Methionine-rich storage protein gene in the rice stem borer, Chilo suppressalis, is expressed during diapause in response to cold acclimation

Gene expressions of acclimatized and non-acclimatized diapausing larvae were examined in Chilo suppressalis using a subtraction technique. A gene encoding a methionine-rich storage protein, CsSP1, was cloned and its complete cDNA sequence was determined. Potentially, CsSP1 encoded a 758-amino acid protein, with a calculated molecular weight of 88.8 kDa. The expression level of CsSP1 was higher in nondiapausing larvae than in diapausing ones. The CsSP1 expression was up-regulated in diapausing larvae when the temperature of cold acclimation was shifted to 5 degrees C. The up-regulated level was maintained at 40 days after incubation at 5 degrees C. In nondiapausing larvae, CsSP1 expression was down-regulated when the temperature was below developmental zero. Involvement of CsSP1 in diapause, cold tolerance acquisition and postdiapause development in C. suppressalis is discussed.     

Decrease in expression of beta-tubulin and microtubule abundance in flight muscles during diapause in adults of Culex pipiens

The cDNA encoding beta-tubulin in the mosquito Culex pipiens has high similarity with the beta-tubulins reported in other insects. In this study, we examine expression of this gene and microtubule abundance in relation to diapause and low temperature. While non-diapausing mosquitoes express beta-tubulin highly in their thoracic muscles, expression is quite low during adult diapause. The abundance of microtubules was also much lower in flight muscles of diapausing adults than in flight muscles from non-diapausing individuals, as confirmed by laser confocal microscopy of tubulins stained using indirect immunofluorescence. Low temperatures decreased microtubule abundance in midguts of non-diapausing mosquitoes, but microtubule abundance in diapausing mosquitoes was already low and remained unchanged by low temperature exposure. Overall, pixel intensity averages were higher in the flight muscles than in the midguts, and again low temperatures decreased microtubule abundance in the flight muscles of non-diapausing females, while levels remained consistently low in diapausing females. These results clearly indicate that a decrease in microtubule abundance is evoked both by the programming of diapause and, in non-diapausing females, by exposure to low temperatures. Quite possibly the reduced microtubule abundance in the flight muscles and reduced expression of beta-tubulin are functionally correlated to the reduction in flight activity that is associated with low temperature and diapause.     

Aedes aegypti transferrin. Gene structure, expression pattern, and regulation

Mosquitoes and all other insects so far examined have an abundant haemolymph transferrin (Tsf). The exact function of these proteins has not been determined, but they may be involved in iron transport, in oogenesis and in innate immune defence against parasites and pathogens. The Tsf gene of Aedes aegypti has been cloned and sequenced. It contains a single small intron, which contrasts it to vertebrate Tsf genes that contain up to sixteen introns. The promoter region of the gene is rich in putative NF-kappaB binding sites, which is consistent with the postulated role of Tsf in insect innate immunity. Tsf message levels are very low in embryos and early larvae, but high in late larvae, pupae and adults. Western blotting experiments revealed high levels of Tsf protein in pupae and adults. Late larvae and ovaries of blood-fed mosquitoes have little intact protein, but two prominent proteolytic degradation products. These may represent biologically active peptides, as has been shown for other organisms. Tsf message is down-regulated by inorganic iron in the diet or environment, but up-regulated by a blood meal in the adult female. The up-regulation following a blood meal may, in part, be due to the decrease in juvenile hormone (JH) that is known to follow blood feeding. Treatment of blood-fed females with methoprene, an analogue of JH, resulted in decrease of the Tsf message.     

Cathepsin L function in insect moulting: molecular cloning and functional analysis in cotton bollworm, Helicoverpa armigera

Moulting is an essential process of insect development but little is known about cysteine proteases in the process. Here, we detail a proteolytic activity profile from fifth larval instar to new pupae of the lepidopteran Helicoverpa armigera. At fifth to sixth instar moulting, the activities were significantly higher than those in non-moulting stages, and were inhibited by the cysteine protease inhibitor, 2S, 3S-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64), or by the cathepsin L-selective inhibitor CLIK148. Further, a 1513 bp cathepsin L cDNA (Har-CL) was isolated from the H. armigera larval cuticle and epidermis layer. Har-CL gene expression, which is correlated closely with ecdysone, was higher during larval moulting. Injection of E-64 or CLIK148 resulted in delayed fifth to sixth instar moulting, suggesting an essential role for cathepsin L in larval moulting.     

Developmental- and tissue-specific expression of an inhibitor of apoptosis protein 1 homologue from Aedes triseriatus mosquitoes

We have identified a homologue of the Drosophila inhibitor of apoptosis protein 1 in Aedes triseriatus mosquitoes (designated AtIAP1). The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA was detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein was detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognized proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 were expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures.