Figure 1.
Schematic diagram of the exon–intron organizations of the putative Tribolium castaneum Knk-family genes.
(A–B) The exon–intron organization of each TcKnk-family- gene was determined by sequence comparison between genomic sequence and the longest available cDNA sequence. (A) Gene organization of TcKnk and TcKnk2; (B) Alternatively spliced forms of TcKnk3 (TcKnk3-FL-1, TcKnk3-FL-2, TcKnk3-5′, TcKnk3-3′-1 and TcKnk3-3′-2). Black closed boxes indicate exons (E) and lines indicate the introns. The gray colored closed box indicates exon 5a for TcKnk3-5′. The open arrowheads represent the open reading frame start codons and * indicates the stop codons. The TcKnk3-3′-transcripts are indicated using broken lines because the precise location of the 5′-ends are unknown.
Figure 2.
Northern blot analysis of TcKnk3 transcripts using 32P-labeled 5′-terminal and 3′-terminal probes.
Ten µg of extracted RNA from: V, TcVer; E9, TcKnk3-exon 9; E5, TcKnk3-exon 5 specific dsRNA-treated pharate adult insects was loaded onto an 1.5% agarose gel, transferred on to the nitrocellulose membrane and hybridized with 32−P-labeled 5′-terminal or 3′-terminal probe as described in Materials and Methods. There is a small amount of cross-hybridization of the 5′-probe and the RNA that gives a strong signal with the 3′-probe (presumed 3′-transcript; left panel, lane marked E5). The indicated sizes of the bands were based on the length of the cloned fragments and assuming a poly-A tail of ∼200 nucleotides.
Figure 3.
Phylogenetic analysis of insect TcKnk2 and TcKnk3.
Protein sequence predicted from the TcKnk3- full length transcript without exon 8a was used for analysis. MEGA 4.0 was used to construct the phylogenetic tree using the neighbor joining method [16]. Bootstrap analyses from 5000 replications are shown by each branch. Drosophila melanogaster (fruit fly) (Knk2, NP_001097889.2; Knk3, NP_001027171.1); Anopheles gambiae (African malaria mosquito) (Knk2, XP_308115.4; Knk3, XP_313250.2); Aedes aegypti (yellow fever mosquito) (Knk2, XP_001657415.1; Knk3, XP_001657668.1); Culex quinquefasciatus (southern house mosquito) (Knk2, XP_001845444.1; Knk3, XP_001864909.1); Pediculus humanus corporis (head louse) (Knk2, XP_002429468.1; Knk3, XP_002425509.1); Tribolium castaneum (red flour beetle) (Knk2, XP_973211.1; Knk3, XP_968712.2); Apis mellifera (honey bee) (Knk2, XP_393508.4; Knk3, XP_003250319.1); Nasonia vitripennis (parasitic wasp) (Knk2, XP_001602771.1; Knk3, XP_001606495.2) and Acyrthosiphon pisum (pea aphid) (Knk2, XP_001946688.2; Knk3, XP_003241759.1); Caenorhabditis elegans (roundworm) (NP_508959.1).
Figure 4.
Domain architecture of putative TcKnk-family proteins from Tribolium castaneum.
Domain analysis was done using SMART protein. Red: Signal peptide; Blue: DM13 domain; Purple: DM13 domain; Green: DOMON domain; Yellow: GPI anchor; Cyan Blue: Trans-membrane domain.
Figure 5.
Developmental stage-specific and tissue-specific expression of TcKnk-family genes by RT-PCR.
(A) Developmental expression profiles of TcKnk (includes data on TcKnk1 taken from [11]) for the purpose of comparison, TcKnk2, TcKnk3-FL, TcKnk3-5′ and TcKnk3-3′ transcripts. cDNAs were prepared from total RNA extracted from whole insects at several developmental stages including E, embryos; YL, young larvae (penultimate instar or younger); ML, mature larvae; PP, pharate pupae; P, pupae; YA, young adults (0 d- old); A, mature adults (10 d-old). (B) Tissue-specific expression of TcKnk-family genes in the feeding stage last instar larvae. M, midgut; H, hindgut; and C, carcass (whole body without gut). T. castaneum ribosomal protein-S6 (TcRpS6) was used as internal loading control for RT-PCR. Results are from 28 and 24 cycles of RT-PCR for TcKnk-family genes and TcRpS6, respectively. TcChs-A (chitin synthase-A) (epidermis-specific) and TcChs-B (chitin synthase-B) (midgut-specific) expression was also measured to rule out cross contamination with RNA from non-targeted tissues.
Figure 6.
Effect of TcKnk2 dsRNA-treatment on the development of T. castaneum.
(A) Injection of dsRNA for TcKnk2 into penultimate instar larvae, last instar larvae and pharate pupae (n = 60) led to lethal phenotype at pupal-adult molt (∼55%) and ∼15% adult hypomorphic phenotype with split elytra. (B) Specific down-regulation of TcKnk2 transcripts by RNAi. dsRNAs (200 ng per insect) for TcKnk2 were injected into pharate pupae. Three days after injection, total RNA was extracted from whole insects of at pupal stage day 3 (n = 3) and used for cDNA synthesis. (C) Specificity of TcKnk2 dsRNA-treatment. Effect of TcKnk2 dsRNA-treatment on transcripts for TcKnk, TcKnk3 and other genes involved in chitin metabolism such as TcChs-A and TcCht5 was checked by RT-PCR. T. castaneum ribosomal protein-S6 (TcRPS6) was used as internal loading control. dsRNA for T. castaneum Vermilion (TcVer) and TcKnk was injected as a control.
Figure 7.
Effect of TcKnk3 dsRNA-treatment on the development of T. castaneum.
(A) dsRNAs specific for different exons and intron of TcKnk3 (exon 6, exon 8, exon 9 and exon 8a) were injected into penultimate instar larvae, last instar larvae and pharate pupae. Approximately 60–100% of the dsRNA-treated insects showed lethal phenotype at pupal-adult molt. Exon 8 and exon 8a dsRNA-treatment led to a weaker phenotype with split elytra. (B) Specific down-regulation of TcKnk3-3′ transcripts by RNAi. dsRNA (200 ng per insect n = 60) for TcKnk3-3′ (corresponding to exon 8a) was injected into pharate pupae. Five days after injection, total RNA was extracted from pharate adult insects (n = 3) and used for cDNA synthesis. Depletion of different transcripts was checked by using pairs of transcript-specific primers (Table S1). For detection of transcripts with or without exon 8a, a pair of forward and reverse primers flanking exon 8a was used (see Table S1). TcRPS6 was used as internal loading control. TcVer dsRNA was injected as a control. RT-PCR for the TcRpS6 transcripts was carried our prior to these analyses to ensure that equal amounts of cDNA templates from different developmental stages were being used in these comparisons. Results are from 28, 28, 28 and 24 cycles of RT-PCR for TcKnk3-FL TcKnk3-5′ and TcKnk3-3′ and TcRpS6, respectively. The RT-PCR products were run on separate gels for each RT-PCR product (with different sizes for each transcript), but the figure as shown is composite showing the relevant regions only to avoid “white space”. The grainy quality of the amplification products for TcKnk3-3′ relative to the other RT-PCR products is due to different camera settings used to reveal the minor band from the alternatively spliced product with exon 8a sequences. (C) TcKnk2 or TcKnk3 (exon-9)-specific dsRNA was injected into last instar larvae and pharate pupae. Four to five days after injections, pharate pupal and pharate adult (n = 5) insects were collected for chitin content analysis by a modified Morgan–Elson method as described earlier [21]. dsRNA for TcVer and TcChs-A were injected as negative and positive controls, respectively. The mean chitin content for TcKnk dsRNA treated insects is adapted from previously published data [11].
Figure 8.
Transmission electron microscopic analysis of TcKnk2 and TcKnk3 (exon 9)-specific dsRNA-treated pharate adult elytra, lateral body wall denticles and tracheal taenidia.
Larvae in the late stages of development were subjected to RNAi using the indicated dsRNA combinations as described in legend to Fig. 7. The dsRNA-treated insects were collected at the pharate adult stage and fixed for TEM analyses as described in the “Materials and methods” section. White brackets point to the area where laminae are found in TcVer control denticles (panel D1) but absent in corresponding sections from insects treated with dsRNA for TcKnk (panel D2) or TcKnk and TcCht5 (panel D3). The arrows in panels D2 and D4 indicate electron-dense material accumulating under the bulges of Velcro-denticles; Arrowheads in the panels of tracheal sections (panels T4 and T6) indicate accumulation of electron-dense material under the taenidiae. They are absent in samples from insects treated with dsRNA both TcKnk2 and TcCht5 (panel T5) or with a mixture of dsRNAs for TcKnk2 and TcCht5 (panel T7). Scale bar, 500 nm.