The Set of Serine Peptidases of the Tenebrio molitor Beetle: Transcriptomic Analysis on Different Developmental Stages

Abstract

Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor.

Keywords: Tenebrio molitor; digestion; expression patterns; phylogenetic analysis; polypeptidases; serine peptidase homologs; serine peptidases.

Figure 1

Total number of SP and SPH genes found in sequenced genomes of insects from different orders. Data on SP are shaded in blue, data on SPH are in yellow, and undifferentiated data on the sum of SP/SPH genes are shaded in green.

Figure 2

Domain organization of 64 trypsins and 10 trypsin-like SPs of T. molitor. Regulatory domains are marked with different shapes and colors. Description for domains: SignalP—signal peptide; TM—transmembrane domain; Clip (B/C/D)—Clip domain; CUB—C1r/C1s, Uegf, Bmp1 domain; TSP—thrombospondin domain; LDL—Low-Density Lipoprotein receptor class A repeat; CBM—Chitin-Binding Domain; SRCR—Scavenger Receptor Cysteine-Rich domain; Fz—Frizzled domain; PAN—Plasminogen-Apple-Nematode domain.

Figure 3

Domain organization of 30 chymotrypsin-like peptidases, 18 elastase-like peptidases, and 15 non-annotated peptidases of T. molitor. Regulatory domains are marked with different shapes and colors. Description for domains: SignalP—signal peptide; TM—transmembrane domain; LDL—Low-Density Lipoprotein receptor class A repeat; Sushi—Sushi domain; GD—Gastrulation Defective domain.

Figure 4

Domain organization of 125 SPHs and 7 polypeptidases of T. molitor. Regulatory domains are marked with different shapes and colors. Description for domains: SignalP—signal peptide; TM—transmembrane domain; Clip—Clip domain; SEA—Sperm protein, Enterokinase, and Agrin domain; LDL—Low-Density Lipoprotein receptor class A repeat; SRCR—Scavenger Receptor Cysteine-Rich domain; Fz—Frizzled domain; Sushi—Sushi domain; EGF—laminin/Epidermal Growth Factor-like domain.

Figure 5

Phylogenetic analysis of 269 SPs and SPHs of T. molitor. Complete protein sequences were aligned using MAFFT. The phylogenetic tree was built in the IQTREE service. Peptidases in the tree are divided into two groups: (A) (red)—SP and SPH without regulatory domains; (B) (blue)—SP and SPH with regulatory domains (including polypeptidases). For the interpretation of the colors of the identifiers, see the legend above.

Figure 6

Heatmaps of stage-specific expression pattern of 269 SP/SPH transcripts of T. molitor. The hierarchical clustering of RPKM values was used to compare the relative expression levels of transcripts from different T. molitor life stages transcriptomes, differentiated into 6 distinct groups. Groups 1–4—SP/SPH without regulatory domains in the propeptide; groups 5–6 have regulatory domains. Group 1 (1a–d) (red)—SPs expressed in feeding stages, group 2 (2a–c) (purple)—SPs expressed at the stages of development, metamorphosis or also at other stages of the life cycle, group 3 (3a–d) (orange)—SPHs expressed at feeding stages; group 4 (4a,b) (yellow)—SPHs expressed at the stages of development, metamorphosis or also at other stages of the life cycle, group 5 (5a–g) (blue)—SPs and SPHs containing clip domains; group 6 (6a–c) (green)—SPs, SPHs, and polypeptidases containing other than clip regulatory domains. The level of mRNA expression is presented as a heatmap from blue to red (log₂(RPKM + 1)). The resulting p-values were adjusted using the Benjamini and Hochberg approach [64]. Values p < 0.05 are colored green, indicating the significance of differences in the expression at different stages of T. molitor development; values from 0.05 to 0.1 are colored yellow; values greater than 0.1 are colored red, showing the unreliability of differences in the expression values at different stages of T. molitor development. The colors of SP/SPH names indicate the types of SPs: trypsins (TRY)—blue, trypsin-like, (TRY-like)—light blue, chymotrypsin-like, (CHYM)—purple, elastase-like, (ELA)—orange, (NA)—non-annotated, grey, pSerp—polypeptidases, TM—transmembrane domain. Designations for regulatory domains: Clip-A—brown; Clip-B—blue; Clip-C—light blue; Clip-D—grey-blue; Sushi—green; GD—red; MSP—blue-green; peptidases with several regulatory domains—dark blue. Life cycle stages: E—egg, LII—second instar larvae, LIV—four instar larvae, EP—early pupa, LP—late pupa, M—male, F—female.