The effect of two proteinase inhibitors, E-64 and the Bowman-Birk inhibitor, on the developmental time and mortality of Acanthoscelides obtectus

An artificial bean seed system was used to evaluate the effects of a cysteine proteinase inhibitor (E-64) and a serine proteinase inhibitor (Bowman-Birk inhibitor) on the developmental time and mortality of the common bean weevil, Acanthoscelides obtectus (Say). These inhibitors were incorporated into artificial bean seeds on which the insect fed. To better understand the mode of action of these inhibitors, free amino acids were also added to the seeds, alone and in combination with the inhibitors. E-64 was found to be highly effective in delaying development and increasing mortality of the insect. Both effects were directly related to the concentration of E-64. Bowman-Birk inhibitor had little effect on these parameters. Assays of gut proteolytic activity of insects reared on artificial seeds with various levels of E-64 demonstrated a direct relationship between E-64 concentration in the diet and reduction of gut proteolytic activity. Free amino acid supplementation to the diet did not prevent inhibition of gut proteolytic activity by E-64, but did reverse its effects on developmental time and mortality, strengthening the hypothesis that E-64 operates by inhibition of essential digestive proteinase activity.

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Résumé Des grains artificiels de haricot ont été utilisés pour évaluer les effets sur la durée de développement et la mortalité d' Acanthoscelides obtectus. Say d'inhibiteurs de la protéinase de la cystéine (E-64) et de la protéinase de la sérine (l'inhibiteur de Bowman-Birk). Ces inhibiteurs avaient été incorporés dans les grains artificiels. Pour mieux comprendre leur voie d'action, des acides aminés libres étaient ajoutés à ces graines, seuls ou combinés aux inhibiteurs. E-64 a très efficacement retardé le développement et accru la mortalité; ces 2 effets étaient liés à sa concentration. L'inhibiteur de Bowman-Birk a eu peu d'effets sur ces paramètres. Des expériences sur l'activité protéolytique du tube digestif d'insectes élevés sur des graines artificielles avec différentes concentrations de E-64 ont montré une relation directe entre la concentration en E-64 et la réduction de l'activité protéolytique. L'addition d'acides aminés libres dans l'aliment n'a pas empêché l'inhibition de l'activité protéolytique par E-64, mais a inversé ses effets sur la durée de développement et la mortalité, renforçant l'hypothèse que E-64 agit en inhibant l'activité protéinase digestive essentielle.

     

Patterns of abundance variation in reef fishes near an artificial reef at Guam

Synopsis Development of the fish community on a submerged 16 m barge and variation in fish abundance on nearby transects were surveyed twice monthly for twenty months. A steady increase in abundance was observed for certain fishes on the barge, whereas a few species exhibited distinct seasonal variation on both the barge and transects. Most of the seasonal species settled between March and May.Some seasonal species appeared to be site selective in their settlement and consequently settled juveniles were clumped in their distribution. An abundance of preferred topographical features may be why settlement was relatively high at the study site and indirectly why predators became significantly (r₃ = 7.67^***, N = 37) more abundant at the study area during the months of maximum prey settlement. Concurrent settlement of several species during the same few months may be important because juveniles become an abundant food source to predators during those few months only. Periodic swamping of predators by abundant juvenile prey may improve the chances for individuals of rarer prey species to be overlooked and therefore be succesfully recruited.     

Application of noninvasive techniques to monitor reproductive function in a brown hyena(Hyena brunnea)

Estrus in a 10-yr-old brown hyena was described by monitoring urinary steroid metabolites. These studies were repeated the succeeding year and combined with ultrasonography, vaginal cytology, and laparoscopy to further aid in identifying natural and induced estrus. Collection of semen from a 14-yr-old male brown hyena was accomplished by electroejaculation. Use of frozen semen once during natural estrus and again during estrus induced with follicle stimulating hormone (FSH) did not result in pregnancy. Further studies with this animal indicate that stimulation with gonadotropin releasing hormone (GNRH), rather than FSH, may be the more appropriate treatment for stimulating ovulation in canids or related species. While this study did not result in conception, a significant step toward that end was realized by utilizing urinary estrogen to monitor the estrus cycle.     

A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus

Relationships among several of the ion movements associated with the acrosome reaction of S. purpuratus were investigated. Egg jelly initiates ⁴⁵Ca²⁺ and ²²Na⁺ uptake, and K⁺ and H⁺ efflux. H⁺ efflux and ²²Na⁺ uptake occur with approximately equivalent stoichiometries as rapidly as the appearance of acrosomal rods, perhaps reflecting a linked process. Most K⁺ loss, as measured either by ⁴²K⁺ efflux or K⁺-ion-selective electrodes, occurs after the acrosome reaction is complete. Since an elevation of seawater K⁺ (from 10 to 15 mM) or the addition of 0.5 mM tetraethylammonium (TEA), an inhibitor of K⁺ channels, inhibits the acrosome reaction half-maximally, K⁺ movements or alterations of K⁺-dependent membrane potentials may regulate the triggering by jelly. Most, but not all, of the ⁴⁵Ca²⁺ influx is inhibited with a mixture of 10 μM FCCP, 1 mM CN^?, and 2 μg/ml oligomycin, suggesting that the mitochondria store most of the Ca²⁺. The extracellular Na⁺ concentration affects Ca²⁺ fluxes: sperm placed into 5 mM Na⁺ seawater have enhanced ⁴⁵Ca²⁺ uptake, but do not undergo the acrosome reaction, unless 30 mM Na⁺ is also added. Low Na⁺ concentrations lead to spontaneous triggering, by allowing for both Ca²⁺ influx and Na⁺-dependent H⁺ efflux. At least one early Ca²⁺ requirement precedes the Na⁺ and H⁺ movements, as inferred from attempts at reversing the inhibitors of jelly induction of the acrosome reaction. When sperm are incubated with jelly in the absence of Ca²⁺, then washed and incubated with jelly in the presence of Ca²⁺, the acrosome reaction is triggered only upon the second incubation. However, when sperm are mixed with jelly in the presence of the other inhibitors (verapamil, TEA, 5 mM Na⁺ seawater, low pH, or elevated K⁺), they are altered so that even upon subsequent washing, jelly-mediated triggering is no longer possible. This suggests the existence of an intermediate state in the reaction pathway, that follows an event for which Ca²⁺ is required, but that precedes the Na⁺ and H⁺ movements, which are inhibited by all inhibitors of the acrosome reaction. These data are used to develop a partial sequence of ionic changes associated with the triggering mechanism.     

In vitro culture of macrobrachium eggs

Causes for the death of the eggs in the prawn Macrobrachium nobilii are: i) shedding of eggs by ovigerous female, and ii) infection by epibionts: a Saprolegnial fungus, bacteria (gram negative) and protozoans (Vorticellids and Paramecium). A cause for the death of freshly hatched larvae of some decapods is the reduction in reserve yolk energy in the larvae hatched in the last few batches. To circumvent these disadvantages, an artificial incubator was designed, in which 70% of the 3-day old eggs can successfully be incubated and hatched simultaneously. The isolted eggs are irrigated with filtered and aerated water over a diaphragm in the incubator; the water flushed from below through the diaphragm in the artificial incubator, sways and keeps the eggs continuously in a suspended motion, simulating the irrigation technique of the mother.Presented in the Second International Symposium on Invertebrate Reproduction held in Davis, California during August, 1979     

Schistosoma mansoni, NIH-Sm-PR-2 strain, in non-susceptible Biomphalaria glabrata: protection by Echinostoma paraensei

Sullivan J. T., Richards C. S., Lie K. J. and Heyneman D. 1981. Schistosoma mansoni, NIH-Sm-PR-2 strain, in non-susceptible Biomphalaria glabrata: Protection by Echinostoma paraensei. International journal for Parasitology11:481–484. Among seven inbred genetic stocks of Biomphalaria glabrata that are non-susceptible for the NIH-Sm-PR-2 strain of Schistosoma mansoni (PR-2), five stocks revert to nearly complete susceptibility when first infected with Echinostoma paraensei. These include both stocks in which PR-2 sporocysts are normally destroyed within 3–7 days, and stocks in which sporocysts often survive undeveloped for at least 3 weeks. Hence, these five stocks are resistant to but physiologically suitable for the development of PR-2. Of the two remaining stocks, one remains partly non-susceptible to PR-2, since less than 50 % of echinostome-infected snails revert to susceptibility, while the other stock remains completely non-susceptible to PR-2 following echinostome infection, due perhaps to a high level of residual resistance and/or unsuitability.     

Dissolved oxygen dependent phosphorus release from profundal sediments of Lake Constance (Obersee)

Phosphorus release rates from profundal sediments of Lake Constance (Obersee) have been determined in D.O., pH regulated sediment-water systems. Above 10% O₂ saturation (> 1.2 ppm D. O.) and with pH as in situ, no net release could be found. Sedimentations of diatom sludge (Asterionella formosa) and carbonate-phosphate coprecipitate (CaCO₃.CaHPO₄) increased the release to 0.5 mg × m^–2 × d^–1 which, however, will not be relevant to the P balance in Obersee. The annual phosphorus accumulation in profundal Obersee and Ueberlingersee is, therefore, observed as due to sinking of phosphorus-bound detritus during the stagnation period.The experimental work was carried out at the Limnological Institute of the University of Freiburg/Breisgau (West Germany) and has been supported by the Industrieverband Körperpflege und Waschmittel e.V. and the Gernan Research Council (DFG)The experimental work was carried out at the Limnological Institute of the University of Freiburg/Breisgau (West Germany) and has been supported by the Industrieverband Körperpflege und Waschmittel e.V. and the Gernan Research Council (DFG)     

Bovine pregnancy and nonreturn rates following artificial insemination using a covered sheath

Contamination transferred into the uterus from external genitalia during artificial insemination (AI) has been hypothesized to cause lowered bovine pregnancy rates (PR). Using aseptic techniques, there is still a possibility of uterine contamination during routine AI. Two experiments were conducted to evaluate the effect of two types of sheath covers (CS) placed over the conventional French Medium Syringe assembly (FMS) used for AI. Their use entailed passing the assembly to the external os of the cervix, pushing the FMS through the CS and manipulating the FMS to the cervical uterine junction in the normal manner. Fifty-six day non-return rate (NRR) in dairy and actual PR in beef cattle were evaluated. In Experiment 1, 30 professional technicians were employed to inseminate 7, 387 dairy cows, while in Experiment 2, six technicians with varying levels of experience inseminated 416 beef cows. Least-squares means for NRR in dairy cattle were 78% using a CS and 79% without. Means for PR in beef cattle were 57% using a CS and 62% without. In Experiment 2, the overall PR was lower in Trial l than in other trials (P<0.05). Since some technicians improved with time, the difference due to trial was attributed to technician variation in gaining experience with a CS. Results indicate that general use of a CS in routine AI of apparently healthy cows will not increase PR.     

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.