An extract of the desert plant Yucca shidigera was assessed for its possible benefit in ruminal fermentation. The extract bound ammonia in aqueous solution when concentrations of ammonia were low (up to 0.4 mM) and when the extract was added at a high concentration to the sample (20%, vol/vol). The apparent ammoniabinding capability was retained after autoclaving and was decreased slightly following dialysis. Acidprecipitated extract was inactive. No evidence of substantial ammonia binding was found at higher ammonia concentrations (up to 30 mM). When Y. shidigera extract (1%, vol/vol) was added to strained rumen fluid in vitro, a small (6%) but significant (P < 0.05) decrease in ammonia concentration occurred, apparently because of decreased proteolysis. Inclusion of Y. shidigera extract (1%, vol/vol) in the growth medium of the rumen bacterium Streptococcus bovis ESI extended its lag phase, while growth of Butyrivibrio fibrisolvens SH13 was abolished. The growth of Prevotella (Bacteroides) ruminicola B14 was stimulated, and that of Selenomonas ruminantium Z108 was unaffected. Protozoal activity, as measured by the breakdown of 14C-leucine-labelled S. ruminantium in rumen fluid incubated in vitro, was abolished by the addition of 1% extract. The antimicrobial activities were unaffected by precipitating tannins with polyvinylpyrrolidone, but a butanol extract, containing the saponin fraction, retained its antibacterial and antiprotozoal effects. Saponins from other sources were less effective against protozoa than Y. shidigera saponins. Y. shidigera extract, therefore, appears unlikely to influence ammonia concentration in the rumen directly, but its saponins have antimicrobial properties, particularly in suppressing ciliate protozoa, which may prove beneficial to ruminal fermentation and may lead indirectly to lower ruminal ammonia concentrations.

A beta-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of beta-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode beta-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a beta-1,4-endoglucanase devoid of a cellulose-binding domain.

Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane (DDT) resistance in the RDDT R strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT R strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2b position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.Keywords: cytochrome P450; mutation; insecticide; resistance; structure.Many cytochrome P450 enzymes are known to be essential for the protection of organisms against xenobiotics. In insects, the involvement of cytochrome P450 enzymes in plant toxin or insecticide resistance has already been suggested or demonstrated [1][2][3][4][5][6][7], although high resistance levels to insecticides still remain unexplained. To date, only three of the cytochrome P450 enzymes linked to resistance have been shown to be able to metabolize insecticides. Two were cloned from the house fly: CYP6A1 metabolizes aldrin, heptachlor [8], terpenoids [9] and diazinon [10] and CYP12A1 metabolizes aldrin, heptachlor, diazinon and azinphosmethyl [11]. The third is CYP6A2 from Drosophila melanogaster. Baculovirus-directed production of wild-type CYP6A2 showed metabolism of cyclodiene and organophosphorous insecticides, but 1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane (DDT) metabolism could not be detected [12]. In addition, sequence polymorphism of CYP6A1 and CYP6D1 has been documented in the house fly, but there is no link between these instances of polymorphism and insecticide resistance [7,13,14]. These results are in contrast with known instances of cytochrome P450 polymorphisms in humans, which are well known to affect the metabolism of drugs [15,16] and even pesticides [17]. In fact, only two examples of pesticide...

A b-1,4-endoglucanase named MI-ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root-knot nematode Meloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living or rumen-associated microorganisms and phytopathogens. In spite of the presence of a cellulose-binding domain at the C-terminus, the ability of MI-ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant±nematode interaction.

The aphid Acyrthosiphon pisum population is composed of different morphs, such as winged and wingless parthenogens, males, and sexual females. The combined effect of reduced photoperiodicity and cold in fall triggers the apparition of sexual morphs. In contrast they reproduce asexually in spring and summer. In our current study, we provide evidence that clonal individuals display phenotypic variability within asexual morph categories. We describe that clones sharing the same morphological features, which arose from the same founder mother, constitute a repertoire of variants with distinct behavioral and physiological traits. Our results suggest that the prevailing environmental conditions influence the recruitment of adaptive phenotypes from a cohort of clonal individuals exhibiting considerable molecular diversity. However, we observed that the variability might be reduced or enhanced by external factors, but is never abolished in accordance with a model of stochastically produced phenotypes. This overall mechanism allows the renewal of colonies from a few adapted individuals that survive drastic episodic changes in a fluctuating environment.[Supplemental material is available online at http://www.genome.org.]Aphids exhibit a complex mode of reproduction, combining parthenogenesis (spring/summer) and sexual activity (fall/winter) in species such as Acyrthociphon pisum (Dixon 1973;Blackman and Eastop 1984). Aphids thus constitute an excellent model system to investigate how this double reproductive system generates polyphenism, a generic concept used to describe the emergence of distinct morphs, such as winged, wingless, sexual female, and male (Blackman and Eastop 1984;Blackman 1987;Muller et al. 2001). Aphid morph distribution, particularly wing dimorphism, is influenced by environmental conditions, such as population density (crowding effects) (Sutherland 1969) and/or host plant vitality, as well as physical parameters including humidity, temperature, and photoperiodicity (Walters and Dixon 1982;Dixon 1998). This raises fascinating questions regarding the outcomes of alternative developmental mechanisms that cause morph switching in a predictable way (Stearns 1989;Nijhout 1999).Some aphid species are ''sexual'' lineages committed exclusively to sexual reproduction, others are ''facultative asexual'' lineages, which alternate between sexual and parthenogenetic modes depending on the season, while some others are obligate parthenogens (Delmotte et al. 2002;Le Trionnaire et al. 2008). This combined double system of reproduction is shared with many other species like Daphnia ( Despite some divergent reports, most authors seem to agree that sexual populations in aphids present a high allelic polymorphism of many genes and predominance of homozygous loci within individuals. In contrast, asexual populations seem to present less allelic polymorphism, but strong heterozygosity at most loci (Delmotte et al. 2002;Kanbe and Akimoto, 2009). It is largely assumed that organisms reproducing asexually should maintain lower g...