Detection of organophosphate and pyrethroid resistance alleles in Czech Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) populations by molecular methods

BACKGROUND: Pyrethroids and organophosphates are the most frequently used insecticides for Colorado potato beetle, Leptinotarsa decemlineata (Say), control in the Czech Republic. Based on molecular methods, organophosphate and pyrethroid resistance alleles have been detected in samples from three sites. The accuracy of restriction fragment length polymorphism (RFLP) and bidirectional polymerase chain reaction amplification of specific alleles (Bi‐PASA) for detection of resistance alleles is compared. RESULTS: Leptinotarsa decemlineata from three sites showed higher frequencies of resistance alleles to organophosphates than to pyrethroids. The rates of occurrence of individuals homozygous resistant (RR) to pyrethroids ranged from 20.0 to 22.9%, while the rates of occurrence of individuals RR to organophosphates ranged from 52.9 to 66.7%. The incidences of individuals with resistance alleles to both organophosphates and pyrethroids ranged from 8.6 to 13.6%. No relationship was found between incidence of the pyrethroid resistance allele and site, while incidence of the organophosphate resistance allele differed significantly according to site. CONCLUSION: Both RFLP and Bi‐PASA were suitable for detecting resistance alleles to pyrethroids, and in most cases also for detecting resistance alleles to organophosphates. In contrast to Bi‐PASA, RFLP was also suitable for samples with lower DNA quality when testing for the resistance allele to pyrethroids. On the other hand, RFLP was not as accurate as Bi‐PASA in detection of the organophosphate resistance allele. Copyright © 2010 Society of Chemical Industry     

The first report on natural Enterocytozoon bieneusi and Encephalitozoon spp. infections in wild East-European House Mice (Mus musculus musculus) and West-European House Mice (M. m. domesticus) in a hybrid zone across the Czech Republic-Germany border

To determine the occurrence of potentially human pathogenic microsporidia (Enterocytozoon bieneusi and Encephalitozoon spp.) in wild mice, we examined 289 East-European House Mice (Mus musculus musculus) and West-European House Mice (M. m. domesticus) trapped at 74 localities in an area across the Czech-German border. Microsporidia were detected at 33 localities, in 34% of M. m. musculus and 33% of M. m. domesticus examined specimens. Single-species infection was detected in 23 mice for E. hellem, 42 mice for E. cuniculi and 25 mice for E. bieneusi. No Encephalitozoon intestinalis positive animals were identified. Moreover, co-infections were detected in 6 animals; E. bieneusi co-existed with E. cuniculi or E. hellem in 3 mice. The natural infection of E. hellem has never been recorded in mice before. No differences were found by a statistical analysis of microsporidia occurrence between the House Mouse subspecies. Although the gender-dependent infestation of microsporidia was statistically supported in M. m. musculus, no significant differences were observed when the occurrence of microsporidia was estimated for all males and females irrespective of the House Mouse subspecies. The results of this report document the low host specificity of detected microsporidia species and imply the importance of synanthropic rodents as a potential source of human microsporidial infection.     

Long-term animal impact modifies potential production of N<Subscript>2</Subscript>O from pasture soil

At cattle overwintering areas, inputs of nutrients in animal excrements create conditions favourable for intensive microbial activity in soil. During nitrogen transformations, significant amounts of N₂O are released, which makes overwintering areas important sources of N₂O emission. In previous studies, however, increasing intensity of long-term cattle impact did not always increase emissions of N₂O from the soil: in some cases, N₂O emissions from the soil were lower at the most impacted area than at the moderately impacted one. Thus, the relationships between the level of long-term animal impact and potential production of N₂O from soil by denitrification were investigated in field and laboratory experiments. Field measurements indicated that the production of N₂O after glucose and nitrate amendments was greater in severely and moderately impacted locations than in an unimpacted location, while differences between the severely and moderately impacted locations were not significant. In laboratory experiments, the potential production of N₂O (measured as anaerobic production of N₂O after addition of glucose and nitrate) was highest in the moderately impacted soil. Surprisingly, potential N₂O production was lower in the most impacted than in the moderately impacted soil, and the net N₂O production in the highly impacted soil was further decreased by a significant reduction of N₂O to N₂. The expected stimulating effect of an increasing ratio of glucose C to nitrate N on the reduction of N₂O to N₂ during denitrification was not confirmed. The results show that cattle increase the denitrification potential of the soil but suggest that the denitrification potential does not increase indefinitely with increasing cattle impact.     

Food selection and internal processing in Archegozetes longisetosus (Acari: Oribatida)

The nutritional biology of the oribatid mite Archegozetes longisetosus Aoki was examined using a control (starvation) and four types of food: Protococcus sp. algae from tree bark, two fungi—Stachybothrys sp. and Alternaria sp.—and filter paper. Direct observations, histology, enzymology and plating techniques were employed to record contact with food, contents and structure of the alimentary tract, presence and viability of bacterial microorganisms inside the mite body, and chitinase and cellulase activity of mite homogenates. Algae were highly palatable, resulting in high apocrine secretion and guts that were continuously full. Initially there was no evidence of chitinase or cellulase production. Chitinase activity started after 10 days, probably due to consumption of fungi that invaded the algal cover, and correlated with the presence of chitinolytic bacteria (Serratia rubidea) in the mite homogenate. Alternaria was grazed intensively, but cell walls of spores and hyphae remained intact and no chitinase was detected suggesting that only cell contents were enzymatically digested. Stachybothrys sp. was rejected as food. The bacterium Alcaligenes faecalis was dominant in homogenates in each of the treatments, but under starvation Achromobacter xylosoxidans became co-dominant. Cellulase activity was not detected in any treatment, but strong chitinase activity was induced with a filter paper (colonized by invasive fungi) diet. Furthermore, bacteria were common in mesenchyme between the internal organs in the filter paper and starvation treatments.     

Characterization of <Emphasis Type="Italic">Agrobacterium</Emphasis> species by capillary isoelectric focusing

Tumorigenic and non-tumorigenic strains of Agrobacterium tumefaciens, A. rhizogenes, A.rubi, and A.vitis were examined using capillary isoelectric focusing, phenotypic determinative tests, PCR and fatty acid analysis. The isoelectric points (pI) of the 40 strains investigated clearly differentiated the strains according to their respective species. The different species were characterized with the following pI values: A. tumefaciens 2.2, A. rhizogenes 4.0, A. rubi 2.15, and A. vitis 2.6. This differentiation corresponded to the phenotypic, PCR and fatty acid characterizations. Strains with the similar chromosomal background but different plasmid content, e.g. A.vitis strain S4, and F2/5 gave the same pI values. Strains of Rhizobium species differed from Agrobacterium strains in their pI values. The advantage of capillary isoelectric focusing over the phenotypic determinative tests, PCR and fatty acid analysis is its speed (15 min), relative simplicity, and the very small amount of chemicals used. This rapid and simple method is a major improvement over the classical methods of separation of Agrobacterium species and should prove useful for rapid characterization of Agrobacterium-like colonies isolated from plant tumours for epidemiological and generic diversity studies.     

Methylglyoxal-induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications

Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial component found in Revamil honey, peptide defensin1, was not identified in manuka honey. The primary aim of the study was to evaluate the content of defensin1 in honeys of different botanical origins and to investigate a presumed effect of reactive MGO on defensin1 and a dominant protein of honey MRJP1 in manuka honey. Immunoblotting of honey samples showed that defensin1 was a regular but quantitatively variable component of honeys. One of the reasons of varying contents of defensin1 in different honeys seems to be constitutive but varying defensin1 expression in individual honeybees in bee populations that we documented on samples of nurse and forager bees by RT-PCR. Comparative analyses of honeys revealed a size modification of defensin1, MRJP1 and probably also α-glucosidase in manuka honey. We further showed that (i) the treatment of purified defensin1 in solution containing high amount of MGO caused a time-dependent loss of its antibacterial activity and (ii) increasing MGO concentrations in a non-manuka honey were connected with a gradual increase in the molecular weight of MRJP1. Obtained results demonstrate that MGO abrogates the antibacterial activity of defensin1 and modifies MRJP1 in manuka honey. We assume that MGO could also have negative effects on the structure and function of other proteins/peptides in manuka honey, including glucose oxidase, generating hydrogen peroxide.     

Pollination dynamics in a Douglas-fir seed orchard as revealed by pedigree reconstruction

• ? Pollination dynamics was studied in a Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seed orchard using 8 nuclear microsatellite markers and pedigree reconstruction.
• ? The seed orchard consisted of 49 parents (clones). Cone-crop management included bloom delay and supplemental mass pollination (SMP) using 12 internal and 4 external pollen donors.
• ? A random sample of 801 bulk seeds was genotyped for both haploid megagametophyte and corresponding diploid embryo.
• ? Using the parental population’s multilocus genotypes, full pedigree reconstruction generated all the information needed to estimate the maternal, paternal, and parental reproductive success, selfing, pollen contamination, and pollination success of the 4 external pollen donors.
• ? Maternal, paternal, and parental reproductive success varied with 80% of gametes being produced by 23, 45, and 37% of the orchard’s parents, respectively, resulting in a drastically reduced effective population size as compared to the census number (14 vs. 53).
• ? Selfing, pollen contamination, and aggregate SMP success (internal and external) were estimated to be 15.2, 10.4, and 15.0%, respectively.
• ? Full pedigree reconstruction was effective in unraveling the orchard’s pollination dynamics and both female and male reproductive success.

     

Stripping Fecundity of Common Bream (Abramis brama L.) from the Rivers Trent and Sow (Nottinghamshire, UK)

Results obtained with common bream (Abramis brama) originating from the rivers Trent and Sow (Great Britain) are presented with particular attention to the egg size and absolute and relative fecundity recorded during stripping of hormonally (pituitary extract) treated females. The average number of eggs obtained by stripping 1 kg of female biomass (RSF) from the river Sow and Trent were 93,642 ± 20,896 and 151,179 ± 25,123, respectively. These values were similar to the respective parameters in bream populations from north-western USSR lakes.