Cloning of heat shock protein genes (hsp70, hsc70 and hsp90) and their expression in response to larval diapause and thermal stress in the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana Géhin, one of the most important pests of wheat, undergoes obligatory diapause as a larva to survive unfavorable temperature extremes during hot summers and cold winters. To explore the potential roles of heat shock proteins (hsp) in this process, we cloned full-length cDNAs of hsp70, hsc70 and hsp90 from S. mosellana larvae, and examined their expression in response to diapause and short-term temperature stresses. Three hsps included all signature sequences of corresponding protein family and EEVD motifs. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known classification of insects. Expression of hsp70 and hsp90 were highly induced by diapause, particularly pronounced during summer and winter. Interestingly, hsp70 was more strongly expressed in summer than in winter whereas hsp90 displayed the opposite pattern. Abundance of hsc70 mRNA was comparable prior to and during diapauses and was highly up-regulated when insects began to enter the stage of post-diapause quiescence. Heat-stressed over-summering larvae (⩾30 °C) or cold-stressed over-wintering larvae (⩽0 °C) could further elevate expression of these three genes, but temperature extremes i.e. as high as 45 °C or as low as −15 °C failed to trigger such expression patterns. Notably, hsp70 was most sensitive to heat stress and hsp90 was most sensitive to cold stress. These results suggested that hsp70 and hsp90 play key roles in diapause maintenance and thermal stress; the former may be more prominent contributor to heat tolerance and the latter for cold tolerance. In contrast, hsc70 most likely is involved in developmental transition from diapause to post-diapause quiescence, and thus may serve as a molecular marker to predict diapause termination.     

Three heat shock proteins are essential for rotifer thermotolerance

Heat shock proteins (HSPs) are important molecules in the stress response of organisms from prokaryotes to mammals, and thus may be useful biomarkers for environmental stress. Here we characterize the functional roles of genes belonging to four distinct families of HSPs (hsp40, hsp60, hsp70, and hsp90) in the monogonont rotifer Brachionus manjavacas. Because B. manjavacas inhabits ponds of varying thermal regimes, including ephemeral ponds that may experience temperature fluctuations, HSP-mediated thermotolerance likely is important to its survival and adaptation. Using interference RNA (RNAi), we provide the first conclusive evidence that HSPs are required for rotifer survival following heat stress. Effective RNAi-mediated suppression of all hsp genes except hsp90 was verified via quantitative PCR. Hsp40, hsp60, and hsp70 are required for rotifer thermotolerance (P < 0.05); however, our data do not indicate hsp90 is essential. Quantitative PCR further revealed immediate up-regulation of hsp40 mRNA following heat stress. Additionally, we demonstrated expression of hsp40 mRNA in multiple tissues using fluorescent in situ hybridization. Our characterization of mRNA expression and functional roles for four distinct hsp genes provides a baseline for molecular-level comparisons of the stress response of rotifers with other taxonomic groups, and the technique for in-depth studies of the role of specific genes in rotifer stress responses. Considering the potential for ambient temperatures to impact species survival, competitive interactions, and body size of individuals, thermotolerance may be an important influence on zooplankton community structure.     

Analysis of digestion of rice planthopper by Pardosa pseudoannulata based on CO-I gene

In order to systematically study the predatory behavior and digestion regularity of spiders, real-time fluorescence quantification PCR technique was used to detect the number of CO-I genes in Pardosa pseudoannulata after it preyed on rice planthoppers in different temperatures within different periods. At 28 °C, 0, 1, 2, 4, 8, 16, and 24 h after P. pseudoannulata preyed on rich planthopper, DNA was extracted from cephalothorax and abdomen of P. pseudoannulata. Routine PCR and real-time fluorescence PCR techniques were employed for CO-I gene amplification. The results show that: The prey liquid was temporarily stored in the sucking stomach of the spider head within 2 h after prey, and gradually transferred to the midgut of the abdomen with the prolongation of time. After 4 h, CO-I gene residues of rice planthopper in the cephalothorax gradually decreased. The CO-I gene of rice planthopper was basically transferred to the abdomen after 16 h. During 0–1 h, food contained in abdominal midgut and other digestive organs was very small, CO-I gene detection was not obvious. Over time, food entered into the midgut from the sucking stomach for digestion. During 2–4 h, CO-I gene amount increased, at 2–4 h, detected CO-I gene residue reached the peak; but rapidly declined after 8, 16, and 24 h, even it is still detectable. The results at different temperatures reveal that: As the temperature increased from 26 °C to 32 °C, CO-I gene residues of rich planthopper in cephalothorax and abdomen of P. pseudoannulata gradually decreased, which indicated that the digestion rate increased with the increase of temperature with some range. However, when the temperature continued to increase to 34 °C, the digestion rate decreased.     

Cloning and expression of heat shock protein genes in two whitefly species in response to thermal stress

Two whitefly species, Trialeurodes vaporariorum and Bemisia tabaci biotype B were shown to have different temperature tolerance and seasonal dynamics. To determine whether this variation in thermal tolerance is related to different expression patterns of heat shock protein (hsp) genes during temperature stress, we obtained complete cDNA sequences for hsp90, hsp70 and hsp20, and analysed their expression profiles across temperature gradients by real‐time quantitative polymerase chain reaction (PCR). Six full‐length cDNAs were cloned and sequenced from these two species. The full‐length cDNAs of hsp90s contain 2166 and 2157 bp open‐reading frames (ORF) which encode proteins with calculated molecular weights of 83 013 and 82 857 Da in T. vaporariorum and B. tabaci, respectively. The 1947 and 1959 bp ORFs of whitefly hsp70s comprise 649 and 653 amino acids with the calculated masses of 70 885 and 71 008 Da in T. vaporariorum and B. tabaci, respectively. Both complete cDNAs of hsp20 of T. vaporariorum and B. tabaci contain 585 bp ORFs and deduced amino acid sequences had molecular weights of 21 559 and 21 539 Da, respectively. The hsp expression profile results showed that temperatures for onset (T_on) or maximal (T_max) induction of hsp expression in T. vaporariorum were generally 2–6°C lower than those in B. tabaci. These results suggest that the T_on (or T_max) of hsps can represent the differences in temperature tolerance of these two whitefly species, and may be used to determine their natural geographical distribution and natural population seasonal dynamics. Significant upregulation of most hsps were observed when temperature stress was lifted, except that hsp70 and hsp20 of B. tabaci did not respond to the cold stress, indicating that response to heat and cold stress may have a different genetic and physiological basis in two whitefly species. These results highlight the importance of understanding the complexity of the heat shock response across multiple isoforms while attempting to link them to whole‐organism traits such as thermal tolerance.     

Qualitative and quantitative analyses of the transpositions of <Emphasis Type="Italic">P</Emphasis> element—based genetic construction into the region of <Emphasis Type="Italic">Drosophila melanogaster hsp70</Emphasis> genes

The hsp70 genes is among the main systems underlying the adaptation of organisms to adverse environmental factors. The ever increasing amount of data in literature demonstrates an important adaptive role of mobile genetic elements in microevolution. Drosophila hsp70 genes are potential target for transpositions of various mobile elements in natural populations. We have analyzed the frequency and localization of a P element—based genetic construction, EPgy2, in the region of Drosophila melanogaster hsp70 genes. A hot spot for the transposition was discovered in the promoter regions of genes hsp70Aa and hsp70Ab. No insertions of this construction in the coding or 3′-flanking regions of hsp70 genes have been recorded. It was demonstrated that the region of 161 to 7800 bp adjacent to the original construction is in certain cases also involved in the transposition. No transpositions of any other mobile elements have been observed. The inserts were shown to change the activity of hsp70 genes and the thermotolerance of transgenic strains.     

Development of the embryonic heat shock response and the impact of repeated thermal stress in early stage lake whitefish (Coregonus clupeaformis) embryos

Lake whitefish (Coregonus clupeaformis) embryos were exposed to thermal stress (TS) at different developmental stages to determine when the heat shock response (HSR) can be initiated and if it is altered by exposure to repeated TS. First, embryos were subject to one of three different TS temperatures (6, 9, or 12 °C above control) at 4 points in development (21, 38, 60 and 70 days post-fertilisation (dpf)) for 2 h followed by a 2 h recovery to understand the ontogeny of the HSR. A second experiment explored the effects of repeated TS on the HSR in embryos from 15 to 75 dpf. Embryos were subjected to one of two TS regimes; +6 °C TS for 1 h every 6 days or +9 °C TS for 1 h every 6 days. Following a 2 h recovery, a subset of embryos was sampled. Our results show that embryos could initiate a HSR via upregulation of heat shock protein 70 (hsp70) mRNA at all developmental ages studied, but that this response varied with age and was only observed with a TS of +9 or +12 °C. In comparison, when embryos received multiple TS treatments, hsp70 was not induced in response to the 1 h TS and 2 h recovery, and a downregulation was observed at 39 dpf. Downregulation of hsp47 and hsp90α mRNA was also observed in early age embryos. Collectively, these data suggest that embryos are capable of initiating a HSR at early age and throughout embryogenesis, but that repeated TS can alter the HSR, and may result in either reduced responsiveness or a downregulation of inducible hsps. Our findings warrant further investigation into both the short- and long-term effects of repeated TS on lake whitefish development.