Insect Bwchem Vol 15 No 4 pp 521 528, 1985 0020-1790/85 $300+000 Printed in Great Britain Pergamon Press Ltd STABILIZATION OF MINERALIZED AND SCLEROTIZED PUPARIAL CUTICLE OF MUSCID FLIES CRAIG R ROSELAND*, MICHAEL J GRODOWITZ*, KARL J KRAMERt~§, THEODORE L HOPKINS* and ALBERTO B BROCE* Departments of Entomology* and Biochemlstryt, Kansas State Umversxty, Manhattan, KS 66506 and ++US Gram Marketing Research Laboratory Agricultural Research Serwce, U S Department of Agriculture, Manhattan, KS 66502, U S A (Recewed 3 June 1984, revised 24 August 1984) A~tract--Calclum, magnesium and phosphorus are the major mineral elements m puparlal exuvme ot the face fly, Mu~ca autumnahs house fly, M domesttca and stable fly, Stomoxys calcttrans, but they are 20-50 times more prevalent m face fly tha m the other two speoes that sclerotxze the puparlum Carbon and mtrogen are approx 5 times more abundant m house fly puparxa than m face fly puparm Face fly puparla contain two and three-fold less total amino acids than the house fly and stable fly, respectively fl-Alanme is a major amino acid m puparml cuticle of the house fly and stable fly, but it is absent m the face fly There is no slgmficant &fference m glucosamlne (chitin) content between the three species Dopamme is the major catechol detected in face fly puparlal cuticle while N-fl-alanyldopamlne (NBAD) is 10 to 15 times more prevalent than other catechols such as dopamme, N-acetyldopamlne (NADA), 3,4-dlhydroxyphenylalanlne (DOPA) and 3,4-&hydroxyphenylacetlc aod (DOPAC) m house fly and stable fly puparml cuticles The latter two species have 75 to nearly 200 times higher levels of extractable catechols than the face fly At the onset of puparmtton, dopamlne and NBAD attain nearly eqmvalent Utres m pupanal cuticles of face fly and house fly, respectively Dopamme subsequently decreases more than 40-fold m the face fly as the cuticle becomes stabdlzed, whde NBAD continues to accumulate m the house fly The house fly covalently incorporates about 150 times more catechols m the puparlum than does the face fly The force required to fracture house fly and stable fly puparla is about three-fold greater than that required to fracture face fly puparla of comparable thickness However, the face fly pupanum attains a strength comparable to those of house fly and stable fly puparm by slgmficantly mcreaslng Its thickness These results demonstrate that &pterans use both catecholammes and minerals for stabdlzatton of pupanal cuticle with the house fly and stable fly relying primarily on sclerotlzatton and the face fly on mmerahzatlon Kel' Word Index Catecholammes, minerals, sclerotlzaUon, pupanatlon, Diptera, tanning, dopamlne, DOPA, N-fl-alanyldopamlne, cuticle, N-acetyldopamme, tyroslne metabolism,/3-alanme, calcium, mag- nesmm, phosphorus, face fly, house fly, stable fly, cutlcular filler protein, mineralization, calcification, glucosamlne, chitin INTRODUCTION Insect cuticle is hardened and stabilized when tyro- sine derivatives are incorporated into the most exter- nal layers of the chitin-protein matrix Qulnonold metabohtes apparently crosshnk proteins and are believed to be the principal means of cuticle sclero- tlzatlon (reviewed by Neville, 1975, Brunet, 1980, Lipke et al, 1983) However, a dehydrating mech- anism involving catechols that impregnate cuticle may also contribute to its stabilization (Fraenkel and Rudall, 1940, Vincent and Hlllerton, 1979) For example, as sclerotlzatlon proceeds, catecholamines extractable in acid have been shown to progressively accumulate in various cuticles (Hopkms et al, 1982, 1984) Typically Diptera metabolize tyroslne to N- acetyldopamlne for the purpose of puparlal tanning (Karlson and Sekerls, I962, Sekens and Herrhch, 1966) The face fly, Musca autumnahs (De Geer), however, appears to be an exception to this mech- ~Send correspondence to K J Kramer, U S Gram Market- lng Research Laboratory, 1515 College Avenue, Man- hattan KS 66502 U S A amsm of puparlal hardening Fraenkel and Hslao (1967) reported that the deposition of calcium salts account for hardening and stabilization of the pupar- lUre in this species Evidence to support their hypoth- esis that calcification supplants sclerotlzatlon in face fly puparla included (1) the presence of high levels of calcium salts, (2) limited decreases in extractable protein in cuticle during puparlal hardening and (3) no decline in tyroslne tltre in whole animals during puparaatlon In contrast, large decreases in tyroslne content and extractable CUtlcular protein occurred in the house fly, Musca domestwa (L), during pupar- latlon Darhngton et al (1983) reexamined the min- eral and organic composition of face fly puparla and observed that, although mineral salts accounted for the bulk of the material in the puparlal cuticle. sclerotlzatlon could not be ruled out since minor amounts of water soluble protein were present after hardening Another significant difference between face fly and house fly puparlal cuticles is the absence of fl-alanlne (3-amino proplomc acid) In the face fly (Bodnaryk, 1972) fl-Alanlne is present in large quantity in house fly puparla and in puparla of other Diptera that normally form dark brown sclero- tlzed cuticle (Dennell, 1958, Fukushl and Sekl, 1965, 521