Ion Transport Peptides and Crustacean Hyperglycemic Hormones

Insect ITPs belong to a large family of peptides that are prominent in crustacean physiological functions: crustacean hyperglycemic hormones (CHHs), molt inhibiting hormones (MIHs), vitellogenesis inhibiting hormones (VIHs), and mandibular organ inhibiting hormones (MOIHs) (reviews: de Kleijn et al., 1998; Van Herp, 1998; Webster, 1998; see also Chapter 3.16). As their names indicate, these large peptides regulate various functions including water homeostasis, energy mobilization, vitellogenesis, and biosynthesis of ecdysteroids and methyl far-nesoate (Keller, 1992; Audsley et al., 1992a, 1992b; Meredith et al., 1996; Wainwright et al., 1996; Liu et al., 1997; Keller et al., 1999).

Since some actions of CHH are associated with crustacean ecdysis, it is given emphasis here. CHH was originally identified in sinus gland extracts of Carcinus maenas as an amidated 72 amino acid peptide (Kegel et al., 1989). Subsequently, numerous related peptides and corresponding precursors and genes were identified in several crustaceans (Weidemann et al., 1989; Tensen et al., 1991; de Kleijn et al., 1994, 1995; Ohira et al., 1997; Gu and Chan, 1998; Gu et al., 2000).

Peptides of the CHH family control multiple functions. For example, CHH stimulates glucose and lipid release into the hemolymph, secretion of digestive enzymes from the hepatopancreas, and ion transport in the gill. This peptide also inhibits production of ecdysteroids and methyl farnesoate from endocrine glands (Keller, 1992; Van Herp, 1998; Spanings-Pierrot et al., 2000). Immunohis-tochemistry revealed that, in several crustaceans, CHH is produced by the eyestalk neuroendocrine system composed of neurosecretory cells in the X-organ and neurohemal sinus gland (Dircksen et al., 1988).

Recent studies showed that CHH immunoreactiv-ity also is produced by a novel endocrine system comprising thousands of endocrine cells in the fore- and hindgut of Carcinus (Chung et al., 1999; Webster et al., 2000). Biochemical and molecular evidence confirmed that this gut immunoreactive peptide and its precursor sequence were identical to CHH produced by the eyestalk neurosecretory system (Chung et al., 1999). Massive release of CHH from gut endocrine cells was detected during ecdysis of Carcinus, causing water and ion uptake necessary for swelling of the animal and rupture of the epidermal lines, followed by ecdysis of the old cuticle and the subsequent increase in size during postecdysis (Chung et al., 1999). CHH-related pep-tides likely control water and ion balance in other arthropods as well, as described in the locust (Phillips et al., 1996, 1998).

Ion transport peptide was originally identified in the storage lobe of locust CC and shown to stimulate salt and water reabsorption, and to inhibit acid

Figure 10 EH-immunoreactivity (EH-IR) in diverse insect species. Hemimetabolous insects usually show a variable number of EH-IR cells in the brain. (a, b) Two brain specimens of the cockroach Nauphoeta show EH-IR in a cluster of six (a) and ten (b) ventromedial (VM) cells. (c) VM cells of the cricket Acheta project axons throughout the entire nerve cord and make prominent arborizations in each ventral ganglion (AG4, arrows). Two pairs of additional dorsolateral neurons are stained in these ventral ganglia (AG4, arrowheads). (d) Six EH-IR neurons were detected in the brain of the bug Pyrrhocoris, but other specimens show 4-9 immunopositive cells. (e) Two pairs of VM cells are stained in the brain of alderfly Sialis and (f) antlion Myrmeleon. Note that a second pair of VM neurons in Myrmeleon is located more posteriorly. (g) Only one pair of EH producing neurons is stained in the dorsomedial brain region of the fruitfly Drosophila. These cells arborize in the brain and project two axons along entire ventral nerve cord (arrows). Scale bar = 100 mm in (a-e); 50 mm in (g).

secretion by the hindgut of Schistocerca gregaria (Audsley et al., 1992a, 1992b). Western blots with antisera to Schistocerca ITP suggest that this peptide is quickly transported from the brain neurosecretory cells into the neurohemal CC-CA. On Western blots, a strongly stained band corresponding to ITP was detected in extracts of two CC-CA, while only a weakly labeled band was visible in extracts of 50 brains (Macins et al., 1999). Using Schistocerca ileal bioassays and Western blots, evidence for the presence of ITP was detected in the CC or brain extracts of orthopteroid insects, including several locusts, cricket A. domestica, cockroach Periplaneta americana, and stick insect Carausius morosus. By the same criteria, ITP was not detected in holometabo-lous insects, such as the moth Spodoptera litura and the fly Neobellieria bullata (Macins et al., 1999). Nevertheless, precursors encoding peptides related to ITP/CHH have been identified in both hemi-and holometabolous insects such as Schistocerca, Locusta, and Bombyx (Meredith et al., 1996; Macins et al., 1999; Endo et al., 2000), and gene sequences encoding putative ITPs became generally accessible after publication of the Drosophila and Anopheles genomes. These precursors and genes encode an amidated 72 amino acid ITP and 1-2 longer free C-terminal ITP-like peptides (Figure 8).

ITP immunoreactivity and mRNA encoding the peptide occur in the CNS of moths. In situ hybridization with anti-sense riboprobes detected ITP mRNA in 5-6 pairs of large neurons in the Bombyx brain (Endo et al., 2000). Immunohistochemical staining with CHH and ITP antisera (Figure 9c; Spalovska-Valachova and Zitnan, unpublished data) showed that these neurons are ipsilateral neurosecre-tory cells (type Ia2). Smaller paired neurons were also stained in each ventral ganglion of Schistocerca and Manduca (Dircksen and Heyn, 1998; Spalovska-Valachova and Zitnan, unpublished data).

At the present time, definitive roles for ITP/CHH-like peptides in insect ecdysis are not well understood. But based on analogy with gut CHH in crustaceans and the demonstrated role of ITP in gut ion transport in locusts, it is speculated that blood-borne ITP may control functions associated with water reabsorption or air inflation, which are necessary preparatory steps for shedding of the old cuticle.

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