副溶血弧菌Ⅲ型分泌系统vscG基因缺失株的构建及其生物学特性

【背景】副溶血弧菌(Vibrio parahaemolyticus)是一种重要的食源性病原微生物,Ⅲ型分泌系统(TypeⅢSecretionSystem,T3SS)在致病过程中发挥重要作用。vscG基因编码的Vsc G蛋白是Ⅲ型分泌系统的伴侣蛋白。vscG基因在副溶血弧菌中的生物学功能尚未确定。【目的】构建副溶血弧菌vscG基因的缺失株和回补株,研究vscG基因对副溶血弧菌生物学特性的影响。【方法】在副溶血弧菌POR-1菌株基础上利用同源重组法构建vscG基因的缺失株ΔvscG和回补株CΔvscG,分析比较POR-1、ΔvscG和CΔvscG在生长性能、生物被膜形成能力、运动性、溶血活性、细胞黏附和细胞毒性等方面存在的差异。【结果】与POR-1菌株相比,缺失株ΔvscG和回补株CΔvscG的生长特性和溶血活性无明显差异,缺失株ΔvscG的生物被膜形成能力下降,运动性较POR-1菌株和回补株CΔvscG增强。细胞感染试验显示,vscG基因的缺失明显降低了副溶血弧菌对HeLa细胞的黏附和毒性作用。【结论】vscG基因影响副溶血弧菌生物被膜的形成和运动性,在该菌黏附细胞和发挥细胞毒性过程中具有重要作用,为进一步探讨副溶血弧菌T3SS的致病机理奠定基础。     

组氨酸氨解酶HutH对副溶血弧菌生物学特性及致病性的影响

组氨酸氨解酶(HutH)作为组氨酸代谢通路上的第一个代谢酶,控制细菌内部组氨酸的代谢。HutH在多数细菌中高度保守,参与细菌的能量代谢平衡。【目的】选取HutH作为研究对象,探究其对副溶血弧菌生物学特性以及致病性的影响。【方法】利用同源重组的方法构建缺失株ΔhutH和回补株CΔhutH。研究HutH对副溶血弧菌生长特性、组氨酸利用能力、组氨酸代谢相关基因表达水平、运动性、生物被膜、环境耐受、细胞毒性以及对小鼠毒力的影响。【结果】与野生型菌株相比,hutH基因缺失不影响副溶血弧菌的生长特性、耐酸耐碱能力、耐盐能力和群集运动。但ΔhutH在组氨酸作为唯一碳源的M9极限培养基中生长受到显著抑制。另外,我们证实,hutH基因缺失使组氨酸代谢操纵子内的相关基因转录水平显著下降,基因VP0889的表达水平提高。hutH基因缺失导致副溶血弧菌生物被膜形成能力下降、泳动能力下降、对HeLa细胞的毒性降低、对ICR小鼠的致死率显著降低。【结论】本研究表明,hutH基因缺失影响副溶血弧菌代谢组氨酸的能力,而且首次证实,HutH在副溶血弧菌的生物被膜形成、运动性和对小鼠毒力等方面具有重要作用,为从调控组氨酸...     

vcrV基因对副溶血弧菌生物学特性及III型分泌系统1效应蛋白易位的影响

【背景】副溶血弧菌(Vibrioparahaemolyticus)具有两套III型分泌系统(typeIIIsecretion system,T3SS)。T3SS1通过向宿主细胞分泌效应蛋白发挥致病作用,vcrV基因位于T3SS1编码基因簇。【目的】以vcrV基因为研究对象,探索其对副溶血弧菌生物学特性及T3SS1致病机制的影响。【方法】以副溶血弧菌POR-1株为参考菌株,利用同源重组技术构建vcrV基因缺失株ΔvcrV和回补株CΔvcrV,比较各菌株在生长性能、生物被膜形成能力、细胞黏附及细胞毒性等生物学特性的差异,应用Western Blot检测T3SS1诱导条件下POR-1、ΔvcrV和CΔvcrV等菌株效应蛋白分泌量,进一步利用具有过表达载体pMMB207-vp1683-CyaA的各菌株侵染HeLa细胞,通过Western Blot检测细胞内效应蛋白VopR(Vp1683)的易位量。【结果】与基础菌株POR-1相比,缺失vcrV基因不影响副溶血弧菌的生长性能、生物被膜形成能力及细胞黏附等生物学特性,显著降低了对HeLa细胞的毒性作用,具有过表达载体的POR-1、ΔvcrV和CΔv...     

双组分系统EnvZ/OmpR促进副溶血弧菌抵抗碱胁迫的作用机制

【目的】探究双组分系统(two-component system,TCS)EnvZ/OmpR对副溶血弧菌(Vibrio parahaemolyticus,VP)抵抗碱胁迫的作用机制。【方法】用SMART在线工具(https://smart.embl.de/)鉴定出副溶血弧菌基因组中的双组分系统EnvZ/OmpR,再利用同源重组技术将envZ和ompR基因分别进行缺失,构建相应回补株,比较各菌株的生长曲线来检测相应基因对细菌适应高渗透胁迫和碱胁迫的作用,并结合qRT-PCR及荧光检测系统,筛选参与EnvZ/OmpR抵抗碱胁迫的下游靶基因,鉴定该双组分系统对下游基因的调控机制。【结果】在副溶血弧菌基因组中鉴定出vp0155/vp0154编码EnvZ/OmpR双组分系统同源蛋白。△ompR菌株在高渗透胁迫和碱胁迫中的生长能力明显弱于野生株,而回补株C△ompR、△envZ和C△envZ菌株生长能力与野生株类似。在△ompR菌株中,孔道蛋白基因vp1218、vp0493、vpa1745、vpa0085与vpa1308的转录水平均明显低于野生株,并且发现这些孔道蛋白基因缺失株(△vpa1308除外)在碱性环境中生长能力均明显弱于野生株。OmpR蛋白可直接抑制调控因子AphB基因转录,而△aphB菌株在碱胁迫中的生长能力明显强于野生株。此外,AphB蛋白可直接抑制孔道蛋白基因vp0493和vpa0085转录。【结论】双组分系统EnvZ/OmpR促进副溶血弧菌抵抗碱胁迫,其中OmpR蛋白可通过抑制调控因子AphB的表达,以促进部分孔道蛋白的表达,从而增强副溶血弧菌抵抗碱胁迫的能力。     

lpxL和lpxM基因对O1、O78血清型禽致病性大肠杆菌毒力的影响

【目的】为探究脂多糖对O1、O78血清型禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)致病作用的影响。【方法】选取负责脂质A生物合成相关基因lpx L和lpx M,利用λ噬菌体的Red同源重组系统分别构建APECE516(O1血清型)和APECE522(O78血清型)缺失株E516Δlpx L、E516Δlpx M、E516Δlpx LΔlpx M、E522Δlpx L、E522Δlpx M和E522Δlpx LΔlpx M,并通过体内外试验对其生物学特性及致病性进行研究。【结果】各菌株生长速度基本一致。E516Δlpx L、E516Δlpx LΔlpx M、E522Δlpx M和E522Δlpx LΔlpx M的抗血清补体杀菌能力和抗鸡巨噬细胞HD-11吞噬能力较野生株显著下降,而缺失株E516Δlpx M、E522Δlpx L与野生株相比无明显差异;半数致死剂量测定结果显示,除E516Δlpx M、E522Δlpx L外,各缺失株毒力降低1000倍左右;SPF鸡体内动态分布试验结果显示,各缺失株在鸡体内定殖能力较野生株显著下降,但回补株的毒力未能恢复至野生株水平。【结论】lpx L和lpx M基因与O1血清型APECE516株和O78血清型APECE522株的毒力有关,但是lpx L和lpx M基因对E516和E522菌株毒力的影响存在差异。     

信号分子AI-2合成关键基因luxS对副溶血性弧菌生物学特性的影响

[背景]副溶血性弧菌是全球范围重要的食源性病原菌,能引起急性肠胃炎。群体感应系统LuxS/AI-2影响细菌的生物学特性,为研究副溶血性弧菌的传播机制和控制技术提供了新的途径。[目的]探讨群体感应信号分子AI-2合成关键基因luxS对海产品中分离的副溶血性弧菌Vp2009027生物学特性的影响。[方法]利用自杀质粒同源重组技术敲除信号分子AI-2合成关键基因luxS,构建副溶血性弧菌Vp2009027的luxS基因缺失株,通过比较野生株与luxS基因缺失株的生长曲线、AI-2活性、运动能力、生物膜形成能力和耐药性,分析LuxS/AI-2系统对副溶血性弧菌生物学特性的影响。[结果]构建了副溶血性弧菌Vp2009027的luxS基因缺失株,野生株和luxS基因缺失株的生长无明显差异,luxS基因的缺失导致AI-2合成受阻、运动能力和生物膜形成能力增强、四环素耐药性降低。[结论]luxS基因对副溶血性弧菌的生物学特性具有重要的调控作用,为进一步研究副溶血性弧菌的传播机制和研发控制技术提供基础。     

T3SS1和T3SS2影响副溶血弧菌生物学特性及细胞致病性的比较

【目的】探究两套Ⅲ型分泌系统T3SS1和T3SS2影响副溶血弧菌生物学特性及细胞致病性的差异和相关性。【方法】以T3SS1和T3SS2主要结构基因vcrD1和vcrD2为研究对象,利用同源重组技术分别构建单基因和双基因缺失株ΔvcrD1、ΔvcrD2、ΔvcrD1-vcrD2,以及互补株CΔvcrD1和CΔvcrD2;分析各菌株的生长特性、生物被膜形成能力、运动性的差异;比较各菌株对细胞毒性以及对细胞炎性因子转录水平的影响。【结果】与野生株相比,各缺失株的生长速度无显著差异。缺失株ΔvcrD1生物被膜形成能力、运动性和细胞毒性均极显著下降;缺失株ΔvcrD2主要表现为细胞炎性因子IL-1β和IL-6转录水平的显著上调,同时对细胞毒性作用下降。双基因缺失株ΔvcrD1-vcrD2在缺失株ΔvcrD1的基础上,生物被膜形成能力、运动性、细胞毒性均进一步显著下降,但在细胞炎性因子的转录水平上,则与ΔvcrD1一致,与野生株相比均无显著差异。【结论】T3SS1和T3SS2对副溶血弧菌生物学特性和细胞致病性的影响存在差异。T3SS1主要影响细菌的生物被膜形成、运动性及细胞毒性作用;T3SS2不影响生物被膜形成、运动性等生物学特性,参与细菌对细胞炎性反应中的负调控作用,同时具有一定的细胞毒性作用。T3SS1有助于副溶血弧菌在环境中的生存,而T3SS2可有利于细菌在宿主体内免疫逃避的过程。T3SS1和T3SS2对副溶血弧菌生物学特性和细胞致病性的影响可能存在一定的相关作用,具体机制有待进一步研究。     

宁波地区环境中副溶血弧菌血清学及毒力相关基因研究

目的了解宁波地区环境来源海产品中副溶血弧菌血清学特点及毒力相关基因分布。方法采集并分离2013年6-10月宁波地区海产品中副溶血弧菌,对其进行O、K抗原血清学分型;并采用PCR或多重PCR的方法来检测溶血素基因(tlh、tdh、trh)、大流行群遗传标志基因(toxRS/new、orf8)和Ⅲ型分泌系统(T3SS1、T3SS2α、T3SS2β)基因。结果从海产品样本中分离鉴定到44株副溶血弧菌的菌株,分属于20种血清型,型别多样,未见优势血清型;溶血素基因检测发现3株tdh+trh-致病性菌株,遗传标志基因检测发现1株tdh+trh-toxRS/new+大流行株,其血清型为O3:K6型;Ⅲ型分泌系统基因检测发现T3SS1基因存在于所有的副溶血弧菌菌株中,而T3SS2α基因则主要分布在tdh+的菌株中。结论宁波地区环境中副溶血弧菌致病性菌株和大流行株的检出,说明该地区具有潜在的食源性疾病爆发的风险。     

外膜蛋白OmpR对副溶血弧菌致病特性的影响

[目的]探究OmpR在副溶血弧菌生物学特性和致病性中发挥的作用。[方法]利用同源重组技术构建了副溶血弧菌ompR基因缺失株(ΔompR)和互补株(CΔompR),分析各菌株的生长特性、运动性和生物被膜形成能力的差异;比较各菌株对细胞黏附、细胞毒性和小鼠致病性的影响。[结果]ompR基因缺失对副溶血弧菌的生长特性、运动性以及细胞毒性无显著影响。但与野生株相比,ΔompR的生物被膜的形成能力显著降低;感染ΔompR的小鼠存活率升高了25%,病变程度更低;ΔompR在小鼠心脏、肝脏和肾脏中的载菌量显著低于野生株,互补株毒力基本恢复至野生株水平。[结论]OmpR参与副溶血弧菌生物被膜形成和致病过程,是副溶血弧菌潜在的毒力因子。     

鼠伤寒沙门菌baeSR基因缺失株的构建及其对抗生素敏感性分析

【背景】沙门菌的多重耐药现象日渐严重,对其耐药机理的研究尤为迫切,双组分系统与细菌耐药性密切相关。【目的】构建鼠伤寒沙门菌baeSR基因缺失株及回补株,探究双组分系统BaeSR对鼠伤寒沙门菌耐药性的影响。【方法】以鼠伤寒沙门菌体外诱导耐药株CR为研究对象,通过自杀质粒p LP12介导的同源重组方法,以氯霉素抗性标记和阿拉伯糖诱导的致死基因vmt进行正、反向双重筛选,获得基因缺失株CRΔbaeSR,并将重组表达质粒pBAD-baeSR转化于CRΔbaeSR构建回补株CR CΔbaeSR。采用微量肉汤稀释法测定11种常见代表药物对野生株、缺失株及回补株的最小抑菌浓度(Minimum inhibitory concentration,MIC),并测定3株菌的生长曲线、运动性及生物膜形成能力。【结果】与野生株相比,环丙沙星(CIP)、恩诺沙星(ENR)、沙拉沙星(SAR)、头孢噻呋(CEF)、庆大霉素(GEN)、阿米卡星(AMK)、安普霉素(APR)对缺失株的MIC值有所下降;缺失株的生长速率稍显缓慢,且最终浓度也相对较低,但并无统计学上的显著差异(P0.05);缺失株的运动性(P0.05)及生物膜形成能力(P0.01)均显著下降。【结论】鼠伤寒沙门菌baeSR基因缺失后,可通过影响其运动性及生物膜形成能力而对抗生素的敏感性产生影响。     

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.     

Structural Peculiarities of Na+,K+-ATPase Isozymes from the Calf Brain

Functionally active preparations of Na⁺,K⁺-ATPase isozymes from calf brain that contain catalytic subunits of three types (1, 2, and 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of their membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na⁺ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na⁺,K⁺-ATPase of the 11 type and minor amounts of isozymes of the 22(1) and the 31(2) type. The axolemma contains 21 and 31 isozymes. A carbohydrate analysis indicated that 11 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the 1 isoform. An enhanced sensitivity of the 3 catalytic subunit of Na⁺,K⁺-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR⁴⁹² Y⁴⁹³ was localized (residue numbering is that of the human 3 subunit). This sequence corresponds to one of the regions of the greatest variability in 1-, 2-, 3-, and 4-subunits, but at the same time, it is characteristic of the 3 isoforms of various species. The presence of the 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na⁺,K⁺-ATPase 31 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the 3 catalytic subunit was shown.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

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.     

The evolution of the Hox cluster: insights from outgroups

Two burgeoning research trends are helping to reconstruct the evolution of the Hox cluster with greater detail and clarity. First, Hox genes are being studied in a broader phylogenetic sampling of taxa: the past year has witnessed important new data from teleost fishes, onychophorans, myriapods, polychaetes, glossiphoniid leeches, ribbon worms, and sea anemones. Second, commonly accepted notions of animal relationships are being challenged by alternative phylogenetic hypotheses that are causing us to rethink the evolutionary relationships of important metazoan lineages, especially arthropods, annelids, nematodes, and platyhelminthes.     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

Elucidation of Structure of Lipid A from the Marine Gram-Negative Bacterium Pseudoalteromonas haloplanktis ATCC 14393T

The chemical structure of lipid A, from the marine -proteobacterium Pseudoalteromonas haloplanktis 14393, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be -1,6-glucosaminobiose 1,4-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3 and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2, respectively.     

Biological control of three floating water weeds,<Emphasis Type="Italic">Eichhornia crassipes,Pistia stratiotes</Emphasis>, and <Emphasis Type="Italic">Salviniamolesta</Emphasis> in the Republic of Congo

Since 1999, four specific weevils (Coleoptera, Curculionidae) were released in the Republic of Congo against three exotic floating water weeds: Neochetina eichhorniae Warner and N. bruchi Hustache against water hyacinth, Neohydronomus affinis Hustache against water lettuce, and Cyrtobagous salviniae Calder and Sands against water fern. Recoveries of exotic weevils were made from all 24 release sites except one, and all four species have established and spread (up to 800 km for water hyacinth weevils). Within a few years of releases, control of water fern and water lettuce was such that fishing and navigation could be resumed, while reductions of water hyacinth populations were only beginning.