Anion conductance across the Drosophila melanogaster Malpighian (renal) tubule was investigated by a combination of physiological and transgenic techniques. Patch-clamp recordings identified clusters of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive “maxi-chloride” channels in a small domain of the apical membrane. Fluid secretion assays demonstrated sensitivity to the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, anthracene-9-carboxylic acid, and niflumic acid. Electrophysiological analysis showed that the calcium-mediated increase in anion conductance was blocked by the same agents. Vibrating probe analysis revealed a small number of current density hot spots, coincident with “stellate” cells, that were abolished by low-chloride saline or the same chloride channel blockers. GAL-4-targeted expression of an aequorin transgene revealed that the neurohormone leucokinin elicits a rapid increase in intracellular calcium levels in stellate cells that precedes the fastest demonstrable physiological effect. Taken together, these data show that leucokinins act on stellate cells through intracellular calcium to increase transcellular chloride conductance through channels. As electrogenic cation conductance is confined to principal cells, the two pathways are spatially segregated in this tissue.

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SUMMARY We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes(Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned,nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

Transport of the prototypical organic cation tetraethylammonium (TEA) by the Malpighian tubules, ureters and gut of Drosophila melanogaster was studied using two novel electrophysiological techniques. Both techniques exploited the high selectivity of the cation exchanger potassium tetra-p-chlorophenylborate for tetraalkylammonium compounds relative to inorganic cations such as K + . In the first technique, TEA fluxes were measured using a non-invasive self-referencing TEA-

Several members of the genus Lavandula produce valuable essential oils (EOs) that are primarily constituted of the low molecular weight isoprenoids, particularly monoterpenes. We isolated over 8,000 ESTs from the glandular trichomes of L. x intermedia flowers (where bulk of the EO is synthesized) to facilitate the discovery of genes that control the biosynthesis of EO constituents. The expression profile of these ESTs in L. x intermedia and its parents L. angustifolia and L. latifolia was established using microarrays. The resulting data highlighted a differentially expressed, previously uncharacterized cDNA with strong homology to known 1,8-cineole synthase (CINS) genes. The ORF, excluding the transit peptide, of this cDNA was expressed in E. coli, purified by Ni-NTA agarose affinity chromatography and functionally characterized in vitro. The ca. 63 kDa bacterially produced recombinant protein, designated L. x intermedia CINS (LiCINS), converted geranyl diphosphate (the linear monoterpene precursor) primarily to 1,8-cineole with K ( m ) and k ( cat ) values of 5.75 μM and 8.8 × 10(-3) s(-1), respectively. The genomic DNA of CINS in the studied Lavandula species had identical exon-intron architecture and coding sequences, except for a single polymorphic nucleotide in the L. angustifolia ortholog which did not alter protein function. Additional nucleotide variations restricted to L. angustifolia introns were also observed, suggesting that LiCINS was most likely inherited from L. latifolia. The LiCINS mRNA levels paralleled the 1,8-cineole content in mature flowers of the three lavender species, and in developmental stages of L. x intermedia inflorescence indicating that the production of 1,8 cineole in Lavandula is most likely controlled through transcriptional regulation of LiCINS.

Background: Lavandula accumulate irregular monoterpenes of unknown biosynthetic origin. Results: We cloned a cis-prenyl diphosphate synthase (cis-PDPS) that produces precursor for irregular monoterpenes in lavenders. Conclusion: Unlike other plants that utilize trans-PDPSs, Lavandula employ a cis-PDPS to initiate the biosynthesis of irregular monoterpenes. Significance: This is the first report of the involvement of a cis-PDPS in irregular monoterpene biosynthesis.

Spotted wing drosophila (SWD), Drosophila suzukii (Matsumura), is an important new invasive pest of stone and berry fruits in North America and current control methods require frequent application of synthetic pesticides. This has created a need for new and environmentally friendly biopesticides for the control of SWD. This paper investigated the potential of nine essential oils from avocado (Persea americana Mill.), neem (Azadirachta indica A. Juss), kukui nut (Aleurites moluccana L.), macadamia nut (Macadamia integrifolia Maiden & Betche), spike lavender (Lavandula latifolia Vill.), Grosso lavandin leaf and flower (Lavandula x intermedia cv 'Grosso'), and Provence lavandin leaf and flower (Lavandula x intermedia cv 'Provence') as well as three major monoterpene constituents of lavender essential oils: 1,8-cineole, 3-carene and linalool for their ability to control SWD through fumigation and contact toxicity assays as well as oviposition deterrent activity. Linalool was found to be the most effective monoterpene in fumigation assays (EC50 1.85 µL/L air) and spike lavender floral essential oil was found to be the most effective whole oil (EC50 3.79 µL oil/L air). In contact toxicity assays 1,8-cineole (EC50 0.67%) was the most effective monoterpene while avocado (EC50 0.54 %) and spike lavender (EC50 0.69%) oils were the most effective whole oils. No significant oviposition deterrent activity was observed. This report indicates that L. latifolia essential oil and commercially available avocado oil are strong lead candidates for management of SWD. Furthermore the activity of L. latifolia essential oil is likely due to the high content of 1,8-cineole and linalool. This is the first report of significant insecticidal activity by these oils and compounds against SWD.