Consequences of 'no-choice, fixed time' reciprocal host plant switches on nutrition and gut serine protease gene expression in Pieris brassicae L. (Lepidoptera: Pieridae).

Rapid adaptive responses were evident from reciprocal host-plant switches on performance, digestive physiology and relative gene expression of gut serine proteases in larvae of crucifer pest P. brassicae transferred from cauliflower (CF, Brassica oleracea var. botrytis, family Brassicaceae) to an alternate host, garden nasturtium, (GN, Tropaeolum majus L., family Tropaeolaceae) and vice-versa under laboratory conditions. Estimation of nutritional indices indicated that larvae of all instars tested consumed the least food and gained less weight on CF-GN diet (significant at p≤0.05) as compared to larvae feeding on CF-CF, GN-GN and GN-CF diets suggesting that the switch to GN was nutritionally less favorable for larval growth. Nevertheless, these larvae, especially fourth instars, were adroit in utilizing and digesting GN as a new host plant type. In vitro protease assays conducted to understand associated physiological responses within twelve hours indicated that levels and properties of gut proteases were significantly influenced by type of natal host-plant consumed, change in diet as well as larval age. Activities of gut trypsins and chymotrypsins in larvae feeding on CF-GN and GN-CF diets were distinct, and represented shifts toward profiles observed in larvae feeding continuously on GN-GN and CF-CF diets respectively. Results with diagnostic protease inhibitors like TLCK, STI and SBBI in these assays and gelatinolytic zymograms indicated complex and contrasting trends in gut serine protease activities in different instars from CF-GN diet versus GN-CF diet, likely due to ingestion of plant protease inhibitors present in the new diet. Cloning and sequencing of serine protease gene fragments expressed in gut tissues of fourth instar P. brassicae revealed diverse transcripts encoding putative trypsins and chymotrypsins belonging to at least ten lineages. Sequences of members of each lineage closely resembled lepidopteran serine protease orthologs including uncharacterized transcripts from Pieris rapae. Differential regulation of serine protease genes (Pbr1-Pbr5) was observed in larval guts of P. brassicae from CF-CF and GN-GN diets while expression of transcripts encoding two putative trypsins (Pbr3 and Pbr5) were significantly different in larvae from CF-GN and GN-CF diets. These results suggested that some gut serine proteases that were differentially expressed in larvae feeding on different species of host plants were also involved in rapid adaptations to dietary switches. A gene encoding nitrile-specifier protein (nsp) likely involved in detoxification of toxic products from interactions of ingested host plant glucosinolates with myrosinases was expressed to similar levels in these larvae. Taken together, these snapshots reflected contrasts in physiological and developmental plasticity of P. brassicae larvae to nutritional challenges from wide dietary switches in the short term and the prominent role of gut serine proteases in rapid dietary adaptations. This study may be useful in designing novel management strategies targeting candidate gut serine proteases of P. brassicae using RNA interference, gene editing or crops with transgenes encoding protease inhibitors from taxonomically-distant host plants.

Eco-friendly synthesis of CuInS2 and CuInS2@ZnS quantum dots and their effect on enzyme activity of lysozyme.

We report on the green and facile aqueous microwave synthesis of glutathione (GSH) stabilized luminescent CuInS2 (CIS, size = 2.9 nm) and CuInS2@ZnS core-shell (CIS@ZnS, size = 3.5 nm) quantum dots (QDs). The core-shell nanostructures exhibited excellent photo- and water/buffer stability, a long photoluminescence (PL) lifetime (463 ns) and high PL quantum yield (PLQY = 26%). We have evaluated the comparative enzyme kinetics of these hydrophilic QDs by interacting them with the model enzyme lysozyme, which was probed by static and synchronous fluorescence spectroscopy. The quantification of the QD-lysozyme binding isotherm, exchange rate, and critical flocculation concentration was carried out. The core-shell QDs exhibited higher binding with lysozyme yielding a binding constant of K = 5.04 × 109 L mol-1 compared to the core-only structures (K = 6.16 × 107 L mol-1), and the main cause of binding was identified as being due to hydrophobic forces. In addition to the enzyme activity being dose dependent, it was also found that core-shell structures caused an enhancement in activity. Since binary QDs like CdSe also show a change in the lysozyme enzyme activity, therefore, a clear differential between binary and ternary QDs was required to be established which clearly revealed the relevance of surface chemistry on the QD-lysozyme interaction.