The ultraspiracle gene of the Spruce Budworm,Choristoneura fumiferana: Cloning of cDNA and developmental expression of mRNA

Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression. Dev. Genet. 22:169–179, 1998. © 1998 Wiley-Liss, Inc.     

Oviposition Behavior of the Eastern Spruce Budworm Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)

We documented 13 behaviors associated with oviposition in unfertilized and fertilized spruce budworm moths, Choristoneura fumiferana, by videotaping active moths with a macro lens. Apart from resting, the most pronounced behaviors were probing, drumming + probing, and egg laying. Probing was a bending of the abdomen and extension of the ovipositor to touch the substrate in a rocking, back and forth motion. Drumming + probing involved tapping of the substrate with the pro- and mesothocacic legs with concurrent bending of the abdomen as above. Egg laying was the actual process of egg deposition onto the needle surface of a balsam fir twig. Both the frequency and the duration of these behavioral elements varied depending on the mating status of the female. Unfertilized females exhibited a higher frequency of probing and egg laying. The duration of probing was longer in unfertilized females, while drumming + probing and egg laying were longer for fertilized females. This study is the basis for future work on the chemosensilla associated with the perception of host-plant surface chemicals by ovipositing females.     

Susceptibility of Various Species of Lepidopterous Pupae to the Entomogenous Nematode Neoaplectana carpocapsae

The susceptibility of certain species of lepidopterous pupae occurring in different ecological situations to the entomogenous nematode, Neoaplectana carpocapsae, was tested. Soil- or litter-pupating lepidopterous insects were not highly susceptible to N. carpocapsae. The most susceptible insect pupating in the soil was Spodoptera exigua with 63% pupal mortality, while Harrisinia brillians, which pupates in litter, had 55% mortality. Other soil- and litter-pupating insects had mortalities of less than 25%. Some insect species that pupate above ground were highly susceptible (> 84% mortality) to N. carpocapsae infection.     

Ultrastructural Effects of a Non-Steroidal Ecdysone Agonist,RH-5992, on the Sixth Instar Larva of the Spruce Budworm,Choristoneura fumiferana

Force feeding of RH-5992 (Tebufenozide), a non-steroidal ecdysone agonist to newly moulted sixth instar larvae of the spruce budworm, Choristoneura fumiferana, (Lepidoptera: Tortricidae) initiates a precocious, incomplete moult. Within 6h post treatment (pt) the larva stops feeding and remains quiescent. Around 12hpt, the head capsule slips partially revealing an untanned new head capsule that appears wrinkled and poorly formed. By 24hrpt, the head capsule slippage is pronounced and there is a mid-dorsal split of the old cuticle in the thoracic region but there is no ecdysis. The larva remains moribund in this state and ultimately dies of starvation and desiccation. The temporal sequence of the external and internal changes of the integument were studied using both scanning and transmission electron microscopy. Within 3hpt, there is hypertrophy of the Golgi complex indicating synthetic activity and soon after, large, putative ecdysial droplets are seen. Within 24h, a new cuticle that lacks the endocuticular lamellae is formed. The formation of the various cuticular components, the degradation of the old cuticle and changes in the organelles of the epidermal cells of the mesothoracic tergite are described. The difference between the natural moult and the one induced by RH-5992 are explained on the basis of molecular events that take place during the moulting cycle. The persistence of this ecdysone agonist in the tissues permits the expression of all the genes that are up-regulated by the presence of the natural hormone but those that are turned on in the absence of the hormone are not expressed.     

Biology of Subanguina picridis,a Potential Biological Control Agent of Russian Knapweed

The knapweed nematode, Subanguina picridis, forms galls on the leaves, stems, and root collar of Russian knapweed, Acroptilon repens. After being revived from a dormant, cryptobiotic state, second-stage juveniles required at least 1 month in a free-living state before becoming infective. Galls were induced on relatively slow-growing host plants that retained their apical meristems at or near the soil surface for 2-5 weeks. Galls developed extensive areas of nutritive tissue. The nematode was introduced from the Soviet Union and released in Canada for the biological control of Russian knapweed.     

Effects of Selected Insecticides and Nematicides on the In Vitro Development of the Entomogenous Nematode Neoaplectana carpocapsae

The effects of organophosphates (mevinphos, phenamiphos, trichlorfon), carbamates (carbofuran, methomyl, oxamyl), a formamidine (chlordimeform), a synthetic pyrethroid (fenvalerate), a chlorinated hydrocarbon (methoxychlor). and an insect growth regulator (diflubenzuron) on in vitro development and reproduction of Neoaplectana carflocapsae were tested by incorporating each chemical into a nematode rearing medium. Organophosphates and carbamates adversely affected development and reproduction at concentrations ≥ 0.1 mg/ml. Phenamiphos was the most toxic, with no nematode reproduction at 0.01 mg/ml. Inoculated infective juveniles developed to adults with some of the organophosphates and carbamates, but limited or no reproduction occurred. Chlordimeform inhibited development at 1.0 mg/ml, while diflubenzuron, fenvalerate, and methoxychlor did not significantly (P > 0.05) reduced reproduction at 1.0 mg/ml. The organophosphate and carbamate nematicides in use for control of plant-parasitic nematodes may be toxic to N. carpocapsae in the soil.     

Evaluation of the Protective and Therapeutic Properties of DBCP for Control of Root-Knot Nematode on Tomato

Twelve soil drenches over a period of 30 days with DBCP concentrations of 40 μg/ml did not completely prevent infection of tomato plants by root-knot nematode juveniles. Repeated DBCP drenches of 40 μg/ml halted gall development during the drenches, but 10 days after drenching was discontinued galls were apparent. DBCP drenches at 200 μg/ml prevented tomato root development, and 40 μg/ml slowed it. Ten μg/ml increased the height of root-knot-infected plants, but not their top weights. Treated plants were lanky. Protective drenches of 2.5 to 40 μg/ml of DBCP decreased nematode populations and increased fruitfulness. DBCP as a therapeutant reduced the incidence of galling on new roots and halted increases in gall size on previously infected roots but did not improve fruitfulness or plant size significantly.     

Influence of Soil pH and Oxygen on Persistence of Steinernema spp.

Survival of infective juveniles of Steinernema carpocapsae and Steinernema glaseri gradually declined during 16 weeks of observation as the tested soil pH decreased from pH 8 to pH 4. Survival of both species of Steinernema dropped sharply after 1 week at pH 10. Survival or S. carpocapsae and S. glaseri was similar at pH 4, 6, and 8 during the first 4 weeks, but S. carpocapsae survival was significantly greater than S. glaseri at pH 10 through 16 weeks. Steinernema carpocapsae and S. glaseri that had been stored at pH 4, 6, and 8 for 16 weeks, and at pH 10 for 1 or more weeks were not infective to Galleria mellonella larvae. Steinernema carpocapsae survival was significantly greater than that of S. glaseri at oxygen:nitrogen ratios of 1:99, 5:95, and 10:90 during the first 2 weeks, and survival of both nematode species declined sharply to less than 20% after 4 weeks. Survival of both nematode species significantly decreased after 8 weeks as the tested oxygen concentrations decreased from 20 to 1%, and no nematode survival was recorded after 16 weeks. Steinernema carpocapsae pathogenicity was significantly greater than that of S. glaseri during the first 2 weeks. No nematode pathogenicity was recorded at oxygen concentrations of 1, 5, and 10% after 2 weeks and at 20% after 16 weeks.     

Ultrastructural Study of the Development of Pleistophora schubergi Zwölfer, 1927 (Protozoa,Microsporida) in Larvae of the Spruce Budworm,Choristoneura fumiferana and Its Subsequent Taxonomic Change to the Genus Endoreticulatus

This study demonstrates that Pleistophora schubergi Zwölfer, 1927, a microsporidium originally isolated from the midgut epithelium of Nygmia phaeorrhoea Don (Euproctis chrysorrhoea L.) and Porthetria dispar L., and subsequently reported in several other insects including the spruce budworm, Choristoneura fumiferana (the host used in this investigation), does not belong in the genus Pleistophora Gurley, 1893. Pleistophora schubergi lacks the major features that are characteristic of Pleistophora typicalis, the type species of this genus. A comparison of ultrastructural observations reported for the type species of the genus Pleistophora, P. typicalis, and our observations of P. schubergi revealed significant differences. A thick (0.5 μm) amorphous coat, derived from parasite secretions and deposited external to the parasite plasmalemma, surrounds all developmental stages in P. typicalis. Double membranes, derived from host rough endoplasmic reticulum cisternae encircle the parasite plasmalemma of all developmental stages in P. schubergi. The sporophorous vesicle encases the spores in P. typicalis, and originates from the parasite-secreted coat that is present around meronts. In P. schubergi, the host endoplasmic reticulum cisternae form the envelope that surrounds the meronts. Moreover, the sporophorous vesicle envelope in P. typicalis persists around groups of spores, while in P. schubergi this envelope breaks easily to release the spores in the host cytoplasm. By comparing the characteristics of the microsporidium found in the spruce budworm with those of the recently created polysporous genera that sporulate within a vesicle, we found that P. schubergi does belong in the new genus Endoreticulatus Brooks et al. 1988, and consequently rename it Endoreticulatus schubergi (Zwölfer, 1927) n. comb.     

wrinkled1: A Novel, Low-Seed-Oil Mutant of Arabidopsis with a Deficiency in the Seed-Specific Regulation of Carbohydrate Metabolism

During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling.     

Cytokine Control of Inflammation and Repair in the Pathology of Multiple Sclerosis

Cytokines are secreted signaling proteins that play an essential role in propagating and regulating immune responses during experimental autoimmune encephalomyelitis (EAE), the mouse model of the neurodegenerative, autoimmune disease multiple sclerosis (MS). EAE pathology is driven by a myelin-specific T cell response that is activated in the periphery and mediates the destruction of myelin upon T cell infiltration into the central nervous system (CNS). Cytokines provide cell signals both in the immune and CNS compartment, but interestingly, some have detrimental effects in the immune compartment while having beneficial effects in the CNS compartment. The complex nature of these signals will be reviewed.     

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca²⁺ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca²⁺ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca²⁺ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.     

Effects of Enzymes,Chemicals, and Tempertaure on Steinernema carpocapsae Attraction to Host Plasma

Migration of exsheathed infective juveniles of Steinernema carpocapsae to plasma of the host insect Spodoptera litura was not affected by treatments with the lectins concanavalin A, soybean agglutinin, or wheat germ agglutinin; with the enzymes neuraminidase, α-mannosidase, lipase, pronase, or phospholipase C; or with cetyl trimethylammonium bromide or spermidine. Treatment with sodium metaperiodate or sodium hypochlorite inhibited nematode attraction towards insect plasma; numbers of randomly wandering nematodes increased. Nematode migration towards the source of attraction was unaffected by temperatures below 33 C but was impaired at 35 and 37 C. The adverse effect of 5 mM and 10 mM NaIO₄ on migratory behavior was reversed 24 hours after rinsing with buffered saline. The effect of NaOCl on nematode behavior was slightly reversible at concentrations of 0.2 and 0.4% (v/v) but apparently irreversible at 0.6 and 1.0%. The effect of heat treatment at 35 and 37 C was reversible.     

SeinFit,a Computer Program for the Estimation of the Seinhorst Equation

A computer program, "SeinFit," was created to determine the Seinhorst equation that best fits experimental data on the relationship between preplant nematode densities and plant growth. Data, which can be entered manually or imported from a text file, are displayed in a data window while the corresponding graph is shown in a graph window. Various options are available to manipulate the data and the graph settings. The best-fitting Seinhorst equation can be calculated by two methods that are both based on the evaluation of the residual sum of squares. Depending on the method, a range of values for different parameters of the Seinhorst equation can be chosen, as well as the number of steps in each range. Data, graphs, and values of the parameters of the Seinhorst equation can be printed. The program allows for quick calculation of the danaage threshold density - one of the parameters of the Seinhorst model. Versions written for Macintosh or DOS-compatible machines are currently available through the Society of Nematologists'' World Wide Web site (http://ianrwww.unl.edu/ianr/plntpath/ nematode/SOFTWARE/nemasoft.htm).     

Evaluation of Cultivars,Experimental Lines and Plant Introduction Collection of Sainfoin for Resistance to Meloidogyne hapla Chitwood

Stands of several cultivars and experimental lines of sainfoin (Onobrychis viciifolia) were severely reduced (92% average loss) in a field naturally infested with Meloidogyne hapla. Stands of two alfalfa cultivars included in the test were unaffected. In studies conducted in the greenhouse with plants inoculated at the time of seeding, average mortality was 55% for sainfoin entries and 7% for Ladak alfalfa. Little mortality occurred when plants were inoculated after establishment. Three months after inoculation, all sainfoin entries were heavily galled (range of 3.3-3.7 on a scale of 1-4) while roots of Ladak were only slightly galled (rating of 1.6). Intermating of plants selected in the field plots for resistance to M. hapla showed a slight increase in resistance. Of the 147 plant introduction lines tested in the greenhouse, none were resistant to M. hapla.     

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.