Enhanced γ-aminobutyric acid levels promote degeneration of silk glands following spermidine supplementation in Bombyx mori

Abstract

Silk glands are modified labial glands that produce silk which has immense commercial importance. Silk is extruded out in liquid form after which the glands undergo autophagy and apoptosis during larval to pupal transition. Biogenic amines, specially spermidine and γ-aminobutyric acid (GABA) are known to play an important role in autophagy. Yet, GABA is not identified in the silk glands till now and therefore its role in autophagy remains unknown. Current study aimed to evaluate role of biogenic amines in the autophagy of silk glands. Fifth instar silkworms were fed with control and spermidine supplemented mulberry leaves under controlled conditions. Qualitative and quantitative analysis of biogenic amines were analyzed in silk glands of control and spermidine fed groups at the end of feeding stage, spinning and pre-pupal stages. Biogenic amines were significantly decreased in the silk glands from feeding stage to non-feeding prepupal stages. Elevated levels of biogenic amines; putrescine, spermidine, and spermine were observed in silk glands at pre-pupal stage in the spermidine fed group. The unknown biogenic amine whose levels were significantly elevated during silk gland degeneration in both control and spermidine fed groups was identified as GABA by spectroscopic techniques. This is the first report of the identification of GABA in the silk glands of Bombyx mori which increased significantly following spermidine supplementation, resulting in elevated levels of calcium deposits, contributing to the early degeneration of the silk glands.

Keywords: Bombyx mori; Autophagy; Biogenic amines; Calcium; GABA; Silk glands; Spermidine.

Fig. 1

Effect of Spd on BAs during feeding (F), spinning (S) and pre pupal (PP) stages. a Representative picture showing morphological changes in the SGs of Con and Spd groups stained with methylene blue. b Quantitative analysis of BA (n = 6). c Qualitative analysis of BA by silica TLC along with marker of known PA mix (M). Unknown band (UB) observed in the samples is shown by an arrow. d Densitometric analysis of Put, Spd and Spm band from TLC plate on PP stage (n = 3). C and S indicate Con and Spd groups respectively. ***p < 0.001, **p < 0.01, and *p < 0.05 are indicative of statistically significant differences

Fig. 2

TLC and HPLC analyses of unknown BA observed in the SG samples on PP stage. a Qualitative analysis of unknown BA by silica TLC on PP stage of Con and Spd treated group along with standard GABA (G). b(i) HPLC analysis of dansylated GABA standard b(ii) HPLC analysis of unknown BA eluted from TLC plate. DNS represents dansyl chloride

Fig. 3

Structural analysis of unknown dansylated BA. a LC–MS spectrum showing fragmentation of unknown BA. b MRM scan of the unknown BA showing transition of m/z 337 → m/z 170.1. c ¹H NMR of the unknown band showing hydrogens of different groups

Fig. 4

Biochemical estimations showing changes in the SGs during F, S, and PP stages. a Quantification of GABA and b Ca²⁺ levels in the SGs of Con and Spd groups. (n = 5). c Alizarin red staining of PSG sections showing Ca²⁺ deposition (white arrowsheads) in the Con and Spd groups. Scale bar represents 50 µM. ***p < 0.001, **p < 0.01 *p < 0.05

Fig. 5

Schematic representation showing the mechanism of spermidine induced silk gland degeneration in B. mori. DAO represents diamine oxidase, and ALDH represents aldehyde dehydrogenase. Green arrows represent increased levels and red arrow indicates decreased levels