172 Experientia 43 (1987), Birkh/iuser Verlag, CHM010 Basel/Switzerland Sclerotization of mosquito cuticle ] M. Sugumaran and V. Semensi Department of Biology, University of Massachusetts at Boston, Harbor Campus, Boston (Massachusetts 02125, USA), 3 September 1985 Summary. The mode of sclerotization of Aedes aegypti pupal and adult cuticle was examined by employing biochemical and radioactive techniques. During larval-pupal metamorphosis, tyrosine is converted to tanning precursors and is incorporated into aryl-amino adducts and fl-crosslinks. The major hydrolysis product of fl-crosslinks in pupal cases is identified to be arterenone. Examination of tanning modes in five different mosquito species shows that the ratio of quinone to fl-sclerotization not only differs within the life stages of the insects, but also differs between species. Key words. Mosquito cuticle; quinone tanning; fl-sclerotization; arterenone; Aedes aegypti. During larval-pupal transformation as well as adult devel- opment, stabilization of insect cuticle in general is achieved by insertion of aromatic bridges between structural protomers and chitin backbone 2. Two models viz., covalent crosslinking and noncovalent hydrophobic interaction have been proposed to account for the hardening and tanning of arthropod cuticle 3-6. The covalent crosslinking hypothesis invokes the reactions of quinones and related reactive intermediates with cuticular com- ponents yielding aryl-protein adducts as well as aryl-protein crosslinks 3-5. The noncovalent crosslinking hypothesis calls for hydrophobic interaction of cuticular components with polymers derived from aromatic compounds 6'7. Recently we provided evidence for the presence of crosslinks in sarcophagid pupal cuticle by employing radioactive tracer tech- nique 8-~~ Since cuticle formation, and in particular sclerotiza- tion of exoskeleton, is a unique process of insects and other arthropods, a clear understanding of the molecular mechanisms of sclerotization could lead to the development of more specific insecticides. Mosquitoes are the vectors for malaria, filariasis, yellow fever, and other dangerous diseases afflicting man. In the past, control of these noxious insects was achieved by organochlorine insecti- cides such as DDT. The development of resistance to such com- pounds by mosquitoes and the persistence of these compounds in the environmnent have created the need for new vector con- trol measures. With this view in mind, we studied the mode of tanning of mosquito cuticle and report some of our results in this communication. Materials and methods. Aedes aegypti eggs were generously do- nated by Dr Andrew Spielman of the Harvard University, School of Public Health, Boston, MA, USA. They were hatched in a beaker of water under vacuum for 20 rain. The larvae were raised in plastic tanks and fed daily with Tetra Min aquarium food. The temperature of the growth chamber was kept at 30 ~ with relative humidity of 60 %. Under these conditions pupation starts in about 6 days. Radioactive tyrosine (2 ~tCi) was administered to mosquito lar- vae as outlined by Zomer and Lipke 11. Isolation of catecho- lamine derivatives from cuticular hydrolyzates was achieved by the method of Murdock and Omar 12. Aryl amino acid adducts were analyzed in cuticular hydrolyzates as described by Suguma- ran and Lipke 8. Quinone to fl-sclerotization ratio was deter- mined by the published procedure ~3, an. Scintillation counting was carried out after mixing aliquots (1 ml) of radioactive sam- ples with 5 ml of aquasol (New England Nuclear Co., Boston, MA, USA) in a Packard Model 3420 liquid scintillation counter. Higher performance liquid chromatography of phenols from mosquitoes was carried out using a high pressure pump (Altex, 2400 8o0 e~ 0 0.5 ! 0 r o4 ~ o.25 < 0 2 4 8 IO 12 14 16 18 20 Time (rain) Figure 1. HPLC separation of radioactive phenols from mosquito larvae. U-[14C]-tyrosine [2 p, Ci] was administered to late fourth instar mosquito larvae as outlined by Zomer and Lipke 11. Prior to pupation the larvae were homogenized in 80 % methanol and centrifuged. The clear super- natant was concentrated and subjected to HPLC analysis as outlined in Materials and Methods. The major radioactive peak was identified to be tyrosine by HPLC, spectral and amino acid analyses. 32 ? O x E >-1( i- ra I-. O E pH 7 PH 12 ! 1 10 20 FRACTION No < 3O Figure 2. Dowex 50 column chromatography of U-[~4C]-tyrosinelabeled cuticle hydrolyzate. 5 mg of U-[lac]-tyrosine labeled cuticle (sp. act. 2930 cpm/mg) was hydrolyzed and chromatographed on Dowex 50-H+ co- lumn using step gradients, as outlined by Sugumaran and Lipke8. The broad peak (pooled fractions) at the pH 7 fraction, which corresponds to unmodified tyrosine, carries approximately 38 % of the total radioactivity loaded on the column. The basic aryl-amino acid adducts appear at pH 12 fraction and carry 36 % of the radioactivity.