Two distinct aphid diapause strategies: slow development or development arrest.

Aphids adapt to unfavourable environmental conditions, such as low temperatures in winter, by laying diapausing eggs that overwinter. Diapause is a stress-resistant and developmentally arrested stage that can be adopted in order to increase the chance of survival in adverse environmental conditions. The diapause process of aphids is still very poorly understood. We followed the development of two species of aphids, Brachycorynella asparagi and Appendiseta robiniae, using the immunostained embryos of the aphids to identify mitotic cell divisions. Two different models of aphid diapause were demonstrated for the first time. In the first strategy, the embryo developed continuously during winter diapause, while in the second case, there was an embryonic arrest. The possibility of slow development of the whole body during diapause is a characteristic feature of aphids. The link to the plant's phenology appears to be a key factor in determining the diapause strategy in aphids.

A Zebrafish/Drosophila Dual System Model for Investigating Human Microcephaly.

Microcephaly presents in neurodevelopmental disorders with multiple aetiologies, including bi-allelic mutation in TUBGCP2, a component of the biologically fundamental and conserved microtubule-nucleation complex, γ-TuRC. Elucidating underlying principles driving microcephaly requires clear phenotype recapitulation and assay reproducibility, areas where go-to experimental models fall short. We present an alternative simple vertebrate/invertebrate dual system to investigate fundamental TUBGCP2-related processes driving human microcephaly and associated developmental traits. We show that antisense morpholino knockdown (KD) of the Danio rerio homolog, tubgcp2, recapitulates human TUBGCP2-associated microcephaly. Co-injection of wild type mRNA pre-empts microcephaly in 55% of KD zebrafish larvae, confirming causality. Body shortening observed in morphants is also rescued. Mitotic marker (pH3) staining further reveals aberrantly accumulated dividing brain cells in microcephalic tubgcp2 KD morphants, indicating that tubgcp2 depletion disrupts normal mitosis and/or proliferation in zebrafish neural progenitor brain cells. Drosophila melanogaster double knockouts (KO) for TUBGCP2 homologs Grip84/cg7716 also develop microcephalic brains with general microsomia. Exacerbated Grip84/cg7716-linked developmental aberration versus single mutations strongly suggests interactive or coinciding gene functions. We infer that tubgcp2 and Grip84/cg7716 affect brain size similarly to TUBGCP2 and recapitulate both microcephaly and microcephaly-associated developmental impact, validating the zebrafish/fly research model for human microcephaly. Given the conserved cross-phyla homolog function, the data also strongly support mitotic and/or proliferative disruption linked to aberrant microtubule nucleation in progenitor brain cells as key mechanistic defects for human microcephaly.

Tweedle proteins form extracellular two-dimensional structures defining body and cell shape in Drosophila melanogaster.

Tissue function and shape rely on the organization of the extracellular matrix (ECM) produced by the respective cells. Our understanding of the underlying molecular mechanisms is limited. Here, we show that extracellular Tweedle (Twdl) proteins in the fruit fly Drosophila melanogaster form two adjacent two-dimensional sheets underneath the cuticle surface and above a distinct layer of dityrosinylated and probably elastic proteins enwrapping the whole body. Dominant mutations in twdl genes cause ectopic spherical aggregation of Twdl proteins that recruit dityrosinylated proteins at their periphery within lower cuticle regions. These aggregates perturb parallel ridges at the surface of epidermal cells that have been demonstrated to be crucial for body shaping. In one scenario, hence, this disorientation of epidermal ridges may explain the squatty phenotype of Twdl mutant larvae. In an alternative scenario, this phenotype may be due to the depletion of the dityrosinylated and elastic layer, and the consequent weakening of cuticle resistance against the internal hydrostatic pressure. According to Barlow's formula describing the distribution of internal pressure forces in pipes in dependence of pipe wall material properties, it follows that this reduction in turn causes lateral expansion at the expense of the antero-posterior elongation of the body.

Uninterrupted Development of Two Aphid Species Belonging to Cinara Genus during Winter Diapause.

Aphids are herbivores carrying the status of major pests for crops and ornamental plants. The many specific biological features of aphids allow them to survive unfavorable environmental conditions. As for other insects, a predominant strategy for aphids surviving winter, is laying diapausing eggs. During diapause, the expression of development may vary between species. Most of the insects stop growing during diapause; however, there is limited information about this process. We immunostained the embryos of aphids in order to detect cell division during diapause. Here, for the first time, we present that two species of aphids belonging to Cinara grow and develop throughout the duration of the winter diapause. Our results showed that the "resting stage" does not occur in embryos of these aphid species. The embryo of C. cupressi and C. juniperi undergoes a type of diapause, with slow growth. It seems that this feature is conducive to the rapid development of embryos in the egg, which may be another specific feature for aphid biology of overwintering.

The putative C-type lectin Schlaff ensures epidermal barrier compactness in Drosophila.

The stability of extracellular matrices is in general ensured by cross-linking of its components. Previously, we had shown that the integrity of the layered Drosophila cuticle relies on the presence of a covalent cuticular dityrosine network. Production and composition of this structure remained unstudied. In this work, we present our analyses of the schlaff (slf) gene coding for a putative C-type lectin that is needed for the adhesion between the horizontal cuticle layers. The Slf protein mainly localizes between the two layers called epicuticle and procuticle that separate from each other when the function of Slf is reduced or eliminated paralleling the phenotype of a cuticle with reduced extracellular dityrosine. Localisation of the dityrosinylated protein Resilin to the epicuticle-procuticle interface suggests that the dityrosine network mediates the adhesion of the epicuticle to the procuticle. Ultimately, compromised Slf function is associated with massive water loss. In summary, we propose that Slf is implied in the stabilisation of a dityrosine layer especially between the epicuticle and the procuticle that in turn constitutes an outward barrier against uncontrolled water flow.

Relationships within aphids Cinara (Cupressobium) (Hemiptera) based on mitochondrial and nuclear DNA sequences.

The relationships between Cinara (Cupressobium) aphids inhabiting woody parts and leaves of conifers belonging to Cupressaceae have been studied using a mitochondrial gene (COI) and a nuclear gene (EF1-α). Based on the COI sequences, genetic distances between species ranged from 5.6 % between Cinara (C.) tujafilina (del Guercio) and Cinara (C.) juniperi (De Geer) to 10.5 % between C. (C.) tujafilina and Cinara (C.) mordvilkoi (Pasek). Genetic distances among EF1-α sequences were lower and showed from 0.1 % between C. cupressi and C. juniperi to 2.3 % between C. tujafilina and C. mordvilkoi. Molecular phylogenetic trees were constructed using the Bayesian inference (BI) phylogenetic analysis and maximum parsimony (MP) criterion. Phylogenetic trees obtained based on COI and EF1-α marker genes created two sister clades. Our results indicate that Cinara (Cupressobium) are a monophyletic group of aphids. Phylogenetic relationships amongst Cupressobium aphids do not result from the association with the host plant, but from the feeding site on the host plant or an ability to change the microhabitat on the plant. As closely related species inhabit similar microhabitats on different host plants, it suggests that the host switching is the main mode of speciation in this subgenus.

Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster.

Exoskeletons stabilize cell, tissue, and body morphology in many living organisms including fungi, plants, and arthropods. In insects, the exoskeleton, the cuticle, is produced by epidermal cells as a protein extracellular matrix containing lipids and the polysaccharide chitin, and its formation requires coordinated synthesis, distribution, and modification of these components. Eventually, the stepwise secretion and sorting of the cuticle material results in a layered structure comprising the envelope, the proteinaceous epicuticle, and the chitinous procuticle. To study the role of chitin during cuticle development, we analyzed the consequences of chitin absence in the embryo of Drosophila melanogaster caused by mutations in the Chitin Synthase-1 (CS-1) gene, called krotzkopf verkehrt (kkv). Our histological data confirm that chitin is essential for procuticle integrity and further demonstrate that an intact procuticle is important to assemble and to stabilize the chitin-less epicuticle. Moreover, the phenotype of CS-1/kkv mutant embryos indicates that chitin is required to attach the cuticle to the epidermal cells, thereby maintaining epidermal morphology. Finally, sclerotization and pigmentation, which are the last steps in cuticle differentiation, are impaired in tissues lacking CS-1/kkv function, suggesting that proper cuticle structure is crucial for the activity of the underlying enzymes.

RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation.

The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitantly with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. We show that both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. Our results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane.

Drosophila p24 homologues eclair and baiser are necessary for the activity of the maternally expressed Tkv receptor during early embryogenesis.

p24 proteins are assumed to play an important role in the transport of secreted and transmembrane proteins into membranes. However, only few cargo proteins are known that partially, but in no case completely require p24 proteins for membrane transport. Here, we show that two p24 proteins are essential for dorsoventral patterning of Drosophila melanogaster embryo. Mutations in the genes, eclair (eca) and baiser (bai), encoding two p24 proteins reduce signalling by the TGF-beta homologue, Dpp, in early embryos. This effect is strictly maternal and specific to early embryogenesis, as Dpp signalling in other contexts is not notably affected. We provide genetic evidence that in the absence of eca or bai function in the oocyte, the maternally expressed type I TGF-beta receptor Tkv is not active. We propose that during early embryogenesis eca and bai are specifically required for the activity of the maternal Tkv, while the zygotic Tkv is not affected in the mutant embryos. Mutations in either eca or bai are sufficient for the depletion of Tkv activity and no enhancement of the phenotypes was observed in embryos derived from oocytes mutant for both genes. The dependence of maternal Tkv protein on the products of p24 genes may serve as an in vivo model for studying p24 proteins.

Krapfen/dMyd88 is required for the establishment of dorsoventral pattern in the Drosophila embryo.

In Drosophila, the dorsoventral axis is set up by the action of the dorsal group of genes and cactus, which have been ordered genetically in a linear pathway. We have identified and characterised krapfen (kra) as a new member of the dorsal-group genes. kra encodes for the Drosophila homologue of MyD88, an adapter protein operating in the mammalian IL-1 pathway. Epistasis experiments reveal that kra acts between the receptor Toll and the cytoplasmic factor Tube. We show that there is a direct interaction between Kra and Tube presumably mediated by the death domains present in both proteins. Tube in turn interacts with its downstream effector Pelle through death domain association. We therefore suggest that upon Toll activation, Kra associates with Pelle and Tube, in an heterotrimeric complex.