Andrew Donini

Analysis of Na+, Cl-, K+, H+ and NH4+ concentration gradients adjacent to the surface of anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes.

SUMMARY Ion concentration gradients adjacent to the surface of the anal papillae of larvae of the mosquito Aedes aegypti were measured using self-referencing ion-selective microelectrodes. The gradients were used to calculate estimates of ion fluxes into and out of the papillae. There was a net influx of Na+, Cl- and K+ from the bathing medium and a net efflux of acid and NH4+. No Ca2+ gradients were detectable. Na+ and Cl- influx occurred against a concentration gradient suggesting active transport. Although Na+, Cl- and NH4+ gradients were uniform along the length of the papillae, the proximal regions of the papillae in vivo revealed significantly higher H+ and K+ gradients compared with distal regions. The calculated ion fluxes at the papillae are sufficient for complete Na+, K+ and Cl- haemolymph replacement in ∼4 h with external ion concentrations of 5 mmol l-1. Ion gradients were also detected adjacent to the surface of isolated papillae; however, Na+ and H+ gradients were higher, and Cl- gradients were lower relative to papillae in vivo. The results support previous findings that the anal papillae of mosquito larvae are important structures for ion regulation, and suggest that these structures may be used for the excretion of nitrogenous waste.

Parallel ionoregulatory adjustments underlie phenotypic plasticity and evolution of Drosophila cold tolerance

Low temperature tolerance is the main predictor of variation in the global distribution and performance of insects, yet the molecular mechanisms underlying cold tolerance variation are poorly known, and it is unclear whether the mechanisms that improve cold tolerance within the lifetime of an individual insect are similar to those that underlie evolved differences among species. The accumulation of cold-induced injuries by hemimetabolous insects is associated with loss of Na+ and K+ homeostasis. Here we show that this model holds true for Drosophila; cold exposure increases haemolymph [K+] in D. melanogaster, and cold-acclimated flies maintain low haemolymph [Na+] and [K+], both at rest and during a cold exposure. This pattern holds across 24 species of the Drosophila phylogeny, where improvements in cold tolerance have been consistently paired with reductions in haemolymph [Na+] and [K+]. Cold-acclimated D. melanogaster have low activity of Na+/K+-ATPase, which may contribute to the maintenance of low haemolymph [Na+] and underlie improvements in cold tolerance. Modifications to ion balance are associated with both phenotypic plasticity within D. melanogaster and evolutionary differences in cold tolerance across the Drosophila phylogeny, which suggests that adaptation and acclimation of cold tolerance in insects may occur through similar mechanisms. Cold-tolerant flies maintain haemolymph osmolality despite low haemolymph [Na+] and [K+], possibly through modest accumulations of organic osmolytes. We propose that this could have served as an evolutionary route by which chill-susceptible insects developed more extreme cold tolerance strategies.

Differential actions of diuretic factors on the Malpighian tubules of Rhodnius prolixus.

SUMMARY The effects of corticotropin-releasing factor (CRF)-related (ZooneDH), calcitonin (CT)-related (RhoprDH31) and kinin-related (leucokinin I) peptides on the ion composition of fluid secreted by upper Rhodnius prolixus Malpighian tubules and on KCl reabsorption by the lower tubules were assessed. ZooneDH stimulated fluid secretion while increasing the [Na+] of secreted fluid at the expense of [K+]. Upper tubules responded to ZooneDH with a characteristic triphasic change in the transepithelial potential (TEP), reminiscent of the response to 5-hydroxytryptamine (5HT). RhoprDH31 produced a small (∼9 mV) lumen-positive shift in TEP of the upper tubule but had no effect on the rate of fluid secretion or ion composition of the secreted fluid. In contrast to 5HT, both peptides failed to activate KCl reabsorption by the lower tubule. Leucokinin I had no effect on the ion composition of fluid secreted by whole or upper Malpighian tubules. We propose that: (1) 5HT and a native CRF-related peptide similar to ZooneDH activate the same second messenger systems and ion transporters in the upper tubule cells; (2) CRF-related peptide is utilized to maintain high rates of fluid secretion during the post-feeding diuresis and is additionally used at times when KCl reabsorption is unnecessary or detrimental. The differential actions of multiple diuretic factors allows for intricate control of ionic and osmotic balance in R. prolixus.

Ammonia transport by terrestrial and aquatic insects

Ammonia, an end product from amino acid and nucleic acid metabolism, is highly toxic for most animals. This review will provide an update on nitrogen metabolism in terrestrial and aquatic insects with emphasis on ammonia generation and transport. Aspects that will be discussed include metabolic pathways of nitrogenous compounds, the origin of ammonia and other nitrogenous waste products, ammonia toxicity, putative ammonia transporters as well as ammonia transport processes known in insects. Ammonia transport mechanisms in the mosquito Aedes aegypti, the tobacco hornworm Manduca sexta and the locust Schistocerca gregaria will be discussed in detail while providing additional, novel data.

Pharmacological characterisation of apical Na+ and Cl– transport mechanisms of the anal papillae in the larval mosquito Aedes aegypti

SUMMARY The anal papillae of freshwater mosquito larvae are important sites of NaCl uptake, thereby acting to offset the dilution of the hemolymph by the dilute habitat. The ion-transport mechanisms in the anal papillae are not well understood. In this study, the scanning ion-selective electrode technique (SIET) was utilized to measure ion fluxes at the anal papillae, and pharmacological inhibitors of ion transport were utilized to identify ion-transport mechanisms. Na+ uptake by the anal papillae was inhibited by bafilomycin and phenamil but not by HMA. Cl– uptake was inhibited by methazolamide, SITS and DIDS but not by bafilomycin. H+ secretion was inhibited by bafilomycin and methazolamide. Ouabain and bumetanide had no effect on NaCl uptake or H+ secretion. Together, the results suggest that Na+ uptake at the apical membrane occurs through a Na+ channel that is driven by a V-type H+-ATPase and that Cl– uptake occurs through a Cl–/HCO3– exchanger, with carbonic anhydrase providing H+ and HCO3– to the V-type H+-ATPase and exchanger, respectively.

Aquaporin homologs and water transport in the anal papillae of the larval mosquito, Aedes aegypti

The hemolymph osmolarity of the freshwater mosquito larvae (Aedes aegypti) is greater than that of their habitat. To combat the influx of water, larvae cycle water entering through the gut and anal papillae to the Malpighian tubules for secretion. The presence of aquaporins (AQPs, water channels) may facilitate the movement of water across these tissues. Tissue distribution of mRNA transcripts of putative aquaporins from mosquito larvae, using quantitative PCR, revealed expression of transcripts in the Malpighian tubules and anal papillae. Four putative aquaporin transcripts are expressed in the Malpighian tubules and provide a basis for further work aimed at discovering the elusive water transporters functioning during diuresis. Transcripts of putative AQPs (AaAQP4 and AaAQP1b) are expressed in the anal papillae. Immunoreactivity to a human AQP1-antibody was found in the anal papillae and mercury inhibits tritiated water uptake in isolated anal papillae. Together, the results suggest that AQP(s) could be responsible for facilitating water transport at the papillae epithelium.

An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito Aedes aegypti

ABSTRACT The transcripts of three putative ammonia (NH3/NH4+) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti. Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na+/K+-ATPase. Ammonium concentration gradients were measured adjacent to anal papillae using the scanning ion-selective electrode technique (SIET) and used to calculate ammonia efflux by the anal papillae. dsRNA-mediated reductions in AeAmt1 decreased ammonia efflux at larval anal papillae and significantly increased ammonia levels in hemolymph, indicating a principal role for AeAmt1 in ammonia excretion. Pharmacological characterization of ammonia transport mechanisms in the anal papillae suggests that, in addition to AeAmt1, the ionomotive pumps V-type H+-ATPase and Na+/K+-ATPase as well as NHE3 are involved in ammonia excretion at the anal papillae. Summary: An animal homolog of plant and bacterial ammonium transporters supports ammonia excretion at the anal papillae of mosquito larvae.

Occluding junctions of invertebrate epithelia

Invertebrate diversity and architecture is immense. This is achieved by the organization and function of four tissue types found in most metazoan phyla—epithelial, connective, muscle and nervous tissue. Epithelial tissue is found in all extant animals (parazoan and metazoan alike). Epithelial cells form cellular sheets that cover internal or external surfaces and regulate the passage of material between separated compartments. The transepithelial movement of biological material between compartments can occur across the transcellular pathway (i.e. across cells) or the paracellular pathway (i.e. between cells) and the latter is regulated by occluding junctions that typically link cells in a subapical domain. In this review, information on occluding junctions of invertebrate epithelia is consolidated and discussed in the context of morphology, ultrastructure and physiology. In addition, an overview of what is currently known about invertebrate occluding junction proteins and their role in maintaining the integrity of invertebrate epithelia and regulating the barrier properties of these tissues is presented.

AedesCAPA-PVK-1 displays diuretic and dose dependent antidiuretic potential in the larval mosquito Aedes aegypti (Liverpool).

This study reveals that AedesCAPA-PVK-1 (GPTVGLFAFPRV-NH(2)) inhibits basal and serotonin stimulated fluid secretion in the Malpighian tubules of larval Aedes aegypti at femtomolar concentrations. Conversely 10(-4)moll(-1) of the peptide stimulated fluid secretion rates. The diuretic effects of 10(-4)moll(-1)AedesCAPA-PVK-1 and antidiuretic effects of 10(-15)moll(-1)AedesCAPA-PVK-1 were abolished by protein kinase A (PKA) and protein kinase G (PKG) inhibition, respectively. Similar to the peptide, 10(-3)moll(-1) cGMP stimulated fluid secretion but doses in the micromolar to nanomolar range inhibited fluid secretion of the Malpighian tubules. Stimulatory effects of cGMP were abolished by PKA inhibition and inhibitory effects of cGMP were abolished by PKG inhibition. Furthermore, the nitric oxide synthase inhibitor l-NAME attenuated the inhibitory effects of AedesCAPA-PVK-1 but did not affect inhibition by cGMP. Based on the results we propose that AedesCAPA-PVK-1 inhibits fluid secretion rates of larval Malpighian tubules via the NOS/cGMP/PKG pathway and that high doses of the peptide lead to diuresis through the cGMP mediated activation of PKA.

Tissue-specific ionomotive enzyme activity and K+ reabsorption reveal the rectum as an important ionoregulatory organ in larval Chironomus riparius exposed to varying salinity

SUMMARY A role for the rectum in the ionoregulatory homeostasis of larval Chironomus riparius was revealed by rearing animals in different saline environments and examining: (1) the spatial distribution and activity of keystone ionomotive enzymes Na+-K+-ATPase (NKA) and V-type H+-ATPase (VA) in the alimentary canal, and (2) rectal K+ transport with the scanning ion-selective electrode technique (SIET). NKA and VA activity were measured in four distinct regions of the alimentary canal as follows: the combined foregut and anterior midgut, the posterior midgut, the Malpighian tubules and the hindgut. Both enzymes exhibited 10–20 times greater activity in the hindgut relative to all other areas. When larvae were reared in either ion-poor water (IPW) or freshwater (FW), no significant difference in hindgut enzyme activity was observed. However, in larvae reared in brackish water (BW), NKA and VA activity in the hindgut significantly decreased. Immunolocalization of NKA and VA in the hindgut revealed that the bulk of protein was located in the rectum. Therefore, K+ transport across the rectum was examined using SIET. Measurement of K+ flux along the rectum revealed a net K+ reabsorption that was reduced fourfold in BW-reared larvae versus larvae reared in FW or IPW. Inhibition of NKA with ouabain, VA with bafilomycin and K+ channels with charybdotoxin diminished rectal K+ reabsorption in FW- and IPW-reared larvae, but not BW-reared larvae. Data suggest that the rectum of C. riparius plays an important role in allowing these larvae to cope with dilute as well as salinated environmental conditions.