The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic and applied silkworm research. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of B. mori lines, stably inheriting single CRISPR/Cas9-induced mutations. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant B. mori lines. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects.

Redox homeostasis is a vital process the maintenance of which is assured by the presence of numerous antioxidant small molecules and enzymes and the alteration of which is involved in many pathologies, including several neurodegenerative disorders. Among the different enzymes involved in the antioxidant response, SOD1 and DJ-1 have both been associated with the pathogenesis of amyotrophic lateral sclerosis and Parkinson's disease, suggesting a possible interplay in their mechanism of action. Copper deficiency in the SOD1-active site has been proposed as a central determinant in SOD1-related neurodegeneration. SOD1 maturation mainly relies on the presence of the protein copper chaperone for SOD1 (CCS), but a CCS-independent alternative pathway also exists and functions under anaerobic conditions. To explore the possible involvement of DJ-1 in such a pathway in vivo, we exposed Drosophila melanogaster to anoxia and evaluated the effect of DJ-1 on fly survival and SOD1 levels, in the presence or absence of CCS. Loss of DJ-1 negatively affects the fly response to the anoxic treatment, but our data indicate that the protective activity of DJ-1 is independent of SOD1 in Drosophila, indicating that the two proteins may act in different pathways.

Single microRNAs are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in Drosophila, ranging from development to behaviour. We investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. The effect of miR-210 in shaping neuronal projections was confirmed in vitro, using a Drosophila neuronal cell line. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development.

Genomic studies suggest an association of circadian clock genes with bipolar disorder (BD) and lithium response in humans. Therefore, we tested mice mutant in various clock genes before and after lithium treatment in the forced swim test (FST), a rodent behavioral test used for evaluation of depressive-like states. We find that expression of circadian clock components, including Per2, Cry1 and Rev-erbα, is affected by lithium treatment, and thus, these clock components may contribute to the beneficial effects of lithium therapy. In particular, we observed that Cry1 is important at specific times of the day to transmit lithium-mediated effects. Interestingly, the pathways involving Per2 and Cry1, which regulate the behavior in the FST and the response to lithium, are distinct as evidenced by the phosphorylation of GSK3β after lithium treatment and the modulation of dopamine levels in the striatum. Furthermore, we observed the co-existence of depressive and mania-like symptoms in Cry1 knock-out mice, which resembles the so-called mixed state seen in BD patients. Taken together our results strengthen the concept that a defective circadian timing system may impact directly or indirectly on mood-related behaviors.

Intronic genes represent ~6% of the total gene complement in Drosophila melanogaster and ~85% of them encode for proteins. We recently characterized the D. melanogaster timeless2 (tim2) gene, showing its active involvement in chromosomal stability and light synchronization of the adult circadian clock. The protein coding gene named 2mit maps on the 11(th) tim2 intron in the opposite transcriptional orientation.

There is evidence of a link between the circadian system and psychiatric diseases. Studies in humans and mammals suggest that environmental and/or genetic disruption of the circadian system leads to an increased liability to psychiatric disease. Disruption of clock genes and/or the clock network might be related to the etiology of these pathologies; also, some genes, known for their circadian clock functions, might be associated to mental illnesses through clock-independent pleiotropy. Here, we examine the features which we believe make Drosophila melanogaster a model apt to study the role of the circadian clock in psychiatric disease. Despite differences in the organization of the clock system, the molecular architecture of the Drosophila and mammalian circadian oscillators are comparable and many components are evolutionarily related. In addition, Drosophila has a rather complex nervous system, which shares much at the cell and neurobiological level with humans, i.e., a tripartite brain, the main neurotransmitter systems, and behavioral traits: circadian behavior, learning and memory, motivation, addiction, social behavior. There is evidence that the Drosophila brain shares some homologies with the vertebrate cerebellum, basal ganglia, and hypothalamus-pituitary-adrenal axis, the dysfunctions of which have been tied to mental illness. We discuss Drosophila in comparison to mammals with reference to the: organization of the brain and neurotransmitter systems; architecture of the circadian clock; clock-controlled behaviors. We sum up current knowledge on behavioral endophenotypes, which are amenable to modeling in flies, such as defects involving sleep, cognition, or social interactions, and discuss the relationship of the circadian system to these traits. Finally, we consider if Drosophila could be a valuable asset to understand the relationship between circadian clock malfunction and psychiatric disease.

Light at night has strong effects on physiology and behavior of mammals. It affects mood in humans, which is exploited as light therapy, and has been shown to reset the circadian clock in the suprachiasmatic nuclei (SCN). This resetting is paramount to align physiological and biochemical timing to the environmental light-dark cycle. Here we provide evidence that light at zeitgeber time (ZT) 22 affects mood-related behaviors also in mice by activating the clock gene Period1 (Per1) in the lateral habenula (LHb), a brain region known to modulate mood-related behaviors. We show that complete deletion of Per1 in mice led to depressive-like behavior and loss of the beneficial effects of light on this behavior. In contrast, specific deletion of Per1 in the region of the LHb did not affect mood-related behavior, but suppressed the beneficial effects of light. RNA sequence analysis in the mesolimbic dopaminergic system revealed profound changes of gene expression after a light pulse at ZT22. In the nucleus accumbens (NAc), sensory perception of smell and G-protein coupled receptor signaling were affected the most. Interestingly, most of these genes were not affected in Per1 knock-out animals, indicating that induction of Per1 by light serves as a filter for light-mediated gene expression in the brain. Taken together we show that light affects mood-related behavior in mice at least in part via induction of Per1 in the LHb with consequences on mood-related behavior and signaling mechanisms in the mesolimbic dopaminergic system.

The domesticated silkworm Bombyx mori has an innate immune system, whose main effectors are the antimicrobial peptides (AMPs). Silkworm strains are commonly grouped into four geographical types (Japanese, Chinese, European and Tropical) and are generally characterised by a variable susceptibility to infections. To clarify the genetic and molecular mechanisms on which the different responses to infections are based, we exposed one silkworm strain for each geographical area to oral infections with the silkworm pathogens Enterococcus mundtii or Serratia marcescens. We detected a differential susceptibility to both bacteria, with the European strain displaying the lowest sensitivity to E. mundtii and the Indian one to S. marcescens. We found that all the strains were able to activate the AMP response against E. mundtii. However, the highest tolerance of the European strain appeared to be related to the specific composition of its AMP cocktail, containing more effective variants such as a peculiar Cecropin B6 isoform. The resistance of the Indian strain to S. marcescens seemed to be associated with its prompt capability to activate the systemic transcription of AMPs. These data suggest that B. mori strains with distinct genetic backgrounds employ different strategies to counteract bacterial infections, whose efficacy appears to be pathogen-dependent.

Mushroom bodies are a higher order center for sensory integration, learning and memory of the insect brain. Memory is generally subdivided into different phases. In the model organism Drosophila melanogaster, mushroom bodies have been shown to play a central role in both short- and long-term memory. In D. melanogaster, the gene 2mit codes a transmembrane protein carrying an extracellular Leucin-rich-repeat domain, which is highly transcribed in the mushroom and ellipsoid bodies of the adult fly brain and has a role in the early phase of memory. Utilizing coimmunoprecipitation experiments and mass spectrometry analyses, we have shown that 2MIT interacts with Arginine kinase in adult fly heads. Arginine kinase belongs to the family of Phosphagen kinases and plays a fundamental role in energy homeostasis. Using the GAL4/UAS binary system, we demonstrated that a downregulation of Arginine kinase mainly driven in the mushroom bodies affects short-term memory of Drosophila adult flies, in a courtship conditioning paradigm. As 2mit c03963 hypomorphic mutants showed comparable results when analyzed with the same assay, these data suggest that 2MIT and Arginine kinase are both involved in the same memory phenotype, likely interacting at the level of mushroom bodies. 2MIT and Arginine kinase are conserved among insects, the implications of which, along with their potential roles in other insect taxa are also discussed.