Rearrangements in the musculature correlate with jumping behaviour in legless Mediterranean fruit fly larvae Ceratitis capitata (Tephritidae).

Larvae of holometabolic insects evolved different crawling strategies depending on the presence or absence of larval legs or life style. A rather unusual mode of locomotion has independently evolved in legless larvae of several dipteran species. Maggots of the Mediterranean fruit fly Ceratitis capitata developed an effective jumping mechanism to increase locomotion speed or to deter predators during the search for suitable pupation sites. Here, we use high-speed videography to visualize even the fastest movements during jump preparation and take-off. Quantification of kinetic and biometric parameters reveal that maggots jump up to 15-fold of their body length from a standing position and gain speed with 27 times the acceleration of gravity. Videos at high spatial resolution show the mechanism of latch formation and release in unprecedented detail. Mouth hooks insert in the caudal segment and raise a cuticular fold that serves as a handle to pressurize the body prior to launch. Since locomotion behaviour should be intrinsically linked to neuromuscular systems, we dissected third instar larvae and determined the precise pattern of abdominal muscles fibres. Compared to non-jumping dipteran larvae, such as Drosophila melanogaster, the overall arrangement is highly similar, but a few muscle fibres show characteristic re-arrangements in orientation and strength that are consistent with a role in bending and jumping. These results suggest that body wall muscles show adaptations to jumping behaviour in Ceratitis larvae, and possibly also in other species with different jumping techniques.

Vitamin D Metabolites in Nonmetastatic High-Risk Prostate Cancer Patients with and without Zoledronic Acid Treatment after Prostatectomy.

There are limited and discrepant data on prostate cancer (PCa) and vitamin D. We investigated changes in three vitamin D3 metabolites in PCa patients after prostatectomy with zoledronic acid (ZA) treatment regarding their metastasis statuses over four years. In 32 patients from the ZEUS trial, 25(OH)D3, 24,25(OH)2D3, and 1,25(OH)2D3 were measured with liquid chromatography coupled with tandem mass spectrometry at four time points. All the patients received daily calcium and vitamin D3. Bone metastases were detected in 7 of the 17 ZA-treated patients and in 5 of the 15 controls (without ZA), without differences between the groups (p = 0.725). While 25(OH)D3 and 24,25(OH)2D3 increased significantly after the study's start, with following constant values, the 1,25(OH)2D3 concentrations remained unchanged. ZA treatment did not change the levels of the three metabolites. 25(OH)D3 and 24,25(OH)2D3 were not associated with the development of bone metastases. In contrast, 1,25(OH)2D3 was also higher in patients with bone metastasis before the study's start. Thus, in high-risk PCa patients after prostatectomy, 25(OH)D3, 24,25(OH)2D3, and 1,25(OH)2D3 were not affected by supportive ZA treatment or by the development of metastasis over four years, with the exception of 1,25(OH)2D3, which was constantly higher in metastatic patients. There might be potential prognostic value if the results can be confirmed.

Relevance of Biotin Deficiency in Patients with Inflammatory Bowel Disease and Utility of Serum 3 Hydroxyisovaleryl Carnitine as a Practical Everyday Marker.

BACKGROUND: Biotin, a water-soluble B vitamin, has demonstrable anti-inflammatory properties. A biotin-deficient diet induced a colitis-like phenotype in mice, alleviable by biotin substitution. Mice with dextran sulfate sodium (DSS)-induced colitis showed biotin deficiency and diminished levels of sodium-dependent multivitamin transporter, a protein involved in biotin absorption. Biotin substitution induced remission by reducing activation of NF-κB, a transcription factor involved in intestinal permeability and inflammatory bowel disease (IBD). We investigated for the first time a possible clinical role of biotin status in IBD. METHODS: In a comparative, retrospective, cross-sectional study, serum samples of 138 patients with IBD (67 female; 72 Crohn's disease (CD), 66 ulcerative colitis (UC)) aged 18-65 years and with a mean age (±SD) of 42.5 ± 14.3 years as well as 80 healthy blood donors (40 female; 40.0 ± 10.0 years; range 20-60 years) were analyzed. Inflammation was defined as hsCRP ≥5 mg/L, and to determine biotin status, serum 3-hydroxyisovaleryl carnitine (3HIVc) levels were measured by LC-MS/MS. RESULTS: A total of 138 patients with IBD (67f; 72CD/66 UC; 42.5 ± 14.3 years) were enrolled: 83/138 had inflammation. Mean serum 3HIVc levels were significantly higher in IBD patients but unaffected by inflammation. Biotin deficiency (95th percentile of controls: >30 nmol/L 3HIVc) was significantly more common in IBD patients versus controls. CONCLUSION: High serum 3HIVc levels and biotin deficiency were associated with IBD but not inflammatory activity or disease type. Our findings suggest biotin may play a role as cause or effect in IBD pathogenesis. Routine assessment and supplementation of biotin may ameliorate IBD and support intestinal integrity.

Expression pattern of CAPA/pyrokinin neuropeptide genes in Remipedia and silverfish: Rapid differentiation after gene duplication in early Hexapoda, followed by strong conservation of newly established features in insects.

Only few genes are known from insects that encode multiple neuropeptides, i.e., peptides that activate different receptors. Among those are the capa and pk genes, which differentiated within Hexapoda following gene duplication. In our study, we focus on the early stages of differentiation of these genes. Specifically: (1) What was the expression pattern of the ancestral capa/pk gene, i.e., prior to gene duplication? (2) What is the expression pattern of capa and pk in silverfish, whose ancestors diverged from Pterygota more than 400 mya? Our results suggest the location and projection of CAPA immunoreactive Va cells in abdominal ganglia (trunk ganglia in Remipedia) are a plesiomorphic trait that was already present in the ancestor of Remipedia and Hexapoda. General features of serial homology such as location of cells bodies, contralateral projection of primary neurites, and presumed peripheral peptide release from segmentally arranged neurohemal release sites could be observed in Remipedia and silverfish, but also in all Pterygota studied so far. Differences are mainly in the specific location of these peripheral release sites. This hypothetical basic pattern of capa/pk neurons underwent modifications in the anterior ganglia of the ventral nerve cord already in Remipedia. In silverfish, as in all Pterygota studied so far, pk expression in the CNS is apparently restricted to the gnathal ganglia, whereas capa expression is typical of abdominal Va cells. Thus, differentiation in the expression pattern of capa and pk genes occurred early in the evolution of Hexapoda; likely soon after the appearance of two separate genes.

Descending octopaminergic neurons modulate sensory-evoked activity of thoracic motor neurons in stick insects.

Neuromodulatory neurons located in the brain can influence activity in locomotor networks residing in the spinal cord or ventral nerve cords of invertebrates. How inputs to and outputs of neuromodulatory descending neurons affect walking activity is largely unknown. With the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunohistochemistry, we show that a population of dorsal unpaired median (DUM) neurons descending from the gnathal ganglion to thoracic ganglia of the stick insect Carausius morosus contains the neuromodulatory amine octopamine. These neurons receive excitatory input coupled to the legs' stance phases during treadmill walking. Inputs did not result from connections with thoracic central pattern-generating networks, but, instead, most are derived from leg load sensors. In excitatory and inhibitory retractor coxae motor neurons, spike activity in the descending DUM (desDUM) neurons increased depolarizing reflexlike responses to stimulation of leg load sensors. In these motor neurons, descending octopaminergic neurons apparently functioned as components of a positive feedback network mainly driven by load-detecting sense organs. Reflexlike responses in excitatory extensor tibiae motor neurons evoked by stimulations of a femur-tibia movement sensor either are increased or decreased or were not affected by the activity of the descending neurons, indicating different functions of desDUM neurons. The increase in motor neuron activity is often accompanied by a reflex reversal, which is characteristic for actively moving animals. Our findings indicate that some descending octopaminergic neurons can facilitate motor activity during walking and support a sensory-motor state necessary for active leg movements.NEW & NOTEWORTHY We investigated the role of descending octopaminergic neurons in the gnathal ganglion of stick insects. The neurons become active during walking, mainly triggered by input from load sensors in the legs rather than pattern-generating networks. This report provides novel evidence that octopamine released by descending neurons on stimulation of leg sense organs contributes to the modulation of leg sensory-evoked activity in a leg motor control system.

Quantification of Biogenic Amines from Individual GFP-Labeled Drosophila Cells by MALDI-TOF Mass Spectrometry.

Cell-cell communication plays a crucial role in orchestrating and modulating neural circuits. To understand such interactions, it is vital to determine and quantify the involved messenger molecules such as neuropeptides and biogenic amines on the level of single cells. In this study, we used single-cell mass spectrometry (SCMS) to qualify and quantify octopamine (OA) and tyramine (TA) from isolated single cells from intact brains of the fruit fly Drosophila melanogaster. Our workflow involved targeted GFP-guided single-cell microdissection, on-plate chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) or 2,5-dimethyl-1 H-pyrrole-3,4-dicarbaldehyde (DPD) for increasing ion stability and ion signal intensity, and isotopically marked internal standards for quantification by MALDI-TOF MS. We were able to determine a limit of detection for OA of 1 fmol/µL, for TA of 2.5 fmol/µL and a lower limit of quantification (LLOQ) of 10 fmol/µL for both substances. SCMS of GFP-labeled somata from ventral midline neurons of the labial neuromere (VMlb) of the gnathal ganglion revealed an OA titer of 17.38 fmol/µL and a TA titer (∼2.5 fmol/µL) lower than the LLOQ, independent of sex. However, using a genetically altered driver line devoid of OA, TßhnM18/Tdc2 > GFP, we confirmed TA in these cells. Furthermore, cold-anesthetization of flies caused a significant increase in OA content in VMlb somata. We compared OA titers of somata from two different OA/TA cell clusters to demonstrate the usefulness of targeted SCMS in advancing our understanding of OA/TA signaling in behavior and physiology. An influence on the detection of neuropeptides by our derivatized SCMS method could be excluded.

Mating-Induced Differential Peptidomics of Neuropeptides and Protein Hormones in Agrotis ipsilon Moths.

In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon, mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity. Here we used next-generation RNA sequencing and a combination of liquid chromatography, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and direct tissue profiling to analyze the transcriptome and peptidome of different brain compartments in virgin and mated males and females of A. ipsilon. We identified 37 transcripts encoding putative neuropeptide precursors and 54 putative bioactive neuropeptides from 23 neuropeptide precursors (70 sequences in total, 25 neuropeptide precursors) in different areas of the central nervous system including the antennal lobes, the gnathal ganglion, and the corpora cardiaca-corpora allata complex. Comparisons between virgin and mated males and females revealed tissue-specific differences in peptide composition between sexes and according to physiological state. Mated males showed postmating differences in neuropeptide occurrence, which could participate in the mating-induced olfactory plasticity.

Neuropeptide Mapping of Dimmed Cells of Adult Drosophila Brain.

Neuropeptides are structurally highly diverse messenger molecules that act as regulators of many physiological processes such as development, metabolism, reproduction or behavior in general. Differentiation of neuropeptidergic cells often corresponds with the presence of the transcription factor DIMMED. In the central nervous system of the fruit fly Drosophila melanogaster, DIMMED commonly occurs in neuroendocrine neurons that release peptides as neurohormones but also in interneurons with complex branching patterns. Fly strains with green fluorescence protein (GFP)-expressing dimmed cells make it possible to systematically analyze the processed neuropeptides in these cells. In this study, we mapped individual GFP-expressing neurons of adult D. melanogaster from the dimmed (c929)>GFP line. Using single cell mass spectrometry, we analyzed 10 types of dimmed neurons from the brain/gnathal ganglion. These cells included neuroendocrine cells with projection into the retrocerebral complex but also a number of large interneurons. Resulting mass spectra not only provided comprehensive data regarding mature products from 13 neuropeptide precursors but also evidence for the cellular co-localization of neuropeptides from different neuropeptide genes. The results can be implemented in a neuroanatomical map of the D. melanogaster brain. Graphical Abstract ᅟ.

Energy Homeostasis Control in Drosophila Adipokinetic Hormone Mutants.

Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based on AKH injections and correlative studies in a broad range of insect species established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone [termed "adipokinetic hormone precursor-related peptide" (APRP)] is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. Here we employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, we provide evidence for a negative autoregulatory loop in Akh gene regulation.

Multiple neuropeptides in the Drosophila antennal lobe suggest complex modulatory circuits.

The fruitfly, Drosophila, is dependent on its olfactory sense in food search and reproduction. Processing of odorant information takes place in the antennal lobes, the primary olfactory center in the insect brain. Besides classical neurotransmitters, earlier studies have indicated the presence of a few neuropeptides in the olfactory system. In the present study we made an extensive analysis of the expression of neuropeptides in the Drosophila antennal lobes by direct profiling using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry and immunocytochemistry. Neuropeptides from seven different precursor genes were unambiguously identified and their localization in neurons was subsequently revealed by immunocytochemistry. These were short neuropeptide F, tachykinin related peptide, allatostatin A, myoinhibitory peptide, SIFamide, IPNamide, and myosuppressin. The neuropeptides were expressed in subsets of olfactory sensory cells and different populations of local interneurons and extrinsic (centrifugal) neurons. In some neuron types neuropeptides were colocalized with classical neurotransmitters. Our findings suggest a huge complexity in peptidergic signaling in different circuits of the antennal lobe.