A Simple Method for Estimating the Spatiotemporal Distribution of Phenoloxidase Proteins in Insect Tissues.

1. Forest entomofauna retain high diversity, and examining beta diversity, or species turnover, among assemblages in a forest community is vital to elucidate the source of this diversity. 2. Under the DIVERSITAS in Western Pacific and Asia–International Biodiversity Observation Year (DIWPA–IBOY) project for simultaneously documenting biodiversity throughout the Western Pacific and Asian Region, 892 lepidopteran species (51 742 specimens) and 355 coleopteran species (11 633 specimens) were collected in 2001 by light traps in a cool–temperate forest in northern Japan. 3. This study evaluated the beta diversity of lepidopteran and coleopteran communities by ecological categories (i.e. trap location, forest strata, sampling days, and months), and assessed the habitat preferences of lepidopteran and coleopteran species. 4. anova ‐like additive apportioning models were used to quantify the beta diversity among the categories. The models simultaneously provide assessments of whether species distributions are biased in favour of particular habitats. 5. Significantly high beta diversity was observed among months for both Lepidoptera and Coleoptera. The category of months corresponded fairly well to the number of specialist species detected in the category, although a remarkably large number of significant specialists in Coleoptera were observed on strata. 6. The high beta diversity and number of specialist species among strata in both communities indicate that stratification between canopy and ground, and seasonal variation, played major roles in species composition and the rich entomofauna in the forest. Highly mobile adults were influenced by the vertical spatial scale, as previously suggested for larvae.

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.

The infectivity of steinernematid nematodes varies with temperature. Five insect species, namely codling moth, Cydia pomonella; mealworm, Tenebrio molitor; blow fly, Sarcophaga bullata; March fly, Bibio marci; and web spinning sawfly, Cephaleia abietis were exposed to a Canadian (# 76) isolate of Steinernema kraussei at 7°C. C. abietis was exposed to the Canadian isolate # D and Czech isolate Vimperk at 4°C. These experiments were done in Petri dishes filled with moist sand, exposure time was 250 h and inoculum ranged from 50 to 800 infective juveniles (IJs). The highest larval mortality occurred in lepidopteran and coleopteran species, C. pomonella (73-100%) and T. molitor (28-70%), respectively. Few dipteran larvae, S. bullata and B. marci, were killed. The mortality of C. abietis larvae ranged from 2% (isolate # 76, at 50 IJs/dish) to 30% (isolate Vimperk, at 400 IJs/dish) at 7°C, but was negligible at 4°C. The intensity of infection expressed as the number of adult nematodes recovered from the insect cadavers, was greatest in C. pomonella where it ranged from 16 to 25% of the initial IJ inoculum, but was almost zero for both fly larval species. When analysed and fitted to the modified Anderson host-parasite model, the data confirmed a significant level of Steinernema parasitism for C. abietis, T. molitor, and C. pomonella larvae at low temperatures, and showed that the two species of fly larvae tested were almost fully resistant to parasitism by S. kraussei. Natural adaptation of the Czech Vimperk isolate to C. abietis may have enhanced this isolate's infectivity when compared with that of the Canadian nematode isolates.

Inter-simple sequence repeat (ISSR) primers designed to anneal to microsatellites were used to obtain deoxyribonucleic acid (DNA) fingerprint profiles to distinguish among 16 established insect cell lines derived from an assortment of lepidopteran, dipteran, and coleopteran species. Three different levels of cell line comparison were made: (1) between parents and their clones, (2) among cell lines derived from different tissues from the same species, and (3) among cell lines derived from different insect species. Of the 16 repeat oligonucleotide primers used in this study, nine primers generated several unique markers to distinguish between parental cell lines and their clones. Four of the 16 primers also generated DNA profiles with a number of unique bands, enabling the distinction among cell lines derived from specific tissues from the same species. In addition, ISSR-generated DNA profiles provided the greatest number of unique markers to distinguish easily among insect cell lines derived from different species.

4 - Phenoloxidases in Insect Immunity

The color patterns on the wings of lepidopterans are among the most striking patterns in nature and have inspired diverse biological hypotheses such as the ecological role of aposomatic coloration, the evolution of mimicry, the role of human activities in industrial melanism, and the developmental basis of phenotypic plasticity. Yet, the developmental mechanisms underlying color pattern development are not well understood for three reasons. First, few mutations that alter color patterns have been characterized at the molecular level, so there is little mechanistic understanding of how mutant phenotypes are produced. Second, although gene expression patterns resembling adult color patterns are suggestive, there are few data available showing that gene products have a functional role in color pattern formation. Finally, because with few exceptions (notably Bombyx), genetic maps for most species of Lepidoptera are rudimentary or nonexistent, it is very difficult to characterize spontaneous mutants or to determine whether mutations with similar phenotypes are because of lesions in the same gene or different genes. Discussed here are two strategies for overcoming these difficulties: germ-line transformation of lepidopteran species using transposon vectors and amplified frequency length polymorphism-based genetic mapping using variation between divergent strains within a species or between closely related and interfertile species. These advances, taken together, will create new opportunities for the characterization of existing genetic variants, the creation of new sequence-tagged mutants, and the testing of proposed functional genetic relationships between gene products, and will greatly facilitate our understanding of the evolution and development of lepidopteran color patterns.

In adult mice the cytochrome P450 Cyp1a1 gene is not constitutively expressed but is highly inducible by foreign compounds acting through the aryl hydrocarbon (Ah) receptor. However, the expression profile of the Cyp1a1 gene in the developing embryo is not well under-stood. Using established transgenic mouse lines where 8.5 kb of the rat CYP1A1 promoter is cloned upstream of the lacZ reporter gene (1), we describe the expression of the CYP1A1-driven reporter gene in all tissues through-out stages E7-E14 of embryonic development. In contrast to the absence of constitutive Cyp1a1 and lacZ transgene expression in tissues of the adult mouse, a constitutive cell-specific and time-dependent pattern of CYP1A1 promoter activity was observed in the embryo. This expression pattern was confirmed as reflecting the endogenous gene by measuring Cyp1a1 mRNA levels and protein expression by immunohistochemistry. The number of cells displaying endogenous CYP1A1 activity could be increased in the embryo upon xenobiotic challenge, but only within areas where the CYP1A1 promotor was already active. When reporter mice were bred onto a genetic background expressing a lower affinity form of the Ah receptor (DBA allele), transgene and murine Cyp1a1 protein expression were both attenuated in the adult mouse liver upon xenobiotic challenge. By comparison, constitutive CYP1A1 promoter activity in the embryo was identical in the presence of either the high or low affinity Ah receptor. These novel data suggest that the Cyp1a1 protein may play a role in murine development and that regulation of the Cyp1a1 gene during this period is either through the action of a high affinity Ah receptor ligand or by an alternative regulatory pathway.

Dragonflies are colorful insects, and recent RNA sequencing studies have identified a number of candidate genes potentially involved in their color pattern formation and color vision. However, functional aspects of such genes have not been assessed due to the lack of molecular genetic tools applicable to dragonflies. We established an electroporation-mediated RNA interference (RNAi) procedure using the tiny dragonfly Nannophya pygmaea Rambur, 1842 (Odonata: Libellulidae) that targets the multicopper oxidase 2 gene (MCO2; also known as laccase2 gene) responsible for cuticular pigmentation in many insects. RNA sequencing of N. pygmaea and genomic survey of the dragonfly Ladona fulva identified four multicopper oxidase family genes: MCO1, MCO2, MCO3 and multicopper oxidase-related protein gene (MCORP). In N. pygmaea, MCO2 was specifically expressed around the cuticular pigmentation period, whereas MCO1 was constantly expressed. MCORP was expressed at adult stages, and MCO3 was scarcely expressed. When we applied in vivo electroporation, final instar larvae injected with MCO2 small interfering RNA became adults with patchy unpigmented regions. RNAi without in vivo electroporation did not affect cuticular pigmentation, suggesting that dragonflies do not show a systemic RNAi response. These results indicate that MCO2 is required for cuticular pigmentation across diverse insects, and highlight the usefulness of the electroporation-mediated RNAi method in dragonflies.

The global status of insect resistance to neonicotinoid insecticides

A field experiment was carried out in 2004 and 2005 to identify the diversity of sunflower (Helianthus annuus L.) pollinators and their influence on seed yield in Makueni district, a semi-arid area in Eastern Kenya. Insect flower visitors were recorded, pollen counted from their body and pollination efficiency index for each visitor determined. Seed yield from plots where insect visitors had access to and where they were denied access was compared. The proportional difference of yield from this pollination scenario was used to estimate monetary net-gain by farmers that could be attributed to insect pollination. In total, individuals belonging to 14 insect species were observed visiting sunflower floral heads. These included six Lepidopteran species, five Hymenopteran species, two Dipteran species, and one Coleopteran species. Apis mellifera L. was the most frequent visitor and had the highest pollination efficiency index. Plots where insect visitors had access produced on average 53% more seed yield compared with plots where insect visitors were excluded. This translates to a net monetary benefit of 51% of the total annual market value of sunflower, accruing to farmers in Makueni district in 2005 due to insect pollination.

This paper deals with a total of 44 species of Coleopterans (Insecta), under 35 genera, belonging to 9 families. These insect species are associated with 7 spp. of cereal crops (of 2 families), 10 spp. of vegetable crops (5 families) and 35 spp. of medicinal plants (14 families), occurring in diverse habitats in vast localities of Jammu, Kashmir and Ladakh regions of J & K State. The Coleopteran species, infesting cereal crops, vegetable crops and medicinal plants accounts for 25%, 36.36% and 50 % respectively of the total Coleopteran- fauna associated with crops and medicinal plants studied. The highest number of species of Coleopterans i.e. 14, pertaining to the family Curculionidae, is associated with host crops (cereals, vegetables) and medicinal plant species. This is followed by family Chrysomelidae and Scarabaeidae, with 11 spp. and 9 spp. respectively. Rest of the Coleopteran families show number of species either 2 spp. or 1 sp., associated with the vegetable crops and medicinal plant species. The family Solanaceae, including vegetable crops and medicinal plants, is affected by highest number of Coleopteran species, 13 (29.54 %). This is followed by cereal crop families, Fabaceae and Poaceae, affected by 6 spp. (13.63 %) each of Coleopteran. An updated systematic checklist of Coleopteran-fauna associated with host cereal and vegetable crops, and medicinal plant species has been given. Apart from this, a Catalogue on Host species -Coleopteran species complex, has been added.

The cytoskeleton of cells in blood vessel walls contains desmin, vimentin, and cytokeratins. The distribution of these proteins in human vessels is not fully known. We have mapped the distribution of intermediate filament proteins in human arterial walls. Monoclonal antibodies targeted at the intermediate filament proteins desmin, vimentin, and cytokeratins were used, and the distribution of these proteins was studied by immunohistochemistry. In the muscular arteries, most smooth muscle cells in the media expressed both desmin and vimentin; in the elastic arteries, the proportion of desmin-labelled cells was lower and preferentially located to the periphery of the media. In general, the desmin immunoreactivity within the intima was weak, but some smooth muscle cells and smooth muscle cells in the musculoelastic layer showed strong immunoreactivity. The vasa vasorum exhibited a heterogeneous desmin-labelling pattern. The vimentin antibodies labelled the endothelium and showed a heterogeneous staining pattern in the other layers of the arterial wall. Cytokeratin was detected in occasional cells in the media of muscular arteries, in many adluminal cells and cell clusters in the coronary intima, and in smooth muscle cells in the media of the elastic arteries. Vimentin is widely distributed in vascular smooth muscle cells, whereas the distribution of desmin and cytokeratin varies. Each artery studied had an intermediate filament pattern typical for the anatomical location. There were no interindividual variations in the distribution of intermediate filament proteins.

Transgenic poplar lines ‘Shanxin’ (Populus davidiana×Populus bolleana) were generated via Agrobacterium-mediated transformation. The transgenic lines carried the expression cassettes of Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3, respectively. The expression levels of the exogenous insect resistance genes in the transgenic lines were determined by Q-PCR and Western blot. Leaves of the transgenic lines were used for insect feeding bioassays on first instar larvae of the gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). At 5 d of feeding, the mean mortalities of larvae feeding on Cry1Ac + SCK and Cry1Ah3 transgenic poplars leaves were 97% and 91%, while mortality on Cry9Aa3 transgenic lines was about 49%. All gypsy moth and fall webworm larvae were killed in 7–9 days after feeding on leaves from Cry1Ac + SCK or Cry1Ah3 transgenic poplars, while all the fall webworm larvae were killed in 11 days and about 80% of gypsy moth larvae were dead in 14 days after feeding on those from Cry9Aa3 transgenic lines. It was concluded that the transgenic lines of Cry1Ac + SCK and Cry1Ah3 were highly toxic to larvae of both insect species while lines with Cry9Aa3 had lower toxicity,and H. cunea larvae are more sensitive to the insecticidal proteins compared to L. dispar. Transgenic poplar lines toxic to L. dispar and H. cunea could be used to provide Lepidoptera pest resistance to selected strains of poplar trees.

A polka-dotted fruit fly, Drosophila guttifera, has a unique pigmentation pattern made of black melanin and serves as a good model system to study color pattern formation. Because of its short generation time and the availability of transgenics, it is suitable for dissecting the genetic mechanisms of color pattern formation. While the ecology and life history of D. guttifera in the wild are not well understood, it is known to be resistant to a mushroom toxin, and this physiological trait is under molecular scrutiny. Pigmentation around crossveins and longitudinal vein tips is common in closely related species of the quinaria group, in addition to which D. guttifera has evolved species-specific pigmentation spots around the campaniform sensilla. Regulatory evolution of the Wnt signaling ligand Wingless, which locally induces pigmentation in the developing wing epithelium, has driven the evolution of distinct aspects of wing and body pigmentation. A melanin biosynthesis pathway gene, yellow, is also involved in the elaboration of these traits, downstream of wingless. Unraveling the detailed mechanism of pigmentation pattern formation of this species sheds light on the general principles of morphological evolution and foreshadows potential parallels with other systems, such as the pigmented wings of butterflies.

Overexpression of a dominant-negative ornithine decarboxylase in mouse skin: effect on enzyme activity and papilloma formation.