Chemical defense is one of the most important traits, which endow insects the ability to conquer a most diverse set of ecological environments. Chemical secretions are used for defense against anything from vertebrate or invertebrate predators to prokaryotic or eukaryotic parasites or food competitors. Tenebrionid beetles are especially prolific in this category, producing several varieties of substituted benzoquinone compounds. In order to get a better understanding of the genetic and molecular basis of defensive secretions, we performed RNA sequencing in a newly emerging insect model, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae). To detect genes that are highly and specifically expressed in the odoriferous gland tissues that secret defensive chemical compounds, we compared them to a control tissue, the anterior abdomen. 511 genes were identified in different subtraction groups. Of these, 77 genes were functionally analyzed by RNA interference (RNAi) to recognize induced gland alterations morphologically or changes in gland volatiles by gas chromatography-mass spectrometry. 29 genes (38%) presented strong visible phenotypes, while 67 genes (87%) showed alterations of at least one gland content. Three of these genes showing quinone-less (ql) phenotypes – Tcas-ql VTGl; Tcas-ql ARSB; Tcas-ql MRP – were isolated, molecularly characterized, their expression identified in both types of the secretory glandular cells, and their function determined by quantification of all main components after RNAi. In addition, microbe inhibition assays revealed that a quinone-free status is unable to impede bacterial or fungal growth. Phylogenetic analyses of these three genes indicate that they have evolved independently and specifically for chemical defense in beetles.

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming ciliary-directed vesicles, through organization of the vesicle fusion machinery at the periciliary membrane.

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in signal transduction. Cilia are anchored inside the cell through basal bodies (BBs), modified centrioles also acting as microtubule-organization centers. Photoreceptors (PRs) are sensory neurons, whose primary cilium forms a highly specialized compartment called the outer segment (OS) responsible for sensing incoming light. Thus, ciliopathies often present with retinal degeneration. Mutations in KIAA0586/TALPID3 (TA3) cause Joubert syndrome, in which 30% of affected individuals develop retinal involvement. To elucidate the function of TALPID3 in PRs, we studied talpid3 zebrafish mutants and identified a progressive retinal degeneration phenotype. The majority of PRs lack OS development due to defects in BB positioning and docking at the apical cell surface. Intracellular accumulation of the photopigment opsin leads to PR cell death of moderate severity. Electroretinograms demonstrate severe visual impairement. A small subset of PRs display normally docked BBs and extended OSs through rescue by maternally-deposited Talpid3. While localization of the small GTPase Rab8a, which plays an important role in BB docking, appears unaffected in talpid3−/− PRs, overexpression of constitutively active Rab8a rescues OS formation, indicating that the role of Ta3 in early ciliogenesis lies upstream of Rab8a activation in PRs.

Cilia sense and transduce sensory stimuli, homeostatic cues and developmental signals by orchestrating signaling reactions. Extracellular vesicles (EVs) that bud from the ciliary membrane have well-studied roles in the disposal of excess ciliary material, most dramatically exemplified by the shedding of micrometer-sized blocks by photoreceptors. Shedding of EVs by cilia also affords cells with a powerful means to shorten cilia. Finally, cilium-derived EVs may enable cell–cell communication in a variety of organisms, ranging from single-cell parasites and algae to nematodes and vertebrates. Mechanistic understanding of EV shedding by cilia is an active area of study, and future progress may open the door to testing the function of ciliary EV shedding in physiological contexts. In this Cell Science at a Glance and the accompanying poster, we discuss the molecular mechanisms that drive the shedding of ciliary material into the extracellular space, the consequences of shedding for the donor cell and the possible roles that ciliary EVs may have in cell non-autonomous contexts.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.