Midgut serine proteinases participate in dietary adaptations of the castor (Eri) silkworm Samia ricini Anderson transferred from Ricinus communis to an ancestral host, Ailanthus excelsa Roxb

Abstract

Dietary change influenced the life-history traits, nutritional utilization, and midgut serine proteinases in the larvae of the domesticated polyphagous S. ricini, transferred from R. communis (common name: castor; family Euphorbiaceae; the host plant implicated in its domestication) to A. excelsa (common name: Indian tree of heaven; family Simaroubaceae; an ancestral host of wild Samia species). Significantly higher values for fecundity and body weight were observed in larvae feeding on R. communis (Scr diet), and they took less time to reach pupation than insects feeding on A. excelsa (Scai diet). Nevertheless, the nutritional index for efficiency of conversion of digested matter (ECD) was similar for larvae feeding on the two plant species, suggesting the physiological adaptation of S. ricini (especially older instars) to an A. excelsa diet. In vitro protease assays and gelatinolytic zymograms using diagnostic substrates and protease inhibitors revealed significantly elevated levels (p ≤ 0.05) of digestive trypsins, which may be associated with the metabolic costs influencing slow growth in larvae feeding on A. excelsa. RT-PCR with semidegenerate serine proteinase gene-specific primers, and cloning and sequencing of 3' cDNA ends identified a large gene family comprising at least two groups of putative chymotrypsins (i.e., Sr I and Sr II) resembling invertebrate brachyurins/collagenases with wide substrate specificities, and five groups of putative trypsins (i.e., Sr III, Sr IV, Sr V, Sr VII, and Sr VIII). Quantitative RT-PCR indicated that transcripts belonging to the Sr I, Sr III, Sr IV, and Sr V groups, especially the Sr IV group (resembling achelase I from Lonomia achelous), were expressed differentially in the midguts of fourth instars reared on the two plant species. Sequence similarity indicated shared lineages with lepidopteran orthologs associated with expression in the gut, protein digestion, and phytophagy. The results obtained are discussed in the context of larval serine proteinases in dietary adaptations, domestication, and exploration of new host plant species for commercial rearing of S. ricini.

Keywords: digestive physiology; domestication; host plant choice; larval gut gene expression; non-mulberry silkworm; nutrition; performance; serine proteinases.

Figure 1

Life history traits of Samia ricini reared on Ricinus communis (Scr) and Ailanthus excelsa (Scai) diets. (A) Survival rate = percentage of insects reaching adult stage; (B) realized fecundity = average number of eggs laid by a moth; (C) fresh weight (g) of pupae; (D) fresh weight (g) of newly eclosed silkmoths; and (E) cocoon shell weight (g). The sample size (n) for each experiment on life history was ≥50; except for realized fecundity where three non-sib females from each diet were used. Bars (Scr, blue; Scai, red) depict mean ± SE. Significant differences at a p-value ≤0.05 are denoted by different letters; and (F) regression plot shows the log-larval fresh weight (mg) in relation to larval duration (days) of S. ricini fed on R. communis (Scr, blue line) and A. excelsa (Scai, red line) diets. The weight of individual larva fed on a Scr diet (blue) and Scai diet (red) is shown as a circle. The relation was linearized using the log-larval weight. R ²-values are provided.

Figure 2

No-choice fixed-time feeding assays were carried out to determine the nutritional indices of third (III), fourth (IV), and fifth (V) instars reared on a Scr diet (blue bars) and Scai diet (red bars). The parameters compared were (A) leaf tissues consumed (PWC, g); (B) larval weight gain (LWG, g); (C) fecal matter produced (FMP, g); (D) efficiency of conversion of digested food, ECD; (E) approximate digestibility, AD; and (F) efficiency of conversion of ingested food, ECI. Bars depict mean ± SE. Significant differences at a p-value ≤ 0.05 are denoted by different letters.

Figure 3

In vitro assays for midgut (A) trypsin and (B) chymotrypsin activities detected per minute per mg total protein in (a) gut extracts (GE) of third (III)-, fourth (IV)-, and fifth (V)-instar Samia ricini larvae fed on Scr and Scai diets. The amidolytic substrates used were BApNA for trypsins and SucAAPFpNA for chymotrypsins along with two plant protease inhibitors, (b) STI and (c) SBBI. Bars depict mean ± SE. Significant differences at a p-value ≤ 0.05 are denoted by different letters. Percent mean trypsin and chymotrypsin activities detected after inhibition by various inhibitors are shown with reference to the total proteolytic activity detected in the absence of inhibitors (GE) taken as 100%. Correlations of the larval weights of third (III), fourth (IV), and fifth (V) instars of S. ricini fed on Scr and Scai diets with (C) midgut trypsin activities and (D) midgut chymotrypsin activities are shown. Kendall tau coefficients (R ²) are provided.

Figure 4

Gelatinolytic zymograms obtained with midgut proteases of (A) third, (B) fourth, and (C) fifth instars of Samia ricini reared on Ricinus communis (Scr) and Ailanthus excelsa (Scai) diets. Differences in activity zones are shown when the midgut samples were incubated with STI, SBBI, TLCK, and E-64 protease inhibitors (1 mg/mL; Sigma-Aldrich; Cat# E3132). Each lane contains equal amounts of total midgut protein. The star-shaped marker denotes a zone of gelatinolytic activity that distinguishes protease complexes (with different mobility and/or inhibitor-susceptibility) detected in gut samples of S. ricini larvae feeding on Scr and Scai diets.

Figure 5

A Bayesian tree based on serine proteases of Samia ricini and closely related homologs identified from BLAST searches ( Table S1 ). Labels for sequences annotated in NCBI as putative trypsins (T), chymotrypsins (C), and/or serine proteases (SP) are blue, green, and red, respectively. Clades containing S. ricini sequences from this study are highlighted in purple. Each label contains an accession number, type of protease, and abbreviated insect and family names. Green and blue circles on various nodes represent sequence lineages with members sharing ≥60% sequence similarity. Bayesian posterior probability values ≥0.5 are shown. Digestive cysteine protease from Bombyx mori (GenBank accession# XP_004926025) was used as the outgroup.

Figure 6

Relative fold gene expression of putative midgut chymotrypsins (Sr1, Sr2) and putative midgut trypsins (Sr3, Sr4, Sr5, Sr7) in fourth-instar Samia ricini reared on Ricinus communis (Scr) and Ailanthus excelsa (Scai) diets. The colors of the histograms for larvae reared on the Scr diet and Scai diet are blue and red, respectively. Decorations within histograms for various genes are as follows: Sr1—diagonal stripes; Sr2—speckles; Sr3—filled dots; Sr4—vertical dashes; Sr5—horizontal stripes; and Sr7—bricks. The data were normalized with respect to levels of elongation factor 1 alpha gene (EF1-α) transcripts and GenBank accession #KX951450 from Scai samples (black bar). Bars depicting the standard error are also shown.