Ian Orchard

Insect diuretic and antidiuretic hormones

Abstract The excretory system of insects comprises the Malpighian tubules and hindgut (ileum and rectum), the functions of which are controlled by diuretic and antidiuretic hormones. Strategies that have been used for the isolation, purification and characterisation of factors (putative hormones) that influence Malpighian tubule secretion and hindgut reabsorption in vitro are reviewed, along with information about their receptors, second messengers, effects on ion transport and structure/activity relationships. This is followed by a detailed description of the immunocytochemical localisation of identified diuretic and antidiuretic factors in neurosecretory cells and in neurohaemal organs from where they might be released into the haemolymph. Consideration is then given to the evidence for these factors functioning as circulating neurohormones to regulate Malpighian tubule secretion and hindgut reabsorption in vivo , and to how their activities are integrated for the maintenance of haemolymph volume and composition. Finally, mention is made of the potential use of diuretic and antidiuretic hormone agonists and/or antagonists as novel insecticides that act by disrupting the endocrine control of the excretory system.

Changes in haemolymph serotonin levels associated with feeding in the blood-sucking bug, Rhodnius prolixus

Abstract The concentration of serotonin in the haemolymph of fifth-instar larvae of Rhodnius prolixus increase quickly after the onset of feeding. In insects fed on either blood or artificial diet the haemolymph serotonin concentration increases from approx. 7 nM in unfed insects to over 100 nM by 5 min after the onset of feeding whereafter it decreases again to low levels at 20 min. If insects are interrupted after 1 or 2 min of feeding the haemolymph serotonin concentration declines rather than following the predictable pattern of increase achieved when feeding is allowed to continue. One source of serotonin appears to be neurohaemal areas lying upon the abdominal nerves, since the intensity of serotonin-like immunoreactivity is greatly reduced in these areas following 15 min of feeding. We conclude that serotonin is a neurohormone in Rhodnius and that feeding is the natural stimulus responsible for its release and subsequent elevated concentration in the haemolymph.

Effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata

Neuropeptides of the cockroach allatostatin (AST) family are known for their ability to inhibit the production of juvenile hormone by the corpora allata of cockroaches. Since their discovery, they have also been shown to modulate myotropic activity in a range of insect species as well as to act as neurotransmitters in Crustaceans and possibly in insects. The midgut of cockroaches contains numerous endocrine cells, some of which produce AST whereas others produce the FMRFamide-related peptide, leucomyosuppressin (LMS). We have determined if ASTs and LMS are also able to influence carbohydrate-metabolizing enzyme activity in the midgut of the cockroach, Diploptera punctata. Dippu-AST 7 stimulates activity of both invertase and alpha-amylase in a dose-dependent fashion in the lumen contents of ligatured midguts in vitro, but not in midgut tissue, whereas the AST analog AST(b)phi2, a cyclopropyl-ala, hydrocinnamic acid analog of Dippu-AST 6, has no effect. Leucomyosuppressin also stimulates enzyme activity in lumen contents only, although the EC50 is considerably greater than for Dippu-AST. Dippu-AST is also able to inhibit proctolin-induced contractions of midgut muscle, and this action had already been described for LMS [18]. Thus, in this organ, AST and LMS have at least two distinct physiological effects.

FMRFamide-related peptides: a multifunctional family of structurally related neuropeptides in insects

Publisher Summary This chapter reviews the information available on the extensive FMRFamide (Phe-Met-Arg-Pheamide)-related peptides (FaRPs) family in insects. The chapter examines the principles behind peptide discovery and the way information can be used as a tool for further investigation. In addition, it puts into perspective the FaRP family from gene to behavior, examining the molecular biology, distribution, receptor activation through to biological activity, and integrative actions on behavior. FMRFamide is regarded as the primary member of an extensive family of peptides with diverse biological activities. The FaRP family may itself be divided into subfamilies, including the extended FMRFamides, FLRFamides, HMRFamides and related peptides. Several molecular biological techniques have been applied to isolate either new members of the FaRP gene family or the homologous gene from a different species using available FaRP-encoding genomic DNA or cDNA as a probe. The most common method is screening of libraries of DNA sequences under conditions of reduced stringency. However, more research is needed on this family of neuropeptides before a clear picture emerges as to their true physiological relevance.

Identified octopaminergic neurons modulate contractions of locust visceral muscle via adenosine 3′,5′-monophosphate (cyclic AMP)

D,L-Octopamine elevates the cyclic AMP content of the lateral oviduct of the locust, Locusta migratoria, in a dose-dependent manner with a threshold of about 10(-8) M. The effect of octopamine is potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The response is specific for octopamine and synephrine with an order of potency being octopamine = synephrine greater than metanephrine greater than tyramine greater than norepinephrine = dopamine = 5-hydroxytryptamine and the effect of octopamine is inhibited by the alpha-adrenergic receptor antagonist phentolamine. The diterpene adenylate cyclase activator forskolin also elevates cyclic AMP levels and IBMX potentiates the action of forskolin. Stimulation of the two identified octopaminergic neurons which project to the lateral oviducts results in an elevation in cyclic AMP and again this effect is blocked by phentolamine. Elevation of cyclic AMP levels in the lateral oviducts by means of IBMX, forskolin or dibutyryl cyclic AMP mimics the physiological effects of octopamine on this preparation. The results indicate that the octopaminergic control of this insects visceral muscle is mediated via cyclic AMP.

Isolation, sequence, and bioactivity of PDVDHVFLRFamide and ADVGHVFLRFamide peptides from the locust central nervous system

The brain and the retrocerebral complex of the locust, Locusta migratoria, were examined for the presence of FMRFamide-related peptides (FaRPs) using an RIA specific for -RFamide. RP-HPLC of these extracts using both C18 and phenyl columns revealed the presence of several FaRPs eluting at different percentages of acetonitrile. The sequences of two peptides were determined. One sequence is identical to the previously described SchistoFLRFamide (PDVDHVFLRFamide), whereas a second peptide is novel and differs from SchistoFLRFamide in positions 1 and 4 (ADVGHVFLRFamide). The bioactivity of these native and synthetic FaRPs on locust oviduct contractions has been examined. Both peptides showed inhibitory activity on the locust oviduct. Truncated versions of PDVDHVFLRFamide revealed that the essential features for inhibition lay in the sequence HVFLRFamide.

Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

SummaryThe presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the α-adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.

The biological activity of diuretic factors in Rhodnius prolixus

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones. In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards. Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.

Isolation of two FMRFamide-related peptides from crayfish pericardial organs

Pericardial organs of the crayfish, Procambarus clarkii, were removed, and material from them was extracted and fractionated using two sequential reverse-phase HPLC columns. Fractions were analysed using a radioimmunoassay (RIA) specific for the C-terminal peptide sequence -Arg-Phe-NH2. The peak immunoreactive fraction from the second column appeared to contain two peptides with the sequences DRNFLRFamide and NRNFLRFamide, based on microsequencing analysis and on the absolute requirement for the amide in the RIA. These two peptides, respectively, have been given the names NF1 and DF2 due to similarity with previously identified lobster peptides F1 and F2. NF1 and DF2 are cardioexcitatory and augment synaptic transmission at a neuromuscular synapse, and evidence is presented for the calcium-dependent release of these peptides from the pericardial organs.

Release of identified adipokinetic hormones during flight and following neural stimulation in Locusta migratoria

Fractionation of methanol extracts of perfusate and haemolymph on thin-layer chromatography was used to separate hormones associated with haemolymph lipid regulation in Locusta. Electrical stimulation of the nervi corporis cardiaci II (NCC II) of isolated corpora cardiaca resulted in the release of three hormones into the perfusate; hypolipaemic hormone and two adipokinetic hormones. The two adipokinetic hormones co-migrated with synthetic adipokinetic hormone (adipokinetic hormone I) and with the RF value similar to Carlsens peptide (adipokinetic hormone II). These two adipokinetic hormones were also present in small amounts in the haemolymph of unflown Locusta, and shown to be released during a 30-min flight. The adipokinetic hormone II fraction from the NCC II-stimulated perfusate and haemolymph also possessed hyperglycaemic activity when assayed in ligated locusts. It is concluded that NCC II controls the release of adipokinetic hormones during flight and that two adipokinetic hormones are released during flight. One of these hormones adipokinetic hormone II also acts as a hyperglycaemic hormone illustrating that a hyperglycaemic hormone is released, during flight.