacchariolentia is raised to the rank of section.     

Changes of dung beetle communities from rainforests towards agroforestry systems and annual cultures in Sulawesi (Indonesia)

Little is known about how tropical land-use systems contribute to the conservation of functionally important insect groups, including dung beetles. In a study at the margin of Lore Lindu National Park (a biodiversity hotspot in Central Sulawesi, Indonesia) dung-beetle communities were sampled in natural forest, young secondary forest, agroforestry systems (cacao plantations with shade trees) and annual cultures (maize fields), each with four replicates (n = 16 sites). At each site we used 10 pitfall traps, baited with cattle dung, along a 100 m transect for six 3-day periods. The number of trapped specimens and species richness at the natural forest sites was higher than in all land-use systems, which did not significantly differ. Each land-use system contained, on average, 75% of the species richness of the natural forest, thereby indicating their importance for conservation. However, a two-dimensional scaling plot based on NESS indices (m = 6) indicated distinct dung beetle communities for both forest types, while agroforestry systems and annual cultures exhibited a pronounced overlap. Mean body size of dung beetles was not significantly influenced by land-use intensity. Five of the six most abundant dung beetle species were recorded in all habitats, whereas the abundance of five other species was significantly related to habitat type. Mean local abundance and number of occupied sites were closely correlated, further indicating little habitat specialisation. The low dung beetle diversity (total of 18 recorded species) may be due to the absence of larger mammals in Sulawesi during historical times, even though Sulawesi is the largest island of Wallacea. In conclusion, the dung beetle fauna of the lower montane forest zone in Central Sulawesi appears to be relatively robust to man-made habitat changes and the majority of species did not exhibit strong habitat preferences.