High-resolution analysis of individual Drosophila melanogaster larvae uncovers individual variability in locomotion and its neurogenetic modulation

Neuronally orchestrated muscular movement and locomotion are defining faculties of multicellular animals. Due to its simple brain and genetic accessibility, the larva of the fruit fly Drosophila melanogaster allows one to study these processes at tractable levels of complexity. However, although the faculty of locomotion clearly pertains to the individual, most studies of locomotion in larvae use measurements aggregated across animals, or animals tested one by one, an extravagance for larger-scale analyses. This prevents grasping the inter- and intra-individual variability in locomotion and its neurogenetic determinants. Here, we present the IMBA (individual maggot behaviour analyser) for analysing the behaviour of individual larvae within groups, reliably resolving individual identity across collisions. We use the IMBA to systematically describe the inter- and intra-individual variability in locomotion of wild-type animals, and how the variability is reduced by associative learning. We then report a novel locomotion phenotype of an adhesion GPCR mutant. We further investigated the modulation of locomotion across repeated activations of dopamine neurons in individual animals, and the transient backward locomotion induced by brief optogenetic activation of the brain-descending ‘mooncrawler’ neurons. In summary, the IMBA is an easy-to-use toolbox allowing an unprecedentedly rich view of the behaviour and its variability of individual larvae, with utility in multiple biomedical research contexts

Characterisation of siderophore producing marine bacteria

Marine bacteria are diverse and highly abundant in the oceans. Under iron-depleted conditions, marine bacteria produce an iron chelator called a siderophore to scavenge for iron. Very little is known about siderophore producing bacteria in the oceans. However, as all marine bacteria are competing for the essential iron molecule, most marine bacteria should produce siderophores. The aims of this thesis were to obtain pure cultures of marine bacteria sampled on a recent international GeoTraces (TAN1109) research cruise and characterise these isolates by phenotypic and molecular methods such as ERIC-PCR, GTG-PCR and restriction enzyme digestion of 16S-23S ISR PCR products. Bacteria were screened for siderophore production by growing pure isolates on CAS agar. Isolates were further dereplicated and identified using 16S rRNA gene sequencing and phylogenetic analysis. Of 188 isolates, 178 were Gram-negative and 10 were Gram-positive. Of all isolates, 179 showed clearing and/or halo formation when grown on CAS agar. For molecular analysis, 91 isolates were chosen based on their ability to form clearings and halos on CAS agar. These included 89 isolates with medium to strong clearing intensities (early and late) as well as 2 with large halo formations only. Of the chosen representatives, three isolates were Gram-positive. GTG-PCR typed 68 of the 91 isolates into 22 clusters (similarity of 70%). ERIC-PCR typed 76 of the 91 isolates into 18 clusters (similarity of 70%). TaqI digestion of 16S-23S-PCR products placed 33 of the 91 isolates into 18 clusters (similarity of 70%). The 16S rRNA gene sequence analysis showed three of the isolates clustered within the class of Bacilli, two clustered within the alpha sub-group of the proteobacteria, and 11 within the gamma sub-group of the proteobacteria. 16S rRNA sequencing of 16 chosen representatives identified four siderophore producing marine bacterial genera across all samples. Sampled isolates showed over 90% sequence similarities to Marinobacter algicola, Halomonas meridiana, Ruegeria scottomollicae, and Bacillus subtilis. MMO3035 is potentially a new Gram-positive siderophore producing marine bacteria belonging to the genus Bacillus. In summary, fingerprint data of ERIC-PCR, GTG-PCR and TaqI digestion of 16S-23S-PCR products suggested that siderophore producing marine bacteria are more phylogenetically diverse and widespread in the environment than current literature indicates. However, 16S rRNA sequencing identified only four siderophore producing marine bacterial genera. This study contributes new research findings to the field of marine microbial ecology and highlights the importance of siderophore producing bacteria in the ocean environment

Characterisation of siderophore producing marine bacteria

Marine bacteria are diverse and highly abundant in the oceans. Under iron-depleted conditions, marine bacteria produce an iron chelator called a siderophore to scavenge for iron. Very little is known about siderophore producing bacteria in the oceans. However, as all marine bacteria are competing for the essential iron molecule, most marine bacteria should produce siderophores. The aims of this thesis were to obtain pure cultures of marine bacteria sampled on a recent international GeoTraces (TAN1109) research cruise and characterise these isolates by phenotypic and molecular methods such as ERIC-PCR, GTG-PCR and restriction enzyme digestion of 16S-23S ISR PCR products. Bacteria were screened for siderophore production by growing pure isolates on CAS agar. Isolates were further dereplicated and identified using 16S rRNA gene sequencing and phylogenetic analysis. Of 188 isolates, 178 were Gram-negative and 10 were Gram-positive. Of all isolates, 179 showed clearing and/or halo formation when grown on CAS agar. For molecular analysis, 91 isolates were chosen based on their ability to form clearings and halos on CAS agar. These included 89 isolates with medium to strong clearing intensities (early and late) as well as 2 with large halo formations only. Of the chosen representatives, three isolates were Gram-positive. GTG-PCR typed 68 of the 91 isolates into 22 clusters (similarity of 70%). ERIC-PCR typed 76 of the 91 isolates into 18 clusters (similarity of 70%). TaqI digestion of 16S-23S-PCR products placed 33 of the 91 isolates into 18 clusters (similarity of 70%). The 16S rRNA gene sequence analysis showed three of the isolates clustered within the class of Bacilli, two clustered within the alpha sub-group of the proteobacteria, and 11 within the gamma sub-group of the proteobacteria. 16S rRNA sequencing of 16 chosen representatives identified four siderophore producing marine bacterial genera across all samples. Sampled isolates showed over 90% sequence similarities to Marinobacter algicola, Halomonas meridiana, Ruegeria scottomollicae, and Bacillus subtilis. MMO3035 is potentially a new Gram-positive siderophore producing marine bacteria belonging to the genus Bacillus. In summary, fingerprint data of ERIC-PCR, GTG-PCR and TaqI digestion of 16S-23S-PCR products suggested that siderophore producing marine bacteria are more phylogenetically diverse and widespread in the environment than current literature indicates. However, 16S rRNA sequencing identified only four siderophore producing marine bacterial genera. This study contributes new research findings to the field of marine microbial ecology and highlights the importance of siderophore producing bacteria in the ocean environment

High-resolution analysis of individual Drosophila melanogaster larvae uncovers individual variability in locomotion and its neurogenetic modulation

Neuronally orchestrated muscular movement and locomotion are defining faculties of multicellular animals. Due to its simple brain and genetic accessibility, the larva of the fruit fly Drosophila melanogaster allows one to study these processes at tractable levels of complexity. However, although the faculty of locomotion clearly pertains to the individual, most studies of locomotion in larvae use measurements aggregated across animals, or animals tested one by one, an extravagance for larger-scale analyses. This prevents grasping the inter- and intra-individual variability in locomotion and its neurogenetic determinants. Here, we present the IMBA (individual maggot behaviour analyser) for analysing the behaviour of individual larvae within groups, reliably resolving individual identity across collisions. We use the IMBA to systematically describe the inter- and intra-individual variability in locomotion of wild-type animals, and how the variability is reduced by associative learning. We then report a novel locomotion phenotype of an adhesion GPCR mutant. We further investigated the modulation of locomotion across repeated activations of dopamine neurons in individual animals, and the transient backward locomotion induced by brief optogenetic activation of the brain-descending 'mooncrawler' neurons. In summary, the IMBA is an easy-to-use toolbox allowing an unprecedentedly rich view of the behaviour and its variability of individual larvae, with utility in multiple biomedical research contexts

Supplementary movie S1 from High-resolution analysis of individual <i>Drosophila melanogaster</i> larvae uncovers individual variability in the neurogenetic modulation of locomotion

A complete sample video from the data set used in Figures 1-3. Displayed are Canton S wildtype larvae tested for their innate odour preference (one of the two Teflon cups contained an odour). The original video is overlaid with the contours (green), head (green dot) and tail (red dot) of all detected larvae, as well as an identification number of each detected object (which may be a larva or a ‘blob’ assembly of several larvae). The white objects on either side are the odour containers (of which one contains an odour and the other one is empty). Note that when larvae collide, red spines indicate the shape model approach used for collision resolution

Supplementary figures from High-resolution analysis of individual <i>Drosophila melanogaster</i> larvae uncovers individual variability in the neurogenetic modulation of locomotion

A PDF file containing Supplementary figures S1-S13, including captions

Isolation, biosynthesis and chemical modifications of Rubterolones A-F:rare tropolone alkaloids from <i>Actinomadura </i>sp 5-2

The discovery of six new, highly substituted tropolone alkaloids, rubterolones A–F, from Actinomadura sp. 5-2, isolated from the gut of the fungus-growing termite Macrotermes natalensis is reported. Rubterolones were identified by using fungus-bacteria challenge assays and a HRMS-based dereplication strategy, and characterised by NMR and HRMS analyses and by X-ray crystallography. Feeding experiments and subsequent chemical derivatisation led to a first library of rubterolone derivatives (A–L). Genome sequencing and comparative analyses revealed their putative biosynthetic pathway, which was supported by feeding experiments. This study highlights how gut microbes can present a prolific source of secondary metabolites.The Daimler Benz foundation to C.B., the Villum Kann Rasmussen Foundation for a Young Investigator Fellowship (VKR10101) to M.P. R.B., the International Leibniz Research School for Microbial and Biomolecular Interactions (ILRS) and School for Microbial Communication (JSMC, DFG).http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1521-37652018-07-12hj2017Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

Supplementary movie S9 from High-resolution analysis of individual <i>Drosophila melanogaster</i> larvae uncovers individual variability in the neurogenetic modulation of locomotion

Animation of a sample R53F07 ChR2-XXL larva. The blue dot marks the position of the head. The filling indicates the tail forward velocity in body lengths per second [bl/s]. At t = 30 s, light stimulation activates R53F07-neurons; at t = 60 s, the light stimulation ends

Supplementary movie S5 from High-resolution analysis of individual <i>Drosophila melanogaster</i> larvae uncovers individual variability in the neurogenetic modulation of locomotion

A complete sample video from [26]. For further details, see Supplementary movie S1

Supplementary movie S9 from High-resolution analysis of individual <i>Drosophila melanogaster</i> larvae uncovers individual variability in locomotion and its neurogenetic modulation

Animation of a sample R53F07 ChR2-XXL larva. The blue dot marks the position of the head. The filling indicates the tail forward velocity in body lengths per second [bl/s]. At t = 30 s, light stimulation activates R53F07-neurons; at t = 60 s, the light stimulation ends