Depolarization of motor nerve terminals by pyrethroids in susceptible and kdr-resistant house flies

When applied at concentrations of one nM or higher to a house fly larval neuromuscular preparation, deltamethrin (DM) and fenvalerate (FV) greatly increased miniature excitatory postsynaptic potential (mepsp) rate and blocked neuromuscular transmission. The DM-induced mepsp discharge was abolished by tetrodotoxin (TTX), removal of Ca²⁺ from the saline, or by application of hyperpolarizing stimuli to the nerve, indicating that it was due to depolarization of the presynaptic terminals. Also, in the presence of TTX, K⁺ depolarization increased mepsp rate at the same external K⁺ concentration before and after DM treatment, confirming that DM released transmitter by depolarizing the nerve terminals rather than by altering the voltage dependence of transmitter release. The potassium channel blocker tetraethylammonium (TEA) increased mepsp rate somewhat, while aconitine (20 μM), which keeps sodium channels open, increased mepsp rate consistently. Pretreatment of nerves with a subthreshold dose of TEA greatly increased the mepsp rate-increasing activity of DM and aconitine, while a subthreshold level of aconitine did not synergize DM. These observations suggest that DM, like aconitine, depolarized nerves by modifying the sodium channels. Knockdown resistant (kdr) larvae were resistant to the depolarizing action of DM and aconitine but not to that of TEA, indicating that the kdr gene produced a modified sodium channel which was less sensitive to the action of pyrethroids and aconitine. During sustained transmitter release by DM, evoked release gradually declined, resulting in a condition called early block in which spontaneous release was high and release could be evoked by electrotonic depolarization of the nerve terminals, but not by a nerve action potential. Early block was probably due to conduction block in the nerve terminals. Early block eventually gave way to late block, characterized by the decline of spontaneous release to subnormal levels and complete failure of evoked release. After late block, the calcium ionophore X-537A could not release transmitter, suggesting that late block was due to depletion of available transmitter. DM did not have a direct effect upon extrasynaptic muscle membrane. However, after late block, muscles were left insensitive to the putative transmitters glutamate and aspartate when these were bath or iontophoretically applied. A low rate of mepsps persisted after late block, indicating that the muscles were still sensitive to the natural transmitters.     

Steady-state and transient analysis of integument penetration by insecticides

Insecticide penetration studies are usually done by applying a finite dose of material to a finite area of the integument in a solvent, such as acetone, that evaporates very quickly. The time-course of penetration is then measured either as the buildup of material inside the insect or as the decline of material recoverable in a surface wash. Pharmacokinetic models are often used to describe such experiments, but identical models can be formulated from diffusion theory. Diffusion theory was applied to obtain equations that describe the steady-state behavior of a single-layer integument with partitioning between integument and solutions, and a double-layer integument with partitioning between layers and between each layer and the adjacent solution. The steady-state flux equations for both models were first-order, and it was shown that this would be the case for steady-state flux through any number of layers. The steady-state analysis has two limitations. First, the assumption of a steady state implies that the concentration gradient across each layer of the integument is linear, and that this could not occur instantaneously. Second, the analysis assumes that material diffuses from a pool on the surface at some external concentration, whereas, ideally, it is applied as a plane source of infinite concentration. If the steady-state assumption is rejected, the diffusion equation can be solved exactly, using Green's function, for diffusion of a topically applied dose across the integument modeled as a homogeneous plate, if the concentration at the internal face is assumed to remain zero. An analytical expression is obtained for concentration as a function of time and position in the integument, and from it are calculated flux into the insect and disappearance of material recoverable in a solvent wash, where material is assumed to be recoverable to an arbitrary depth. For small values of recovery depth, recovery falls precipitously following application, then progressively more slowly as the experiment proceeds. This phenomenon has been seen in much published data, and is explained adequately for the first time by the theory presented here. Predicted flux into the insect rises with a delay and then falls, but its fall is not exponential, as is often assumed. Peak flux is predicted to be directly related to the diffusion coeficient. The impact of these results on the design of penetration experiments is discussed.     

Metaflumizone is a novel sodium channel blocker insecticide

Metaflumizone is a novel semicarbazone insecticide, derived chemically from the pyrazoline sodium channel blocker insecticides (SCBIs) discovered at Philips-Duphar in the early 1970s, but with greatly improved mammalian safety. This paper describes studies confirming that the insecticidal action of metaflumizone is due to the state-dependent blockage of sodium channels. Larvae of the moth Spodoptera eridania injected with metaflumizone became paralyzed, concomitant with blockage of all nerve activity. Furthermore, tonic firing of abdominal stretch receptor organs from Spodoptera frugiperda was blocked by metaflumizone applied in the bath, consistent with the block of voltage-dependent sodium channels. Studies on native sodium channels, in primary-cultured neurons isolated from the CNS of the larvae of the moth Manduca sexta and on Para/TipE sodium channels heterologously expressed in Xenopus (African clawed frog) oocytes, confirmed that metaflumizone blocks sodium channels by binding selectively to the slow-inactivated state, which is characteristic of the SCBIs. The results confirm that metaflumizone is a novel sodium channel blocker insecticide.     

Anti-Phytophthora cinnamomi activity of Phlomis purpurea plant and root extracts

Phlomis purpurea (Lamiaceae), found in Quercus suber and Quercus ilex ssp. rotundifolia forest habitats in southern Portugal, is a non-host for the oomycete Phytophthora cinnamomi, the main biotic factor involved in cork oak and holm oak decline in the Iberian Peninsula. The effect of P. purpurea crude ethanol root extract was evaluated in vitro on P. cinnamomi mycelial growth, sporangial production, zoospore release and germination as well as on chlamydospore production and viability. The protection of cork oak against infection by the pathogen was also evaluated in planta. At 10 mg ml^?1, in vitro inhibition of the pathogen structures was 85–100 %. In addition, P. purpurea plants were shown to protect Q. suber and Q. ilex from P. cinnamomi infection and to reduce the inoculum potential in glasshouse trials, indicating the ability to reduce root infection by the pathogen. The results suggest that P. purpurea has the potential to reduce disease spread and that their root extracts could provide candidate substances for control of the important pathogen, P. cinnamomi.     

Identification of a mannose‐binding lectin‐like protein recognized by the anti‐CD25 antibody in haemocytes from Cherax quadricarinatus

The crustacean haemolymph contains three main cell populations; however, it is not clear which mechanisms participate in the regulation of cells related to innate immunity. This work aimed to identify potential interleukin‐like receptors that could regulate cellular responses in Cherax quadricarinatus. By histochemical analysis with murine anti‐CD25 staining (targeting the α‐chain of the IL‐2 receptor), we identified that this antibody recognizes cytoplasmic granules in semigranular and granular haemocytes. In haemocytes stimulated with phorbol myristate acetate (PMA), increased fluorescence was observed in these cytoplasmic granules, whereas staining with a human IL‐2 antibody after stimulation with 1–10 ng/ml PMA revealed no overexpression of the receptor or oxidative burst in haemocytes. Two‐dimensional Western blot analysis of haemocyte lysates showed that anti‐CD25 identified a 27.4‐kDa protein with an isoelectric point (pI) of 7.7 and a 46‐kDa protein with a pI of 6.9. De novo sequencing of these proteins identified that they had 32% homology with a mannose‐binding lectin (MBL) from Pacifastacus leniusculus. Our results indicate that a mannose‐binding lectin‐like protein could exert a protective effect that prevents damage from other activated immune responses.     

Life strategies of cephalopod paralarvae in a coastal upwelling system (NW Iberian Peninsula): insights from zooplankton community and spatio‐temporal analyses

The early life stages of cephalopods ‐ octopods, squids, sepiolids and ommastrephids ‐, are uncommon in zooplankton samples and little is known about their life strategies. Accordingly, cephalopod paralarvae were examined in the upwelling ecosystem of the Ría de Vigo (NW Spain) at night from 2008 to 2010. Multivariate analyses and generalized linear models (GLMs) were used to explore relationships between cephalopod paralarvae and the zooplankton communities that they inhabited in 2008. In addition, the foraging strategy and prey preferences of Octopus vulgaris paralarvae within these communities were determined. Multivariate and GLM results showed a strong association of cephalopod paralarvae with coastal and frontal zooplankton communities. Octopus paralarvae were shown to be specialist predators with a strong preference for decapod zoeae in each of the communities examined. Using the three years of sampling, GLM analyses of paralarval spatio‐temporal variations in relation with the upwelling strength showed a positive relationship with upwelling intensity for O. vulgaris and sepiolids, as well as contrasting temporal, horizontal and vertical distributions for the different paralarvae analysed. Under strong upwelling events, Octopus paralarvae were more abundant in surface waters, whereas the abundance of loliginids and sepiolids was higher in the water column. This vertical behaviour in conjunction with the physical conditions of the Western Iberian Upwelling ecosystem suggests the coexistence of two different life strategies: a coastal strategy displayed by loliginid and sepiolid paralarvae that are retained over the shelf, and an oceanic strategy displayed by O. vulgaris paralarvae that are dispersed far from the shelf.     

Identifying suitable temperate grass species and cultural practices for herbage production in the mountain regions of North Vietnam

In North Vietnam, during winter, shortage of herbage constrains the profitability of dairy farming. The first objective of this study was to evaluate the potential of alternative temperate (C₃) forage species, namely common oat ( Avena sativa L.), lop-sided oat ( Avena strigosa Schreb.), Italian ryegrass ( Lolium multiflorum L.) and a commercial forage mixture (Avex), to address the shortage of herbage during winter in the mountain regions of North Vietnam. The second objective was to measure the effects of cultural practices (sowing rate, harvest interval and irrigation level) on yield of dry matter (DM), chemical composition, digestibility of DM and metabolizable energy (ME) concentration of herbage from the best adapted C₃ species previously tested (lop-sided oat). Four experiments were conducted. Oat species proved to be the best adapted species and produced the highest annual yield of herbage (7600 kg DM ha⁻¹) with a high nutritive value. Yields of DM of lop-sided oat were not affected by sowing rate of seed but increased as the length of harvest intervals increased from 30 to 45 and 60 d ( P  <   0·001). Crude protein concentration, digestibility of DM and ME concentration decreased as the harvest interval increased ( P  <   0·01). Irrigation increased the yields of DM of lop-sided oats by 1·3 ( P  <   0·05) but had no effect on the nutritive value of herbage. It is concluded that both common oat and lop-sided oat are suitable species to provide herbage in the winter for the mountain regions of North Vietnam.     

Ozone and Nitrogen Dioxide Levels Monitored in an Urban Area (Ciudad Real) in central-southern Spain

This work describes the evolution of NO₂ and O₃ levels from January to December of 2007, covering the four seasonal periods in the urban air of Ciudad Real in the central-southern Spain. The measurements were carried out by means of passive samplers (Radiello® samplers). Eleven samples were collected weekly, placed at different monitoring site locations. The data indicate that the mean levels obtained during this period for O₃ and NO₂ were 38.5?±?3.5 and 20.8?±?3.8 μg/m³, respectively. These measurements were compared with other studies in Ciudad Real. Meteorological conditions (temperature, pressure, humidity relative, wind speed and direction) were also investigated.     

Molecular epidemiology of foot-and-mouth disease virus type A in South America

A databank of 78 VP(1) complete sequences of type A foot-and-mouth disease virus (FMDV) from South American isolates was constructed. Forty-nine samples corresponded to FMDV that circulated between the years 1999-2008, mainly in Venezuela, where most type A outbreaks have occurred lately and twenty-nine to strains historically relevant for the continent. The phylogenetic analysis showed that all South American FMDV belonged to the Euro-SA topotype. Sixteen subgenotypes could be identified, based on a 15% nucleotide divergence cut-off criterion: eight are extinguished, three were active until the year 2002 and the remaining five circulated in Venezuela during the years 2001-2007, illustrating the potential for FMDV diversification under appropriate selective pressure. The last emergencies reported in already-free areas of Colombia in 2004 and 2008 were closely related to isolates acting in Venzuela. Evidence of positive selection over codon 170, within the immunogenic site 4 of VP1 protein, was recorded. A codon deletion in amino acid position 142, within the G-H loop, was found in some isolates within subgenotypes 14, 15 and 16. Conversely amino acid deletion 197 was restricted to all isolates within a particular genetic cluster. The present work is the first comprehensive phylogenetic analysis of FMDV type A in South America, filling a gap of knowledge with respect to both, historical and acting viruses. The results provided evidence that supports the ecosystem dynamics in the region, and also served as an input to establish genetic links of emergencies in already-declared free areas, highlighting the need for strengthening control activities.