小菜蛾中肠Polycalin蛋白的基因克隆、表达及与Cry1Ac毒素的结合特性分析

【目的】Polycalin蛋白是一种新发现的Bt毒素受体,可能与昆虫对Bt的抗性有关。本研究旨在明确小菜蛾Plutella xylostella Polycalin蛋白与Bt Cry1Ac毒素的关系。【方法】本研究通过PCR结合RACE技术从小菜蛾3龄幼虫中肠克隆获得Polycalin蛋白基因的全长cDNA序列,采用实时定量PCR技术研究Polycalin蛋白在小菜蛾不同发育阶段、4龄幼虫不同组织及用不同浓度Cry1Ac毒素饲喂3龄幼虫后的表达量。提取小菜蛾幼虫中肠的刷状缘膜囊泡(BBMV),运用合成多肽的方法制备Polycalin蛋白抗体,利用Western blot和Ligand blot技术对小菜蛾Polycalin蛋白进行鉴定,分析其与Cry1Ac毒素的结合特性。【结果】克隆得到的小菜蛾Polycalin蛋白基因的cDNA序列全长为9 102 bp(GenBank登录号:MF138149),其中,开放阅读框为8 778 bp,编码2 925个氨基酸;预测蛋白质分子量为326.38 kD,等电点为4.39;在推导的氨基酸序列中,前20个氨基酸为N-末端信号肽序列,含有14个N-糖基化位点,67个O-糖基化位点,6个脂质运载蛋白特征序列,6个脂质运载蛋白位点和13个类脂质运载蛋白结构域,并且在第20和21位(TSG-QVV)氨基酸之间存在一个裂解位点,在C-末端存在2个GPI结合位点,具有Bt毒素受体的特征。该蛋白在幼虫期的表达量较高,尤其在3龄幼虫体内表达量最高,在蛹和成虫中的表达量最低;在4龄幼虫的头、胸、腹中均有表达,在幼虫腹部中的表达量最高;3龄幼虫取食不同浓度的Cry1Ac毒素后,Polycalin蛋白的表达量下降,且Cry1Ac毒素浓度越高,下降越明显。通过Western blot检测到小菜蛾中肠刷状缘膜囊泡(BBMV)上存在大约300 kD的Polycalin蛋白条带;Ligand blot实验证明了小菜蛾Polycalin蛋白能与Cry1Ac毒素结合。【结论】本研究首次初步明确了小菜蛾Polycalin蛋白具有与Bt毒素结合的特性,为研究Bt对昆虫的作用机理和利用Bt防治害虫提供一定的理论基础和参考价值。     

鳞翅目昆虫氨肽酶N与Bt毒素的结合及其与Bt抗性的关系

随着Bt Cry作物在我国的广泛应用和推广,靶标害虫对其抗性风险已成为Bt Cry作物生态安全研究的重要内容.氨肽酶N(Aminopeptidase N,APN)是位于昆虫中肠刷状缘膜囊泡(Brush Border Membrane Vesicles,BBMV)上Bt Cry毒素重要的受体蛋白之一,它与Bt Cry毒素...     

RNAi介导的棉铃虫氨肽酶N基因Haapnl和钙粘蛋白基因Ha_BtR沉默对Cry1Ac毒力的影响

氨肽酶N(aminopeptidase N,APN)和钙粘蛋白(cadherin)是存在于鳞翅目昆虫中肠刷状缘膜囊(brush border membrane vesicles,BBMV)上Bt毒素Cry1A的受体.本实验将棉铃虫Helicoverpa armigera氨肽酶N1基因Haapnl和钙粘蛋白基因Ha_BtR双链RNA(dsRNA)注入棉铃虫4龄幼虫体内,以研究这两种受体基因沉默后对Cry1Ac毒力的影响.结果表明:注射dsRNA(1 μg/头)进行基因沉默后,Haapnl mRNA表达量比注射缓冲液(elution solution,ES)的对照下降了30%～49%,Ha_BtR mRNA表达量下降了30%～37%.注射Haapnl dsRNA的幼虫在40和70 μg/cm2 Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫,而在100和170 μg/cm2 Cry1Ac原毒素处理下两者死亡率无显著差异;Cry1Ac活化毒素以及原毒素对注射Ha_BtR dsRNA幼虫与注射ES幼虫的毒力均无显著差异.当同时注射Haapnl及Ha_BtR dsRNA后,干扰后的幼虫对Cry1Ac活化毒素和原毒素的敏感性均显著下降.本研究进一步证明了棉铃虫Haapnl和Ha_BtR均是Bt毒素Cry1Ac的功能受体,这两种受体蛋白共同参与Cry1Ae的毒杀作用过程.该结果也提示.Haapnl或Ha_BtR基因产生突变都可能导致棉铃虫对CrylAc产生抗性.     

大弹涂鱼和中华乌塘鳢肠刷状缘膜消化酶活性的比较

采用酶学分析的方法,研究了大弹涂鱼和中华乌塘鳢肠刷状缘膜的麦芽糖酶、蔗糖酶、乳糖酶、海藻糖酶、纤维二糖酶、碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶等8种消化酶的活性。结果表明:1)大弹涂鱼肠Ⅱ刷状缘膜的麦芽糖酶、蔗糖酶、乳糖酶、海藻糖酶和纤维二糖酶等5种二糖酶的比活力均显著高于肠Ⅰ和肠Ⅲ(P<0·05);中华乌塘鳢肠Ⅰ刷状缘膜除乳糖酶外,其余4种二糖酶的比活力均显著高于肠Ⅱ和肠Ⅲ(P<0·05);大弹涂鱼肠Ⅲ碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶等3种消化酶的比活力均显著高于肠Ⅰ和肠Ⅱ(P<0·05);中华乌塘鳢肠Ⅱ的这3种消化酶的比活力均显著高于肠Ⅰ和肠Ⅲ(P<0·05);2)大弹涂鱼各段肠刷状缘膜的5种二糖酶的比活力均显著高于中华乌塘鳢(P<0·05),前者肠刷状缘膜碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶等3种消化酶的比活力整体上也稍微高于后者。由此说明:8种消化酶的活性在大弹涂鱼和中华乌塘鳢肠刷状缘膜中的分布模式明显不同,大弹涂鱼和中华乌塘鳢对二糖的消化和吸收的主要部位分别是在肠Ⅱ和肠Ⅰ,而二者对蛋白质、脂类和无机盐等营养吸收的主要部位分别是在肠Ⅲ和肠Ⅱ;大弹涂鱼和中华乌塘鳢肠刷状缘膜的5种二糖酶的活性与两者的食性关系密切,而碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶等3种消化酶的活性与两者的食性并无密切的相关性。     

昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展

鳞翅目昆虫中肠上皮细胞刷状缘膜(BBM)上的Bt杀虫晶体蛋白毒素受体氨肽酶N(APN)的结构和位点密度的改变是昆虫对Bt毒素的主要抗性机制之一,该文简要综述了APN受体的研究进展。每种昆虫中肠上皮细胞中有数种APNs,彼此间同源性较高,其中部分APNs为crylA家族毒素的功能性受体。不同种类昆虫的APNs受体,甚至同一种昆虫的不同类型APNs,其所结合的毒素种类可能不同。APNs决定该昆虫对crylA类毒素的敏感程度差异。有些抗性昆虫的APNs基因编码区发生了多个点突变。     

苏云金芽胞杆菌CrylA δ-内毒素对家蚕中肠柱状上皮细胞上Mg~(2 )腺苷三磷酸酶活性的影响(英文)

本文研究了来自苏云金芽胞杆菌库斯塔克亚种HD 1菌株的CrylAδ 内毒素对家蚕中肠柱状上皮细胞Mg2 腺苷三磷酸酶活性的影响。细胞化学定位结果表明 ,该酶在家蚕中肠刷状缘的微绒毛膜上分布丰富、活性强。通过强迫饲喂过量CrylA毒素 ,中肠上皮细胞在短时间内出现了明显的病理特征 ,然而在肿胀、泡沫化的微绒毛膜乃至细胞碎片上Mg2 腺苷三磷酸酶仍具有很高的活性 ,而未观察到毒素对该酶的抑制作用。这说明Mg2 腺苷三磷酸酶可能不是δ 内毒素的作用靶子 ,也不是毒素所影响的天然的离子选择性通道。     

利用RNAi技术沉默小菜蛾类钙粘蛋白基因

RNA干扰（RNA interference, RNAi）是一种调控基因表达的方法, 其通过体外合成一段与内源靶基因同源的双链RNA（dsRNA）或siRNA, 导入生物体内, 使内源靶基因中同源mRNA降解, 从而达到阻抑基因表达的目的。类钙粘蛋白（cadherin-like protein）是位于昆虫中肠刷状缘膜囊(brush border membrane vesicles, BBMV)上与钙粘蛋白(cadherin)结构相似的物质, 是多种昆虫体内Bt杀虫蛋白的受体。本研究利用基因特异引物通过RT-PCR扩增了小菜蛾类钙粘蛋白基因的2个片段（CAD1和CAD2）, 合成相对应的双链RNA（double-stranded RNA, dsRNA）; 并将dsRNA通过显微注射导入小菜蛾3龄幼虫体内, 测定了不同靶位点、不同剂量、不同检测时间对目的基因mRNA表达量的影响。结果表明: 将70 nL CAD1对应的dsRNA注射到幼虫体内48 h后, 基因表达量显著下降, 72 h后恢复。免疫印迹检测结果表明, 类钙粘蛋白在注射dsRNA 48 h后幼虫BBMV中的含量明显下降。本实验成功实现了小菜蛾类钙粘蛋白基因的沉默, 该体系的成功建立为利用RNAi技术分析小菜蛾及其他鳞翅目昆虫基因的功能提供了参考。     

棉铃虫幼虫中肠细胞脂筏的制备及鉴定

脂筏是细胞膜上富含胆固醇、鞘脂类和糖基磷脂酰肌醇锚着蛋白的去垢剂不溶性微结构域,被认为是多种细胞膜孔毒素在细胞表面形成寡聚体的平台。为研究脂筏与Bt毒素在细胞膜上形成寡聚体膜孔的关系,本文对棉铃虫Helicoverpa armigera幼虫中肠脂筏的制备与鉴定方法进行了研究。根据脂筏在低温（4℃）下不溶于去垢剂的特性,采用Triton X-100处理棉铃虫幼虫中肠刷状缘膜囊泡,溶解非脂质筏成分,以OptiPrep为介质进行密度梯度离心,分离去垢剂不溶组分,成功地得到了棉铃虫幼虫中肠上皮细胞的脂筏。再以脂筏的特有化学成分神经节苷脂GM1作为脂筏的标志分子,利用霍乱毒素β亚基能与GM1特异性结合的特性,以辣根过氧化物酶标记的霍乱毒素β亚基用点印迹法化学发光检测神经节苷脂的分布,从而对脂筏进行定性鉴定。结果表明我们建立的脂筏制备方法简便、易行,比传统的蔗糖梯度离心法大大缩短了制备所需时间。     

细胞膜毒素对线粒体上钙通道的抑制

本文探讨膜毒素对鼠肝线粒体Ca~(++)传递和Ca~(++)结合亲和力的影响。当膜毒素的浓度为7.14毫微克分子/毫克线粒体蛋白时,处理过的线粒体传递Ca~(++)能力下降至原来一半左右。本实验做Ca~(++)结合膜毒素处理线粒体的Scatchard图呈直线(K_d=48.2μM,结合Ca~(++)数目N=341毫微克分子/毫克线粒体蛋白)。就是说,膜毒素抑制线粒体高亲和力Ca~(++)结合部位,而不影响低亲和力Ca~(++)结合部位。我们认为膜毒素作用位点在于线粒体高亲和力Ca~(++)结合部位。     

苏云金芽孢杆菌杀虫晶体蛋白与DNA分子的相互作用

苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,简称Ｂｔ)在形成芽孢的同时能够产生伴孢晶体 ,其中含有一种或几种杀虫晶体蛋白 (ＩＣＰｓ,ＩｎｓｅｃｔｉｃｉｄａｌＣｒｙｓｔａｌＰｒｏｔｅｉｎｓ) ,即δ 内毒素[1] 。伴孢晶体进入敏感昆虫的消化道后发生溶解并释放出 2 7～ 1 40ｋＤ的原毒素。在中肠蛋白酶的作用下 ,原毒素被激活为 2 3～ 70ｋＤ的毒性多肽[2～ 4 ] 。随后毒素与中肠刷状缘膜泡 (ＢＢＭＶ ,ＢｒｕｓｈＢｏｒｄｅｒＭｅｍｂｒａｎｅＶｅｓｉｃｌｅ)上的特异受体发生结合并且在细胞膜上形成孔道 ,破坏细胞…     

Cross-resistance studies of Cry1Ac-resistant strains of Helicoverpa armigera (Lepidoptera: Noctuidae) to Cry2Ab

Bioassays and binding tests between Cry toxins (CrylAa, CrylAb, CrylAc, and Cry2Ab) and brush-border membrane vesicles (BBMVs) from larvae of a Bacillus thurningiensis (Bt)-susceptible (96S) and two CrylAc-resistant strains (BtR and LFR10) were conducted for investigating cross-resistance of CrylAc-resistant strains to Cry2Ab in Helicoverpa armigera (Hiibner) (Lepidoptera: Noctuidae). The resistance ratio (RR) values of the BtR and LFR10 strains to CrylAc and Cry2Ab were 2,971- and 1.1-fold and 253- and 1.0-fold, respectively, indicating that there was no cross-resistance to Cry2Ab. The binding experiments between Cry toxins and BBMVs from BtR, LFR10, and 96S larvae showed that all of the toxins could bind with these BBMVs, but the Cry2Ab could not displace 125I labeled CrylAc and CrylAb. The same results were observed in reciprocal binding tests, demonstrating that CrylA and Cry2Ab had different binding sites in H. armigera and providing a potential mechanism for the lack of cross-resistance between CrylA and Cry2Ab toxins. These results suggest that the transgenic cotton, Gossypium hirsutum L., expressing CrylAc and Cry2Ab genes may be deployed for management of CrylAc resistant H. armigera.     

BINDING ASSAY OF BACILLUS THURINGIENSIS CRY1AC INSECTICIDAL CRYSTAL PROTEINS IN DIAMONDBACK MOTH*

Abstract Binding assay was performed with NTP-labeled Bacillus thuringiensis activated toxin Cry 1Ac on midgut brush border membrane vesicles (BBMVs) prepared from the whole larvae of dia-mondback moth Plutella xylostella. We conclude that the BBMVs can be isolated properly and Cry 1Ac toxin binds saturably to BBMVs.     

Study of the irreversible binding of Bacillus thuringiensis Cry1Aa to brush border membrane vesicles from Bombyx mori midgut

The binding of Bacillus thuringiensis δ-endotoxin to brush border membrane vesicles (BBMVs) from the target insect larval midgut comprises with not only a reversible but also an irreversible component. The irreversible binding of δ-endotoxin is thought to be a pathologically important factor. Here, we studied the irreversible binding of Cry1Aa to the BBMVs of Bombyx mori. The ¹²⁵I-labeled Cry1Aa bound to the solubilized brush border membrane (BBM) through rapid dissociation only, unlike the binding to BBMVs, indicating that the toxin bound to the solubilized BBM through only a reversible process. Low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the toxin bound irreversibly to BBMVs formed an oligomer of 220 kDa, whereas that bound reversibly to the solubilized BBM did not oligomeraize. When the ¹²⁵I-labeled Cry1Aa bound irreversibly to the BBMVs was digested by proteinase K, approximately 40% of the toxin observed to be resistant to proteinase K. The molecular mass of the toxin resistant to proteinase K was 60 kDa, suggesting that the irreversible binding comprise two forms. These results support the notion that the irreversible binding of the toxin to BBMVs is due to the insertion of the toxin into the lipid bilayers and oligomerization to form channels.     

Binding of the CryIVD Toxin of Bacillus thuringiensis subsp. israelensis to Larval Dipteran Midgut Proteins

Ligand-blotting experiments on dipteran brush border membrane vesicles (BBMVs) showed binding of CryIVD toxin of Bacillus thuringiensis subsp. israelensis to proteins of 148 kDa in Anopheles stephensi and of 78 kDa in Tipula oleracea, both species being susceptible to CryIVD. Binding of CryIVD with BBMVs of A. stephensi resulted in a stronger signal than with BBMVs of T. oleracea. Likewise, larvae of A. stephensi are 10,000-fold more susceptible to the CryIVD toxin than are larvae of T. oleracea. Binding was also found with six proteins ranging in size from 48 to 110 kDa in BBMVs from the lepidopteran species Manduca sexta, but CryIVD was not toxic for M. sexta larvae. No binding of trypsinated CryIVD to BBMV proteins was observed. With the lepidopteran-specific toxin CryIA(b), no binding to dipteran BBMVs was found. Binding of CryIA(b) to nine different BBMV proteins ranging in size from 71 to 240 kDa was observed in M. sexta. The major binding signal was observed with a protein of 240 kDa for CryIA(b).     

N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis.

Proteins synthesized by the bacterium Bacillus thuringiensis are potent insecticides. When ingested by susceptible larvae they rapidly lyse epithelial cells lining the midgut. In vitro the toxins lyse certain insect cell lines and show saturable, high-affinity binding to brush-border membrane vesicles (BBMVs) prepared from insect midguts. We observed that the sugar N-acetyl galactosamine (GalNAc) specifically decreased the cytolytic activity of a CryIA(c) toxin towards Choristoneura fumiferana CF1 cells, completely abolished toxin binding to Manduca sexia BBMVs, partially inhibited binding to Heliothis virescens BBMVs and had no apparent effect on binding to Pieris brassicae BBMVs. In ligand blotting experiments the toxin bound proteins of 120 kDa in M. sexta, 125 kDa in P. brassicae and numerous proteins in H. zea. Toxin binding to these proteins was specifically inhibited by GalNAc. The toxin binding proteins of M. sexta and H. zea also bound the lectin soybean agglutinin. Taken together these findings suggest that N-acetyl galactosamine might be a component of a CryIA(c) toxin receptor of CF1 cells and of at least two of the insects tested.     

Selective antagonism to the cadherin BT-R1 interferes with calcium-induced adhesion of epithelial membrane vesicles

BT-R(1) is a member of the cadherin superfamily of proteins and is expressed in the midgut epithelium of Manduca sexta during larval development. Previously, we showed that calcium ions influence the structure and stability of BT-R(1) on brush border membrane vesicles (BBMVs) prepared from M. sexta midgut epithelium. In the present study, the effects of calcium and Cry1Ab toxin, produced by Bacillus thuringiensis, on the adhesive properties of BBMVs were investigated. Addition of calcium to a suspension of BBMVs promoted adhesion and aggregation of the vesicles. Treatment of BBMVs with trypsin or lowering the pH (pH 4.0) of the BBMV suspension abolished calcium-induced vesicle aggregation, whereas treatment with deglycosylating enzymes did not affect the aggregation of vesicles, indicating that adhesion and clustering of BBMVs involves protein-protein interactions. Preincubation of BBMVs with Cry1Ab toxin, which specifically binds to BT-R(1) with high affinity and disrupts the midgut epithelium of M. sexta, caused a 50% decrease in calcium-induced vesicle aggregation. The inhibitory effects of the Cry1Ab toxin on BBMV aggregation was blocked completely when the toxin was preincubated with a peptide containing the toxin-binding site of BT-R(1). Cry3A toxin, which is similar in molecular structure to Cry1Ab but does not bind to BT-R(1) and is not toxic to M. sexta larvae, did not affect BBMV aggregation. The results of this study demonstrate that the adhesive function of BT-R(1) is compromised by the Cry1Ab toxin, which acts as a selective antagonist, and supports the notion that BT-R(1) is critical in preserving the integrity of larval midgut epithelium in M. sexta.     

Resistance to Bacillus thuringiensis toxin Cry2Ab in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia

Transgenic cotton, Gossypium hirsutum L., expressing the crylAc and cry2Ab genes from Bacillus thuringiensis (Bt) Berliner variety kurstaki in a pyramid (Bollgard II) was widely planted for the first time in Australia during the 2004-2005 growing season. Before the first commercial Bollgard II crops, limited amounts of cotton expressing only the crylAc gene (Ingard) was grown for seven seasons. No field failures due to resistance to CrylAc toxin were observed during that period and a monitoring program indicated that the frequency of genes conferring high level resistance to the CrylAc toxin were rare in the major pest of cotton, Helicoverpa armigera (Htibner) (Lepidoptera: Noctuidae). Before the deployment of Bollgard II, an allele conferring resistance to Cry2Ab toxin was detected in field-collected H. armigera. We established a colony (designated SP15) consisting of homozygous resistant individuals and examined their characteristics through comparison with individuals from a Bt-susceptible laboratory colony (GR). Through specific crosses and bioassays, we established that the resistance present in SP15 was due to a single autosomal gene. The resistance was recessive. Homozygotes were highly resistant to Cry2Ab toxin, so much so, that we were unable to induce significant mortality at the maximum concentration of toxin available. Homozygotes also were unaffected when fed leaves of a cotton variety expressing the cry2Ab gene. Although cross-resistant to Cry2Aa toxin, SP15 was susceptible to CrylAc and to the Bt product DiPel.     

Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.     

一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响

为探讨苏云金芽孢杆菌Bacillus thuringiensis（Bt）杀虫晶体蛋白与昆虫细胞的相互作用,以Bt Cry1Ac毒素和对该毒素敏感的粉纹夜蛾Trichoplusia ni离体细胞BTI-TN-5B1-4为材料,研究了一些化学物质对Cry1Ac毒素与昆虫离体细胞相互作用的影响.结果表明：N-糖基化抑制剂衣霉素、蛋白质合成抑制剂放线菌酮、胞吞作用抑制剂莫能菌素和胰蛋白酶预处理,都能不同程度地提高BTI-TN-5B1-4细胞对Cry1Ac毒素的敏感性,其中胰蛋白酶预处理的作用最明显;而N-乙酰半乳糖胺不能抑制Cry1Ac毒素对这种离体细胞的毒力.     

Effects of Bt cotton and crylac toxin on survival and development of pink bollworm (Lepidoptera: Gelechiidae).

We evaluated the effects of Bacillus thuringiensis (Bt) toxin CrylAc on survival and development of a susceptible strain and laboratory-selected resistant strains of pink bollworm, Pectinophora gossypiella (Saunders). For susceptible and resistant strains tested on artificial diet, increases in CrylAc concentration reduced developmental rate and pupal weight. In greenhouse tests, survival of resistant larvae on transgenic cotton that produces CrylAc (Bt cotton) was 46% relative to their survival on non-Bt cotton. In contrast, Bt cotton killed all susceptible larvae tested. F1 hybrid progeny of resistant and susceptible adults did not survive on Bt cotton, which indicates recessive inheritance of resistance. Compared with resistant or susceptible larvae reared on non-Bt cotton, resistant larvae reared on Bt cotton had lower survival and slower development, and achieved lower pupal weight and fecundity. Recessive resistance to Bt cotton is consistent with one of the basic assumptions of the refuge strategy for delaying resistance to Bt cotton. Whereas slower development of resistant insects on Bt cotton could increase the probability of mating between resistant adults and accelerate resistance, negative effects of Bt cotton on the survival and development of resistant larvae could delay evolution of resistance.