Metagenomic analyses highlight the symbiotic association between the glacier stonefly Andiperla willinki and its bacterial gut community.

The glacier stonefly Andiperla willinki is the largest metazoan inhabiting the Patagonian glaciers. In this study, we analysed the gut microbiome of the aquatic nymphs by 16S rRNA gene amplicon and metagenomic sequencing. The bacterial gut community was consistently dominated by taxa typical of animal digestive tracts, such as Dysgonomonadaceae and Lachnospiraceae, as well as those generally indigenous to glacier environments, such as Polaromonas. Interestingly, the dominant Polaromonas phylotypes detected in the stonefly gut were almost never detected in the glacier surface habitat. Fluorescence in situ hybridization analysis revealed that the bacterial lineages typical of animal guts colonized the gut wall in a co-aggregated form, while Polaromonas cells were not included in the aggregates. Draft genomes of several dominant bacterial lineages were reconstructed from metagenomic datasets and indicated that the predominant Dysgonomonadaceae bacterium is capable of degrading various polysaccharides derived from host-ingested food, such as algae, and that other dominant bacterial lineages ferment saccharides liberated by the polysaccharide degradation. Our results suggest that the gut bacteria-host association in the glacier stonefly contributes to host nutrition as well as material cycles in the glacier environment.

Sp-tetraKCNG: A novel cyclic nucleotide gated K(+) channel.

The sequence of a novel cGMP-regulated, tetrameric, K(+) selective channel (Sp-tetraKCNG) was discovered in the sea urchin Strongylocentrotus purpuratus. The Sp-tetraKCNG is a single polypeptide made of four KCNG domains similar to voltage-dependent Na(+) and Ca(2+) channels. Each KCNG domain has six transmembrane segments (S1-S6), the ion pore having the K(+) selectivity signature GYGD and a cyclic nucleotide-binding domain (CNBD). This novel channel is evolutionary located between K(+)-selective and voltage-dependent EAG channels and voltage-independent cationic CNG channels. Bilayer reconstitutions demonstrate such a cGMP-regulated K(+) selective channel in sea urchin spermatozoa.

Bridging behavior and physiology: ion-channel perspective on mushroom body-dependent olfactory learning and memory in Drosophila.

An important body of evidence documents the differential expression of ion channels in brains, suggesting they are essential to endow particular brain structures with specific physiological properties. Because of their role in correlating inputs and outputs in neurons, modulation of voltage-dependent ion channels (VDICs) can profoundly change neuronal network dynamics and performance, and may represent a fundamental mechanism for behavioral plasticity, one that has received less attention in learning and memory studies. Revisiting three paradigmatic mutations altering olfactory learning and memory in Drosophila (dunce, leonardo, amnesiac) a link was established between each mutation and the operation of VDICs in Kenyon cells, the intrinsic neurons of the mushroom bodies (MBs). In Drosophila, MBs are essential to the emergence of olfactory associative learning and retention. Abnormal ion channel operation might underlie failures in neuronal physiology, and be crucial to understand the abnormal associative learning and retention phenotypes the mutants display. We also discuss the only case in which a mutation in an ion channel gene (shaker) has been directly linked to olfactory learning deficits. We analyze such evidence in light of recent discoveries indicating an unusual ion current profile in shaker mutant MB intrinsic neurons. We anticipate that further studies of acquisition and retention mutants will further confirm a link between such mutations and malfunction of specific ion channel mechanisms in brain structures implicated in learning and memory.

Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic.

Fertility is a highly complex and regulated phenomenon essential for the survival of any species. To identify Drosophila fertility-specific neural networks, we used a GAL4/UAS enhancer trap genetic screen that selectively inactivates groups of neurons. We identified a GAL4 line (bwktqs) that has a female sterile phenotype only when it expresses the tetanus toxin light chain (TeTxLC). These flies lack oviduct contraction, lay almost no eggs, sperm accumulate in the oviducts, and fewer than normal are seen in the storage organs. In insects, two neuroactive substances are important for oviduct contraction: octopamine (OA), a monoamine that inhibits oviduct contraction, and glutamate (Glu), a neurotransmitter that induces contraction. It is known that octopaminergic neurons of the thoracic abdominal ganglion (TAG) modulate oviduct contraction, however, the glutamatergic neurons that innervate the oviduct have not been identified yet and the interaction between these two neuroactive substances is not well understood. Immunostaining experiments revealed that the bwktqs line trapped an octopaminergic neural network that innervates the genital tract. We show that wt like oviduct contraction in TeTxLC-inactivated flies can only be rescued by simultaneous application of Glu and OA suggesting that the abdominal bwktqs neurons are both octopaminergic and glutamatergic, the use of an agonist and an antagonist for Glu receptors as well as their direct visualization confirmed its participation in this phenomenon. Our work provides the first evidence that adult abdominal type II visceral innervations co-express Glu and OA and allows us to re-evaluate the previous model of neuronal network controlling insect oviduct contraction.

Drosophila melanogaster Scramblases modulate synaptic transmission.

Scramblases are a family of single-pass plasma membrane proteins, identified by their purported ability to scramble phospholipids across the two layers of plasma membrane isolated from platelets and red blood cells. However, their true in vivo role has yet to be elucidated. We report the generation and isolation of null mutants of two Scramblases identified in Drosophila melanogaster. We demonstrate that flies lacking either or both of these Scramblases are not compromised in vivo in processes requiring scrambling of phospholipids. Instead, we show that D. melanogaster lacking both Scramblases have more vesicles and display enhanced recruitment from a reserve pool of vesicles and increased neurotransmitter secretion at the larval neuromuscular synapses. These defects are corrected by the introduction of a genomic copy of the Scramb 1 gene. The lack of phenotypes related to failure of scrambling and the neurophysiological analysis lead us to propose that Scramblases play a modulatory role in the process of neurotransmission.

Cholesterol-depleting compounds modulate K+-currents in Drosophila Kenyon cells.

Sterol-enriched lipid rafts have been involved in Drosophila membrane signalling such as Hedgehog targeting and glutamate receptor ligand-affinity regulation. Here, we show that the voltage-dependent K(+) currents expressed by the intrinsic neurons of the Mushroom bodies are upward-modulated by compounds that remove sterols from the plasma membrane. Modulation seems to rely on a fast-exchanging sterol-pool, which more strongly affects the slowly inactivating current. Our results provide the first evidence that sterols influence the operation of voltage-gated ion channels in Drosophila neurons and strengthen the importance of lipid rafts in this biological model.

Shal and shaker differential contribution to the K+ currents in the Drosophila mushroom body neurons.

Shaker, a voltage-dependent K+ channel, is enriched in the mushroom bodies (MBs), the locus of olfactory learning in Drosophila. Mutations in the shaker locus are known to alter excitability, neurotransmitter release, synaptic plasticity, and olfactory learning. However, a direct link of Shaker channels to MB intrinsic neuron (MBN) physiology has not been documented. We found that transcripts for shab, shaw, shaker, and shal, among which only Shaker and Shal have been reported to code for A-type currents, are present in the MBs. The electrophysiological data showed that the absence of functional Shaker channels modifies the distribution of half-inactivation voltages (V(i1/2)) in the MBNs, indicating a segregation of Shaker channels to only a subset (approximately 28%) of their somata. In harmony with this notion, we found that approximately one-fifth of MBNs lacking functional Shaker channels displayed dramatically slowed-down outward current inactivation times and reduced peak-current amplitudes. Furthermore, whereas all MBNs were sensitive to 4-aminopyridine, a nonspecific A-type current blocker, a subset of neurons (approximately 24%) displayed little sensitivity to a Shal-specific toxin. This subset of neurons displaying toxin-insensitive outward currents had more depolarized V(i1/2) values attributable to Shaker channels. Our findings provide the first direct evidence that altered Shaker channel function disrupts MBN physiology in Drosophila. To our surprise, the experimental data also indicate that Shaker channels segregate to a minor fraction of MB neuronal somata (20-30%), and that Shal channels contribute the somatic A-type current in the majority of MBNs.

Synaptic vesicle pools and plasticity of synaptic transmission at the Drosophila synapse.

Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus, this synapse offers an excellent model for studies of the molecular mechanism of synaptic transmission and synaptic plasticity. Two synaptic vesicle (SV) pools have been identified and characterized using a fluorescent styryl dye, FM1-43, to stain SVs. They are termed the exo/endo cycling pool (ECP), which corresponds to the readily releasable pool (RRP) defined electrophysiologically, and the reserve pool (RP). These two pools were identified first in a temperature-sensitive paralytic mutant, shibire, and subsequently confirmed in wild-type larvae. The ECP participates in synaptic transmission during low frequency firing of presynaptic nerves and locates in the periphery of presynaptic boutons in the vicinity of release sites, while SVs in the RP spread toward the center of boutons and are recruited only during tetanic stimulation. These two pools are separately replenished by endocytosis. Cyclic AMP facilitates recruitment of SVs from the RP to the ECP. Activation of presynaptic metabotropic glutamate receptors recruits SVs from the RP and enhances SV release by elevation of the cAMP level. Memory mutants that have defects in the cAMP/PKA cascade, dunce and rutabaga, exhibit reduced levels of recruitment of synaptic SVs from the RP to the ECP and have limited short-term synaptic plasticity. SV mobilization between the two pools could be a key step for changes in synaptic efficacy. Since a variety of mutants that have distinct defects in synaptic transmission are available for detailed studies of synaptic function, this direction of approach in Drosophila seems promising.

Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila.

Although synapses are assembled in a highly regulated fashion, synapses once formed are not static structures but continue to expand and retract throughout the life of an organism. One second messenger that has been demonstrated to play a critical role in synaptic growth and function is cAMP. Here, we have tested the idea that signaling through the heterotrimeric G protein, Gs, plays a coincident role with increases in intracellular Ca(+2) in the regulation of adenylyl cyclases (ACs) during synaptic growth and in the function of synapses. In larvae containing a hypomorphic mutation in the dgs gene encoding the Drosophila Gs alpha protein, there is a significant decrease in the number of synaptic boutons and extent of synaptic arborization, as well as defects in the facilitation of synaptic transmission. Microscopic analysis confirmed that Gs alpha is localized at synapses both pre- and postsynaptically. Restricted expression of wild-type Gs alpha either pre- or postsynaptically rescued the mutational defects in bouton formation and defects in the facilitation of synaptic transmission, indicating that pathways activated by Gs alpha are likely to be involved in the reciprocal interactions between pre- and postsynaptic cells required for the development of mature synapses. In addition, this Gs alpha mutation interacted with fasII, dnc, and hyperexcitability mutants in a manner that revealed a coincident role for Gs alpha in the regulation of cAMP and FASII levels required during growth of these synapses. Our results demonstrate that Gs alpha-dependent signaling plays a role in the dynamic cellular reorganization that underlies synaptic growth.