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β-Glucosidases from saliva and gut tissue of <i>Z. filipendulae</i> do not hydrolyse linamarin and lotaustralin.

<p><b>A.</b> Hydrolysis of cyanogenic glucosides (CNglcs) with corresponding HCN release is visualized by Feigl-Anger paper, or <b>B.</b> via fluorescence of methylumbelliferone, the hydrolysis product of the generic substrate 4-methylumbelliferyl β-D-glucopyranoside (MUG; in black). The β-glucosidases (BGDs) extracted from the saliva and gut are active enzymes as they hydrolyse MUG (<b>B.</b>), as well as prunasin in case of the gut β-glucosidase (<b>A.</b>). Importantly, the two CNglcs linamarin and lotaustralin present in the food plant <i>L. corniculatus</i> (indicated by *) are not hydrolysed by the saliva and gut β-glucosidases. Linamarin and lotaustralin are neither hydrolysed if tested individually (<b>A.</b>, top), nor hydrolysed if tested using a <i>cyd2</i> leaf macerate (<b>A.</b>, bottom). A macerate of <i>L. japonicus cyd2</i> mimics digestion of a leaf containing linamarin and lotaustralin, but does not release HCN as it lacks the corresponding BGD.</p

HCN emission from <i>L. corniculatus</i> leaf macerates is strongly reduced in the highly alkaline midgut.

<p>The pH of <i>L. corniculatus</i> leaf macerates is slightly acidic (5.9±0.1 SD, N = 10, green dotted line), whereas the pH measured in the midgut lumen of <i>Z. filipendulae</i> larvae is highly alkaline (10.6±0.1 SD, N = 11, blue dotted line). HCN emission from leaf disc macerates is highest at pH 5–6, which matches the pH of <i>L. corniculatus</i> leaf macerates. However, HCN emission is significantly reduced under highly alkaline conditions at pH 10–11 present in the midgut lumen of <i>Z. filipendulae</i> larvae (one-tailed Student’s t-test, P<0.001). Each data point represents the mean (±SE) of ten independent incubations, i.e. 90 leaf discs were analysed in total.</p

Saliva extracts of <i>Z. filipendulae</i> do not inhibit plant cyanogenesis.

<p>Feigl-Anger paper showing HCN emission over time from leaf macerates of <i>L. corniculatus</i> and <i>L. japonicus</i> (wild-type MG-20) incubated with either: insect saliva of <i>Z. filipendulae</i> larvae, water or heat-inactivated saliva as control (latter only on MG-20). When leaf macerates of both <i>Lotus</i> species are mixed with insect saliva, HCN emission increases at a similar rate as the leaf macerate incubated with water or heat-inactivated saliva.</p

The mandible morphology of <i>Z. filipendulae</i> enables leaf-snipping to ingest and digest large leaf fragments.

<p><b>A.</b> Larva of <i>Z. filipendulae</i> feeding on its host plant <i>L. corniculatus</i>, which contains the cyanogenic glucosides linamarin and lotaustralin. The mouthparts including the mandibles are indicated by an arrowhead. The larva is ∼ 2.5 cm long. <b>B.</b> Frontal-ventral view of the head with the two mandibles laying partly over each other. The distance between the bases of both mandibles is ∼ 600 µm (arrowheads). The leaf-processing area of the mandible is indicated by a dashed line. Both mandibles are partly covered by the labrum in a closed position. <b>C.</b> The right mandible viewed dorsally showing a round, concave and non-toothed shape with a length of ∼ 400 µm and a width of ∼ 300 µm. The leaf-processing area is indicated by a dashed line. <b>D.</b> The larval gut content shows that ingested <i>L. corniculatus</i> leaf fragments are relatively large and match the dimensions and morphology of the two mandibles. <b>E.</b> Detail of a representative <i>L. corniculatus</i> leaf fragment from the larval gut which is ∼ 550×450 µm - a similar size is retained in the frass (<b>F.</b>)<b>.</b></p

Chemical Defense Balanced by Sequestration and <i>De Novo</i> Biosynthesis in a Lepidopteran Specialist

<div><p>The evolution of sequestration (uptake and accumulation) relative to <i>de novo</i> biosynthesis of chemical defense compounds is poorly understood, as is the interplay between these two strategies. The Burnet moth <i>Zygaena filipendulae</i> (Lepidoptera) and its food-plant <i>Lotus corniculatus</i> (Fabaceae) poses an exemplary case study of these questions, as <i>Z. filipendulae</i> belongs to the only insect family known to both <i>de novo</i> biosynthesize and sequester the same defense compounds directly from its food-plant. <i>Z. filipendulae</i> and <i>L. corniculatus</i> both contain the two cyanogenic glucosides linamarin and lotaustralin, which are defense compounds that can be hydrolyzed to liberate toxic hydrogen cyanide. The overall amounts and ratios of linamarin and lotaustralin in <i>Z. filipendulae</i> are tightly regulated, and only to a low extent reflect the ratio in the ingested food-plant. We demonstrate that <i>Z. filipendulae</i> adjusts the <i>de novo</i> biosynthesis of CNglcs by regulation at both the transcriptional and protein level depending on food plant composition. Ultimately this ensures that the larva saves energy and nitrogen while maintaining an effective defense system to fend off predators. By using <i>in situ</i> PCR and immunolocalization, the biosynthetic pathway was resolved to the larval fat body and integument, which infers rapid replenishment of defense compounds following an encounter with a predator. Our study supports the hypothesis that <i>de novo</i> biosynthesis of CNglcs in <i>Z. filipendulae</i> preceded the ability to sequester, and facilitated a food-plant switch to cyanogenic plants, after which sequestration could evolve. Preservation of <i>de novo</i> biosynthesis allows fine-tuning of the amount and composition of CNglcs in <i>Z. filipendulae</i>.</p></div

Biosynthesis of cyanogenic glucosides in <i>Z. filipendulae</i>.

<p>R represents a methyl group in linamarin and an ethyl group in lotaustralin.</p

Tissue specific localization of the cyanogenic glucoside biosynthetic pathway in <i>Z. filipendulae</i> larvae.

<p>Tissue localization of CYP405A2 in a representative larva determined by immunolocalization on 100 µm transverse larval sections using fluorescein isothiocyanate (FITC) labeled polyclonal antibodies and analyzed using fluorescence microscopy. A) Background control labeling with FITC as observed without primary antibodies. B) Polyclonal antibodies raised against CYP405A2. cII, small (type II) cuticular cavity; ep, epidermis; fb, fat body; hf, hair follicle; sc, sensory cell; and sh, sensory hair (seta). Scale bars: 100 µm.</p

Tissue specific cyanogenic glucoside biosynthetic gene expression in <i>Z. filipendulae</i> larvae.

<p>Tissue localization of <i>CYP405A2</i>, <i>CYP332A3</i> and <i>UGT33A1</i> in a representative larva determined by <i>in tube in situ</i> PCR analysis on 80 µm transverse sections using tetramethylrhodamine isothiocyanate (TRITC) and analyzed using fluorescence microscopy. A–D) Controls to visualize the different cell types analyzed using light microscopy, with 4.7 ms exposure time. E) Negative control excluding primers in the PCR reaction, visualized with red light excitation and 370.4 ms exposure time. F) Negative control visualized with red light excitation and 960.8 ms exposure time. G–I) Expression of CNglc biosynthetic genes as monitored by TRITC labeling, visualized with red light excitation and 960.8 ms exposure time. J) Expression of <i>UGT33A1</i> as monitored by TRITC labeling, visualized with red light excitation and at 370.4 ms exposure time. br, basal ring; cII, small (type II) cuticular cavity; ep, epidermis; fb, fat body; hf, hair follicle; lc, lamellate cuticle; sc, sensory cell; and sh, sensory hair (seta). Scale bars: 100 µm.</p

<i>Z. filipendulae</i> larval anatomy.

<p>A) Larva releasing defense droplets, containing CNglcs, from large (type I) cuticular cavities following physical irritation. B) Graphical representation of cross section of fifth instar larva. C) Close-up of larval integument with cavities and hairs. br, basal ring; cI, large (type I) cuticular cavity; cII, small (type II) cuticular cavity; ep, epidermis; fb, fat body; gu, gut; ha, haemolymph; hf, hair follicle; in, integument; lc, lamellate cuticle; Mt, Malpighian tubules; sc, sensory cell; and sh, sensory hair (seta). Drawings were made by Kirsten Lehrmann Madsen.</p