Characterization and functional analysis of hsp18.3 gene in the red flour beetle, Tribolium castaneum

Abstract

Small heat shock proteins (sHSPs) are diverse and mainly function as molecular chaperones to protect organisms and cells from various stresses. In this study, hsp18.3, one Tribolium castaneum species-specific shsp, has been identified. Quantitative real-time polymerase chain reaction illustrated that Tchsp18.3 is expressed in all developmental stages, and is highly expressed at early pupal and late adult stages, while it is highly expressed in ovary and fat body at the adult period. Moreover, it was up-regulated 4532 ± 396-fold in response to enhanced heat stress but not to cold stress; meanwhile the lifespan of adults in ds-Tchsp18.3 group reduced by 15.8% from control group under starvation. Laval RNA interference (RNAi) of Tchsp18.3 caused 86.1% ± 4.5% arrested pupal eclosion and revealed that Tchsp18.3 played an important role in insect development. In addition, parental RNAi of Tchsp18.3 reduced the oviposition amount by 94.7%. These results suggest that Tchsp18.3 is not only essential for the resistance to heat and starvation stress, but also is critical for normal development and reproduction in T. castaneum.

Keywords: Tchsp18.3; Tribolium castaneum; development; reproduction; stress.

Figure 1

Amino acid sequence alignment of Tchsp18.3 and five selected shsps from Bombyx mori, Apis mellifera, Anopheles gambiae and Drosophila melanogaster. Identical residues in the sequences are in black and similar residues are in gray. Secondary structures, as indicated by arrows (β‐sheets) above and line (α‐crystallin domain) below the alignment, were predicted and numbered according to Aevermann and Waters (2008) and Li et al. (2009).

Figure 2

The expression patterns of Tchsp18.3 in different developmental stages and various late adult tissues. (A) Different developmental stages are EE: early eggs; LE: late eggs; EL: early larvae; LL: late larvae; EP: early pupae; LP: late pupae; EA: early adults; LA: late adults. (B) Various tissues from late adults are head, epidermis, gut, fat body, accessory gland, testis and ovary. Tribolium ribosomal protein S3 (rps3) transcript with the same complementary DNA (cDNA) template served as an internal control. Different lowercase letters indicate statistically significant differences (P < 0.05).

Figure 3

The expression patterns of Tchsp18.3 under cold (4°C) and heat (45°C) treatment, with an ambient temperature 25°C serving as control. Tribolium ribosomal protein S3 (rps3) transcript with the same complementary DNA (cDNA) template served as an internal control. Different lowercase letters indicate statistically significant differences (P<0.05).

Figure 4

Effect of Tchsp18.3 on starvation treatment. (A) Time course analysis of Tchsp18.3 expression under starvation stress. Different lowercase letters indicate statistically significant differences (P < 0.05). (B) The differential survival rates of wild type (WT), IB and ds‐Tchsp18.3 beetles under starvation. (C) Mean survivals (days) of WT, IB and ds‐Tchsp18.3 beetles under starvation. WT, wild type, beetles received a non‐injection; IB, beetles injected with physiological buffer; ds‐Tchsp18.3, beetles injected with double‐stranded RNA in 2‐day‐old pupae. *P < 0.05; **P < 0.001.

Figure 5

RNA interference (RNAi) phenotype of Tchsp18.3. (A) RNAi resulted in lethal phenotypes in pupal/adult stages. P7D, 7‐day‐old pupae; A1D, 1‐day‐old adult. (B) Expression levels of knocking down Tchsp18.3 using larval RNAi. (C) Mortality of wild type (WT), IB and ds‐Tchsp18.3 beetles. WT, wild type, beetles received a non‐injection; IB, beetles injected with physiological buffer; ds‐Tchsp18.3, beetles injected with double‐stranded RNA in late larval stage. **P < 0.001.

Figure 6

Effect of Tchsp18.3 on fertility. Wild type (WT), beetles received a non‐injection; IB, beetles injected with physiological buffer; ds‐Tchsp18.3, beetles injected with double‐stranded RNA in 2‐day‐old pupae. ds‐Tchsp18.3♂ × WT♀, ds‐Tchsp18.3 male crossed with WT female adult; WT♂ × ds‐Tchsp18.3♀, WT male crossed with ds‐Tchsp18.3 female adult. **P < 0.001.