Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause

A comparison of the cDNA sequences （1 056 bp） of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog orB. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of lisp40 gene, and its role in egg diapause induction in B. mori.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein （HSP） genes （hsp70, hsc70, hsp90） were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus （Homoptera： Delphacidae）, which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepidopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock （40 ℃） upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4℃ did not change the expression levels of any hsp in either species.     

Effect of pine foliage damage on the incidence of larval diapause in the pine caterpillar Dendrolimus punctatus （Lepidoptera： Lasiocampidae）

The pine caterpillar Dendrolimus punctatus （Walker） with a larval facultative diapause is one of the most destructive insect pests of the pine tree Pinus massoniana in China. The larvae feeding on pine trees with different damage levels were studied to determine the induction of diapause under both laboratory and field conditions. Developmental duration of larvae before the third instar was the longest when fed with 75%-90% damaged needles, followed by 25%-40% damaged needles and intact pine needles, whereas mortalities did not differ among different treatments under the conditions of 25℃ and critical photoperiod 13.5：10.5 L：D. At 25 ℃, no diapause was induced under 15：9 L：D, whereas 100% diapause occurred under 12：12 L：D regardless of the levels of needle damage. Incidences of larvae entering diapause when they were fed with intact, 25%-40% and 75%- 90% damaged pine needles were 51.7%, 70.8% and 81% under 13.5：10.5 L：D, respectively. Similar results were obtained in the field experiment. Incidence of diapause was significantly different among the pine needle damage levels of pine trees when the photoperiod was close to the critical day length, indicating that the effect of host plants on diapause induction was dependent on the range of photoperiod. The content of amino acid and sugar decreased and tannin increased in pine needles after feeding by the pine caterpillars, suggesting that changed levels of nutrients in damaged needles or a particular substance emitted by damaged pine trees was perhaps involved in the diapause induction of the pine caterpillar.     

Effects of photoperiod and temperature on diapause induction in Conogethes punctiferalis （Lepidoptera： Pyralidae）

The yellow peach moth, Conogethes punctiferalis （Guenee）, a multivoltine species that overwinters as diapausing larvae, is one of the most serious insect pests on maize in China. Effect of photoperiod and temperature on larval diapause was examined under empirical laboratory conditions. Short-day treatments caused larval diapause at 25℃, and the critical photoperiod was between 12 and 13 h （or 12 h 51 min） light per day. No sensitive instar was identified for diapause induction under alternated short- （L ： D 11 ： 13 h） and long-day （L ： D 14 ： 10 h） treatments at different larval stages. However, accumulative treatment of three instars and 10 d under short-day treatment was required for the induction of 50% larval diapause. All larvae entered diapause at 20℃, whereas less than 3% did so at 30℃, irrespective of the long- or short-day treatment. Furthermore, under the short-day treatment, more than 90% of larvae went into diapause with temperatures ≤ 25℃, but less than 17% did so at 28℃. In contrast, under the long-day treatment, less than 19% of larvae went into diapause with temperatures ≥23 ℃. The forward shift （5℃） of critical temperature under the long-day regime demonstrated the compensatory effect of temperature and photoperiod on diapause induction. In conclusion, C. punctiferalis had a temperature-dependent type Ⅰ photoperiodic diapause response; there was no sensitive instar for diapause determination, but the photoperiodic accumulation time countermeasures both of the short-day cycles and the number ofinstars exposed, and the photoperiodic diapause response, was a temperature-compensated phenomenon.     

Genome‐wide identification and characterization of HSP gene superfamily in whitefly (Bemisia tabaci) and expression profiling analysis under temperature stress

Heat shock proteins (HSP)are essential molecular chaperones that play important roles in the stress stimulation of insects.Bemisia tabaci,a phloem feeder and invasive species,can cause extensive crop damage through direct feeding and transmission of plant viruses.Here we employed comprehensive genomics approaches to identity HSP superfamily members in the Middle East Asia Minor 1 whitefly genome.In total,we identified 26 Hsp genes,including three Hsp90,17 Hsp70,one Hsp60 and five sHSP (small heat shock protein)genes.The HSP gene superfamily of whitefly is expanded compared with the other five insects surveyed here.The gene structures among the same families are relatively conserved.Meanwhile,the motif compositions and secondary structures of BtHsp proteins were predicted.In addition,quantitative polymerase chain reaction analysis showed that the expression patterns of BtHsp gene superfamily were diverse across different tissues of whiteflies.Most Hsp genes were induced or repressed by thermal stress (40℃)and cold treatment (4℃)in whitefly.Silencing the expression of BtHsp70-6 significantly decreased the survival rate of whitefly under 45℃.All the results showed the Hsps conferred thermo-tolerance or cold-tolerance to whiteflies that protect them from being affected by detrimental temperature conditions.Our observations highlighted the molecular evolutionary properties and the response mechanism to temperature assaults of Hsp genes in whitefly.     

PHOTOPERIODIC CONTROL OF ADULT DIAPAUSE IN CHRYSOPERLA SINICA （TJEDER） （NEUROPTERA： CHRYSOPIDAE） -I. CRITICAL PHOTOPERIOD AND SENSITIVE STAGES OF ADULT DIAPAUSE INDUCTION

Photoperiodic sensitivity for diapause induction of the green lacewing, Chrysoperla sinica (Tjeder) was examined at 22℃. The adult diapause of C. sinica was induced by short-day photoperiods, and the critical photoperiod for its induction was between 12.5L-11.5D and 13L-11D.Adults developed without diapause under long-day conditions, and entered diapause under short-day conditions. The adult stage was the uppermost sensitive stage for adult diapause induction, adults could go into diapause only when the emerging adults were under diapause-inducing short-day photoperiods. The short-day photoperiodic experience by transferring between 15L: 9D and 9L: 15D at preimaginal stages did not result in adult diapause under 15L: 9D photo regime, although some treatments extended the pre-oviposition period in adult stage. The results showed that the 3rd instar larvae and pre-pupae were more sensitive to the photoperiodic change from 15L: 9D to 9L: 15D photo regime than the other preimaginal stages.     

Diapause affects cuticular hydrocarbon composition and mating behavior of both sexes in Drosophila montana

Envipnmental cues,mainly photoperiod and temperature,are known to control female adult reproductive diapause in several insect species.Diapause enhances female survival during adverse conditions and postpones progeny production to the favorable season.Male diapause(a reversible inability to inseminate receptive females)has been studied much less than female diapause.However,if the males maximized their chances to fertilize females while minimizing their energy expenditure,they would be expected to be in diapause at the same time as females.We investigated Drosophila montana male mating bchavior under short-day conditions that induce diapause in females and found the males to be reproductively inactive.We also found that males reared under long-day conditions(reproducing individuals)court reproducing postdiapause fermales,but not diapausing ones.The diapausing fies of both sexes had more long-chain and less short-chain hydrocarbons on their cuticle than the reproducing ones,which presumably increase their survival under stressful conditions,but at the same time decrease their attractiveness.Our study shows that the mating behavior of females and males is well coordinated during and afier overwintering and it also gives support to the dual role of insect cuticular hydrocarbons in adaptation and mate choice.     

Molecular mechanism of diapause in insect (I) --Expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori

The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.     

Melatonin in the seasonal response of the aphid Acyrthosiphon pisum

Aphids display life cycles largely determined by the photoperiod.During the warm long-day seasons.most aphid species reproduce by viviparous parthenogenesis.The shortening of the photoperiod in autumn induces a switch to sexual reproduction.Males and sexual females mate to produce overwintering resistant eggs.In addition to this full life cycle(holocycle),there are anholocyelic lineages that do not respond to changes in photoperiod and reproduce continuously by parthenogenesis.The molecular or hormonal events that trigger the scasonal response(i.c,induction of the sexual phenotypes)are still unknown.Although circadian synthesis of melatonin is known to play a key role in vertebrate photoperiodism,the involvement of the circadian clock and/or of the hor-mone melatonin in insect seasonal responses is not so well established.Here we show that melatonin levels in the aphid Acyrthosiphon pisum are significantly higher in holocyclice aphids reared under short days than under long days,while no differences were found between anholoeyelic aphids under the same conditions.We also found that melatonin is localized in the aphid suboesophageal ganglion(SOG)and in the thoracic ganglionic mass(TGM).In analogy to vertcbrates,insect-type arylalkxylamine N-acetyltransferases(i-AANATs)are thought to play a key role in melatonin synthesis.We measured the expression of four I-AANAT genes identified in A.pisum and localized two of them in situ in the insect central nervous systems(CNS).Levels of expression of these genes were compatible with the quantities of melatonin observed.Moreover,like melatonin,expression of these genes was found in the SOG and the TGM.     

Structure and expression of a rice hsp70 gene

A genomic hsp70 gene was isolated from a rice IR36 genomic library and 4 794 bp of the gene have been sequenoed. The 5' flanking region of the gene contained a putative TATA box and a typical heat shock element sequence 5'-CTcgGAAccTTCgAG-3'. The amino acid sequence of the rice HSP70 deduced from the coding region shared 84%-92% homologies with those of HSP70s from other plant species. An intron 1939bp long was identified in the coding region at the codon specifying amino acid 72 (Asp), the similar position introns occurring in other intron-containing hsp70 genes. In addition, another intron of 57 bp was found in the 3'-untranslated region in the rice hsp70 gene. Southern blot hybridization showed that rice hsp70 gene family contained at least three members. Analysis of the RNA leveis with the gene-specific and non-specific probes revealed that the rice hsp70 gene expressed at normal temperature and the expression was enhanced by heat shock treatment.     

Cloning and characterization of Methoprene-tolerant(Met)and Kruppel homolog 1(Kr-h1)genes in the wheat blossom midge,Sitodiplosis mosellana

Juvenile hormone(JH),a growth regulator,inhibits ecdysteroid-induced meta-morphosis and controls insect development and diapause.Methoprene-tolerant(Met)and Krippel homolog I(Kr-h1)are two proteins involved in JH action.To gain some insight into their function in development of Sitodiplosis mosellana,an insect pest undergoing obligatory larval diapause at the mature 3rd instar stage,we cloned full-length complemen-tary DNAs of Met and Kr-h1 from this specics.SmMet encoded a putative protein,which contained three domains typical of the bHLH-PAS family and eight conserved amino acid residues important for JH binding.SmKr-h1 encoded a protein showing high sequence homology to its counterparts in other specics,and contained all eight highly conserved Zn-finger motifs for DNA-binding.Expression patterns of SmMet and SmKr hl were de-velopmentally regulated and JH III responsive as well.Their mRNA abundance increased as larvae entered carly 3rd instar,pre-diapause and maintenance stages,and peaked during post-diapause quiescence,a pattern correlated with JH titers in this species.Different from reduced expression of SmMer,SmKr-h1 mRNA increased at mid-to-late period of post-diapause development.Topical application of JH II on diapausing larvac also induced the two genes in a dose-dependent manner.Expression of SmuMer and SmKr-h1 clearly declined in the pre-pupal phase,and was significantly higher in female adults than male adults.These results suggest that JH-responsive SmMet and SmKr-h1 might play key roles in diapause induction and maintenance as well as in post-diapause quiescence and adult reproduction,whereas metamorphosis from larvae to pupac might be correlated with their reduced expression.     

Host plant mediation of diapause induction in the cabbage beetle, Colaphellus bowringi Baly （Coleoptera： Chrysomelidae）

The cabbage beetle, Colaphellus bowringiBaly （Coleoptera： Chrysomelidae）, is a serious pest of crucifers in China, undergoing an imaginal summer and winter diapause in the soil. The effects of host plants on diapause incidence were tested in the beetle. The ratio of adults entering diapause was significantly low when they fed on the mature leaves of Chinese cabbage Shanghaiqin （Brassica chinesis var communis） than those feeding on Chinese cabbage Suzhouqin （Brassica chinesis var communis）, radish （Raphanus sativus var longipinnatus） and stem mustard （Brassicajuncea vat tumida） at 25℃ combined with 13：11 （L： D） h. Fewer adults entered diapause on young leaves compared to physiologically aged and mature radish leaves at 25℃ combined with 13：11 （L： D） h. The effect of host plant species on diapause induction was also evident under continuously dark rearing conditions or at different photoperiods. These experimental results demonstrate that host plant mediation of diapause induction exists in the cabbage beetle. However, at temperatures ≤20℃ or photoperiods of 16：8 （L： D） h combined with 25℃, all individuals entered diapause regardless of the host plants, indicating that the effects of host plants on diapause induction could be expressed only within a limited range of temperatures and photoperiods.     

Evolutionary conservation and DNA binding properties of the Ssh7 proteins from Sulfolobus shibatae

The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both n     

Computational identification and evolutional analysis of Piwi subfamily in 11 Drosophila species

The complete genome sequences of 11 Drosophila species provide an opportunity to investigate the gene family evolution in closely related species. Drosophila Piwi subfamily, including three members, piwi, Aub and Ago3, has attracted increasing attention as it participates in the biogenesis of piRNA. Here, we identified 33 Piwi homologs from the genome of 11 Drosophila species. The full-length cDNA sequences ofpiwi and Aub genes were obtained by using New GENSCAN Web Server. The Ago3 homologs were difficult regarding full-length information because they had long introns. The genomic structure of Piwi subfamily genes are highly conserved among diverse Drosophila species. Insect piwi and Aub genes have long first introns. The average length of the first intron is 1 284 bp for piwi and 840 bp for Aub, which is much larger than those of other introns （93 bp for Piwi and 54 bp for Aub）. However, this phenomenon is not observed in mammalian piwi genes. We also found that there were abundant repeat sequences in both exons and introns of insect Ago3 genes. Due to recent insertions of long terminal repeat elements in four Drosophila species, part of the third introns exhibit higher conservation than adjacent exons and other introns. An evolutional tree created by Minimum Evolution method indicates that mammalian piwi genes are more closely related to the insect Ago3 Piwi subfamily.