Michael J. O’Donnell

Insect Excretory Mechanisms

Publisher Summary This chapter discusses three separate aspects of excretion, as first proposed by Maddrell: removal of molecules that are undesirable or perhaps even poisonous at all except very low concentrations; excretion of molecules that are not toxic but merely useless, and that would become obstructive if allowed to accumulate; and excretion of molecules that are useful or essential but are present to excess. This includes excretion of water and physiological ions in some circumstances. The chapter attempts to link recent advances in the understanding of the physiology, biochemistry, genetics, molecular biology, and chemical ecology of insect excretory mechanisms to the fundamental principles of design established several years ago. For example, the primary ATP-dependent transporter in insect epithelia was referred to in the literature until the early 1990s as the electrogenic alkali cation pump. The theme of this chapter is that studies of insect excretory mechanisms frequently provide illustrations of the Krogh principle; for a large number of problems, there will be some animal of choice or a few such animals on which it can be most conveniently studied. Adaptations to extreme environments, coupled with the large number of unusual food sources exploited by insects and their extraordinary diversity, provides a fertile ground for research into excretory mechanisms.

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.

Identification, spatial expression analysis and functional characterization of a pyrokinin-1 receptor in the Chagas' disease vector, Rhodnius prolixus.

The capability or capa gene, encodes a pyrokinin-related peptide (known as pyrokinin-1, PK1) that contains the consensus carboxy-terminal sequence of WFGPRL-NH(2). Although the CAPA precursor polypeptide in Rhodnius prolixus yields the anti-diuretic hormone, RhoprCAPA-α2, no function has yet been elucidated for the pyrokinin-1 peptide, RhoprCAPA-αPK1. In order to elucidate the possible physiological roles of the PK1-related peptides in R. prolixus, we have isolated and functionally characterized the PK1 receptor, RhoprPK1-R. Additionally, we have determined a set of three optimal reference genes to utilize for normalization of data obtained when carrying out spatial expression analyses via quantitative reverse transcriptase PCR (RT-qPCR) in various tissues of fifth instar R. prolixus. The RhoprPK1-R expression profile differs strikingly from the receptor for the anti-diuretic hormone RhoprCAPA-α2, which is localized mainly to gut epithelial tissues. Instead, RhoprPK1-R expression in fifth instar stage insects was identified in tissues that are not involved in osmotic and ionic balance, including the prothoracic glands, male reproductive tissues and a pooled sample composed of fat body, dorsal vessel, abdominal nerves and female reproductive tissues. Thus, this research establishes novel possibilities for the physiological roles of the pyrokinin-related peptides in this medically relevant disease vector.

Secretion of water and ions by malpighian tubules of larval mosquitoes: effects of diuretic factors, second messengers, and salinity.

The effects of changes in the salinity of the rearing medium on Malpighian tubule fluid secretion and ion transport were examined in larvae of the freshwater mosquito Aedes aegypti and the saltwater species Ochlerotatus taeniorhynchus. For unstimulated tubules of both species, the K+ concentration of secreted fluid was significantly lower when larvae were reared in 30% or 100% seawater (O. taeniorhynchus only), relative to tubules from freshwater‐reared larvae. The Na+ concentration of secreted fluid from unstimulated tubules of O. taeniorhynchus reared in 30% or 100% seawater was higher relative to tubules from freshwater‐reared larvae. The results suggest that changes in salinity of the larval rearing medium lead to sustained changes in ion transport mechanisms in unstimulated tubules. Furthermore, alterations of K+ transport may be utilized to either conserve Na+ under freshwater (Na+‐deprived) conditions or eliminate more Na+ in saline (Na+‐rich) conditions. The secretagogues cyclic AMP [cAMP], cyclic GMP [cGMP], leucokinin‐VIII, and thapsigargin stimulated fluid secretion by tubules of both species. Cyclic AMP increased K+ concentration and decreased Na+ concentration in the fluid secreted by tubules isolated from O. taeniorhynchus larvae reared in 100% seawater. Interactions between rearing salinity and cGMP actions were similar to those for cAMP. Leucokinin‐VIII and thapsigargin had no effect on secreted fluid Na+ or K+ concentrations. Results indicate that changes in rearing medium salinity affect the nature and extent of stimulation of fluid and ion secretion by secretagogues.

The Heterodimeric Glycoprotein Hormone, GPA2/GPB5, Regulates Ion Transport across the Hindgut of the Adult Mosquito, Aedes aegypti

A family of evolutionarily old hormones is the glycoprotein cysteine knot-forming heterodimers consisting of alpha- (GPA) and beta-subunits (GPB), which assemble by noncovalent bonds. In mammals, a common glycoprotein hormone alpha-subunit (GPA1) pairs with unique beta-subunits that establish receptor specificity, forming thyroid stimulating hormone (GPA1/TSHβ) and the gonadotropins luteinizing hormone (GPA1/LHβ), follicle stimulating hormone (GPA1/FSHβ), choriogonadotropin (GPA1/CGβ). A novel glycoprotein heterodimer was identified in vertebrates by genome analysis, called thyrostimulin, composed of two novel subunits, GPA2 and GPB5, and homologs occur in arthropods, nematodes and cnidarians, implying that this neurohormone system existed prior to the emergence of bilateral metazoans. In order to discern possible physiological roles of this hormonal signaling system in mosquitoes, we have isolated the glycoprotein hormone genes producing the alpha- and beta-subunits (AedaeGPA2 and AedaeGPB5) and assessed their temporal expression profiles in the yellow and dengue-fever vector, Aedes aegypti. We have also isolated a putative receptor for this novel mosquito hormone, AedaeLGR1, which contains features conserved with other glycoprotein leucine-rich repeating containing G protein-coupled receptors. AedaeLGR1 is expressed in tissues of the alimentary canal such as the midgut, Malpighian tubules and hindgut, suggesting that this novel mosquito glycoprotein hormone may regulate ionic and osmotic balance. Focusing on the hindgut in adult stage A. aegypti, where AedaeLGR1 was highly enriched, we utilized the Scanning Ion-selective Electrode Technique (SIET) to determine if AedaeGPA2/GPB5 modulated cation transport across this epithelial tissue. Our results suggest that AedaeGPA2/GPB5 does indeed participate in ionic and osmotic balance, since it appears to inhibit natriuresis and promote kaliuresis. Taken together, our findings imply this hormone may play an important role in ionic balance when levels of Na+ are limited and levels of K+ are in excess – such as during the digestion and assimilation of erythrocytes following vertebrate blood-feeding by females.

Transport of H+, Na+ and K+ across the posterior midgut of blood-fed mosquitoes (Aedes aegypti)

Following ingestion of a blood meal, the adult female mosquito undergoes a massive diuresis during which Na(+), Cl(-) and water are secreted at high rates by the Malpighian tubules. In the hours following completion of diuresis, digestion of the K(+)-rich blood cells provides a source of energy as well as amino acids for proteins in the developing eggs. Although the transport of inorganic ions by the Malpighian tubules of blood-fed mosquitoes has been extensively characterized, relatively little is known of the epithelial transport mechanisms responsible for movement of Na(+), H(+), and K(+) across the posterior midgut. In this paper we have used the Scanning Ion-selective Electrode Technique (SIET) to measure the basal (unstimulated) rates of transport of K(+), Na(+) and H(+) across the isolated posterior midgut at intervals after the blood meal. We have also measured luminal concentrations of Na(+) and K(+) and the transepithelial electrical potential at the same time points and have calculated the electrochemical potentials for Na(+), K(+) and H(+) across the midgut. SIET measurements reveal absorption (lumen to bath) of Na(+) and H(+) and secretion of K(+) for the first 2h after blood-feeding. By 24h after the meal, absorption of Na(+) and H(+) remains active while there is an electrochemical gradient favouring absorption of K(+). Inhibition by ouabain and Ba(2+) suggest a role for the Na(+)/K(+)-ATPase and K(+) channels in absorption of Na(+) and K(+), respectively. Inhibition of H(+) absorption by acetazolamide implicates carbonic anhydrase in transepithelial H(+) transport.

Accumulation and excretion of morphine by Calliphora stygia, an Australian blow fly species of forensic importance.

This study examined the ability of the forensically important blow fly, Calliphora stygia to actively excrete morphine, thereby maintaining a low morphine level within its body when fed on a diet containing morphine at low (7pmolg(-1)) and high (17.5pmolg(-1)) concentrations. Morphine was accumulated within the bodies of maggots (≈70% within the tissues) at concentrations which were lower than that of the meat (3-24%). The morphine content of the initial developing stages (second and third instar maggots) maintained on the high morphine diet was higher than those on the low morphine diet. Morphine was cleared from the body with negatively exponential kinetics (High morphine group: Morphine (pmolg(-1) wet weight)=8425e(-0.014t). Low morphine group: Morphine (pmolg(-1) wet weight)=2180e(-0.010t)). Clearance constants for morphine by animals in both groups were similar and thus both groups had a similar ability to excrete morphine. The Malpighian tubules of maggots were able to actively secrete morphine using a transport mechanism that transports small type II organic cations, such as morphine and quinine. The rate of morphine secretion by the Malpighian tubules could explain the clearance of the drug by the maggots. As the morphine was transported across the Malpighian tubules cells, a significant proportion was metabolised into a compound that is yet to be fully characterised.

Natriuresis and diuretic hormone synergism in R. prolixus upper Malpighian tubules is inhibited by the anti-diuretic hormone, RhoprCAPA-α2

Insects contain an array of hormones that coordinate the actions of the excretory system to achieve osmotic and ionic balance. In the hematophagous insect, Rhodnius prolixus, two diuretic hormones have been identified, serotonin (5HT) and a corticotropin releasing factor-related peptide (RhoprDH), and both lead to an increase in fluid secretion by Malpighian tubules (MTs). However, only 5HT activates reabsorption by the lower MTs to recover K(+) and Cl(-). An anti-diuretic hormone (RhoprCAPA-α2) is believed to coordinate the cessation of the rapid diuresis following blood meal engorgement. However, the role of RhoprCAPA-α2 on fluid secretion by MTs stimulated by RhoprDH was previously unknown. Here we demonstrate that, unlike the inhibitory effect on 5HT-stimulated secretion by MTs, RhoprCAPA-α2 does not inhibit secretion stimulated by RhoprDH although it does abolish the synergism that occurs between the two diuretic hormones. In addition, we show that the natriuresis elicited by either diuretic hormone is reduced by RhoprCAPA-α2. Using electrophysiological tools, we investigate the possible mechanism by which this complex regulatory pathway is achieved. Analysis of the pH of secreted fluid as well as the triphasic response in transepithelial potential in MTs treated with diuretic hormones, suggests that RhoprCAPA-α2 does not inhibit the V-type H(+) ATPase. Taken together, these results indicate that RhoprCAPA-α2 functions to reduce the rapid diuresis following blood feeding, and in addition, it inhibits the natriuresis associated with diuretic hormone stimulated MTs. This may reflect an important regulatory mechanism related to the slow diuresis that occurs as the K(+)-rich blood cells are digested.

Potassium fluxes across the blood brain barrier of the cockroach, Periplaneta americana

Potassium fluxes across the blood-brain barrier of the cockroach Periplaneta americana were measured using the scanning ion-selective microelectrode technique. In salines containing 15 mM or 25 mMK(+), an efflux of K(+) from the ganglia of isolated nerve cords was counterbalanced by an influx across the connectives. Metabolic inhibition with CN(-) resulted in an increase in K(+) efflux across both the ganglia and the connectives. Depletion of K(+) by chilling the nerve cords in K(+)-free saline was associated with subsequent K(+) influx across the connectives in K(+)-replete saline at room temperature. There were dramatic increases in K(+) efflux across both ganglia and connectives when the nerve cords were exposed to the pore-forming antibiotic amphotericin B. K(+) fluxes across the ventral nerve cord were also altered when paracellular leakage was augmented by transient exposure to 3M urea. K(+) efflux was reduced by the K(+) channel blockers Ba(2+) and tetraethylammonium or by exposure to Ca(2+)-free saline and K(+) efflux from the ganglia was increased by addition of ouabain to the bathing saline. The results provide direct support for a model proposing that K(+) is cycled through a current loop between the ganglia and the connectives and that both the Na(+)/K(+)-ATPase and K(+) channels are implicated in extracellular K(+) homeostasis within the central nervous system.

Characterization of transepithelial transport of salicylate by the Malpighian tubules of Drosophila melanogaster and the effects of changes in fluid secretion rate

Abstract A radioisotope tracer technique is used to study mechanisms and regulation of transepithelial transport of the plant allelochemical salicylate by the Malpighian tubules of Drosophila melanogaster. Transepithelial transport of salicylate is nearly abolished in Na+‐free saline, and inhibited by ouabain, low K+ or K+‐free bathing saline. In addition, the carboxylates probenecid, unlabelled salicylate, fluorescein, and p‐aminohippuric acid (PAH) significantly inhibit transepithelial transport of salicylate. The sulphonates taurocholate and phenol red also inhibit transepithelial transport of salicylate, whereas amaranth has no effect. Stimulation of fluid secretion by cAMP, cGMP or leucokinin I increases transepithelial transport of salicylate, particularly when the concentration of salicylate in the bathing saline is high. The correlation between the fluid secretion rate and transepithelial transport of salicylate shows that 64% of the changes in salicylate transport can be explained on the basis of changes in fluid secretion rate. The results show that naturally‐occurring plant secondary metabolite salicylate is transported into the lumen of the Mapighian tubules of D. melanogaster by a mechanism similar to that previously described for the prototypical organic anions PAH and fluorescein. In addition, the transepithelial transport of salicylate increases in response to increases in fluid secretion rate.