Molecular characterization of an ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia nonagrioides

We obtained a full-length cDNA encoding a carboxylesterase in Sesamia nonagrioides. The complete cDNA sequence is comprised of 1838 bp with an open reading frame encoding 576 amino acid residues with predicted molecular mass of 64.24 kDa. The deduced amino acid sequence showed high identity to JHE-Related of Trichoplusia ni (65% amino acid identity) and 49-46% amino acid identity to JHEs of other lepidopterans and contained all five functional motifs of insect JHEs. The gene has been termed as SnJHE-Related (SnJHER) to denote its similarity to other insect JHE genes and the occurrence of an unusual cysteine residue immediately adjacent to the catalytic serine, instead of the conventional alanine residue. Phylogenetic analyses localised SnJHER together with TnJHER in a branch of the lepidopteran's JHEs group, with other carboxylesterases (COEs) occuring in separated groups. The JH analog methoprene did not affect the expression of SnJHER in contrast to other insect JHEs. Additionally, ecdysteroid analogs induced SnJHER gene expression. The SnJHER mRNA levels were higher in long-day non-diapausing larvae than in short-day diapausing ones. In the fifth instar of non-diapausing and ninth instar of diapausing larvae, the SnJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) non-diapausing larval instar, SnJHER mRNA levels peaked in the intermolt period but were lower than during the fifth instar.     

Developmental expression and stress induction of glutathione S-transferase in the spruce budworm, Choristoneura fumiferana

Developmental and stress-induced expression of Choristoneura fumiferana glutathione S-transferase (CfGST) mRNA and protein were examined using Northern blots and Western blots. High levels of CfGST mRNA and protein were detected in 1st instar larvae and diapausing 2nd instar larvae. Expression of CfGST gradually decreased during larval development from 3rd to 5th instar, after which the expression increased once again, reaching peak levels in 6th instar larvae. CfGST mRNA and protein were undetectable in the pupal stage. Exposure to low temperature did not induce an increase in CfGST expression. Feeding on balsam fir foliage resulted in an increase in the expression of CfGST as compared to larvae that fed on artificial diet. The bacterial insecticide, Bacillus thuringiensis delta-endotoxin (Bt), the non-steroidal ecdysone analog, tebufenozide, and the synthetic pyrethroid, permethrin, induced the expression of CfGST mRNA in 5th instar larvae, whereas the chitin synthesis inhibitor, diflubenzuron, did not have any such effect. These results suggest that CfGST plays an important role in detoxifying various allelochemicals and insecticides in the spruce budworm. The developmental expression pattern strongly suggests that in addition to detoxification, CfGST might be involved in other functions.     

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.     

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.     

Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens

Two actin genes cloned from Culex pipiens L. are upregulated during adult diapause. Though actins 1 and 2 were expressed throughout diapause, both genes were most highly expressed early in diapause. These changes in gene expression were accompanied by a conspicuous redistribution of polymerized actin that was most pronounced in the midguts of diapausing mosquitoes that were exposed to low temperature. In nondiapausing mosquitoes reared at 25 degrees C and in diapausing mosquitoes reared at 18 degrees C, polymerized actin was clustered at high concentrations at the intersections of the muscle fibers that form the midgut musculature. When adults 7-10 days post-eclosion were exposed to low temperature (-5 degrees C for 12 h), the polymerized actin was evenly distributed along the muscle fibers in both nondiapausing and diapausing mosquitoes. Exposure of older adults (1 month post-eclosion) to low temperature (-5 degrees C for 12 h) elicited an even greater distribution of polymerized actin, an effect that was especially pronounced in diapausing mosquitoes. These changes in gene expression and actin distribution suggest a role for actins in enhancing survival of diapausing adults during the low temperatures of winter by fortification of the cytoskeleton.     

Desiccation resistance in pre-diapause, diapause and post-diapause larvae of Choristoneura fumiferana (Lepidoptera: Tortricidae)

Desiccation resistance was examined in pre-diapause, diapause and post-diapause larvae of the spruce budworm, Choristoneura fumiferana (Clemens), in terms of passive water evaporation under three desiccation conditions: freeze-drying, desiccant-drying at 2 degrees C and desiccant-drying at 18 degrees C. Diapausing second instar larvae and post-diapause non-feeding second instar larvae showed strongest desiccation resistance: a significant amount of water was retained after repeated drying under desiccating conditions, while pre-diapause first instar larvae and post-diapause feeding second instar larvae lost almost all their water content after one or two drying cycles. A hibernaculum covering had no effect on water evaporation. While dead larvae tended to lose significantly more water than their living counterparts, particularly among first instar larvae, such an impact was much weaker among diapausing second instar larvae. Desiccation resistance was lost when post-diapause second instar larvae were allowed access to water while the level of desiccation resistance was maintained or enhanced when the larvae did not have access to water. These results are discussed in the context of overwintering ecology of the species and possible mechanisms for the desiccation resistance are also discussed.     

Differential expression of two HSP70 transcripts in response to cold shock,thermoperiod, and adult diapause in the Colorado potato beetle

Partial clones for two members of Leptinotarsa decemlineata inducible 70kDa heat shock protein family (LdHSP70A and B) were developed using RT-PCR. LdHSP70A, but not LdHSP70B, was upregulated during adult diapause. The ability of L. decemlineata to express these two genes in response to subzero temperatures depended on the thermal history of the beetles. Chilling diapausing beetles increased the rate at which both LdHSP70A and B were expressed following a cold shock at -10 degrees C. Following cold shock at -10 degrees C, LdHSP70B expression peaked after 3h at 15 degrees C for chilled diapausing individuals, decreasing to near background levels by the sixth hour. In contrast, nonchilled diapausing beetles expressed their highest level of LdHSP70B only after 6h at 15 degrees C. Diapausing beetles exposed to a thermoperiod with a mean temperature of either 0 or -2.5 degrees C expressed significantly higher levels of both LdHSP70A and B than beetles exposed to constant 0 or -2.5 degrees C. These results demonstrate that the expression of LdHSP70A and B is differentially regulated in response to diapause and environmental conditioning.     

Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.     

Expression of larval hemolymph proteins (Lhp) genes and protein synthesis in the fat body of greater wax moth (Galleria mellonella) larvae during diapause

When one-day-old, last instar Galleria mellonella larvae are exposed to 18 degrees C they enter diapause and cease further development for several months. During diapause a group of proteins (72-84 kDa) synthesized in the fat body and secreted into the hemolymph is markedly elevated. Partial sequencing of the N-terminus of two proteins from this group confirmed their identity with larval hemolymph proteins (LHP) belonging to the family of hexameric storage proteins. The expression of two Lhp genes of known sequence (Lhp76 and Lhp82) were monitored in both diapausing and non-diapausing individuals. The expression of both genes and subsequent synthesis of the proteins (LHP76 and LHP82) is maintained until at least 90-100 days of diapause.     

Effects of diapause and cold-acclimation on the avoidance of freezing injury in fat body tissue of the rice stem borer, Chilo suppressalis Walker

Overwintering freeze-tolerant larvae of Chilo suppressalis can survive at -25 degrees C, but non-diapausing larvae cannot. We reported earlier that to prevent intracellular freezing, which causes death in overwintering larvae of the Saigoku ecotype distributed in southwestern Japan, water leaves and glycerol enters fat body cells through water channels during freezing. However, it is still unclear how diapause and low-temperature exposure are related to the acquisition of freeze tolerance. We compared the extent of tissue damage, accumulation of glycerol, and transport of glycerol and water in fat body tissues between cold-acclimated and non-acclimated non-diapausing and diapausing larvae. The tissue from cold-acclimated diapausing larvae could survive only when frozen in Grace's insect medium with 0.25 M glycerol at -20 degrees C. The protection provided by glycerol was offset by mercuric chloride, which is a water-channel inhibitor. Fat body tissue isolated from non-acclimated diapausing larvae was injured by freezing even though glycerol was added to the medium, but the level of freezing injury was significantly lower than in non-diapausing larvae. Radiotracer assays in cold-acclimated diapausing larvae showed that during freezing, water left the cells into the medium and glycerol entered the cells from the medium at the same time. Therefore, in Saigoku ecotype larvae of the rice stem borer, both diapause and cold-acclimation are essential to accumulate glycerol and activate aquaporin for the avoidance of freezing injury.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

Temperature-dependent regulatory mechanism of larval development of the wax moth (Galleria mellonella)

The mechanisms underlying larval diapause in the wax moth (Galleria mellonella) is one of the most throughly studied aspects. At the low temperature of 18 degrees C, the last instar larvae did not pupate but transferred to 30 degrees C they initiated development and pupation in a circadian manner. Different types of surgical manipulations including head-ligation, nerve cord-severance, implantation of the brain, prothoracic glands, accompanied with ecdysteroid titre measurements indicated that diapausing arrest of larval development at 18 degrees C might be due to the nervous inhibition of their prothoracic glands.     

Diapause phenomena in non-diapausing last instar larvae,pupae, and pharate adults of the Colorado beetle

From the first day of the last (fourth) larval instar no trace of juvenile hormone (JH) can be detected in the haemolymph by Galleria bioassay. Three specific diapause proteins, which are also found in diapausing adults, appear in the haemolymph. These proteins disappear towards the end of the pupal stage. Study of the ultrastructure of the fat body revealed the formation from lysosomes of proteinaceous bodies which are also characteristic for adult diapause. The behaviour of last instar larvae and pupae resembles that of prediapausing and diapausing adults respectively. Injection of synthetic JH delays the appearance of the diapause proteins in the haemolymph and of proteinaceous bodies in the fat body for 2 to 3 days. The absence of JH seems to trigger off these diapause phenomena.     

Identification, characterization, and developmental regulation of two storage proteins in the bamboo borer Omphisa fuscidentalis

Two insect storage proteins, OfSP1 (75 kDa) and OfSP2 (72 kDa), were purified using three different chromatographies from the hemolymph of Omphisa fuscidentalis larvae during diapause, and their genes were cloned. OfSP1 and OfSP2 concentrations in the hemolymph were high during diapause. During pupation, OfSP1 levels decreased in the male hemolymph and disappeared from the female hemolymph. OfSP1 and OfSP2 mRNA levels in the fat bodies were low during the third instar, but increased greatly during the fourth and fifth larval instars. During diapause, mRNA expression continued at a lower level than during the feeding period. The injection of 20-hydroxyecdysone (20E) into diapausing larvae caused an increase in OfSP1 and OfSP2 mRNA levels 2-3 days post-injection, followed by a decrease in expression until pupation, which occurred 2-4 days thereafter. When larvae were treated with juvenile-hormone analog (JHA), OfSP1 and OfSP2 mRNA levels gradually decreased until the onset of pupation. In Omphisa, OfSP1 and OfSP2 proteins are produced and released by the larval fat bodies in the fourth and fifth-instar larvae, and the proteins accumulate in the hemolymph until the insects enter diapause. OfSP1 may be reabsorbed by the fat bodies at the end of diapause for subsequent re-use during pupation.     

Diapause and development in the tobacco budworm,Heliothis virescens: A comparison of haemolymph ecdysteroid titres

The signal to induce diapause in H. virescens comes early in development (prior to the third instar in most insects), but the signal to break diapause can come shortly after entrance into diapause at pupation. Haemolymph ecdysteroid titres in both diapause-bound and non-diapause-bound Heliothis virescens larvae were similar in the first two thirds of the last-larval instar, when similar changes in morphology and behaviour occurred. However, the number of stepwise increases in titre and the timing of the steps was different in the two groups of larvae. Haemolymph ecdysteroid titres in the last third of the instar were approx, five times higher in non-diapause than in diapause-bound larvae. In diapausing pupae, haemolymph ecdysteroid titres dropped to levels found in larvae which had completed two thirds of the last instar. When diapausing pupae were warmed to break diapause, haemolymph ecdysteroid titres rose again. However, 2 of the 4 high ecdysteroid levels detected in pupae developing after diapause break were considerably lower than those detected for non-diapause pupae.     

Effect of an ecdysteroid agonist, tebufenozide, on the completion of diapause in susceptible and resistant strains of the codling moth, Cydia pomonella

Codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae) larvae kept under a short photoperiod enter a diapause which can be broken by exposure to 25°C following an extended chilling period. We investigated the effects of the ecdysteroid agonist tebufenozide on the diapause of this species, both on a laboratory strain susceptible to insecticides and two strains selected for resistance to diflubenzuron or deltamethrin. The treatment induced moulting by breaking diapause of the susceptible strain when applied at the end of the growth period, or by reducing the pre-emergence period when applied after chilling. The strains exhibiting resistance in the neonate instar were also resistant to the ecdysteroid agonist applied to diapausing larvae, indicating that mechanisms of resistance remained active during this arrest of development. High doses of tebufenozide were lethal for the most resistant insects, but neither reduced the pupation delay nor produced the larval-pupal moults which occur in the susceptible strain. As diapausing larvae of C. pomonella can be easily collected, the expression of resistance to tebufenozide both in the target instar, i.e. neonates, and during diapause may contribute to early detection of resistance to this insecticide in field populations.