Rafael Cantera

The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae.

Malaria transmission depends on the competence of some Anopheles mosquitoes to sustain Plasmodium development (susceptibility). A genetically selected refractory strain of Anopheles gambiae blocks Plasmodium development, melanizing, and encapsulating the parasite in a reaction that begins with tyrosine oxidation, and involves three quantitative trait loci. Morphological and microarray mRNA expression analysis suggest that the refractory and susceptible strains have broad physiological differences, which are related to the production and detoxification of reactive oxygen species. Physiological studies corroborate that the refractory strain is in a chronic state of oxidative stress, which is exacerbated by blood feeding, resulting in increased steady-state levels of reactive oxygen species, which favor melanization of parasites as well as Sephadex beads.

Real-time, in vivo analysis of malaria ookinete locomotion and mosquito midgut invasion

Invasion of the Anopheles mosquito midgut by the Plasmodium ookinete is a critical step in the malaria transmission cycle. We have generated a fluorescent P. berghei transgenic line that expresses GFP in the ookinete and oocyst stages, and used it to perform the first real‐time analysis of midgut invasion in the living mosquito as well as in explanted intact midguts whose basolateral plasma membranes were vitally stained. These studies permitted detailed analysis of parasite motile behaviour in the midgut and cell biological analysis of the invasion process. Throughout its journey, the ookinete displays distinct modes of motility: stationary rotation, translocational spiralling and straight‐segment motility. Spiralling is based on rotational motility combined with translocation steps and changes in direction, which are achieved by transient attachments of the ookinetes trailing end. As it moves from the apical to the basal side of the midgut epithelium, the ookinete uses a predominant intracellular route and appears to glide on the membrane in foldings of the basolateral domain. However, it traverses serially the cytoplasm of several midgut cells before entering and migrating through the basolateral intercellular space to access the basal lamina. The invaded cells commit apoptosis, and their expulsion from the epithelium invokes wound repair mechanisms including extensive lamellipodia crawling. A ‘hood’ of lamellipodial origin, provided by the invaded cell, covers the ookinete during its egress from the epithelium. The flexible ookinete undergoes shape changes and temporary constrictions associated with passage through the plasma membranes. Similar observations were made in both A. gambiae and A. stephensi, demonstrating the conservation of P. berghei interactions with these vectors.

Drosophila tracheal morphogenesis: intricate cellular solutions to basic plumbing problems☆

The cellular architecture of tubular organs suggests striking similarities in the mechanisms of tubulogenesis between species. The formation of the Drosophila respiratory organ (trachea) highlights the basic principles of branch patterning and tube growth that generate a highly elaborate but stereotyped epithelial tubular network. Oriented cell migration, changes in cell shape, selective growth of the apical cell membrane and intracellular lumen formation are essential events in this process. These morphogenetic processes build four structurally distinct classes of tubes that facilitate optimal airflow and gas exchange with target tissues. The molecular players in these plots include attractant and repellent signals, differentiation factors that cause a high diversity of cell fates within the epithelium, and determinants of tube formation and dimensions.

Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I

SummaryAn antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electronmicroscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hindgut.

Grainy head controls apical membrane growth and tube elongation in response to Branchless/FGF signalling.

Epithelial organogenesis involves concerted movements and growth of distinct subcellular compartments. We show that apical membrane enlargement is critical for lumenal elongation of the Drosophila airways, and is independently controlled by the transcription factor Grainy head. Apical membrane overgrowth in grainy head mutants generates branches that are too long and tortuous without affecting epithelial integrity, whereas Grainy head overexpression limits lumenal growth. The chemoattractant Branchless/FGF induces tube outgrowth, and we find that it upregulates Grainy head activity post-translationally, thereby controlling apical membrane expansion to attain its key role in branching. We favour a two-step model for FGF in branching: first, induction of cell movement and apical membrane growth, and second, activation of Grainy head to limit lumen elongation, ensuring that branches reach and attain their characteristic lengths.

Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca

The glandular cells of the corpus cardiacum of the locust Locusta migratoria, known to synthesize and release adipokinetic hormones (AKH), are contacted by axons immunoreactive to an antiserum raised against the locust neuropeptide locustatachykinin I (LomTK I). Electron-microscopical immunocytochemistry reveals LomTK immunoreactive axon terminals, containing granular vesicles, in close contact with the glandular cells cells. Release of AKH I from isolated corpora cardiaca of the locust has been monitored in an in vitro system where the amount of AKH I released into the incubation saline is determined by reversed phase high performance liquid chromatography with fluorometric detection. We could show that LomTK I induces release of AKH from corpora cardiaca in a dose-dependent manner when tested in a range of 10-200 microM. This is thus the first clear demonstration of a substance inducing release of AKH, correlated with the presence of the substance in fibers innervating the AKH-synthesizing glandular cells, in the insect corpora cardiaca.

Sleep, clocks, and synaptic plasticity

Sleep is widely believed to play an essential role in synaptic plasticity. However, the precise mechanisms governing this presumptive function are largely unknown. There is also evidence for independent circadian oscillations in synaptic strength and morphology. Therefore, synaptic changes observed after sleep reflect interactions between state-dependent (e.g., wake versus sleep) and state-independent (circadian) processes. In this review we consider how sleep and biological clocks influence synaptic plasticity. We discuss these findings in the context of current plasticity-based theories of sleep function and propose a new model that integrates circadian and brain-state influences on synaptic plasticity.

Smt3 is required for Drosophila melanogaster metamorphosis.

Sumoylation, the covalent attachment of the small ubiquitin-related modifier SUMO to target proteins, regulates different cellular processes, although its role in the control of development remains unclear. We studied the role of sumoylation during Drosophila development by using RNAi to reduce smt3 mRNA levels in specific tissues. smt3 knockdown in the prothoracic gland, which controls key developmental processes through the synthesis and release of ecdysteroids, caused a 4-fold prolongation of larval life and completely blocked the transition from larval to pupal stages. The reduced ecdysteroid titer of smt3 knockdown compared with wild-type larvae explains this phenotype. In fact, after dietary administration of exogenous 20-hydroxyecdysone, knockdown larvae formed pupal cases. The phenotype is not due to massive cell death or degeneration of the prothoracic glands at the time when puparium formation should occur. Knockdown cells show alterations in expression levels and/or the subcellular localisation of enzymes and transcription factors involved in the regulation of ecdysteroid synthesis. In addition, they present reduced intracellular channels and a reduced content of lipid droplets and cholesterol, which could explain the deficit in steroidogenesis. In summary, our study indicates that Smt3 is required for the ecdysteroid synthesis pathway at the time of puparium formation.

The Drosophila Nora virus is an enteric virus, transmitted via feces.

The biology of the Drosophila viruses has not been intensely investigated. Here we have investigated the biology of the Nora virus, a persistent Drosophila virus. We find that injected Nora virus is able to replicate in the files, reaching a high titer that is maintained in the next generation. There is a remarkable variation in the viral loads of individual flies in persistently infected stocks; the titers can differ by three orders of magnitude. The Nora virus is mainly found in the intestine of infected flies, and the histology of these infected intestines show increased vacuolization. The virus is excreted in the feces and is horizontally transmitted. The Nora virus infection has a very mild effect on the longevity of the flies, and no significant effect on the number of eggs laid and the percent of eggs that develop to adults.

Muscle Structure and Innervation Are Affected by Loss of Dorsal in the Fruit Fly,Drosophila melanogaster ☆

In Drosophila, the Rel-protein Dorsal and its inhibitor, Cactus, act in signal transduction pathways that control the establishment of dorsoventral polarity during embryogenesis and the immune response during postembryonic life. Here we present data indicating that Dorsal is also involved in the control of development and maintenance of innervation in somatic muscles. Dorsal and Cactus are colocalized in all somatic muscles during postembryonic development. In larvae and adults, these proteins are distributed at low levels in the cytoplasm and nuclei and at much higher levels in the postsynaptic component of glutamatergic neuromuscular junctions. Absence of Dorsal, in homozygous dorsal mutant larvae results in muscle misinsertions, duplications, nuclear hypotrophy, disorganization of actin bundles, and altered subcellular distribution of Cactus. Some muscles show very abnormal neuromuscular junctions, and some motor axon terminals are transformed into growth cone-like structures embedded in synaptotagmin-enriched vesicles. The detailed phenotype suggests a role of Dorsal signalling in the maintenance and plasticity of the NMJ.