Homology between the seed cytolysin enterolobin and bacterial aerolysins

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian treeEnterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins fromAeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences ofA. hydrophila andA. sobria aerolysins showed several similar regions with many residue identites. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.     

Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA

Summary Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R², IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.     

Roles for structural and temporal shelter-changing by fern-feeding lepidopteran larvae

Summary Larvae of the pyralid moth, Herpetogramma aeglealis, construct feeding shelters upon the Christmas fern (Polystichum acrostichoides). Field and laboratory study involving 532 shelters showed that as the larvae mature, they sequentially inhabit approximately 5 shelters of 3 distinct types, constructed at night on different fronds of the same plant. The bundle shelter, simple and ephemeral, is first to be inhabited and constructed. The fiddlehead shelter which houses slightly older larvae strongly resembles contemporaneously emerging frond fiddleheads. The final shelter form, the globe, is a silk-bound ball of leaflets at the frond tip. An individual larva usually constructs 3 globe shelters on different fronds of the same plant before completing its development. As shelter sites, sterile Polystichum fronds are chosen preferentially over fertile fronds. The bundle and fiddlehead shelter forms, less abundant, appear cryptic to humans and perhaps to other vertebrates. The final globe shelter form is larger and quite conspicuous. However, the persistence of empty globe shelters left on the plant as the larva moves to a new one may serve to make searching for larvae less profitable for potential predators and parasites. We suggest that the energetic costs of constructing and occupying multiple shelters may be offset by circumvention of reduced frond palatability and reduced exposure to predators and parasites.     

Effects of defoliation on seed protein concentration in normal and high protein lines of soybean

Two high (NC106, NC111) and two normal (NC103, NC107) seed protein concentration lines, derived from two different recurrent selection populations of soybean (Glycine max L. Merr.) were subjected to partial defoliation at beginning seed fill (R5) under outdoor pot culture and field conditions. The aim of this study was to test the hypothesis that capacity to store N in vegetative organs and/or to mobilize that N to reproductive organs is associated with the high seed protein concentration trait. Symbiotic N₂ fixation was the sole source of N in the pot experiment and the major source of N (met > 50% of the N requirement) in the low N soil used in the field experiment. Seed protein concentration and seed yield at maturity in both experiments and N accumulation and mobilization between R5 and maturity in the pot experiment were measured. The four genotypes did not differ significantly with respect to the amount of N accumulated before beginning seed fill (R5). Removal of up to two leaflets per trifoliolate leaf at R5 significantly decreased the seed protein concentration of NC107/111 but had no effect on this trait in NC103/106. Defoliation treatments significantly decreased seed yield, whole plant N accumulation (N₂-fixation) during reproductive growth and vegetative N mobilization of all genotypes. Differences in harvest indices between the high and low protein lines accounted for approximately 35% of the differences in protein concentration. The two normal protein lines mobilized more vegetative N to the seed (average. 5.26 g plant^–1) than the two high protein lines (average. 4.28 g plant^–1). The two high seed protein lines (NC106, NC111) exhibited significantly different relative dependencies of reproductive N accumulation on vegetative N mobilization, 45% vs. 29%, in the control treatment. Whereas, NC103 with normal and NC106 with high seed protein concentration exhibited similar relative dependencies of reproductive N accumulation on vegetative N mobilization, (47% vs. 45%). Collectively, these results indicate that N stored in shoot organs before R5 and greater absolute and relative contribution of vegetative N mobilization to the reproductive N requirement are not responsible for the high seed protein concentration trait.Abbreviations DAT days after transplanting - R5 fifth reproductive stage according to Fehr and Caviness, 1977 Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Behavioural response of Heterorhabditis megidis towards plant roots and insect larvae

The behavioural response of infective juveniles (IJs) of Heterorhabditis megidis (strain NLH-E87.3) to cues from roots of strawberry (Fragaria x ananassa Duch.), thuja (Thuja occidentalis L.) and to larvae of the black vine weevil, Otiorhynchus sulcatus, was studied. Choice assays were conducted in an Y-tube olfactometer filled with moist sand. Infective juveniles were activated by the presence of intact roots of both strawberry and thuja plants. Some nematodes aggregated in the compartments with roots but most moved away from the roots to the opposite side. Given a choice, IJs showed a preference for strawberry roots above O. sulcatus larvae. No difference in preference was observed between thuja roots and O. sulcatus larvae. The combination of strawberry roots with vine weevil larvae was preferred above roots alone. In the assays with thuja roots and larvae versus thuja roots alone, however, IJs were stimulated to move but showed preference for the opposite compartment away from the arms with roots and larvae. Nematodes responded differently to mechanically damaged roots as opposed to roots damaged by vine weevil larvae. In assays with damaged thuja roots, IJs were most attracted by the roots damaged by larvae, whereas in the strawberry assays IJs showed a clear preference for the mechanically damaged roots. When challenged with a choice between strawberry and thuja roots, IJs moved preferentially to strawberry than to thuja roots. A preference for the combination of strawberry roots plus larvae over the thuja roots plus larvae was also observed.     

Effects of chlorogenic acid-and tomatine-fed caterpillars on the behavior of an insect predator

If generalist insect predators are a selective force contributing to patterns of feeding specialization by insect herbivores, then predators should be deterred from eating allelochemical-fed prey. The attack and feeding behaviors of naive predators (Podisus maculiventris stinkbugs) reared on control caterpillars (Manduca sexta) fed plain diet were compared to experienced predators reared on caterpillars fed tomato allelochemicals. Tomatine-fed prey were found more quickly by both naive and tomatine-experienced predators, and chlorogenic acid-experienced predators were more stimulated to begin searching for prey. However, experienced predators were less likely to attack both chlorogenic acidfed and tomatine-fed caterpillars than were naive predators. These results indicate that allelochemical-fed prey were easier for predators to locate, but allelochemical-containing prey often deterred predation by experienced predtors.     

Cytosolic and nuclear localization of the cytolytic and insecticidal plant protein enterolobin

Enterolobin is a 52 905 Da cytolytic, inflammatory and insecticidal protein obtained from seeds of the Brazilian tree Enterolobium contortisiliquum. It shares structural and functional similarities with aerolysins, pore-forming toxins from Aeromonas bacteria. These characteristics suggest that enterolobin might play a defence role in the plant. In order to determine the localization and distribution of this phytocytolysin in the seed cells, immunocytochemical labelling experiments were carried out using rabbit anti-enterolobin serum and colloidal gold conjugated with goat anti-rabbit IgG. Light microscopy results indicated that labelling was widely distributed in the cytosol (33%) of cotyledon cells and in the nucleus (67%). Electron microscopy results confirmed the presence of enterolobin in both the cytosol and nucleus. Putative nuclear localization signals (NLS) were found in the sequence of enterolobin.Keywords: Enterolobium contortisiliquum, plant cytolysin, cytolytic protein, immunocytolocalization, nuclear localization signal      

Intrinsic glycosylation potentials of insect cell cultures and insect larvae

Summary The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared. TheSpodoptera frugiperda (Sf21) andTrichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked oligosaccharides. The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types. Similarly, theT. ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides. Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus inT. ni larvae was modified with complex oligosaccharide having sialic acid linked α(2–6) to galactose.     

Phylogenetic affiliation of Aeromonas culicicola MTCC 3249(T) based on gyrB gene sequence and PCR-amplicon sequence analysis of cytolytic enterotoxin gene

We determined the gyrB gene sequences of all 17 hybridizations groups of Aeromonas. Phylogenetic trees showing the evolutionary relatedness of gyrB and 16S rRNA genes in the type strains of Aeromonas were compared. Using this approach, we determined the phylogenetic position of Aeromonas culicicola MTCC 3249(T), isolated from midgut of Culex quinquefasciatus. In the gyrB based-analysis A. culicicola MTCC 3249(T) grouped with A. veronii whereas, it grouped with A. jandaei in the 16S rRNA based tree. The number of nucleotide differences in 16S rRNA sequences was less than found with the gyrB sequence data. Most of the observed nucleotide differences in the gyrB gene were synonymous. The Cophenetic Correlation Coefficient (CCC) for gyrB sequences was 0.87 indicating this gene to be a better molecular chronometer compared to 16S rRNA for delineation of Aeromonas species. This strain was found to be positive for the cytolytic enterotoxin gene. PCR-Amplicon Sequence Analysis (PCR-ASA) of this gene showed that the isolate is affiliated to type I and is potentially pathogenic. These PCR-ASA results agreed in part with the gyrB sequence results.     

Effect of some crude and azadirachtin-enriched neem (Azadirachta indica) seed kernel extracts on larvae of Aedes aegypti

Bioassays against larvae of Aedes aegypti were conducted with neem seed kernel extracts obtained by extraction with water and organic solvents. Permanent exposure of fourth instar larvae to treated water resulted in a conspicuous growth-disrupting effect, mainly during imaginal development. The effectiveness of the extracts increased with decreasing polarity of the solvents used for extraction. Three neem seed kernel extracts caused an extreme prolongation of the larval period when first instar larvae were continuously exposed to treated water until adult emergence. The time necessary for lethal action of neem seed kernel extracts to set in was similar to that reported for some synthetic IGR's.

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Zusammenfassung Zehn mit Wasser und verschiedenen organischen Lösungsmitteln hergestellte Niem-Samen-Extrakte wurden auf ihre Wirkung auf Larven der Gelbfiebermücke Aedes aegypti untersucht. Dauerhaltung der Viertlarven in behandeltem Wasser führte zu einer beträchtlichen wachstumshemmenden Wirkung, die hauptsächlich während der Imaginalentwicklung in Erscheinung trat. Der Wirkungsgrad der Extrakte steigerte sich im biotest mit abnehmender Polarität der bei der Extraktion verwendeten Lösungsmittel. Drei Extrakte (ANSKE, AZT-VR-K-E und MTB/H₂O-K-NR-E) deren Wirkung auch auf Erstlarven untersucht wurde, verursachten erhebliche Entwicklungsverzögerungen wenn die Larven bis zum Erscheinen der Imagines behandeltem Wasser ausgesetzt waren. Die von den Extrakten verursachte Mortalität trat zu einem ähnlichen Zeitpunkt ein, wie er für einige synthetische Insektenwachstumshemmer berichtet wird.

     

Cry1C蛋白对4种棉花害虫的致死效果

【背景】苏云金芽胞杆菌(Bt)属革兰氏阳性细菌,是目前世界上应用最广泛的杀虫剂。来自Bt的Cry1Ac基因自1997年商业化以来,其产生的蛋白对棉铃虫、红铃虫等鳞翅目害虫起到了很好的控制作用。但该基因对甜菜夜蛾、斜纹夜蛾、玉米螟等害虫的控制作用较差,因此,需要寻找新的Bt基因亚型控制上述害虫。【方法】将Cry1C蛋白加到人工饲料上形成药膜后,接入低龄幼虫进行生物测定,以常规不加毒蛋白的饲料为对照,计算1、3、5、7 d后不同害虫的校正死亡率。【结果】随着Cry1C蛋白浓度的增加及时间的延长,甜菜夜蛾和斜纹夜蛾的校正死亡率逐渐升高,Cry1C浓度为10μg·m L-1时,处理7 d后,甜菜夜蛾和斜纹夜蛾的校正死亡率分别为100%和85.42%,表明甜菜夜蛾比斜纹夜蛾更敏感,而棉铃虫和玉米螟对Cry1C蛋白的敏感性较差。【结论与意义】Cry1C基因可作为控制棉田甜菜夜蛾和斜纹夜蛾的有价值的候选基因,但是对棉铃虫和玉米螟无效。该研究为筛选新的Bt基因亚型用于防治不同的棉花害虫提供了数据参考。     

Isozyme and seed protein phylogeny of the genusCitrullus (Cucurbitaceae)

Electrophoretic analysis of 26 enzyme coding genes was conducted on accessions of threeCitrullus species and the relatedPraecitrullus fistulosus andAcanthosicyos naudinianus. The isozyme phylogeny of the genusCitrullus and the related species was constructed based on pairwise measurements of the respective genetic distances between the species and races.P. fistulosus andA. naudinianus form two distinct outgroups toCitrullus which is characterized by two main clusters: The first includes twoC. colocynthis races and the second,C. lanatus andC. lanatus var.citroides, which are more closely related to each other than they are toC. ecirrhosus. The isozyme phylogeny is consistent with the variability in six seed protein bands and with the crossability relations among the examined species.     

Partial recovery of cell membrane-bounded human interleukin-2 fusion protein from insect cell debris by using various detergent extractions

We previously found that the human interleukin-2 (hIL-2) fused with green fluorescent protein (GFP) mainly remained in the insect cell debris after disruption due to the highly hydrophobic property of hIL-2 itself. Even though the significant GFPuv/hIL-2 fusion proteins were associated with cell membrane fractions, these were still functionally active. Therefore, to increase the total product yield, we performed partial recovery of the cell membrane-bounded hIL-2 fusion protein from the insoluble cell debris using several non-ionic, zwitterionic, and anionic detergents.     

Toxicity of insecticides to obliquebanded leafroller, Choristoneura rosaceana, larvae and adults exposed previously to neem seed oil

Adult and sixth instar obliquebanded leafroller, Choristoneura rosaceana (Harris), exposed previously as fifth instar larvae to sub-lethal concentrations of neem, Azadirachta indica A. Juss., seed oil contained in artificial diet were more susceptible to topically applied pyrethroid, carbamate and organophosphate insecticides than C. rosaceana reared on control diets. Increased susceptibility to insecticides did not result from reduced vigour, as measured by reductions in adult or larval weights, but is instead correlated with previously demonstrated reductions in detoxication enzyme activities. The modest increases in susceptibilities, not exceeding ca. 4.5-fold, for C. rosaceana exposed to neem were achieved with subjects from a susceptible laboratory colony; a larger response could be expected for resistant field populations having elevated detoxication enzyme levels. Neem-based insecticides could be useful tools for the management of orchard pests that have developed resistance to synthetic neurotoxins. These findings also contribute to a better understanding of the numerous physiological changes that occur in larval Lepidoptera following exposure to neem extracts.     

Silk filaments enhance the settlement of stream insect larvae

The recombination that follows natural hybridization may produce hybrid genotypes with traits that are intermediate or extreme relative to the parental species, and these traits may influence the relative fitness of the hybrids. Here we examined leaf ecophysiological traits that may influence fitness patterns in a natural plant hybrid zone. We compared the biochemical photosynthetic capacity of Ipomopsis aggregata, I. tenuituba, and early generation hybrids, as well as their photosynthetic responses to varying light and temperature, two abiotic factors found to differ among sites along the hybrid zone. In general, ecophysiological traits expressed in these plants were consistent with their natural habitat, even when grown under common greenhouse conditions. I. tenuituba reached higher photosynthetic rates (A) at higher light levels than I. aggregata, and also had a higher optimal temperature for photosynthesis (T _opt). This suite of traits may reflect adaptations to the more exposed, rocky sites where I. tenuituba is found, compared to the more vegetated, mesic I. aggregata site. Hybrids had characters that were largely intermediate or tenuituba-like, but particular individual hybrids were extreme for some traits, including light saturation level, light-saturated A, and T _opt. Many of these traits are consistent with adaptations reported for plants found in warm, dry sites, so they may put certain hybrids at an advantage at the relatively xeric center of the natural hybrid zone.     

鳞翅目昆虫中肠Bt杀虫蛋白受体研究进展

杀虫晶体蛋白(insecticidal crystal proteins,ICPs;含有Cry和Cyt 2大家族)和营养期杀虫蛋白(vegetative insecticidal proteins,Vips)等Bt杀虫蛋白可有效防治鳞翅目害虫,其中Cry应用最广泛。然而,一些地区的鳞翅目害虫已对Bt杀虫蛋白产生了抗性。目前,普遍认为鳞翅目昆虫中肠受体与Bt杀虫蛋白结合能力的改变是导致其对Bt杀虫蛋白产生抗性的最主要因素。在鳞翅目昆虫中,Cry受体是研究得最为透彻的Bt受体,已经被证实的有氨肽酶N、钙黏蛋白、碱性磷酸酶和ABC转运蛋白等。Vips杀虫蛋白类与鳞翅目昆虫中肠受体的结合方式与Cry杀虫蛋白相似,但结合位点与Cry杀虫蛋白不同。本文从结构特点、作用机制及不同鳞翅目昆虫间的表达差异等角度对以上4种鳞翅目昆虫中肠Bt受体进行了综述,并提出如下展望:(1)以棉铃虫或小菜蛾等鳞翅目昆虫为农业害虫模式生物进行深入研究,阐明其对Bt杀虫蛋白产生抗性的机制,为研究其他鳞翅目农业害虫对Bt杀虫蛋白产生抗性的机制提供理论借鉴;(2)鉴于在不同鳞翅目昆虫间,中肠Bt受体与Bt杀虫蛋白结合存在差异,且同一Bt杀虫蛋白与鳞翅目昆虫Bt受体并不专一性结合,Bt杀虫蛋白多基因组合策略是较为有效的田间鳞翅目昆虫防治策略,是今后一段时间内Bt杀虫蛋白应用的发展方向。     

Characterization of growth and Oryctes rhinoceros nudivirus production in attached cultures of the DSIR-HA-1179 coleopteran insect cell line

The DSIR-HA-1179 coleopteran cell line is a susceptible and permissive host to the Oryctesrhinoceros nudivirus (OrNV), which has been used as a biocontrol agent against the coconut rhinoceros beetle (Oryctes rhinoceros); a pest of palms in the Asia-Pacific region. However, little is known about growth and metabolism of this cell line, knowledge of which is necessary to develop an in vitro large-scale OrNV production process. The strong anchorage-dependent characteristics of the cell line, its particular fragility and its tendency to form dense clumps when manipulated, are the most likely reasons that have precluded further development of the cell line. In order to characterize DSIR-HA-1179 cells, there was first a need for a reliable technique to count the cells. A homogenous cell suspension suitable for enumeration could be produced by treatment with TrypLE Express™ with optimum mean time for cell release calculated as 30 min. The cell line was adapted to grow in four serum-supplemented culture media namely TC-100, IPL-41, Sf-900 II and Sf-900 III and cell growth, glucose consumption, lactate and ammonia production were assessed from static-batch cultures. The maximum viable cell density was reached in Sf-900 II (17.9 × 10⁵ cells/ml), with the maximum specific growth rate observed in this culture medium as well (0.0074 h⁻¹). Higher production of OrNV was observed in IPL-41 and TC-100 (4.1 × 10⁷ TCID₅₀/ml) than in cultures infected in Sf-900 III (2.0 × 10⁷ TCID₅₀/ml) and Sf-900 II (1.4 × 10⁷ TCID₅₀/ml). At the end of the growth period, glucose was completely consumed in cultures grown in TC-100, while remained in excess in the other three culture media. The cell line produced lactate and ammonia to very low levels in the TC-100 culture medium which is a promising aspect for its cultivation at large-scale.     

Recombinant protein production in insect larvae: host choice,tissue distribution,and heterologous gene instability

The expression of the fluorescent protein, DsRed, facilitates the optimization of protein production in orally-infected whole larvae. Trichoplusia ni was shown to be a much better host for recombinant AcMNPV compared to four other noctuid Lepidopteran species achieving 100% infectivity at the minimal tested dose. The highest density of marker protein was found in endothelial and tracheal cells, fat body, and hemocytes. Trichoplusia ni larvae possessed visually detected color over sequential passages of oral infection until the sixth round. Western blot analysis confirmed the progressive decrease of both tetramer and monomer forms of DsRed. The intact DsRed gene and promoter region was present in late passages, but viral population carrying the heterologous gene had dropped more than 2-logs after the fifth round while the amount of total viral DNA remained unchanged over sequential passages. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An alternative to conventional insect marking procedures: detection of a protein mark on pink bollworm by ELISA*

Four experiments were conducted to examine the feasibility of marking pink bollworm, Pectinophora gossypiella (Saunders) with rabbit immunoglobulin G (IgG) protein for mark-release-recapture studies. Pink bollworm were internally marked by feeding larvae an enriched rabbit IgG diet or externally marked by submerging pupae and spraying adults. Individuals were then assayed for the presence of rabbit IgG by sandwich enzyme-linked immunosorbent assay (ELISA) using anti-rabbit IgG. The internal marker was retained in larvae and retained in prepupae and pupae, but not in adults. A second experiment showed that rabbit IgG was retained on adults that were externally marked as pupae. A third series of tests examined the feasibility of externally marking adults with rabbit IgG. Rabbit IgG was retained on externally marked adults for six days in the field. Protein was retained on marked moths in the laboratory after they were captured on and removed from sticky traps. Finally, laboratory tests showed that large groups of externally marked moths transferred rabbit IgG to unmarked moths, but individual males do not readily transfer the protein to unmarked females in small vials.