Nucleotide and deduced amino acid sequence of BSFL CTSL.

The dotted line indicates the putative signal peptide sequence (Met1-Ala16), and the putative mature domain is underlined (Pro175-Pro337). The triad of conserved catalytic active sites (Cys146, His285, and Asn306) is in circled frames, and Gln140 of the oxyanion hole is in the diamond frame. Five S2 subsites are defined by residues Leu190, Met191, Ala257, Leu283, and Gly286 and indicated by the square frames. The asterisk marks the stop codon at the end of the open reading frame.</p

Morphological organization and immunohistochemical localization of CTSL in BSFL.

The histology of BSFL is presented in longitudinal sections (A, B, C) and cross sections (D, E, F). H&E staining revealed the main structure of the fifth instar of BSFL(A, D). BSFL were stained with naïve mouse serum as a negative control (B, E). BSFL were stained by chromogenic IHC methods following injection of mice anti-BSFL CTSL serum (C, F). Positive signals were revealed in brown, while hematoxylin was used as a counterstain. H, head. (Bar = 500 μm).</p

Titer determination of anti-CTSL polyclonal antibodies by ELISA.

After the final immunization, antiserum was serially diluted, and the absorbance values were measured. (TIF)</p

SDS-PAGE of purified recombinant CTSL.

M: Protein molecular weight marker; lane 1, total protein extracted from E. coli BL21 (DE3); lane 2: E. coli containing pET-24a-CTSL without IPTG induction; lane 3: E. coli containing pET-24a-CTSL with IPTG induction; lane 4: soluble protein; lane 5: inclusion bodies; lane 6: purified recombinant CTSL protein. (TIF)</p

Unrooted phylogenetic tree of CTSL and other homologs.

The neighbor-joining algorithm constructed the tree using the Mega11 program based on the multiple sequence alignments by Clustal Omega. The branches indicate bootstrap values of 1,000 replicates (%). The scale bar corresponds to 0.05 estimated amino acid substitutions per site. The amino acid sequences of all species were obtained from the NCBI database. GenBank accession numbers are as follows: Drosophila bipectinata (XP017106640.1), Drosophila eugracilis (XP017068923.1), Drosophila serrata (XP020800944.1), Anopheles gambiae (XP001689282.1), Aedes aegypti (XP001655999.2), Culex quinquefasciatus (XP001848344.1), Macrobrachium rosenbergii (AJP62583.2), Penaeus monodon (AME17651.1), Danio rerio (CAA69623.1), and Homo sapiens (NP001244901.1).</p

Alignment of amino acid sequences of CTSL from representative arthropods and vertebrates.

Asterisks (*) indicate amino acids identical in all species. Dashes (–) denote gaps. The CTSL family signatures, including ERFNIN, GNYD, and GCNGG are shaded.</p

Relative expression levels of CTSL mRNA in the different larval stages.

Relative mRNA expression (2 -ΔΔct) levels of CTSL were detected using SRBR green assay and normalized to β-actin. The fold-change of CTSL expression was calculated for the 3rd, 4th, 5th, and 6th instars and compared with the 2nd instar. Bars represent mean ± S.E. (n = 3). Letters indicate significant differences (p < 0.05).</p

Dynamic expression of CTSL mRNA in response to <i>E</i>. <i>coli</i> challenge.

Relative mRNA expression (2 -ΔΔct) levels of CTSL were detected using SRBR green assay and normalized to β-actin in the 2nd (A), 3rd (B), 4th (C), 5th (D), and 6th (E) instars. The fold-change of CTSL expression was calculated at 1.5, 3, 6, 9, and 12 h post-stimulation with E. coli and compared with 0 h (negative control was non-treated BSFL). Bars represent mean ± S.E. (n = 3). Statistical significance is indicated with lowercase letters (p < 0.05).</p

Primers for RACE and quantitative real-time PCR used in this study.

Primers for RACE and quantitative real-time PCR used in this study.</p

Dynamic proteolytic activity of CTSL in response to <i>E</i>. <i>coli</i> challenge.

Percentage of CTSL activity in the fifth instar under E. coli challenge compared to negative control (cysteine proteinase inhibitor E-64). Error bars represent mean ± S.E. (n = 5). Statistical significance is indicated with different lowercase letters (p < 0.05).</p