Multilevel studies on the two phenological forms of Large Blue (Maculinea arion) (Lepidoptera: Lycaenidae)

The main goal of our research was to study comprehensively the differences between the two phenological forms of the socially parasitic and globally threatened Large Blue (Maculinea arion) in the Carpathian Basin using four character sets (mitochondrial sequences, allozymes, male genitalia and wing morphometrics). Comparative analyses of distance matrices, phylogenetic trees and ordination patterns have been applied. The genetic and morphometric patterns revealed by our studies were discordant. While we experienced a significant differentiation between the ‘spring’ and ‘summer type’ of M. arion in both wing and genital traits, the two phenological forms did not show any genetic differentiation on two mitochondrial loci and in allozymes. At the same time, all individuals were infected by Wolbachia. Although certain wing traits may not represent reliable tracers of phylogeny because of the particular adaptive significance, the wing characteristics involved in our research are probably determined genetically. Additionally, the significant differentiation of male genitalia also indicates incipient prezygotic isolation arising from phenological differentiation between the ‘spring and summer arion’. It is possible that all extant differences between the two forms are attributable to (1) different host‐ant use, (2) incipient speciation, (3) cytoplasmatic incompatibility (CI) by Wolbachia or the combination of these factors. In addition, discordant results indicate that the combined use of different approaches and data sets is strictly necessary to clarify systematic and evolutionary relationships.     

Control of tsetse and trypanosomiasis transmission in Uganda by applications of lambda-cyhalothrin

The pyrethroid insecticide lambda-cyhalothrin was evaluated in field trials against Glossina f.fuscipes and sleeping sickness transmission in Iyolwa sub-county, Tororo District, Uganda. The insecticide was applied selectively to the resting-sites of tsetse, by bush-spraying, using 10% wettable powder (10WP) formulation at an application rate of 11.6 g a.i./ha over an area of 28 km2, or by a 2% Electrodyn formulation (2ED) applied at 0.9 g a.i./ha over 30 km2. In a third trial area of 32 km2, 215 pyramidal traps treated with lambda-cyhalothrin 100 mg/m2 were set. The best impact was obtained with 10WP lambda-cyhalothrin which eliminated tsetse within 1-2 months, whereas G.f.fuscipes persisted at very low density in part of the area treated with 2ED lambda-cyhalothrin. In both treated areas the numbers of human sleeping sickness cases fell to no more than one per month, compared with four to twelve per month previously. The overall rate of cattle trypanosomiasis (T.brucei and T.vivax) was also reduced slightly. Insecticide-treated traps remained fully effective for at least 6 months under field conditions and catches were reduced 20-90-fold. These results in the control of tsetse and trypanosomiasis transmission lead us to recommend lambda-cyhalothrin for tsetse control operations.     

Conservation of two predator species for biological control ofChrysophtharta bimaculata (Col.: Chrysomelidae) in tasmanian forests

Chrysophtharta bimaculata (Olivier) (Coleoptera: Chrysomelidae) is a major defoliator of regeneration eucalypt trees in Tasmania causing a significant reduction in height and diameter increment of trees which reduces wood volume per hectare. A study to conserve and enhance the efficiency of coccinellid species chieflyCleobora mellyi (Mulsant) (Coleoptera: Coccinellidae), and the cantharid,Chauliognathus pulchellus (Macleay) (Coleoptera: Cantharidae), for the biological control ofC. bimaculata was conducted in young regeneration forests in southern Tasmania from 1991–92. Cantharid adults and coccinellid adults and larvae feed onC. bimaculata eggs and, to a lesser extent, young larvae. The study found that coccinellids were more active throughout the egg and early (1^st and 2^nd) stage ofC. bimaculata. The cantharid, however was active only during the egg stage of the prey and then disappeared from the plantation. The coccinellids were therefore the most useful predators, but their population declined when the prey reached the 3^rd and 4^th stages. As shortage of food may account for this decline, supplementary food was provided in the form of sucrose sprays or sugar granules at a feeding station. This resulted in the retention of both predators and particularly the coccinellids and enhanced their efficacy.     

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle

The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.     

Alginate embedding and subsequent sporulation of in vitro-produced Conidiobolus thromboides hyphae using a pressurised air-extrusion method

Conidiobolus thromboides is an entomophthoralean fungus with potential as a biological control agent of aphids. However, its application in biological control is limited due to its formulation requirements. The objective of this study was to develop and optimise a novel air-extrusion method to embed C. thromboides hyphae at high density in alginate pellets. An orthogonal experimental design was used to investigate selected combinations of parameters known to affect hyphal density within pellets. The diameter of pellets produced, and the calculated density of hyphae within them, ranged from 0.18 ± 0.09 to 3.17 ± 0.06 mm and from 0.02 to 350.56 mg/mm³ respectively. These data were used to predict the optimal parameter combination to deliver the greatest density of hyphae of C. thromboides per pellet: 1% sodium alginate, a 1:2 ratio of hyphae to sodium alginate, an orifice diameter of 0.232 mm and an air pressure of 0.05 MPa. Pellets made under the optimal conditions predicted produced a mean total of 4.3 ± 0.6 × 10⁵ conidia per pellet at 100% relative humidity which was significantly greater than the mean total number of conidia produced from infected aphid cadavers of comparable size (9.35 ± 0.85 × 10⁴) (p < 0.001). In conclusion, air-extrusion embedding appears to be a promising method for formulating in vitro-produced hyphae of C. thromboides for use in biological control.     

Responses of production and storage walnut pests to Bacillus thuringiensis insecticidal crystal protein fragments

Recent advances in genetic engineering have provided the opportunity to induce walnut plants to produce Bacillus thuringiensis Berliner insecticidal crystal protein fragments (ICPFs) for insect control. We studied the effects of two ICPFs CryIA(b) and CrylA(c) previously shown to be encoded by the cryIA(b) and cryIA(c) genes in the B. thuringiensis strains HD-1 and HD-73, respectively. The lethal effects on larvae of codling moth, Cydia pomonella (L.), navel orangeworm, Amyelois transitella (Walker), and the major postharvest pest Indianmeal moth, Plodia interpunctella (Hübner), were investigated. Both proteins were toxic to the three species tested. Indianmeal moth larvae were the most susceptible and navel orangeworm the least; CryIA(b) was generally more toxic to navel orangeworm. Similar relationships resulted when ICPFs were incorporated into the diet. Both ICPFs caused decreased rate of development of navel orangeworm. Effects on pupal weight occurred only at the highest concentration (100 ng/cm²). Neither ICPF affected frequency of mating or fecundity. In addition to the lethal effects, the extended development times observed could have considerable effects on the population dynamics of the navel orangeworm and possibly other species.     

Intra- and interbreed genetic variations of mitochondrial DNA major non-coding regions in Japanese native dog breeds [Canis familiaris)

Mitochondrial DNA (mtDNA) major non-coding regions were amplified from 73 dogs of eight Japanese native dog breeds and from 21 dogs of 16 non-Japanese dog breeds by the polymerase chain reaction and their DNA sequences were determined. A total of 51 nucleotide positions within the non-coding region (969–972 base pairs) showed nucleotide variations of which 48 were caused by transition. These nucleotide substitutions were abundant in the region proximate to tRNA^Pro. In addition to the nucleotide substitutions, the dog mtDNA D-loop sequences had a heteroplasmic repetitive sequence (TACACGTÀCG) involving size variation. The DNA sequences of the non-coding region were classified into four different groups by phylogenetic analysis and the deepest branchpoints of this dog phylogeny was calculated to about 100 000 years before the present. Phylogenetic analysis showed that Japanese native dog breeds could not be clearly delimited as distinct breeds. Many haplotypes found in members of some clustering groups were seen in each dog breed, and interbreed nucleotide differences between Japanese dog breeds were almost the same as the intrabreed nucleotide diversities.