Identification of an allatostatin from the tobacco hornworm Manduca sexta.

Abstract

A peptide (Manduca sexta ailatostatin) that strongly inhibits juvenile hormone biosynthesis in vitro by the corpora allata from fifth-stadium larvae and adult females has been purified from extracts of heads of pharate adult M. sexta by a nine-step purification procedure. The primary structure of this 15-residue peptide has been determined: pGlu-Val-ArgPhe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-OH, where pGlu is pyroglutamate). To our knowledge, this neurohormone has no sequence similarity with any known neuropeptide from other organisms. The synthetic free acid and amide forms showed in vitro activity indistihable from that of native M. sexta aliatostatn. The EDsO of synthetic M. sexta allatostatin on early fifth stadium larval corpora allata in vitro was =2 nM. This inhibition was reversible. In a cross-species study, M. sexta allatostatin also inhibited the corpora allata of adult female Heliothis virescens but had no effect on the activity of corpora allata ofadult females of the beetle Tenebrio molitor, the grasshopper Melanoplus sanguinipes, or the cockroach Periplaneta americana.Juvenile hormone (JH), which is synthesized and released by the retrocerebral corpora allata (CA), plays a vital role in insect development, primarily in the control of metamorphosis, adult sexual maturation, and reproduction (1, 2). Environmental and physiological factors influence neurosecretory centers in the brain that affect the activity of CA through peptidergic materials either transported directly to the CA by axons or released into the blood (3). These neuropeptides may be stimulatory (allatotropins, e.g., refs. 4-8) (27). However, instead of assaying for JH production by chloroform extraction followed by TLC analysis, we used a rapid isooctane partitioning assay for JH (28). In the cross-species studies, we used the same in vitro technique (one or two pair of CA per incubation).Extraction and Preliminary Purification (Steps 14). Thirty thousand trimmed heads of M. sexta (fresh weight, "'.1460 g) were processed in three batches of 10,000. Each batch was defatted by homogenizing in 2 liters of ice-cold acetone and filtered. Residues were extracted with 1.4 liters of 1 M HOAc/20 mM HCO (containing 0.1 mM phenylmethylsulfonyl fluoride and 0.01 mM pepstatin A, prepared immediately before use) and centrifuged at 10,000 x g for 30 min at 40C. The pellet was extracted and centrifuged twice using a total volume of 2.8 liters of the same solution. The combined supernatants were stirred with swollen SP-Sephadex C-25 resin (300 ml) overnight. The resin was allowed to settle for 2 hr, subsequently poured into a Bio-Rad column (50 x 300 mm), and equilibrated with 1 M HOAc. Material was eluted from the column sequentially with 1-liter volumes of 0.05 M NH4OAc (pH 4.0) and 0.05 M, 0.1 M, 0.2 M, 0.4 M, and 0.8 M NH4OAc (pH 7.0). The 0.1 M and 0.2 M fractions, which had M. sexta AS (Mas-AS) activity, were applied directly to 10 g of reversed-phase Vydac C4 packing material (20-30 pAm, contained in a 75-ml polypropylene syr...

Immunostaining for allatotropin and allatostatin-A and -C in the mosquitoes Aedes aegypti and Anopheles albimanus

“…AS-C was originally identified in M. sexta (Kramer et al 1991). In this species and other moths, it inhibits JH biosynthesis (Kramer et al 1991;McNeil and Tobe 2001).…”

“…In this species and other moths, it inhibits JH biosynthesis (Kramer et al 1991;McNeil and Tobe 2001). In D. melanogaster, an AS-C homolog slows heart contraction (Price et al 2002).…”

“…Allatostatins (AS) and allatotropins (AT) are structurally diverse peptides that were first described as modulators of juvenile hormone (JH) biosynthesis in the corpora allata (CA) of a number of insect species (Kataoka et al 1989;Woodhead et al 1989;Kramer et al 1991;Lorenz and Hoffmann 1995;Gilbert et al 2000). AS and AT have subsequently been recognized as having multiple physiological effects, controlling processes such as heart rate and gut motility, nutrient absorption, migratory preparedness, and modulation of the circadian cycle (Bendena et al 1999;Nässel 2002;Petri et al 2002;Elekonich and Horodyski 2003).…”

Allatostatin A-like immunoreactivity in the nervous system and gut of the larval midge, Chironomus riparius (Meigen): Modulation of hindgut motility, rectal K+ transport and implications for exposure to salinity

“…In adult insect females, juvenile hormones can also regulate reproductive activities such as the maturation of oocytes. Synthesis of juvenile hormones is inhibited by allatostatins (ASTs), members of a large and diverse family of peptides that are structurally characterized into three distinct groups (Gäde and Goldsworthy, 2003).The C-type allatostatins with an N-terminal pGlu residue and a free C terminus have been discovered in the moth Manduca sexta, the fly Drosophila melanogaster and the mosquito Aedes aegypti (Kramer et al, 1991;Williamson et al, 2001;Li et al, 2006). The B-type allatostatins all have a common Trp-(X6)-Trp-amide C terminus and were first discovered in the cricket (Wang et al, 2004).…”

Comparison of the Allatostatin Neuropeptide Precursors in the Distantly Related Cockroaches

“…IJnivcrsiry, Kingston, Ontario, Canada K7L 3N6 in corpora allata 161. Similarly, a non-arnidated 15-amino-acid allatostatin from M. sexfa inhihits juvenile hormonc biosynthesis only in Icpidoptera corpora allata [7]. 'I'hc Mcr/idiic(i allatostalin is dislinct in sequence and has no similarity to the allatostatins purified Tumi thc cockroach.…”