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The sesquiterpenoid juvenile hormone (JH) regulates insect development and reproduction. Most insects produce only one chemical form of JH, but the Lepidoptera produce four derivatives featuring ethyl branches. The biogenesis of these JHs requires the synthesis of ethyl-substituted farnesyl diphosphate (FPP) by FPP synthase (FPPS). To determine if there exist more than one lepidopteran FPPS, and whether one FPPS homolog is better adapted for binding the bulkier ethyl-branched substrates/products, we cloned three lepidopteran FPPS cDNAs, two from Choristoneura fumiferana and one from Pseudaletia unipuncta. Amino acid sequence comparisons among these and other eukaryotic FPPSs led to the recognition of two lepidopteran FPPS types. Type-I FPPSs display unique active site substitutions, including several in and near the first aspartate-rich motif, whereas type-II proteins have a more "conventional" catalytic cavity. In a yeast assay, a Drosophila FPPS clone provided full complementation of an FPPS mutation, but lepidopteran FPPS clones of either type yielded only partial complementation, suggesting unusual catalytic features and/or requirements of these enzymes. Although a structural analysis of lepidopteran FPPS active sites suggested that type-I enzymes are better suited than type-II for generating ethyl-substituted products, a quantitative real-time PCR assessment of their relative abundance in insect tissues indicated that type-I expression is ubiquitous whereas that of type-II is essentially confined to the JH-producing glands, where its transcripts are approximately 20 times more abundant than those of type-I. These results suggest that type-II FPPS plays a leading role in lepidopteran JH biosynthesis in spite of its apparently more conventional catalytic cavity., ((c) 2006 Wiley-Liss, Inc.)

Chemical ecology is the study of how particular chemicals are involved in interactions of organisms with each other and with their surroundings. In order to reduce insect attack, plants have evolved a variety of defence mechanisms, both constitutive and inducible, while insects have evolved strategies to overcome these plant defences (such as detoxification enzymes). A major determinant of the influence of evolutionary arms races is the strategy of the insect: generalist insect herbivores, such as Myzus persicae aphid, need more complex adaptive mechanisms since they need to respond to a large array of different plant defensive chemicals. Here we studied the chemical ecology of M. persicae associated with different plant species, from Brassicaceae and Solanaceae families. To identify the involved adaptation systems to cope with the plant secondary substances and to assess the differential expression of these systems, a proteomic approach was developed. A non-restrictive approach was developed to identify all the potential adaptation systems toward the secondary metabolites from host plants. The complex protein mixtures were separated by two-dimensional electrophoresis methods and the related spots of proteins significantly varying were selected and identified by mass spectrometry (ESI MS/MS) coupled with data bank investigations. Fourteen aphid proteins were found to vary according to host plant switch; ten of them were down regulated (proteins involved in glycolysis, TCA cycle, protein and lipid synthesis) while four others were overexpressed (mainly related to the cytoskeleton). These techniques are very reliable to describe the proteome from organisms such as insects in response to particular environmental change such as host plant species of herbivores.

Lectins are carbohydrate binding proteins which are widely distributed in nature: they have been isolated from microorganisms, fungi, plants and animals. Many of these proteins were tested for their potential biocide effect on lot of pests. Indeed, lectins can cause dramatic changes in the cellular morphology and metabolism, particularly on the digestive system of insect having ingested them, by lectin binding to membrane glycosyl groups of the digestive tract cells. A fungal lectin, namely Xerocomus Chrysenteron lectin (XCL) was previously purified and was shown to be toxic to several pests including aphids. At the cell level, an increase in the endocytosis, the induction of morphological changes such as the actin cytoskeleton shape was determined. In this work, the recombinant XCL was produced and was tested for its potential aphicide effect on Myzus persicae, a polyphagous aphid found on more than 400 host plant species and transmitting more than 100 viral diseases. We developed bioassays using different artificial diets incorporating a broad range of XCL concentrations (from 10 microgx ml(-1) to lmg.ml(-1)) to assess the potential negative effects of XCL on the development and reproduction of the M. persicae aphid. Significant mortality rates, changes of developmental durations and nymph production were observed depending on the XCL concentration in the artificial diet. Concanavalin A lectin was also used in a new range of experiments to compare the effects of the two lectins on the aphid biological parameters. According to the observed dose responses toward the XCL and Con-A lectins included in the artificial diet and the action mode of this kind of proteins, the perspectives of lectin use in pest control will be discussed.

A field experiment designed to assess the biodiversity related to nettle strips closed to crops, and more particularly the aphid and related beneficial populations, was established in experimental farm located in Gembloux (Belgium). Margin strips of nettle (Urtica dioica) closed to wheat (Triticum aestivum), green pea (Pisum sativum) and rape (Brassicae napus) fields were investigated. The diversity, abundance of aphids and related predators were analysed according to the plant crop species and the differential pesticide application (treated plot and control). Insects were visually observed every week during all the cultivation season. Two main families of aphidophagous predators were found in all field crops and nettle, the Coccinellidae and Syrphidae. The diversity of the aphidophagous predators was shown to be higher on nettle than in field crops, particularly the Chrysopidae, the Anthocoridae and the Miridae. However, a striking difference of ladybird abundance was observed according to the aphid host plant. In one side, Coccinella septempunctata was much more abundant on Acyrthosiphon pisum infested green pea than on the other host plant species. At the opposite, higher occurrence of Harmonia axyridis was observed on the aphid infested nettle plants than on the crop plants. In particular, none of H. axyridis was found in wheat crop. Also, more than only a significant positive correlation between predator and aphid abundance, specialised relations between particular aphid species and some so-called generalist predators was determined in the fields. Finally, intraguild interactions between the aphidophagous predators was assessed and shown that only a significant negative correlation between Episyrphus balteatus and H. axyridis related to the nettle aphid, Micrlophium carnosum, was observed. The relative distribution of the ladybirds, namely C. septempunctata and H. axyridis according to the host plant, nettle strips and crop plots was discussed in relation to integrated pest management approach.

Several aphidophagous beneficials such as parasitoids and predators are known to respond positively to aphid infested plants. Semiochemicals from the latter association play an important role in the foraging of predators in a tritrophic approach. In this work, three host plants infested with green peach aphid, Myzus persicae Sulzer were used to study the effect of prey density and aphid colony location on plant toward Episyrphus balteatus female. Their reproductive behaviour and efficiency (in terms of fecundity) were observed in net cages. Three kinds of experiments were performed using a disc leaf on agar diet in small Petri dishes under different conditions: 1) leaf disc of broad bean (Vicia faba) were infested with different aphid prey densities, 2) three host plants (Vicia faba, Solanum tuberosum, Solanum nigrum) infested with 3 different aphid prey densities and 3) three different heights of aphid colony location on V. faba plant infested with constant aphid density were also tested using leaf disc system. Aphid-free leaf discs were also used as control for predator behaviour observations. Oviposition rates of hoverfly on leaf disc system vary significantly with prey aphid densities. The means of eggs per laying were 0.9, 5.3, and 31.2 for 0, 10 and 100 aphid densities respectively. E. balteatus females were also able to evaluate and adjust oviposition rates according to different aphid prey densities/host plant. The 5 and 20 cm stakes were the most attractive heights of aphid colony location for the hoverfly oviposition. The means of eggs per laying were 16.7, 18.5 and 5.8 for 5, 20 and 40cm heights respectively. Our leaf disc system was found to be a practical and efficient way to assess chemical cues from aphids according to different conditions on the hoverfly reproductive behaviour.

Although, beetles of the genus Tribolium first evolved as saprophylic insects, they have adapted to the stored products environment for several thousand years. In this study reproductive strategies are described for eight species of Tribolium that are known to occur in this environment. Experiments were conducted under the same conditions for every species, and several life history traits, including egg mass, adult mass, developmental time and fecundity were examined and compared among these species. Common reproductive strategies were not found among the different species and univariate analysis highlighted strong differences between the species for most of the traits investigated. Some species showed reproductive traits that are likely to give a fitness advantage in the environment of stored products. Multivariate statistical analysis allowed the detection of different sub-groups with respect to their reproductive strategy. Adult mass and egg-to-adult developmental time discriminated between groups. Intraspecific allometric relationships were further investigated but only a few correlations appeared to be significant.

This is the first report of an insect esterase efficiently expressed in the methylotrophic yeast Pichia pastoris (so far insect esterases have been produced only in the baculovirus system). Having isolated a Tribolium castaneum carboxylesterase cDNA (TCE), we were initially unable to express it in Escherichia coli or P. pastoris despite significant transcription levels. As codon usage bias is different in T. castaneum and P. pastoris, we assumed this was a possible explanation for the translational barrier observed in yeast. Accordingly, we designed and constructed by recursive PCR a synthetic TCE gene (synTCE) optimized for heterologous expression in P. pastoris, i.e., a gene in which certain TCE codons are replaced with synonymous codons 'preferred' in P. pastoris. When the altered gene was placed under the control of either the P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase (AOX1) promoter and introduced on an expression vector into P. pastoris, its product was produced intracellularly. We also successfully explored the possibility of obtaining a secreted product: P. pastoris cells expressing an in-frame fusion of synTCE with the alpha-factor secretion signal under the control of the GAP promoter were found to secrete the recombinant esterase into the external medium (to a concentration of 7 mg/L). In addition to this demonstration of TCE production in yeast, our results suggest that the GAP promoter could advantageously replace the AOX1 promoter as a driver of synTCE expression. TCE specific activity was approximately 5 U/mg when p-nitrophenyl acetate was used as substrate.

In this article, two theories are unified to investigate the effect of hydrodynamics on a specific bioprocess: the network-of-zones (NOZ) hydrodynamic structured modeling approach (developed by several researchers but applied to only a few bioprocesses) and the effectiveness factor eta approach. Two process scales were investigated (20 and 500 L), and for each, hydrodynamics were quantified using an NOZ validated by homogeneity time measurements. Several impeller combinations inducing quite different hydrodynamics were tested at the 20-L scale. After this step, effectiveness factors were determined for each fermentation run. To achieve this, a perfectly mixed microbial kinetic model was evaluated by using simple Monod kinetics with a fed-batch mass balance. This methodology permitted determination of the effectiveness factor with more accuracy because of the relation with the perfect case deduced from the Monod kinetics. It appeared that for the small scale, eta decreased until reaching a value of approx 0.7 (30% from the ideal case) for the three impeller systems investigated. However, stirring systems that include hydrofoils seemed to maintain higher effectiveness factors during the course of the fermentation. This effect can be attributed to oxygen transfer performance or to homogenization efficiency exhibited by the hydrofoils. To distinguish the oxygen transfer from the homogenization component of the effectiveness factor, these phenomena were analyzed separately. After determining the evolution of etaO2 linked to oxygen transfer for each of the fermentation runs, the NOZ model was employed to quantify substrate gradient appearance. After this step, another effectiveness factor, etamix, related to mixing was defined. Consequently, it is possible to distinguish the relative importance of the mixing effect and oxygen transfer on a given bioprocess. The results have highlighted an important scale effect on the bioprocess that can be analyzed using the NOZ model.

Glutathione S-transferases (GST) in insects play an important role in the detoxification of many substances including allelochemicals from plants. Induction of GST activity in Myzus persicae in response to secondary metabolites from Brassica plants was determined using different host plant species and confirmed using artificial diet with pure allelochemicals added. The 2,4-dinitro-1-iodobenzene (DNIB) was found to be a useful substrate for identifying particular GSTs in insects. GSTs from M. persicae were purified using different affinity chromatography columns and related kinetic parameters were calculated. GST isoenzymes were characterised using electrophoretic methods. Although SDS-PAGE results indicated similarity among the purified enzymes from each affinity column, biochemical studies indicated significant differences in kinetic parameters. Finally, the GST pattern of M. persicae was discussed in terms of insect adaptation to the presence of plant secondary substances such as the glucosinolates and the isothiocyanates, from Brassicaceae host plants.

Essential oils from seven plants species currently found in Cameroon were extracted by steam distillation and tested for their insecticidal activities against Sitophilus zeamats Motsch. Responses varied with the test applied and the plant species. For the contact toxicity, the acetone was used in order to dilute the pure essential oil. Formulations of 1% of essential oils of Xylopia aethiopica and Ocimum gratissimum were the most toxic and led to 96 and 98% of mortality respectively after 24 h. There was no death in control (0% of mortality). Fumigation test were done by applying 300 microl of pure essential oils in the bottom of closed 800 ml glass containers. In that case, the essential oil of Hyptis spicigera was the most toxic (fumigant) after 48 hours of fumigation followed by Annona seregalensis and Xylopia aethiopica 96 and 95% of mortality respectively. Some of the essential oils of plant species tested are promising for pest control in farmer granaries. However, further investigations are to be done on formulation and side effects.

The volatiles released from several aphid and host plant species, alone or associated, were studied for their infochemical role in prey location. Using a four-arm olfactometer, the attraction of several combinations of three aphid (Myzus persicae, Acyrthosiphon pisum, and Brevicoryne brassicae) and three plant (Vicia faba, Brassica napus, and Sinapis alba) species toward Adalia bipunctata larvae and adults was observed. Both predatory larvae and adults were attracted only by A. pisum and M. persicae when they were crushed, whatever the host plant. (E)-beta-farnesene, the aphid alarm pheromone, was the effective kairomone for the ladybird. Plant leaves alone (V. faba, B. napus, and S. alba) or in association with nonstressed whole aphids (the three species) did not have any attraction for the predator. The B. brassicae specialist aphid is the only prey that was not attracted to A. bipunctata larvae and adults, even if they were crushed. Release of B. brassicae molecules similar to the host plant allelochemicals was demonstrated by GC-MS analysis. The lack of behavioral response of the ladybird at short distance toward the cruciferous specialist aphid was related only to the absence of (E)-beta-farnesene in the aphid prey volatile pattern.

Glutathione S-transferases (GST) play an important role in the detoxification of many substances including organic pollutants and plant secondary metabolites. We compared the GST of two syrphid species, the aphidophagous Syrphus ribesii and the saprophagous Myathropa florea to assess the relation between feeding type and GST patterns. Differences between the GST of the hoverfly species were observed after purification by affinity chromatography, SDS-PAGE and kinetic studies. While the specific activities of the purified enzymes were different, the purification yields were similar. The variation in specific activities was related to the presence of different isoenzymes in both syrphid species by SDS-PAGE. While two bands of 24 and 32 kDa were observed for M. florea, one more band of 26 kDa was present in S. ribesii. When a range of substrate and glutathione concentrations was tested, differences in Km and Vmax between the glutathione S-transferases from both hoverfly species were also observed. These results are discussed in terms of adaptations to the feeding habit and the habitat of the two syrphid species.

Malathion-specific resistance in the red flour beetle, Tribolium castaneum, is widespread and stable in natural populations even in the absence of pesticide exposure. To understand this stability, both resistant and susceptible males were placed in competition for susceptible female fertilization. Females were then isolated and their progeny was tested for malathion susceptibility. Male reproductive success was estimated for populations originating from different geographic areas and for isogenic strains. In most cases, resistant males had a greater reproductive success rate than susceptibles. The results suggest that male reproductive success is not traded against the selection for malathion resistance, even resistant individuals are at an advantage for this fitness trait. This absence of fitness cost may be the result of postselection of (1) modifier gene which ameliorate the fitness of resistant individuals or (2) nondeleterious resistance gene. Resistant phenotype superiority could be due to increased male mating success, improved ability of resistant males in sperm competition, female mate choice, and/or cryptic female choice of resistance gene(s). The effect of male phenotypic frequency on male reproductive success was also examined. We observed that male fertilization success is frequency dependent and inversely related to their frequency. However, this "rare male" advantage did not counteract the superiority of the resistant males.

Despite that resistance frequency is assumed to decline when selective pressure is relaxed, the stability of resistance frequency has been observed in some insects in the absence of insecticide. In the red flour beetle, Tribolium castaneum, the first case of malathion-resistance was reported in the early 1960s. The malathion-specific resistant phenotype has now almost completely replaced the susceptible one in red flour beetle populations. In the present study, several life-history traits that could influence the fitness of the insects were compared between insecticide-susceptible and malathion-specific resistant populations of the red flour beetle. On average, egg fertility and egg-to-adult development time did not differ between susceptible and resistant populations. However, the fecundity of resistant females was greater than that of susceptible ones. Generally, differences in development time between insecticide resistant and susceptible populations are considered as having more effect on fitness than do differences in fecundity. However, the observed increased female fecundity may participate, in combination with the previously observed increased male reproductive success, to the development and the stability of malathion-specific resistance in T. castaneum.

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.

Myrosinase from Brevicoryne brassicae was purified by ammonium sulfate fractionation, dialysis, and chromatography on a DEAE column. The chromatography yielded a single peak and a 115.6-fold purification. Further FPLC gel filtration gave a single peak at 120 kDa. Denaturing SDS/PAGE of the protein revealed a single band at 60 kDa, indicating that the native B. brassicae myrosinase is a dimer. Kinetic parameters towards 8 glucosinolates were calculated. Strong differences of V(max) and K(m) were observed depending on the substrate. Degradation products of each glucosinolate were identified and quantified by GC-MS and GLC-FID, respectively. Using both crude aphid homogenates and purified myrosinase, two unique hydroxyglucosinolates, 3-butenyl- and benzyl-isothiocyanates were identified from progoitrin ((2S)-2-hydroxybut-3-enyl-glucosinolate) and sinalbin (4-hydroxybenzyl-glucosinolate) degradation respectively. Addition of ascorbic acid to the reaction mixtures containing sinalbin and progoitrin caused the production of hydroxylated degradation products usually associated with plant myrosinase metabolisation. The occurrence of the myrosinase system in B. brassicae is discussed in terms of similar allelochemical adaptation between the herbivore and its host plant., (Copyright 2002 Wiley-Liss, Inc.)

Flour beetles are cosmopolitan and common pests in grain stores and flour mills. Their ability to exploit a wide variety of stored products has contributed to their status as major pests of stored food. Although it was previously reported that the same aggregation pheromone, 4,8-dimethyldecanal (DMD), is shared by three flour beetles species (Tribolium castaneum, T. confusum, and T. freemani), the volatiles released by the other Tribolium species associated with stored products have not yet been examined. In the present study, the volatiles produced by males and females of eight Tribolium species were examined by solid phase microextraction (SPME). SPME samples were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). Experiments were conducted to identify volatiles emitted by the adults of different Tribolium species and to determine whether DMD is a common aggregation pheromone. We observed that DMD is not a common pheromone of the eight species tested, but is common to T. castaneum, T. confusum, T. freemani, and T. madens. Two other volatiles were detected, 1-pentadecene, which is shown here to be a common semiochemical of flour beetles, and 1,6-pentadecadiene, which was detected in five species (T. audax, T. brevicornis, T. destructor, T. freemani, and T. madens).

Isoenzymes of glutathione S-transferase (GST) in adult Adalia bipunctata, an aphidophagous predator, were studied. Cytosolic GST activity was studied in each beetle developmental stage. The highest activities towards both 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dinitro-1-iodobenzene (DNIB) occurred in adults. The enzyme distribution was investigated in adults. While most of the enzymatic activity was found in the abdomen (40-50 and 34-63% respectively) using several concentrations of both CDNB and DNIB, significant differences were observed for the head and the thorax depending on the substrate. Activities were more abundant in the thorax with DNIB (37-47%) compared to the 13-19% obtained with CDNB. Some GST activity was also detected in the elytra. GSTs were purified by epoxy-activated Sepharose 6B affinity chromatography and applied to an HPLC column to determine the native molecular weight (69 kDa). Three isoenzymes were separated by chromatofocusing at pH ranges 7-4. Three bands with molecular mass from 23 to 26 kDa were visualised on SDS-PAGE. Their isoelectric points were 6.66, 6.36, and 6.21. The substrate specificities and the kinetic parameters (Vm and Km) of the isoenzymes showed large differences depending on the isoenzyme. Arch., (Copyright 2002 Wiley-Liss, Inc.)

Surfactin C14, surfactin C15, and iturin C15 are lipopeptides purified from Bacillus subtilis (S499 strain). They were incorporated to artificial diet of the fruit fly Drosophila melanogaster (Meigen) (Diptera, Drosophilidae) to assess their potential insecticide activity. Surfactins with long fatty acid chain (C14 and C15) showed insecticide effect on the fruit fly, D. melanogaster. On the contrary, iturin was not toxic to fruit fly D. melanogaster. At 100 ppm, surfactin C14 and C15 showed respectively 85.4 and 92.6% adults mortality after one-day exposure. F1 progeny fly emergence inhibition by C14 and C15 were respectively 79.8% and 91.3%. To check whether the biocide activity of lipopeptides was due to their surface-active properties, detergent Triton X100, SDS, CTAB and Tween 80 were tested. No adult mortality was recorded with the detergents but Triton X100 and SDS showed F1 progeny emergence inhibition similar to that of surfactins. We showed that there was a dose-response activity with surfactin C15.

In vitro enzyme activity of head homogenates from adults of Coccinella septempunctata, Chrysoperla carnea and Forficula auricularia originated from different habitats in Belgium (wheat, barley, rye, set-aside fields and experimental orchard, uncultivated area) were investigated in presence of insecticide active ingredients. Using the procedure of Ellman, I50 (M) and Ki (M-1 min-1) values were established. The beneficial insects showed the least susceptibility to diazinon and the differences between their measured values were not remarkable. Paraoxon was extremely toxic to the AChE of F. auricularia but Ch. carnea and C. septempunctata were similarly more tolerant to this organophosphate. In the case of malaoxon earwig and green lacewing AChEs were much more sensitive than AChE of the ladybird beetle. Measuring the carbaryl inhibition, F. auricularia was the least tolerant. The susceptibility of ladybird AChE differed highly from that of both species. According to the measured values, the green lacewing was less tolerant than the ladybird beetle but more tolerant compared with the common earwig. Summarizing our biochemical results, the order of susceptibility of beneficial insects to insecticides investigated was the following F. auricularia > Ch. carnea > C. septempunctata.

Vegetable open fields areas have been increasing for the last decade in Wallonia (South part of Belgium), mainly in Hesbaye. To be in accordance with quality standards, especially in terms of agrochemical residues (R.M.L.), biological pest control was developed and reduces the insecticide use, leading to have safer fresh products. Carrot represents an important cultivated species in Wallonia. To asses the impact of close habitat on both pest (mainly aphids) and beneficial insects, carrot fields were investigated during all the production duration in 2000. Twelve fields between Waremme and Hannut were visited weekly from June to October. Insects were caught using yellow traps and determined to the family level. Approximately 90,000 insects belonging to 109 families were identified. Significant differences linked to field closed habitat were observed on 31 families. An increase of biodiversity in term of family number near set-asides and woody borders was observed. Evaluation of pest and beneficial diversity and density in vegetable crops was discussed to promote future IPM program.

The movement and dispersion of Coccinella septempunctata and its efficacy as aphid control agent over large areas is not really understood because of the difficulty in identifying the origins of predators. To quantify the genetic diversity within the species and monitor the spatial foraging, populations were sampled from Belgium and analysed for RAPD DNA variation. Twenty decamer primers generated more than hundred polymorphic RAPD bands and pairwise distances were calculated between populations according to Nei and Li, then used to construct a radial neighbour-joining dendrogram and examine intra- and inter-population variance coefficients, by analysis of molecular variation (AMOVA). This study shows that while a number of factors can complicate the use and interpretation of RAPD fragments as genetic markers, RAPD analysis can be a valuable technique for studies of intra-specific genetic variation in C. septempunctata.

Malathion resistance in the red flour beetle, Tribolium castaneum (Herbst), is a worldwide problem and is very stable once it becomes widespread in natural populations. In the absence of insecticide the proportion of resistant phenotypes may rapidly decline but the development of resistance does not always involve reduced fitness. Malathion-specific resistance in T. castaneum seems not to involve any loss of fitness in laboratory or field conditions. Susceptible beetles were in competition with resistant beetles at different initial frequencies and modifications of susceptible gene frequency were estimated in these laboratory populations over 10 generations. A significant decrease in susceptible gene frequency was observed in Tribolium populations over time. The selection coefficient of the susceptible allele was estimated and the fitness of susceptible alleles in all tests was observed to range from 0.89 to 0.93 compared with the fitness of resistant genotypes, which was assumed to be 1. Data provided evidence that the resistant strains exhibited fitness advantages in the absence of malathion. We also compared the biotic potential (fecundity and developmental time) of the susceptible strain, the homozygous malathion-specific resistant strain, and their hybrids. Malathion-specific resistant strains showed an 8 -23% increase in biotic potential relative to the susceptible strain. These findings are consistent with those of malathion-specific resistance in T. castaneum; the fitness of the insects seems independent of the genetic background and the fitness of the resistant insects is not affected by this resistance mechanism.

Three Brassicaceae species, Brassica napus (low glucosinolate content), Brassica nigra (including sinigrin), and Sinapis alba (including sinalbin) were used as host plants for two aphid species: the generalist Myzus persicae and the specialist Brevicoryne brassicae. Each combination of aphid species and prey host plant was used to feed the polyphagous ladybird beetle, Adalia bipunctata. Experiments with Brassicaceae species including different amounts and kinds of glucosinolates (GLS) showed increased ladybird larval mortality at higher GLS concentrations. When reared on plants with higher GLS concentrations, the specialist aphid, B. brassicae, was found to be more toxic than M. persicae. Identification of GLS and related degradation products, mainly isothiocyanates (ITC), was investigated in the first two trophic levels, plant and aphid species, by high-performance liquid chromatography and gas chromatography-mass spectrometry, respectively. While only GLS were detected in M. persicae on each Brassicaceae species, high amounts of ITC were identified in B. brassicae samples (allyl-ITC and benzyl-ITC from B. nigra and S. alba, respectively) from all host plants. Biological effects of allelochemicals from plants on predators through aphid prey are discussed in relation to aphid species to emphasize the role of the crop plant in integrated pest management in terms of biological control efficacy.

We carried out a cross-sectional study in 12 rural villages in order to identify the risk factors for Kashin-Beck disease in Tibet. Children aged 5-15 years (n=575) were examined and their corresponding houses were visited. Samples were collected in order to study fungal contamination of stored grain and the organic matter content of drinking water. Multivariate analysis was performed using logistic regression and population attributable fractions were computed to estimate the impact of each factor. The following variables were independently associated with the disease: age, gender, low socio-economic status, indicators of a poorly diversified diet, iodine deficiency and small water container size (with higher organic matter levels in small containers). Selenium deficiency was severe in all study subjects. The degree of fungal contamination of barley grain was related to the highest percentage of cases (65%) in a sample of the study population. Higher urinary iodine levels were not associated with decreasing prevalence rates when Alternaria sp. was isolated. The data that we report supports the hypothesis that Kashin-Beck disease occurs as a consequence of oxidative damage to cartilage and bone cells when associated with decreased antioxidant defence. Another mechanism that may coexist is bone remodelling stimulated by thyroid hormones whose actions can be blocked by certain mycotoxins.

A crucial aspect of predator oviposition behaviour is host plant choice, especially in hoverflies where the newly hatched offspring are unable to move a great distance to search for the appropriate prey. Such offspring must generally feed on the host plant aphids previously selected by the mother. Some factors involved in the selection of the oviposition site of Episyrphus balteatus De Geer include aphids associated to chemical stimuli, aphid colony size and host plant characteristics. Here we tested the hypothesis that there will not only be a rank order hierarchy of preference for aphid prey species reared on the same host plant but that a similar hierarchy of different host plant of one aphid species could be established. Therefore we compared the number of eggs laid on different combinations of host plant and aphid species. Vicia faba L., secondary metabolites free, Brassica napus L. and Sinapis alba L., containing low and high levels of glucosinolates respectively were used. The latter compounds are well known allelochemicals from Brassicaceae having a strong influence on specialist and generalist insects from both phytophagous and entomophagous levels. These experiments enhance the importance of tritrophic interactions in biological control of pests by underlining the host plant influence on aphidophagous predators, either directly or through the odours emitted by the phytophagous prey.

Kashin-Beck disease occurs in several villages of Tibet; however, its local importance varies greatly. The ecoclimatological as well as the phytogeographical framework of the studied area are presented. An ecological approach based upon the ethno-ecosystem concept was carried out in the vicinity of each village. This study identifies 18 vegetation units on a structural basis; they were named for the dominant plants of each unit. Half of them belonged to the aquatic milieu. The different factors controlling their distribution were also identified. Particular attention will be paid to the links between man and the environment, particularly regarding alternative food intakes and water supply access.

Aphidophagous predators such as hoverfly and ladybird beetles are effective biological agents to control aphid pests in perennial and annual cultivated species. Introduction and conservation of beneficial insects are two ways to increase natural control of pests. Whether massive releases of entomophagous insects are expensive and time consuming, the preservation of predator natural populations can be expected by reducing and by adapting chemical treatments in crop fields. Vegetable cultivated areas increased in Belgium for several years, mainly Fabaceae species such as peas and beans. In this work, the evolution of ladybird species population was assessed from May to June in broad bean fields (Vicia faba L.) between Waremme and Hannut, in Hesbaye. Weekly, the aphid and aphidophagous beetle populations were collected from yellow traps and determined on plants by visual observations. Even if five ladybird species were identified, three of them represented more than 95% of the collected insects (Coccinella septempunctata L., Propylea quatuor-decimpunctata L. and Psyllobora vingintiduopunctata L.). Evolution of coccinellid populations during the cultivation season was discussed in relation to the presence of potential aphid preys and the agrochemical treatments which were applied. Integrated pest management in vegetable fields constitute a reliable way to increase the quality level of fresh vegetables in terms of pesticide residue limitations.

The contamination of drinking water by organic acids, selenium deficiency and the ingestion of fungal mycotoxins are the three main aetiological factors in the development of Kashin-Beck disease. An avian tibial chondrodysplasia induced by mycotoxins has been reported. Deoxynivalenol (DON) is one of many mycotoxins produced by the most common contaminating species of fungi. The pattern of skeletal malformations induced by its administration intraperitoneally to pregnant mice is reported. Costo-vertebral segmentation abnormalities were the main deformities observed. The chondrodysplasia previously described was not seen.

In order to clarify the association between mycotoxin-producing fungi in food and Kashin-Beck disease (KBD), we examined the occurrence and contamination levels of fungi in samples of barley grain, from KBD-affected families and from unaffected families in endemic areas. A control area without the occurrence of KBD served as reference. The first results obtained in 1995 showed that total mesophilic fungal contamination of barley grain was consistently higher in families with KBD. Trichothecium roseum (Pers) Link ex gray, Dreschlera Ito and Alternaria Nees ex Fr. were the three most common fungi significantly associated with KBD. In 1996 we again observed a significant difference between affected and non-affected families, especially with Trichothecium roseum and Ulocladium Preuss. On this basis, measures to prevent KBD were suggested and a preventive program has been set up since 1998 in 20 new villages.

Mycotoxins are naturally occurring toxic chemical compounds produced by fungi infesting agricultural crops both during their growth and storage. Such secondary metabolites, when ingested, can produce toxic syndromes in humans. As it has been suggested that mycotoxins might be involved in the development of Kashin-Beck disease (KBD), we undertook a survey of barley grains of KBD-affected families and non-affected families in that country. We found, by thin layer chromatography, a hitherto unknown metabolite of Alternaria sp. This was especially common on the barley grains of KBD-affected families.

A radiological study of osteoarticular changes in Kashin-Beck Disease (KBD) was undertaken on the appendicular skeleton in 105 patients with KBD, in 31 healthy subjects living in an endemic area and in 30 healthy subjects living in a non-endemic area. The bone age was delayed in all three populations with no significant difference between the three studied Tibetan populations. Radiological changes occur in 56% of patients with KBD, and are usually bilateral. An analysis of the distribution of lesions shows a proximo-distal gradient. The changes are more common in the distal aspect of the limb and the lower limb is involved more commonly than the upper limb. The foot and ankle are involved in 89.5% of cases. The radiological changes and their distribution might be explained by the hypothesis of inhibition of angiogenesis by mycotoxins, exacerbated by chemical and physical environmental factors.

There is increasing evidence that normal male reproductive function can be disrupted by exposure to pollutants in the environment that can exogenously mimic, antagonize or block sex-hormone function. One possible consequence of exposure to these xenobiotics is disruption to spermatogenesis, but results thus far provide only indirect and inconsistent evidence. In this study we exposed adult male guppies (Poeciliidae: Teleostei) to environmentally relevant levels of the common xenobiotics tributyltin (11.2-22.3 ngl-1) and bisphenol A (274-549 micrograms l-1) in experimental aquaria. After 21 days of exposure, we found significant declines (by 40-75%) in total sperm counts for male fishes exposed to tributyltin and bisphenol A compared with controls. This short-term decline in sperm count is unlikely to be the result of endocrine-mediated alteration of the germ line, and we found no change in testis size or sperm lengths between treatments. However, Sertoli cells, which facilitate the transport of maturing sperm into the testicular deferent duct (where they are stored prior to ejaculation), are directly sensitive to xenobiotic action and it is therefore possible that spermatogenesis was inhibited through in vivo interference with normal Sertoli-cell function.

The differential inhibition of acetylcholinesterase (AChE) by organophosphate (OP) and carbamate (C) is followed by the distinct duration of exposure effect on common carp AChE. Hence, in the present study in vivo exposure period effect and in vitro concentration-response of chlorfenvinphos, chlorpyrifos diazinon, and carbofuran were investigated on Cyprinus carpio L. AChE. Individuals of 1-year-old carp were exposed for 96 h to different concentrations of insecticides, after which AChE activity was measured in the brain. The highest concentrations of carbofuran (2.44 mg x L(-1)), chlorfenvinphos (2.9 mg x L(-1)), and diazinon (2.5 mg x L(-1)) killed all the test animals after only 4 h, although there was no statistically significant difference from the control group's brain AChE activity. The lowest concentration significantly inhibited brain AChE after 96 h. Chlorfenvinphos was the most potent inhibitor in vivo and chlorpyrifos the least active inhibitor after 96 h of exposure time. In vitro experimentation with the same pesticide indicated that several concentrations inhibited 50% of the AChE activity (I50), ranging from 4.1x10(-7) to 8.12x10(-4) M in both single inhibitory action and joint inhibitory effect. The results suggest that in biomonitoring programs carp brain AChE can be a good diagnostic tool for chronic OP nd C pollution.

Biological activity of leaves, fruits and extract of the African shrubBoscia senegalensis (PERS.) LAM. ex Poir. was evaluated against five stored-grain insects. When added to cowpeas at 2-4% (w/w), fresh ground fruits and leaves caused 80-100% mortality inCallosobruchus maculants (F.) adults and significantly reduced both emergence and damage of the F1 progeny. Acetone fruit extract exhibited a potent fumigant effect onProstephanus truncatus HORN, C.maculatus, andSitotroga cerealella OLIV.; with LT50 values of 3.8, 2.3, and below 1.5 hr, respectively. LC50 determination forB. senegalensis fruits and leaves as well as pure methylisothiocyanate (MITC) onTribolium castaneum HERBST,Sitophilus zeamais MOTSCH. andC. maculatus showed a differential response of the insects to plant parts or MITC. Quantitative dosage ofBoscia active components and LC50 values obtained for the plant tissues, compared to those of pure molecules, indicate that the biological activity ofB. senegalensis is due to the liberation of MITC from a glucosinolate precursor glucocapparin contained inBoscia fruits and leaves.