Calcium flashes orchestrate the wound inflammatory response through DUOX activation and hydrogen peroxide release

A crucial early wound response is the recruitment of inflammatory cells drawn by danger cues released by the damaged tissue. Hydrogen peroxide (H2O2) has recently been identified as the earliest wound attractant in Drosophila embryos and zebrafish larvae. The H2O2 signal is generated by activation of an NADPH oxidase, DUOX, and as a consequence, the first inflammatory cells are recruited to the wound within minutes. To date, nothing is known about how wounding activates DUOX. Here, we show that laser wounding of the Drosophila embryo epidermis triggers an instantaneous calcium flash, which travels as a wave via gap junctions several cell rows back from the wound edge. Blocking this calcium flash inhibits H2O2 release at the wound site and leads to a reduction in the number of immune cells migrating to the wound. We suggest that the wound-induced calcium flash activates DUOX via an EF hand calcium-binding motif and thus triggers the production of the attractant damage cue H2O2. Therefore, calcium represents the earliest signal in the wound inflammatory response

A Single Nucleotide Polymorphism within the Novel Sex-Linked Testis-Specific Retrotransposed PGAM4 Gene Influences Human Male Fertility

The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes.We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo.These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility

Does Sex Trade with Violence among Genotypes in Drosophila melanogaster?

The evolutionary forces shaping the ability to win competitive interactions, such as aggressive encounters, are still poorly understood. Given a fitness advantage for competitive success, variance in aggressive and sexual display traits should be depleted, but a great deal of variation in these traits is consistently found. While life history tradeoffs have been commonly cited as a mechanism for the maintenance of variation, the variability of competing strategies of conspecifics may mean there is no single optimum strategy. We measured the genetically determined outcomes of aggressive interactions, and the resulting effects on mating success, in a panel of diverse inbred lines representing both natural variation and artificially selected genotypes. Males of one genotype which consistently lost territorial encounters with other genotypes were nonetheless successful against males that were artificially selected for supernormal aggression and dominated all other lines. Intransitive patterns of territorial success could maintain variation in aggressive strategies if there is a preference for territorial males. Territorial success was not always associated with male mating success however and females preferred ‘winners’ among some male genotypes, and ‘losers’ among other male genotypes. This suggests that studying behaviour from the perspective of population means may provide limited evolutionary and genetic insight. Overall patterns of competitive success among males and mating transactions between the sexes are consistent with mechanisms proposed for the maintenance of genetic variation due to nonlinear outcomes of competitive interactions

A CRISPR screen identifies a pathway required for paraquat-induced cell death

Paraquat, a herbicide linked to Parkinson's disease, generates reactive oxygen species (ROS), which causes cell death. Because the source of paraquat-induced ROS production remains unknown, we conducted a CRISPR-based positive-selection screen to identify metabolic genes essential for paraquat-induced cell death. Our screen uncovered three genes, POR (cytochrome P450 oxidoreductase), ATP7A (copper transporter), and SLC45A4 (sucrose transporter), required for paraquat-induced cell death. Furthermore, our results revealed POR as the source of paraquat-induced ROS production. Thus, our study highlights the use of functional genomic screens for uncovering redox biology

Pleiotropic Effects of DDT Resistance on Male Size and Behaviour

Understanding the evolution and spread of insecticide resistance requires knowing the relative fitness of resistant organisms. In the absence of insecticides, resistance is predicted to be costly. The Drosophila melanogaster DDT resistance allele (DDT-R) is associated with a male mating cost. This could be because resistant males are generally smaller, but DDT-R may also alter courtship behaviours. Here we tested for body size and courtship effects of DDT-R on mating success in competitive and non-competitive mating trials respectively. We also assessed relative aggression in resistant and susceptible males because aggression can also influence mating success. While the effect of DDT-R on male size partly contributed to reduced mating success, resistant males also had lower rates of courtship and were less aggressive than susceptible males. These differences contribute to the observed DDT-R mating costs. Additionally, these pleiotropic effects of DDT-R are consistent with the history and spread of resistance alleles in nature

Immunolocalization of the short neuropeptide F receptor in queen brains and ovaries of the red imported fire ant (Solenopsis invicta Buren)

<p>Abstract</p> <p>Background</p> <p>Insect neuropeptides are involved in diverse physiological functions and can be released as neurotransmitters or neuromodulators acting within the central nervous system, and as circulating neurohormones in insect hemolymph. The insect short neuropeptide F (sNPF) peptides, related to the vertebrate neuropeptide Y (NPY) peptides, have been implicated in the regulation of food intake and body size, and play a gonadotropic role in the ovaries of some insect species. Recently the sNPF peptides were localized in the brain of larval and adult <it>Drosophila</it>. However, the location of the sNPF receptor, a G protein-coupled receptor (GPCR), has not yet been investigated in brains of any adult insect. To elucidate the sites of action of the sNPF peptide(s), the sNPF receptor tissue expression and cellular localization were analyzed in queens of the red imported fire ant, <it>Solenopsis invicta </it>Buren (Hymenoptera), an invasive social insect.</p> <p>Results</p> <p>In the queen brains and subesophageal ganglion about 164 cells distributed in distinctive cell clusters (C1-C9 and C12) or as individual cells (C10, C11) were immuno-positive for the sNPF receptor. Most of these neurons are located in or near important sensory neuropils including the mushroom bodies, the antennal lobes, the central complex, and in different parts of the protocerebrum, as well as in the subesophageal ganglion. The localization of the sNPF receptor broadly links the receptor signaling pathway with circuits regulating learning and feeding behaviors. In ovaries from mated queens, the detection of sNPF receptor signal at the posterior end of oocytes in mid-oogenesis stage suggests that the sNPF signaling pathway may regulate processes at the oocyte pole.</p> <p>Conclusions</p> <p>The analysis of sNPF receptor immunolocalization shows that the sNPF signaling cascade may be involved in diverse functions, and the sNPF peptide(s) may act in the brain as neurotransmitter(s) or neuromodulator(s), and in the ovaries as neurohormone(s). To our knowledge, this is the first report of the cellular localization of a sNPF receptor on the brain and ovaries of adult insects.</p

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca2+ with high affinity on one side of the membrane, and releases the bound Ca2+ on the other side of the membrane to yield a high free Ca2+ gradient. These features are a basic requirement for cellular Ca2+ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes

Targeted Manipulation of Serotonergic Neurotransmission Affects the Escalation of Aggression in Adult Male Drosophila melanogaster

Dopamine (DA) and serotonin (5HT) are reported to serve important roles in aggression in a wide variety of animals. Previous investigations of 5HT function in adult Drosophila behavior have relied on pharmacological manipulations, or on combinations of genetic tools that simultaneously target both DA and 5HT neurons. Here, we generated a transgenic line that allows selective, direct manipulation of serotonergic neurons and asked whether DA and 5HT have separable effects on aggression. Quantitative morphological examination demonstrated that our newly generated tryptophan hydroxylase (TRH)-Gal4 driver line was highly selective for 5HT-containing neurons. This line was used in conjunction with already available Gal4 driver lines that target DA or both DA and 5HT neurons to acutely alter the function of aminergic systems. First, we showed that acute impairment of DA and 5HT neurotransmission using expression of a temperature sensitive form of dynamin completely abolished mid- and high-level aggression. These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships. We showed next that manipulation of either 5HT or DA neurotransmission failed to duplicate this phenotype. Selective disruption of 5HT neurotransmission yielded flies that fought, but with reduced ability to escalate fights, leading to fewer dominance relationships. Acute activation of 5HT neurons using temperature sensitive dTrpA1 channel expression, in contrast, resulted in flies that escalated fights faster and that fought at higher intensities. Finally, acute disruption of DA neurotransmission produced hyperactive flies that moved faster than controls, and rarely engaged in any social interactions. By separately manipulating 5HT- and DA- neuron systems, we collected evidence demonstrating a direct role for 5HT in the escalation of aggression in Drosophila

Distribution of the Octopamine Receptor AmOA1 in the Honey Bee Brain

Octopamine plays an important role in many behaviors in invertebrates. It acts via binding to G protein coupled receptors located on the plasma membrane of responsive cells. Several distinct subtypes of octopamine receptors have been found in invertebrates, yet little is known about the expression pattern of these different receptor subtypes and how each subtype may contribute to different behaviors. One honey bee (Apis mellifera) octopamine receptor, AmOA1, was recently cloned and characterized. Here we continue to characterize the AmOA1 receptor by investigating its distribution in the honey bee brain. We used two independent antibodies produced against two distinct peptides in the carboxyl-terminus to study the distribution of the AmOA1 receptor in the honey bee brain. We found that both anti-AmOA1 antibodies revealed labeling of cell body clusters throughout the brain and within the following brain neuropils: the antennal lobes; the calyces, pedunculus, vertical (alpha, gamma) and medial (beta) lobes of the mushroom body; the optic lobes; the subesophageal ganglion; and the central complex. Double immunofluorescence staining using anti-GABA and anti-AmOA1 receptor antibodies revealed that a population of inhibitory GABAergic local interneurons in the antennal lobes express the AmOA1 receptor in the cell bodies, axons and their endings in the glomeruli. In the mushroom bodies, AmOA1 receptors are expressed in a subpopulation of inhibitory GABAergic feedback neurons that ends in the visual (outer half of basal ring and collar regions) and olfactory (lip and inner basal ring region) calyx neuropils, as well as in the collar and lip zones of the vertical and medial lobes. The data suggest that one effect of octopamine via AmOA1 in the antennal lobe and mushroom body is to modulate inhibitory neurons

Mapping Peptidergic Cells in Drosophila: Where DIMM Fits In

The bHLH transcription factor DIMMED has been associated with the differentiation of peptidergic cells in Drosophila. However, whether all Drosophila peptidergic cells express DIMM, and the extent to which all DIMM cells are peptidergic, have not been determined. To address these issues, we have mapped DIMM expression in the central nervous system (CNS) and periphery in the late larval stage Drosophila. At 100 hr after egg-laying, DIMM immunosignals are largely congruent with a dimm-promoter reporter (c929-GAL4) and they present a stereotyped pattern of 306 CNS cells and 52 peripheral cells. We assigned positional values for all DIMM CNS cells with respect to reference gene expression patterns, or to patterns of secondary neuroblast lineages. We could assign provisional peptide identities to 68% of DIMM-expressing CNS cells (207/306) and to 73% of DIMM-expressing peripheral cells (38/52) using a panel of 24 markers for Drosophila neuropeptide genes. Furthermore, we found that DIMM co-expression was a prevalent feature within single neuropeptide marker expression patterns. Of the 24 CNS neuropeptide gene patterns we studied, six patterns are >90% DIMM-positive, while 16 of 22 patterns are >40% DIMM-positive. Thus most or all DIMM cells in Drosophila appear to be peptidergic, and many but not all peptidergic cells express DIMM. The co-incidence of DIMM-expression among peptidergic cells is best explained by a hypothesis that DIMM promotes a specific neurosecretory phenotype we term LEAP. LEAP denotes Large cells that display Episodic release of Amidated Peptides