[Phylogenetic relationship among some groups of orthopteran based on complete sequences of the mitochondrial COI gene].

Complete sequences of the COI gene were used to reconstruct the phylogenetic relationship among 56 species from Orthoptera. We also analyzed the reliability of Orthoptera phylogenetic relationship using translated amino acid sequences of the COI genes. The COI sequences were divided into three data sets on the basis of different codon positions to calculate the Partitioned Bremer support (PBS), and to test the phylogenetic signal in different codon positions of protein-coding genes. The result supports the monophyly of Caelifera and Ensifera; but the monophyly of Acrididae, Catantopidae, Oedipodidae, Arcypteridae and Gomphoceridae are not supported. The P-distances among families vary from 0.107 to 0.153, which are smaller than those of other families, being consist with the classification that these 5 families should be merged into one family (Acrididae). Chrotogonidae and Pyrgomorphidae belong to the superfamily Pyrgomorphoidea. Pamphagidae should be a family alone consistent with Otte's taxonomic system. According to the PBS values, the 3rd and 1st codon positions contribution more for the Phylogenetic tree branches than the 2nd, and longer sequences contain more informative sites. We further demonstrated that it is feasible for phylogenetic studies at family level to use the genetic distances among COI sequences from different species of Orthopera.

Age- and density-dependent prophylaxis in the migratory, cannibalistic Mormon cricket Anabrus simplex (Orthoptera: Tettigoniidae).

As a result of the increased potential for disease transmission, insects are predicted to show an increased constitutive immunity when crowded. Cannibalistic aggressive interactions further increase the risk of wounding and pathogen transmission in crowds. Nymphal Mormon crickets Anabrus simplex Haldeman were collected in Montana and reared in the laboratory either solitarily or at densities similar to that experienced by Mormon crickets in migratory bands. As teneral adults, solitarily-reared Mormon crickets tended to have greater phenoloxidase activity than those reared in groups. Sampling enzyme activity a second time when the adults were nearing reproductive maturity, group-reared Mormon crickets had elevated levels of prophenoloxidase and encapsulated foreign objects faster than solitarily-reared insects. Rearing density did not have a significant effect on either the darkness of the cuticle or antibacterial activity. This is the first report of age-related responses of adult insect immunity to crowding.

Ontogenetic changes in immunity and susceptibility to fungal infection in Mormon crickets Anabrus simplex.

Insects have innate immunity that may be weakened by resource allocation to growth. I measured enzymatic immunity, encapsulation response, and susceptibility to fungal infection in Mormon crickets of known age. Although the concentrations of circulating spontaneous and total phenoloxidase (PO) increased with age from the most recent molt in late instar nymphs (5th, 6th, and 7th) and 0-5 day old adults, mean values did not differ between stadia, indicating that circulating PO titers are knocked back with each molt. In contrast, encapsulation rate increased throughout nymphal development and adult maturation. No longer required to molt, adult PO titers increased steadily with age. Survivorship also increased with the age at which Metarhizium acridum fungus was applied to adults. I conclude that immunity relevant to defense against fungi continues to develop well into the adult stage. With each molt setting the insects back in circulating PO titers, very young adults are much like nymphs in enzymatic immunity.

Comparative allozyme analysis of two geographic populations of Pararcyptera microptera meridionalis in China.

The genetic structure of the two populations of Pararcyptera microptera meridionalis (Ikonn.) from Hebei and Liaoning in China was analyzed with horizontal starch gel electrophoresis. Among 15 loci of 11 enzymes identified in zymograms, Adk-1, Fbp-1, Mdh-2 and G3pd-1 showed low variability with few alleles. Higher allelic polymorphisms were observed at Fbp-2, Mdh-1 and Me-1. The two populations demonstrated high percentage of polymorphic loci (93.3% and 100.0%) but low observable overall heterozygosity (0.061 and 0.086), that could be attributed to heterozygote deficiencies, which led to the genotype frequency deviating from Hardy-Weinberg expectations. It is reasoned that the strong movement capability of the insect makes the individuals likely to be exposed to drastically varied environments, which tends to maintain dynamic equilibrium of genetic polymorphisms. The F-statistics between the two populations was comparatively smaller ( F(st) = 0.084), but larger when compared with those in migratory locusts like Locusta migratoria manilensis. Nei's genetic identity (I) and Roger's genetic distance (D) also showed close genetic relationship of the two populations by their high genetic identity (I = 0.904) and small genetic distance (D = 0.256). However,considerably qualitative and quantitative differences were noted at loci Acp-1 (F(st) = 0.462) and Pgi-1 (F(st) = 0.182).

Characterization of a hybrid zone between two chromosomal races of the weta Hemideina thoracica following a geologically recent volcanic eruption.

Two chromosomal races (2n=17 and 2n=15; XO) of the weta Hemideina thoracica meet at the centre of a volcanic region in North Island, New Zealand. Five independent polymorphic genetic markers showed broadly coinciding, steep frequency clines from north to south across this zone beside the flooded crater, Lake Taupo. Three unlinked nuclear gene markers provide estimates of zone width that are at least twice the width of the chromosomal and mitochondrial clines, with cline centres displaced at least 2.5 km. The different zone widths and centres suggest that this hybrid zone is a semipermeable barrier reducing the introgression of the chromosomal markers more than genic markers. We estimate that this species of weta must have a dispersal rate of at least 100 m per generation using the time since the last Taupo eruption (1850 years ago), which covered an area of about 20 000 km2 with pyroclastic flow.

Genetic structure and phyletic relationships of eastern Mediterranean Bacillus atticus brunner (Insecta Phasmatodea): a biochemical study.

The allozymic characterization of several new Croatian, Greek, and Turkish samples thought to belong to different subspecies of Bacillus atticus or to atticus-like taxa is given. Several allelic combinations (zymotypes) were observed among both diploid and triploid samples; the occurrence of highly different levels of heterozygosity for the same locus among populations is also common. The biochemical-genetic features of the numerous zymotypes are interpreted on the basis of the recently assessed cytology of their parthenogenetic reproduction. Biochemical and meiotic features also allow one to suggest that both diploid and triploid cytotypes of B. atticus are more likely interracial hybrids in origin. The new triploid Greek samples show only small genetic distances from the Turkish triploid and diploid ones; also, they do not show clear-cut morphological differences, so that all triploids and Turkish diploid samples are together referred to as B. a. carius. On the other hand, all Croatian, Greek, and Italian diploids appear to belong to the same electrophoretic cluster, biochemically differentiated at a subspecific level from B. a. carious. This newly defined comprehensive group of diploid samples, which also morphologically show gradual patterns of variation, is referred to as B. a. atticus.

Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT.

Enhancement of firefly luciferase activity by cytidine nucleotides.

The temporal pattern of light production by firefly luciferase depends on the ATP concentration. With low concentrations of ATP a constant production of light occurred while at high concentrations of ATP (greater than 10 microM) there was a flash of light followed by a decline in light production. This time course of light production with high ATP concentrations was changed from the flash pattern to a pattern with a constant production of light by several cytidine nucleotides. CTP, CDP, dCTP, dCDP, dideoxyCTP, periodate-oxidized CTP and CDP, and the etheno derivatives of CTP and CDP produced that change. CMP, cytidine, CDP-glycerol, CDP-glucose, CDP-ethanolamine, and benzoylbenzoylCTP either were inhibitory to firefly luciferase or were not effective in changing the flash time course. Coenzyme A and related compounds also changed the time course of light production. The changes in time course produced by either cytidine nucleotides or CoA were inhibited by desulfoCoA. These compounds apparently enhanced light production by promoting the dissociation of the inhibitory product, oxidized luciferin, from the enzyme. When the activating compounds were used with high concentrations of ATP, the sensitivity of assay for firefly luciferase was increased. This increased sensitivity is important when using the firefly luciferase gene as a reporter.

The delta 12-desaturase from the house cricket, Acheta domesticus (Orthoptera: Gryllidae): characterization and form of the substrate.

A novel delta 12-desaturase from animals, which converts oleic acid (18:1n-9) to linoleic acid (18:2n-6), was characterized in the house cricket, Acheta domesticus. The delta 12-desaturase product, linoleic acid, was determined by silver nitrate thin-layer chromatography, radio-gas-liquid chromatography and radio-high-performance liquid chromatography with the latter being used for routine analyses. Enzyme activity was located in the microsomal fraction of whole insect homogenates. NADPH or NADH was required for activity, with NADPH being the more efficient electron donor. In short incubation times with oleoyl-CoA as substrate, the highest amount of product, linoleic acid, was found as linoleoyl-CoA. With longer incubation periods, most of the linoleic acid was recovered in the polar lipid fraction containing phospholipid. Preincubation of the microsomal preparation in the absence of NADPH, which allowed 90% of the oleoyl moiety to be transacylated into complex lipid, resulted in no detectable desaturation upon addition of NADPH. These data indicate that the oleic acid moiety used as substrate was in the form of a CoA derivative and not in the form of a phospholipid, as it is for the plant delta 12-desaturase. This is the first characterization of a delta 12-desaturase from an animal system and the first report of a delta 12-desaturase that uses oleoyl-CoA as substrate.

[NADH- resp. NADPH-oxidase-activity in the blood cells of hemi- and holometabolic insects (author's transl)]. / NADH-bzw. NADPH-Oxidase-Aktivität in den Blutzellen hemi- und holometaboler Insekten

In the hemocytes of a control group and a group of immunised insects an enhanceable NADH-or NADPH-oxidase-activity is demonstrated histochemically which, according to KLEBANOFF, has a share in the development of an intracellular antibacterial activity via the synthesis of H2O2.