The tissue expression pattern of <i>O. chinensis Hsp</i>s in 5th instar nymphs.

<p>The bars represent the mean ± SE (n = 3) of target gene mRNA expression in different tissues. The small letters on the bars indicate significant differences between tissues (Duncan’s multiple comparison, <i>P</i>< 0.05). BR: brain; EP: epidermis; FG: foregut; GC: gastric caecum; MG: midgut; HG: hindgut; MT: Malpighian tubule; FB: fat body. The mRNA level in the brain was arbitrarily taken as 1.0.</p

mRNA expression of <i>O. chinensis Hsps</i> after chronic Cd exposure.

<p>Wheat seedlings cultured in distilled water were used as a control. During this experiment, significant numbers of insect deaths did not occur. The bars represent the mean ± SE (n = 3) of target gene mRNA expression after chronic Cd exposure. The small letters on the bars represent significant differences between different Cd concentrations after the insects fed on dietary Cd (Duncan’s multiple comparison, <i>P</i>< 0.05). The value of the control was arbitrarily taken as 1.0.</p

mRNA expression of <i>O. chinensis Hsps</i> 6 h after exposure to different Cd concentrations.

<p>The bars represent the mean ± SE (n = 3) of target gene mRNA expression in 5th instar nymphs 6 h after the injection of different Cd concentrations. The small letters on the bars represent significant differences between different Cd concentrations (Duncan’s multiple comparison, <i>P</i>< 0.05). Distilled water was used as a control. The value of the control was arbitrarily taken as 1.0.</p

A complete list of species used in the phylogenetic analysis and accession numbers for Hsp sequences.

<p>A complete list of species used in the phylogenetic analysis and accession numbers for Hsp sequences.</p

The developmental expression pattern of <i>O. chinensis Hsp</i>s in eggs, nymphs and adults.

<p>The bars represent the mean ± SE (n = 3) of target gene mRNA expression at different developmental stages. The different small letters on the bars indicate significant differences between developmental stages (Duncan’s multiple comparison, <i>P</i>< 0.05). EG: eggs; 1st: first instar nymphs; 2nd: second instar nymphs; 3rd: third instar nymphs; 4th: fourth instar nymphs: 5th: fifth instar nymphs; AD: adults. The mRNA level in the eggs was arbitrarily taken as 1.0.</p

Characteristics of the four <i>Hsps</i> in <i>O</i>. <i>chinensis</i>.

<p>Characteristics of the four <i>Hsps</i> in <i>O</i>. <i>chinensis</i>.</p

Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper <i>Oxya chinensis</i>

<div><p>Heat shock proteins (<i>Hsps</i>) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the <i>Grp78</i> (glucose-regulated protein 78), <i>Hsp70</i>, <i>Hsp90</i>, and <i>Hsp40</i> genes from the Chinese rice grasshopper <i>Oxya chinensis</i>. The full-length cDNA sequences of <i>OcGrp78</i>, <i>OcHsp70</i>, <i>OcHsp90</i>, and <i>OcHsp40</i> contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these <i>Hsp</i> genes in different tissues and developmental stages. The mRNAs encoding these four <i>Hsp</i> genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four <i>Hsps</i> in <i>O</i>. <i>chinensis</i> subjected to Cadmium (Cd) stress. <i>OcGrp78</i>, <i>OcHsp70</i>, <i>OcHsp90</i>, and <i>OcHsp40</i> mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, <i>OcHsp70</i> was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased <i>OcGrp78</i>, <i>OcHsp90</i>, and <i>OcHsp40</i> expression. However, dietary Cd induced a significant reduction of <i>OcHsp70</i> expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four <i>Hsps</i> genes, especially <i>OcHsp70</i>, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that <i>OcHsp70</i> is not suitable for use as a molecular marker of chronic Cd contamination.</p></div

mRNA expression of <i>O. chinensis Hsp</i>s at different times after acute Cd exposure.

<p>The bars represent the mean ± SE (n = 3) of target gene mRNA expression in 5th instar nymphs 0 h, 2 h, 6 h, 12 h, 24 h and 48 h after the injection of 2.7 mg·kg^-1 Cd. The small letters on the bars indicate significant differences between the different treatment times (Duncan’s multiple comparison, <i>P</i>< 0.05). The mRNA level at 0 h was arbitrarily taken as 1.0.</p

Cadmium tolerance of <i>E. coli</i> BL21 cells expressing <i>OcMT1</i> and <i>OcMT2</i>.

<p>A: OcMT1, B: OcMT2. Bacterial growth curve of <i>E. coli</i> cells transformed with pET-28a, pET-28a-OcMT and pET-28a-OcMT-IPTG. pET-28a is an “empty” vector; pET-28a-OcMT group is transformed with the <i>OcMT1</i> or <i>OcMT2</i> gene without IPTG; pET-28a-OcMT-IPTG group is transformed with the <i>OcMT1</i> or <i>OcMT2</i> gene with IPTG. Five microliters of CdCl₂ was added into medium when bacteria were grown to OD600 = 0.6. All bacteria were grown for 11 h. Concentration gradient of CdCl₂ were 0, 0.82, 1.74, 3.27 mM.</p

Mortalities of <i>O. chinensis</i> injected with different heavy metals after <i>OcMT1</i> and <i>OcMT2</i> gene silencing by RNAi.

<p>A: <i>OcMT1</i> RNAi; B: <i>OcMT2</i> RNAi. Insects were injected <i>with OcMT1</i> and <i>OcMT2</i> dsRNA, and the control groups were injected with the same amount of ds<i>GFP</i>. After 24 h, at least 100 insects were randomly selected, and 4 µL of a concentration gradient of CdCl₂ (0.87, 1.74, 2.61, 3.48, 4.35 mM), CuCl₂ (8.79, 11.73, 14.67, 17.61, 20.55 mM) and ZnSO₄ (15.65, 19.13, 22.61, 26.09, 29.57 mM) solution were injected. The control groups were injected with distilled water. Mortalities were recorded at 48 h after the injections of the metal solutions. An asterisk (*) on the error bars indicates significant differences (<i>P</i><0.05, Tukey's HSD test; n = 3).</p