Simon G. Webster

Recent advances in arthropod endocrinology

Part I. Moulting, Metamorphosis and Reproduction: 1. Structures, functions and occurence of insect allatostatic peptides R. J. Weaver, J. P. Edwards, W. G. Bendena, and S. S. Tobe 2. Neuropeptides inhibiting growth and reproduction of crustaceans S. G. Webster 3. Molecular, cytological and physiological aspects of the crustacean hyperglycemic hormone family F. Van Herp 4. Endocrine effectors in insect vitellogenesis X. Belles 5. Endocrine regulation of development and reproduction in Acarines L. O. Lomas, and H. H. Rees 6. Ecdysteroid synthesis in the crustacean Y-organ: role of cyclic nucleotides and Ca 2+ D. Sedlmeier, and A. Seinsche 7. Regulation of steroidogenesis: role of transaldolase in crab moulting glands F. Lachaise, and G. Somme Part II. Control of Intermediary Metabolism, Ion and Water Balance: 8. New perspectives on the structures, assays and actions of locust adipokinetic hormones M. J. Lee, and G. J. Goldsworthy 9. Signal transduction of adipokinetic hormone W. J. A. Van Marrewijk, and D. J. Van der Horst 10. The regulation of primary urine production in insects G. M. Coast 11. Locust ion transport peptide (ITP): function, structure, DNA and expression J. E. Phillips, J. Meredith, N. Audsley, M. Ring, A. Macins, H. Brock, D. Theilmann, and D. Littleford Part III. Myotropic and Myoininhibitory Arthropod Neuropeptides: Structures and Functions?: 12. The dipteran Leu-callatostatins: structural and functional diversity in an insect neuroendocrine peptide family H. Duve, A. Thorpe, A. H. Johnsen, J-L. Maestro, A. G. Scott, and P. D. East 13. An insect peptide family in search of functions the tachykinin-related peptides D. R. Nassel, C. T. Lundquist, J. E. Muren, and A. S. A. Winther 14. The distribution, biological activity, and pharmacology of SchistoFLRFamide and related peptides in insects I. Orchard, and A. B. Lange 15. Ontogenetic, phylogenetic and physiological aspects of the conserved crustacean cardioactive peptide (CCAP) neural networks in arthropods H. Dirksen 16. Control of the insect oviduct: the role of the neuropeptide CCAP in the tobacco hornworm, Manduca sexta A. K. Marshall, and S. E. Reynolds Part IV. Peptidases, Peptide and Pseudopeptide Mimetics: Toward New Strategies of Insect Pest Control?: 17. Insect angiotensim-converting enzyme: comparative biochemistry and evolution R. E. Isaaac, D. Coates, T. A. Williams, and L. Schoofs 18. Mimetic analogues of the myotropic diuretic insect kinin neuropeptide family R. J. Nachman, G. M. Holman, and G. M. Coast.

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

SummaryBy use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.

Dissociation of Circadian and Circatidal Timekeeping in the Marine Crustacean Eurydice pulchra

Summary Background Tidal (12.4 hr) cycles of behavior and physiology adapt intertidal organisms to temporally complex coastal environments, yet their underlying mechanism is unknown. However, the very existence of an independent “circatidal” clock has been disputed, and it has been argued that tidal rhythms arise as a submultiple of a circadian clock, operating in dual oscillators whose outputs are held in antiphase i.e., ∼12.4 hr apart. Results We demonstrate that the intertidal crustacean Eurydice pulchra (Leach) exhibits robust tidal cycles of swimming in parallel to circadian (24 hr) rhythms in behavioral, physiological and molecular phenotypes. Importantly, ∼12.4 hr cycles of swimming are sustained in constant conditions, they can be entrained by suitable stimuli, and they are temperature compensated, thereby meeting the three criteria that define a biological clock. Unexpectedly, tidal rhythms (like circadian rhythms) are sensitive to pharmacological inhibition of Casein kinase 1, suggesting the possibility of shared clock substrates. However, cloning the canonical circadian genes of E. pulchra to provide molecular markers of circadian timing and also reagents to disrupt it by RNAi revealed that environmental and molecular manipulations that confound circadian timing do not affect tidal timing. Thus, competent circadian timing is neither an inevitable nor necessary element of tidal timekeeping. Conclusions We demonstrate that tidal rhythms are driven by a dedicated circatidal pacemaker that is distinct from the circadian system of E. pulchra, thereby resolving a long-standing debate regarding the nature of the circatidal mechanism.

Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae (Giles).

SUMMARY Mosquito natriuretic peptide (MNP), an uncharacterised peptide from the yellow fever mosquito, Aedes aegypti, acts via cyclic AMP to stimulate secretion of Na+-rich urine by opening a Na+ conductance in the basolateral membrane of Malpighian tubule principal cells. Corticotropin releasing factor (CRF)-related peptides and calcitonin (CT)-like diuretic peptides use cyclic AMP as a second messenger and were therefore considered likely candidates for MNP. BLAST searches of the genome of the malaria mosquito Anopheles gambiae, gave sequences for the CRF-related peptide Anoga-DH44 and the CT-like peptide Anoga-DH31, which were synthesised and tested for effects on Malpighian tubules from An. gambiae and Ae. aegypti, together with 8-bromo-cyclic AMP. The cyclic AMP analogue stimulated secretion of Na+-rich urine by An. gambiae Malpighian tubules, reproducing the response to MNP in Ae. aegypti. It also depolarised the principal cell basolateral membrane voltage (Vb) while hyperpolarising the transepithelial voltage (Vt) to a similar extent. Anoga-DH44 and Anoga-DH31 stimulated production of cyclic AMP, but not cyclic GMP, by Malpighian tubules of An. gambiae. Both peptides had diuretic activity, but only Anoga-DH31 had natriuretic activity and stimulated fluid secretion to the same extent as 8-bromo-cyclic AMP. Likewise, Anoga-DH31 reproduced the effects of cyclic AMP on tubule electrophysiology, whereas Anoga-DH44 initially hyperpolarised Vb and depolarised Vt, which is the opposite of the effect of Anoga-DH31. Anoga-DH44 and Anoga-DH31 were also tested for effects on fluid secretion and ion transport by Ae. aegypti tubules. As in An. gambiae, the CRF-related peptide Anoga-DH44 had a non-specific effect on the transport of Na+ and K+, whereas the CT-like peptide Anoga-DH31 specifically stimulated transepithelial Na+ transport. We conclude that the CT-like peptide Anoga-DH31 is the previously uncharacterised mosquito natriuretic peptide.

Identification and developmental expression of mRNAs encoding crustacean cardioactive peptide (CCAP) in decapod crustaceans

SUMMARY Full-length cDNAs encoding crustacean cardioactive peptide (CCAP) were isolated from several decapod (brachyuran and astacuran) crustaceans: the blue crab Callinectes sapidus, green shore crab Carcinus maenas, European lobster Homarus gamarus and calico crayfish Orconectes immunis. The cDNAs encode open reading frames of 143 (brachyurans) and 139-140 (astacurans) amino acids. Apart from the predicted signal peptides (30-32 amino acids), the conceptually translated precursor codes for a single copy of CCAP and four other peptides that are extremely similar in terms of amino acid sequence within these species, but which clearly show divergence into brachyuran and astacuran groups. Expression patterns of CCAP mRNA and peptide were determined during embryonic development in Carcinus using quantitative RT-PCR and immunohistochemistry with whole-mount confocal microscopy, and showed that significant mRNA expression (at 50% embryonic development) preceded detectable levels of CCAP in the developing central nervous system (CNS; at 70% development). Subsequent CCAP gene expression dramatically increased during the late stages of embryogenesis (80-100%), coincident with developing immunopositive structures. In adult crabs, CCAP gene expression was detected exclusively in the eyestalk, brain and in particular the thoracic ganglia, in accord with the predominance of CCAP-containing cells in this tissue. Measurement of expression patterns of CCAP mRNA in Carcinus and Callinectes thoracic ganglia throughout the moult cycle revealed only modest changes, indicating that previously observed increases in CCAP peptide levels during premoult were not transcriptionally coupled. Severe hypoxic conditions resulted in rapid downregulation of CCAP transcription in the eyestalk, but not the thoracic ganglia in Callinectes, and thermal challenge did not change CCAP mRNA levels. These results offer the first tantalising glimpses of involvement of CCAP in environmental adaptation to extreme, yet biologically relevant stressors, and perhaps suggest that the CCAP-containing neurones in the eyestalk might be involved in adaptation to environmental stressors.

Amino acid sequences of both isoforms of crustacean hyperglycemic hormone (CHH) and corresponding precursor-related peptide in Cancer pagurus.

Both isoforms of the crustacean hyperglycemic hormone (CHH) and corresponding crustacean hyperglycemic hormone precursor-related peptide (CPRP) derived from HPLC-purified sinus gland extracts from the edible crab Cancer pagurus were fully characterised by microsequencing and mass spectrometry. The amino acid sequences of the CHH isoforms were almost identical except that the N-terminus of the minor isoform (CHH-I), was glutamine rather than pyroglutamate in the major isoform (CHH-II). Both CHH isoforms were of similar biological activity, as tested by in vivo hyperglycemia bioassays and in vitro repression of ecdysteroid synthesis. Comparison with other published CHH and CPRP sequences show that for crabs, these peptides form a distinct group, that the presence of CHH isoforms with free and blocked N-termini seems unique to crabs. It is argued that this phenomenon reflects a slow post-translational modification in sinus gland neurosecretory terminals. This study appears to complete the entire sinus gland inventory of functionally and structurally characterised CHH-related peptides in a crab.

Determination of the amino acid sequence of the moult-inhibiting hormone from the edible crab, Cancer pagurus

Putative moult-inhibiting hormone (MIH) from sinus glands of the edible crab Cancer pagurus was characterized by high-performance liquid chromatography, followed by fractional bioassay (inhibition of ecdysteroid synthesis by Y-organs) and immunoassay (using antisera raised against Carcinus MIH). This peptide was fully sequenced by automated Edman degradation of endoproteinase-derived fragments. C. pagurus MIH is a 78 residue peptide (M(r) 9194), with free N- and C-termini and three intrachain disulphide bridges. Comparison with previously published MIH sequences confirms a high degree of sequence identity (c. 80%), supporting the view that brachyurans (crabs), possess distinct, structurally similar MIH neuropeptides.

Expression and release patterns of neuropeptides during embryonic development and hatching of the green shore crab, Carcinus maenas

Crustacean ecdysis is controlled by at least three neuropeptides: moult-inhibiting hormone (MIH), which represses ecdysteroid synthesis; crustacean hyperglycaemic hormone (CHH), which not only influences ecdysteroid synthesis but also water uptake during moulting; and crustacean cardioactive peptide (CCAP), which is involved in stereotyped ecdysis behaviour. During embryonic development, moulting takes place in the egg, but there is little information regarding developmental expression of these neuropeptides during this period or during hatching – an event that is analogous to eclosion in insects. To address this problem, we determined expression profiles of MIH and CHH mRNA by quantitative RT-PCR, together with developmental peptide expression studies [confocal immunocytochemistry (ICC) and radioimmunoassay (RIA)]. Likely homologous events relating to neuropeptide surges of both CHH and CCAP were seen during larval hatching, when compared to the adult moult, and cell-specific copy concentration of both MIH and CHH mRNAs was identical to that of the adult during late embryonic development. We measured parallel mRNA and peptide expression of two neuropeptides (red pigment-concentrating hormone RPCH) and pigment-dispersing hormone (PDH) during development, as these have roles as neuromodulators and as classical neurohormonal roles. For MIH and CHH, gene expression was in accordance with peptide expression, but novel sites of CHH expression were found (abdominal peripheral neurones), the expression and release patterns of which may be related to larval eclosion and water uptake necessary for eggshell rupture and hatching. For RPCH and PDH, gene transcription and peptide expression were not in accordance. A significant contribution of maternally derived (non-translated) PDH mRNA to the embryo was seen, and for RPCH, high-level mRNA and peptide expression during late embryogenesis is related to a long ignored, but potentially important release site – the enigmatic post-commissural organs – which are the most prominent structures expressing RPCH during late embryogenesis.

Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression.

Development and reproduction of crustaceans is regulated by a combination of neuropeptide hormones, ecdysteroids (moulting hormones) and the isoprenoid, methyl farnesoate (MF), the unepoxidised analogue of insect juvenile hormone-III (JH-III). MF and the ecdysteroids are respectively synthesised under the negative control of the sinus gland-derived mandibular organ-inhibiting hormones (MO-IHs) and moult-inhibiting hormone (MIH) that are produced in eyestalk neural ganglia. Previous work has demonstrated the existence of two isoforms of MO-IH, called MO-IH-1 and -2, that differ by a single amino acid in the mature peptide and one in the putative signal peptide. To study the structural organisation of the crab MIH and MO-IH genes, a genomic DNA library was constructed from DNA of an individual female crab and screened with both MO-IH and MIH probes. The results from genomic Southern blot analysis and library screening indicated that the Cancer pagurus genome contains at least two copies of the MIH gene and three copies of the MO-IH genes. Upon screening, two types of overlapping genomic clone were isolated. Each member of one type of genomic clone contains a single copy of each of the convergently transcribed MO-IH-1 and MIH genes clustered within 6.5kb. The other type contains only the MO-IH-2 gene, which is not closely linked to an MIH gene. There are three exons and two introns in all MIH and MO-IH genes analysed. The exon-intron boundary of the crab MIH and MO-IH genes follows Chambons rule (GT-AG) for the splice donor and acceptor sites. The first intron occurs within the signal peptide region and the second intron occurs in the coding region of the mature peptide. Sequence analysis of upstream regions of MO-IH and MIH genes showed that they contained promoter elements with characteristics similar to other eukaryotic genes. These included sequences with high degrees of similarity to the arthropod initiator, TATA box and cAMP response element binding protein. Additionally, putative CF1/USP and Broad Complex Z2 transcription factor elements were found in the upstream regions of MIH and MO-IH genes respectively. The implications of the presence of the latter two putative transcription factor binding-elements for control of expression of MIH and MO-IH genes is discussed. Phylogenetic analysis and gene organisation show that MO-IH and MIH genes are closely related. Their relationship suggests that they represent an example of evolutionary divergence of crustacean hormones.

Is crustacean hyperglycaemic hormone precursor-related peptide a circulating neurohormone in crabs?

Abstract. Sites of synthesis and release patterns of crustacean hyperglycaemic hormone precursor-related peptide (CPRP) were investigated with those of crustacean hyperglycaemic hormone (cHH), in order to determine whether this precursor-related peptide satisfies certain criteria necessary for its definition as a secretable, circulating hormone. Using the edible crab, Cancer pagurus, sites of CPRP synthesis were determined by immunohistochemistry and release patterns of both peptides were determined in vivo and in vitro by radioimmunoassay of haemolymph and eyestalk superfusates. Both peptides were co-released from sinus glands (SGs) following potassium-evoked depolarization of isolated eyestalk preparations. However, stress-evoked in vivo release resulted in apparent non-stoichiometric circulating peptide profiles. This phenomenon is explained by notable differences in clearance rates of the peptides in haemolymph. In contrast to cHH, CPRP is very slowly degraded in vivo. Although CPRP is clearly a circulating peptide, whose release is concomitant with that of cHH, physiologically pertinent roles for this molecule remain to be discovered.