Comparative analysis of neonicotinoid binding to insect membranes: I. A structure-activity study of the mode of [3H]imidacloprid displacement in Myzus persicae and Aphis craccivora

Neonicotinoids bind selectively to insect nicotinic acetylcholine receptors with nanomolar affinity to act as potent insecticides. While the members of the neonicotinoid class have many structural features in common, it is not known whether they also share the same mode of binding to the target receptor. Previous competition studies with [3H]imidacloprid, the first commercialised neonicotinoid, indicated that thiamethoxam, representing a novel structural sub-class, may bind in a different way from that of other neonicotinoids. In the present work we analysed the mode of [3H]imidacloprid displacement by established neonicotinoids and newly synthesized analogues in the aphids Myzus persicae Sulzer and Aphis craccivora Koch. We found two classes of neonicotinoids with distinct modes of interference with [3H]imidacloprid, described as direct competitive inhibition and non-competitive inhibition, respectively. Competitive neonicotinoids were acetamiprid, nitenpyram, thiacloprid, clothianidin and nithiazine, whereas thiamethoxam and the N-methyl analogues of imidacloprid and clothianidin showed non-competitive inhibition. The chloropyridine or chlorothiazole heterocycles, the polar pharmacophore parts, such as nitroimino, cyanoimino and nitromethylene, and the cyclic or acyclic structure of the pharmacophore were not relevant for the mode of inhibition. Consensus structural features of the neonicotinoids were defined for the two mechanisms of interaction with [3H]imidacloprid binding. Furthermore, two sub-classes of non-competitive inhibitors can be discriminated on the basis of their Hill coefficients for imidacloprid displacement. We conclude from the present data that the direct competitors share the binding site with imidacloprid, whereas non-competitive compounds, like thiamethoxam, bind to a different site or in a different mode.     

Comparative analysis of neonicotinoid binding to insect membranes: II. An unusual high affinity site for [3H]thiamethoxam in Myzus persicae and Aphis craccivora

Neonicotinoids represent a class of insect-selective ligands of nicotinic acetylcholine receptors. Imidacloprid, the first commercially used neonicotinoid insecticide, has been studied on neuronal preparations from many insects to date. Here we report first intrinsic binding data of thiamethoxam, using membranes from Myzus persicae Sulzer and Aphis craccivora Koch. In both aphids, specific binding of [3H]thiamethoxam was sensitive to temperature, while the absolute level of non-specific binding was not affected. In M persicae, binding capacity (Bmax) for [3H]thiamethoxam was ca 450 fmol mg(-1) of protein at 22 degrees C and ca 700 fmol mg(-1) of protein at 2 degrees C. The negative effect of increased temperature was reversible and hence not due to some destructive process. The affinity for [3H]thiamethoxam was less affected by temperature: Kd was ca 11 nM at 2 degrees C and ca 15 nM at 22 degrees C. The membranes also lost binding sites for [3H]thiamethoxam during prolonged storage at room temperature, and upon freezing and thawing. In A craccivora, [3H]thiamethoxam was bound with a capacity of ca 1000 fmol mg(-1) protein and an affinity of ca 90 nM, as measured at 2 degrees C. Overall, the in vitro temperature sensitivity of [3H]thiamethoxam binding was in obvious contrast to the behaviour of [3H]imidacloprid studied in parallel. Moreover, the binding of [3H]thiamethoxam was inhibited by imidacloprid in a non-competitive mode, as shown with M persicae. In our view, these differences demonstrate that thiamethoxam and imidacloprid, which represent different structural sub-classes of neonicotinoids, do not share the same binding site or mode. This holds also for other neonicotinoids, as we report in a companion article.     

Evaluation of hemato-immunological parameters during the reproductive cycle of the scallop Nodipecten nodosus in association with a carotenoid-enriched diet

The cultivation of scallops Nodipecten nodosus is a promising activity emerging in Brazil. The purpose of this study was to characterize the immune system of N. nodosus and evaluate the modulation of some hemato-immunological parameters during the reproductive cycle, in association with an astaxanthin-enriched diet. It was hypothesized that a supplementation on astaxanthin could enhance scallop immune system and minimize stress of reproduction. Scallops were separated in different groups: juveniles (J), adults (A), sexually mature (M), and recently spawned (S) animals. The last two groups were fed standard (M and S) or astaxanthin-enriched (Ma and Sa) diets. Scallop hemolymph contained two hemocyte populations: hyaline (HH) and granular hemocytes (GH). Antimicrobial peptides, similar to mussel defensins and mytilins, were found by immunodetection within the GH granules, even though the scallop hemolymph did not exhibit significant antimicrobial activity against different bacteria, including marine vibrios. Scallop hemocytes were able to phagocytose zymosan and produce reactive oxygen intermediates (ROI–NBT reduction). The number of circulating hemocytes (Neubauer chamber) varied from 12 to 26.10⁶ cells mL^− 1, and the GH was always the predominant cell type (67–99%). The plasma of N. nodosus contained lectins, specific to galactose and sialoconjugates, and their agglutinating activity was partially calcium-dependent. A phenoloxidase (PO) activity (146–446 U min^− 1 mg^− 1) was observed in the scallop hemolymph. However, this activity was not induced by trypsin or components of microorganism surface, and was strongly enhanced by alkaline pH (≥ 8.5). The total protein concentration of the plasma varied from 240 to 660 μg mL^− 1. In general terms, all examined hemato-immunological parameters (hemograms, superoxide anion production, PO activity, lectin titers and total protein concentration) had a similar profile during all the scallop reproductive cycle. Their levels increased significantly from juveniles to adults (except PO activity), and declined markedly (immune depletion) in the sexually mature scallops. After spawning, the animals had a tendency to recover the standard levels of their immune parameters. Apparently, the astaxanthin-enriched diet had no effect on the tested immune parameters except for a slight influence on the scallop immune-oxidative reactions (ROI production). The results obtained in this study suggested the occurrence of a general immune depletion in the sexually mature scallops, confirming that the reproductive stage is a critical period in scallop life.     

The Anti‐microbial Activity of Electrolysed Oxidizing Water against Microorganisms relevant in Veterinary Medicine

Standards of the German Association of Veterinary Medicine (DVG) for the evaluation of chemical disinfectants were used to assess the anti‐microbial efficacy of electrolysed oxidizing water (EOW). Enterococcus faecium, Mycobacterium avium subspecies avium, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans were exposed to anode EOW (pH, 3.0 ± 0.1; oxidation‐reduction potential (ORP), +1100 ± 50 mV; free chlorine, 400 ± 20 mg/l Cl₂) and combined EOW (7 : 3 anode : cathode, v/v; pH, 8.3 ± 0.1; ORP, 930–950 mV; free chlorine, 271 ± 20 mg/l Cl₂). In water of standardized hardness (WSH), all bacterial strains were completely inactivated by a 30 min exposure to maximum 10.0% anode EOW (～40.0 mg/l Cl₂) or 50.0% combined EOW (～135.5 mg/l Cl₂). The sensitivity ranking order for anode EOW to the bacterial test strains was P. mirabilis > S. aureus > M. avium ssp. avium > E. faecium > P. aeruginosa. P. mirabilis and S. aureus decreased to undetectable levels after 5 min of exposure to 7.5% anode EOW (～30.0 mg/l Cl₂). Candida albicans was completely inactivated by a 5‐min exposure to 5.0% anode EOW. Both, anode and combined EOW exhibited no anti‐microbial activities in standardized nutrient broth or after addition of 20.0% bovine serum to the WSH. Further research is necessary to evaluate the efficacy of EOW as a disinfectant under operating conditions in animal production facilities.     

Genetic diversity and structure of Ethiopian,Yemen and Brazilian C<Emphasis Type="Italic">offea arabica</Emphasis> L. accessions using microsatellites markers

Genetic diversity among 115 coffee accessions from the Coffea Germplasm Collection of IAC was assessed using SSR markers. The germplasm represents 73 accessions of Coffea arabica derived from spontaneous and subspontaneous plants in Ethiopia and Eritrea, species center of origin and diversity, 13 commercial cultivars of C. arabica developed by the Breeding Program of IAC, 1 accession of C. arabica cv. ‘Geisha’, 13 accessions of C. arabica from Yemen, 5 accessions of C. eugenioides, 4 accessions of C. racemosa and 6 accessions of C. canephora. Genetic analysis was performed using average number of alleles per locus (A), proportion of polymorphic loci (P), Shannon’s genetic index (H′ and G′_ST) and clustering analysis. All evaluated species were distinguished by a cluster analysis based on Jaccard’s coefficient. Differentiation between the cultivated plants of C. arabica and accessions derived from spontaneous and subspontaneous plants was observed. Spontaneous and subspontaneous accessions from Ethiopia were separated according to the geographical origin: east and west of the Great Rift Valley. Cultivated plants showed a low genetic diversity with a division in two groups: accessions from Yemen (H′=0,028) and Brazilian commercial cultivars (H′=0,030). The results agreed with previously reported narrow genetic basis of cultivated plants of C. arabica and supported the hypotheses about domestication of the species. This study also showed a significant genetic diversity among accessions from Ethiopia and Eritrea present in the Germplasm Collection of IAC. This diversity is specially observed in accessions from Sidamo (H′=0,143), Kaffa (H′=0,142) and Illubabor (H′=0,147) indicating their importance as source of genetic variability for coffee breeding programs.     

Are ricefields a good alternative to natural marshes for waterbird communities in the Camargue, southern France?

Rice cultivation has frequently been suggested to provide an important wetland habitat for waterbirds. However, in contrast to most other regions of the Mediterranean, the Camargue still has a substantial amount of surface area of natural marshes. Thus, we compared the abundance, species richness, and community composition of waterbirds in ricefields and natural marshes of the Camargue, during a one-year study. Based on surveys conducted at 4–7 day intervals in 1997–1998, our results suggest that natural marshes had substantially greater abundance of waterbirds, with ca. 99% of the individuals having been observed in natural marshes. Estimates of species richness and associated parameters further indicated that ricefields were clearly less rich than natural marshes. Although our results were rather striking for the Camargue, they may not be applicable to other regions because of the relatively high availability of natural marshes and differences in management of ricefields.     

Sequestering ability of butylated hydroxytoluene,propyl gallate,resveratrol, and vitamins C and E against ABTS,DPPH, and hydroxyl free radicals in chemical and biological systems

The antioxidant capacity of butylated hydroxytoluene (BHT; 2,6-di-tert-butyl-p-cresol), propyl gallate (3,4,5-trihydroxybenzoic acid n-propyl ester), resveratrol (trans-3,4',5-trihydroxystilbene), and vitamins C (l-ascorbic acid) and E [(+)-alpha-tocopherol] was studied in chemical and biological systems. The chemical assays evaluated the capacity of these antioxidants to sequester 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS.) and 1,1 diphenyl-2-picrylhydrazyl (DPPH.). A new colorimetric method to determine hydroxyl radical scavenging is also described. The biological tests use the eucaryotic cells of Saccharomyces cerevisiae treated with the antioxidants in the presence of the stressing agents apomorphine, hydrogen peroxide, and paraquat dichloride (methylviologen; 1,1'-dimethyl-4,4'-bipyridinium dichloride). The results in chemical systems showed that all of the antioxidants were able to significantly inhibit the oxidation of beta-carotene by hydroxyl free radicals. The assays in yeast showed that the antioxidant activity of the tested compounds depended on the stressing agent used and the mechanism of action of the antioxidant.     

Antioxidant activity of the flavonoid hesperidin in chemical and biological systems

The antioxidant hesperidin, a major flavonoid in sweet orange and lemon, was evaluated using chemical and biological systems. The chemical assay evaluates the hesperidin capacity to sequester 1,1-diphenyl-2-picrylhydrazyl (DPPH*). Biological studies were done using the eukaryotic cells of superoxide-dismutase proficient and deficient strains of Saccharomyces cerevisiae treated with hesperidin and the stressing agents hydrogen peroxide or paraquat (methylviologen; 1,1'-dimethyl-4,4'-bipyridinium dichloride). Hesperidin was able to reduce significantly the level of the free radical DPPH* with similar efficacy of trolox (positive control). When the yeast cells were exposed to the flavonoid hesperidin before the stressing agents, there was a significant increase in the survival of all strains. Paraquat induced higher catalase and superoxide dismutase than did hydrogen peroxide, which only increased catalase activity. Previous addition of hesperidin to these treatments was able to reduce significantly both enzymatic levels. These observations clearly demonstrate that hesperidin provides strong cellular antioxidant protection against the damaging effects induced by paraquat and peroxide hydrogen.     

Chemical composition and antioxidant activity of yerba-mate (Ilex paraguariensis A.St.-Hil., Aquifoliaceae) extract as obtained by spray drying

Yerba-mate or mate? (Ilex paraguariensis A.St.-Hil., Aquifoliaceae) leaves are typically used for their stimulant, antioxidant, antimicrobial, and diuretic activity, presenting as principal components polyphenolic compounds. In this study, the objective was to develop a yerba-mate dry extract by using spray drying technology and to evaluate the dry extract antioxidant activity and chemical composition. The results obtained by means of the DPPH assay show that the extract presents an IC(50) of 2.52 mg/mL. The yerba-mate spray-dried extract presents high catalase-like activity, suggesting that it is a strong free-radical scavenger. The antioxidant activity as expressed as catalase-like activity was related to total polyphenol content. In addition, the results show that the spray-dried extract presents high polyphenol content, namely, high concentrations of caffeic acid (1.54 mg/g), 5-caffeoylquinic acid (91.40 mg/g), rutin (5.38 mg/g), and total phenolics (178.32 mg/g), which justifies its high antioxidant activity.