Biocatalytic potential of Pseudolycoriella CAZymes (Sciaroidea, Diptera) in degrading plant and fungal cell wall polysaccharides

Abstract

Soil biota has a crucial impact on soil ecology, global climate changes, and effective crop management and studying the diverse ecological roles of dipteran larvae deepens the understanding of soil food webs. A multi-omics study of Pseudolycoriella hygida comb. nov. (Diptera: Sciaroidea: Sciaridae) aimed to characterize carbohydrate-active enzymes (CAZymes) for litter degradation in this species. Manual curation of 17,881 predicted proteins in the Psl. hygida genome identified 137 secreted CAZymes, of which 33 are present in the saliva proteome, and broadly confirmed by saliva CAZyme catalytic profiling against plant cell wall polysaccharides and pNP-glycosyl substrates. Comparisons with two other sciarid species and the outgroup Lucilia cuprina (Diptera: Calliphoridae) identified 42 CAZyme families defining a sciarid CAZyme profile. The litter-degrading potential of sciarids corroborates their significant role as decomposers, yields insights to the evolution of insect feeding habits, and highlights the importance of insects as a source of biotechnologically relevant enzymes.

Keywords: Biocatalysis; Mycology; Plant biology.

Graphical abstract

Figure 1

The Psl. hygida genome (A) Psl. hygida life cycle. See STAR Methods for further details. (B) Manually corrected Hi-C heatmap. The four main red squares in the diagonal correspond to the four Psl. hygida chromosomes. Chromosomes A, B, C, and X are indicated at the top and right end of the image. Scaffolds that were not integrated into the main chromosome-length scaffolds are presented in the extreme lower right square. (C) Distribution of repetitive sequences in the Psl. hygida genome. Repeat classes that represent less than 1% of the genome were grouped in the “Other” category. (D) BUSCO assessment results for the final genome assembly and the predicted gene set. The coloring scheme for the bar segments is shown in the inset of the panel. (E) Number of genes functionally annotated in each database, as indicated on the left hand of the panel. A total of 17,881 genes were annotated in the Psl. hygida genome. See also Figures S1, S3, and S4 and Tables S1–S5 and S8.

Figure 2

CAZymes distribution in the predicted Psl. hygida gene set and Gene Ontology (GO) annotation of the saliva proteome (A) Sunburst chart showing the distribution of manually curated CAZymes in the predicted Psl. hygida gene set. The inner circle shows the CAZyme classes, and the outer circle shows the numbers of annotated CAZymes found in the named families within each class. The asterisks denote CAZymes that were also identified in the Psl. hygida saliva proteome (Table S9). Those predicted CAZyme families with a single representative are shown alongside the open brackets. (B) The bars show the GO terms annotated in the saliva proteome in the Biological Process category. The labels on each bar give the percentage of each term. GO terms with less than 1% are not shown and correspond to 10.48% of the annotated proteins. (GH) Glycoside Hydrolase; (GT) Glycosyl Transferase; (AA) Auxiliary Activities; (CBM) Carbohydrate-Binding Module; (CE) Carbohydrate Esterase; (PL) Polysaccharide Lyase. See also Figure S4 and Tables S9 and S10.

Figure 3

Catalytic activity of the Psl. hygida saliva (A) Catalytic activity of saliva measured against polysaccharides at pH 5 (blue bars) and pH 8 (orange bars). The polysaccharides are as indicated in the graph. Error bars show the mean ± SD of 3 measurements from each of the two biological replicates. (B) Catalytic activity of saliva measured against synthetic p-nitrophenol monosaccharide derivatives at pH 5 (blue bars) and pH 8 (orange bars). Individual substrates are as shown in the graph. Error bars show the mean ± SD of 3 measurements from each of the two biological replicates. (C) Chromatograms using LC-MS detection of the retention times of chitin oligosaccharides of chitin samples (black lines) and after treatment with saliva (red lines). The degree of polymerization and m/z ratios of the individual chitooligosaccharides are as indicated in the relevant panel. See also Figures S6–S10 and Tables 1, S9 and S10.

Figure 4

CAZyme families and occurrences identified in the genomes of three sciarids and L. cuprina The number of enzymes in the CAZymes classes of: (A) auxiliary activities (AA). (B) carbohydrate-binding modules (CBM). (C) carbohydrate esterases (CE). (D) glycosyl transferases (GT). (E) polysaccharide lyases (PL). (F) glycosyl hydrolases (GH). Each bar includes the number of enzymes found in the given CAZy family in the four genomes analyzed, Psl. hygida (blue), B. cellarum (orange), B. tilicola (gray), and L. cuprina (yellow). For clarity, the GT (panel D) and GH (panel F) families are separated into two bar charts showing the less (i) and more (ii) numerous enzymes with suitably adjusted x axis scales. See also Table S9.

Figure 5

Comparative analysis of the number of CAZy families found in the sciarid and L. cuprina genomes (A) Venn diagram showing the number of CAZy families in the genomes of Psl. hygida, B. tilicola, and B. cellarum. (B) Venn diagram showing the number of CAZy families present in the L. cuprina genome and in a combined dataset of all CAZy families present in the genomes of Psl. hygida, B. tilicola, and B. cellarum (All sciarids). See also Table S9.