Electroporation-mediated RNA Interference Method in Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, ...

The University of Tokyo

National Institute of Advanced Industrial Science and Technology (AIST)

the university of tokyo

The adult honeybee (Apis mellifera L.) mushroom bodies (MBs, a higher center in the insect brain) comprise four subtypes of intrinsic neurons: the class-I large-, middle-, and small-type Kenyon cells (lKCs, mKCs, and sKCs, respectively), and class-II KCs. Analysis of the differentiation of KC subtypes during metamorphosis is important for the better understanding of the roles of KC subtypes related to the honeybee behaviors. In the present study, aiming at identifying marker genes for KC subtypes, we used a cDNA microarray to comprehensively search for genes expressed in an MB-preferential manner in the honeybee brain. Among the 18 genes identified, we further analyzed three genes whose expression was enriched in the MBs: phospholipase C epsilon (PLCe), synaptotagmin 14 (Syt14), and discs large homolog 5 (dlg5). Quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of PLCe, Syt14, and dlg5 was more enriched in the MBs than in the other brain regions by approximately 31-, 6.8-, and 5.6-fold, respectively. In situ hybridization revealed that expression of both Syt14 and dlg5 was enriched in the lKCs but not in the mKCs and sKCs, whereas expression of PLCe was similar in all KC subtypes (the entire MBs) in the honeybee brain, suggesting that Syt14 and dlg5, and PLCe are available as marker genes for the lKCs, and all KC subtypes, respectively. In situ hybridization revealed that expression of PLCe is already detectable in the class-II KCs at the larval fifth instar feeding stage, indicating that PLCe expression is a characteristic common to the larval and adult MBs. In contrast, expression of both Syt14 and dlg5 became detectable at the day three pupa, indicating that Syt14 and dlg5 expressions are characteristic to the late pupal and adult MBs and the lKC specific molecular characteristics are established during the late pupal stages.

Analysis of the Differentiation of Kenyon Cell Subtypes Using Three Mushroom Body-Preferential Genes during Metamorphosis in the Honeybee (Apis mellifera L.).

The saw-toothed grain beetle,  (Silvanidae), is a cosmopolitan stored-product pest. Early studies on  in the 1930s described the presence of peculiar bacteriomes harboring endosymbiotic bacteria in the abdomen. Since then, however, the microbiological nature of the symbiont has been elusive. Here we investigated the endosymbiotic system of  in detail. In the abdomen of adults, pupae, and larvae, four oval bacteriomes were consistently identified, whose cytoplasm was full of extremely elongated tubular bacterial cells several micrometers wide and several hundred micrometers long. Molecular phylogenetic analysis identified the symbiont as a member of the , in which the symbiont was the most closely related to the endosymbiont of a grain pest beetle,  (Bostrichidae). The symbiont was detected in developing embryos, corroborating vertical symbiont transmission through host generations. The symbiont gene showed AT-biased nucleotide composition and accelerated molecular evolution, plausibly reflecting degenerative evolution of the symbiont genome. When the symbiont infection was experimentally removed, the aposymbiotic insects grew and reproduced normally, but exhibited a slightly but significantly more reddish cuticle and lighter body mass. These results indicate that the symbiont of  is not essential for the host's growth and reproduction but contributes to the host's cuticle formation. Symbiont genome sequencing and detailed comparison of fitness parameters between symbiotic and aposymbiotic insects under various environmental conditions will provide further insights into the symbiont's biological roles for the stored-product pest. Some beetles notorious as stored-product pests possess well-developed symbiotic organs called bacteriomes for harboring specific symbiotic bacteria, although their biological roles have been poorly understood. Here we report a peculiar endosymbiotic system of a grain pest beetle, , in which four oval bacteriomes in the abdomen are full of extremely elongated tubular bacterial cells. Experimental symbiont elimination did not hinder the host's growth and reproduction, but resulted in emergence of reddish beetles, uncovering the symbiont's involvement in host's cuticle formation. We speculate that the extremely elongated symbiont cell morphology might be due to the degenerative symbiont genome deficient in bacterial cell division and/or cell wall formation, which highlights an evolutionary consequence of intimate host-symbiont coevolution.

A Novel, Extremely Elongated, and Endocellular Bacterial Symbiont Supports Cuticle Formation of a Grain Pest Beetle.

In an attempt to establish an experimental dragonfly model, we developed a laboratory rearing system for the blue-tailed damselfly, Ischnura senegalensis. Adoption of multi-well plastic plates as rearing containers enabled mass-rearing of isolated larvae without cannibalism and convenient microscopic monitoring of individual larvae. Feeding Artemia brine shrimps to younger larvae and Tubifex worms for older larvae resulted in low mortality, synchronized ecdysis, and normal development of the larvae. We continuously monitored the development of 118 larvae every day, of which 49 individuals (41.5%) reached adulthood. The adult insects were fed with Drosophila flies in wet plastic cages, attained reproductive maturity in a week, copulated, laid fertilized eggs, and produced progeny. The final larval instar varied from 9 to 12, with the 11 instar (56.5%) and the 12 instar (24.2%) constituting the majority. From the 1 instar to the penultimate instar, the duration of each instar was relatively short, mainly ranging from three to 11 days. Afterwards, the duration of each instar was prolonged, reaching 7-25 days for the penultimate instar and 14-28 days for the final instar. Some larvae of final, penultimate and younger instars were subjected to continuous and close morphological examinations, which enabled developmental staging of larvae based on size, shape, and angle of compound eyes and other morphological traits. This laboratory rearing system may facilitate the understanding of physiological, biochemical, and molecular mechanisms underlying metamorphosis, hormonal control, morphogenesis, body color polymorphism, and other biological features of dragonflies.

Laboratory Rearing System for Ischnura senegalensis (Insecta: Odonata) Enables Detailed Description of Larval Development and Morphogenesis in Dragonfly.

Many animals change their body color for visual signaling and environmental adaptation. Some dragonflies show wax-based color change and ultraviolet (UV) reflection, but the biochemical properties underlying the phenomena are totally unknown. Here we investigated the UV-reflective abdominal wax of dragonflies, thereby identifying very long-chain methyl ketones and aldehydes as unique and major wax components. Little wax was detected on young adults, but dense wax secretion was found mainly on the dorsal abdomen of mature males of  and , and pruinose wax secretion was identified on the ventral abdomen of mature females of  and . Comparative transcriptomics demonstrated drastic upregulation of the  gene, a member of the fatty acid elongase gene family, whose expression reflected the distribution of very long-chain methyl ketones. Synthetic 2-pentacosanone, the major component of dragonfly's wax, spontaneously formed light-scattering scale-like fine structures with strong UV reflection, suggesting its potential utility for biomimetics.

Molecular basis of wax-based color change and UV reflection in dragonflies.

Cockroaches are commonly found in human residences and notorious as hygienic and nuisance pests. Notably, however, no more than 30 cockroach species are regarded as pests, while the majority of 4,500 cockroaches in the world are living in forest environments with little relevance to human life. Why some cockroaches have exceptionally adapted to anthropic environments and established pest status is of interest. Here we investigated the German cockroach , which is a cosmopolitan pest species, and the forest cockroach , which is a wild species closely related to . In contrast to easy rearing of , laboratory rearing of  was challenging-several trials enabled us to keep the insects for up to three months. We particularly focused on the distribution patterns of specialized cells, bacteriocytes, for harboring endosymbiotic , which has been suggested to contribute to host's nitrogen metabolism and recycling, during the postembryonic development of the insects. The bacteriocytes were consistently localized to visceral fat bodies filling the abdominal body cavity, where a number of single bacteriocytes were scattered among the adipocytes, throughout the developmental stages in both females and males. The distribution patterns of the bacteriocytes were quite similar between  and , and also among other diverse cockroach species, plausibly reflecting the highly conserved cockroach- symbiotic association over evolutionary time. Our study lays a foundation to experimentally investigate the origin and the processes of urban pest evolution, on account of possible involvement of microbial associates.

Bacteriocytes and  Endosymbionts of the German Cockroach , the Forest Cockroach , and Other Cockroach Species.

The order Odonata (dragonflies and damselflies) comprises diurnal insects with well-developed vision, showing diverse colors in adult wings and bodies. It is one of the most ancestral winged insect groups. Because Odonata species use visual cues to recognize each other, color patterns have been investigated from ecological and evolutionary viewpoints. Here we review the recent progress on molecular mechanisms of pigmentation, especially focused on light-blue coloration. Results from histology and pigment analysis showed that ommochrome pigments on the proximal layer and pteridine pigments on the distal layer of the epidermis are essential for light-blue coloration. We also summarize genes involved in the biosynthesis of three major insect pigments conserved across insects and discuss that gene-functional analysis deserves future studies.

Genta Okude

Department of Biological Sciences,
Graduate School of Science,
Department of Biological Sciences, Graduate School of Science

The University of Tokyo

Electroporation-mediated RNA Interference Method in Odonata

Genta Okude

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences,Graduate School of Science,Department of Biological Sciences, Graduate School of Science

The University of Tokyo

Electroporation-mediated RNA Interference Method in Odonata

Department of Biological Sciences,
Graduate School of Science,
Department of Biological Sciences, Graduate School of Science