Insects and bamboos
Five internodes (length: mean ± SD = 44.8 ± 1.1 cm, n = 5; diameter in the middle part of internodes: 21.4 ± 0.8 mm, n = 5) of five living mature culms of P. simonii bamboo were sampled at Kawaminami, Miyazaki Prefecture, Japan [32°9′ N, 131°29′ E] on 6 June, 2019. Per internode, four semi-cylindrical strips (ca. 15 × 2 cm) were made and stored at − 25 °C until use.
To obtain fungus-free larvae of D. bucculenta, we sampled five beetle eggs from P. simonii bamboo obtained at Toyota, Aichi Prefecture, Japan [35°9′ N, 137°13′ E] on 9 May, 2019 in the laboratory from ovipositing females collected at Kawaminami on 10 and 11 April, 2019. The eggs were immersed in 99.5% ethanol for 10 s followed by 70% ethanol for 10 s for surface sterilization and then individually placed on potato dextrose agar (PDA) (Difco, Detroit, MI, USA) plates. The plates were incubated at 25 °C in the dark until 30 days after larval hatching to confirm the absence of the formation of yeast or other microbial colonies. Consequently, all five larvae hatched successfully and aseptically.
The bamboo used in this study was morphologically identified using the literature29. This is native to the study areas and no other host bamboo species are distributed there29. Therefore, no voucher specimen of this bamboo has been deposited in a publicly available herbarium. No specific permits were required for the described field studies. The location is not privately-owned or protected in any way. The field studies did not involve endangered or protected species. All applicable international, national, and/or institutional guidelines for the care and use of animals and plants were followed. This study is reported in accordance with ARRIVE guidelines.
Component analyses of bamboo tissues
For YP and LP, the yeast W. anomalus originating from D. bucculenta in Kawaminami (strain: DBL05Kawaminami) was cultured on a 9-cm PDA plate to obtain enough biomass for further experiments. Afterwards, yeast cells were suspended in ca. 10 mL of sterilized water, and were inoculated on the inner surface of the autoclaved internode strips using an autoclaved tissue paper immersed with the yeast suspension. For LP, additionally, the fungus-free 2nd instar larvae (weight: mean ± SD = 2.4 ± 0.4 mg, n = 5) were individually placed on the yeast-inoculated strips. Each of these yeast-inoculated and yeast-and-larva-inoculated strips was then put in a sterilized test tube (3.0 cm in diameter and 20 cm tall) with moistened cotton placed at the bottom. Each of the test tubes was covered with a sterilized polypropylene cap, sealed with Parafilm Sealing Film (Pechiney Plastic Packaging, Chicago, IL, USA) on which three small holes were made using a fire-sterilized insect pin to avoid oxygen shortage, and individually put in a plastic zipper bag. These yeasts and insects were incubated at 25 °C in the dark for 47 days for YP (n = 5), and 47 (n = 4) and 73 (n = 1) days until these larvae reached adulthood for LP (adult elytral length: mean ± SD = 9.2 ± 0.4 mm, n = 5). Microbial contamination was invisible to the naked eye.
For FP, YP and LP, the inner surface (up to 0.3 mm in thickness, dry weight: 336 to 935 mg) of a strip was sampled using a small U-shaped gouge. In the case of FX, first, the pith of a strip was completely removed, and then xylem tissue (up to 0.5 mm in thickness, dry weight: 729 to 872 mg) was sampled using a small U-shaped gouge. These tissues were individually sampled from five strips derived from five different internodes for each tissue type.
Samples were extracted by aqueous ethanol and hydrolyzed by sulfuric acid with reference to the literature30,31,32 as follows. Four types of samples were freeze-dried and pulverized using a rotor-speed mill (Fritsch, PULVERISETTE 14, 0.2 mm mesh). About 80 mg of powdered sample was extracted using 5-mL 80% ethanol aqueous solution (aq.) at 63 °C three times. The volume of the extracts was adjusted to 25 mL, filtered, and analyzed using ion exchange chromatography measurements (extractable sugar analysis). Their extracted residues were hydrolyzed using sulfuric acid as follows: 50-mg samples were immersed in 1.64-g 72% sulfuric acid aq. at 30 °C for 2 h, boiled in 39.4-g 3% sulfuric acid aq. for 4 h, and filtered to collect sulfuric acid residues as sulfuric acid lignin fractions. The volumes of the filtrates were fixed to 100 mL, passed through a sulfuric acid-removing filter (DIONEX OnGuard IIA), and submitted to ion exchange chromatography measurements (structural sugar analysis). For the uronic acid measurements, the sulfuric acid-removing filter was not used.
Ion exchange chromatography measurements were conducted using a DIONEX ICS-3000 apparatus. The measurement conditions were as follows: column, CarboPac PA-1 (2.0 mm I.D. × 250 mm L, Dionex corp.); flow rate, 0.3 mL min−1; column temperature, 30 °C; injection volume, 25 µL; eluent, H2O (solvent A), 100 mM NaOHaq. (solvent B), aqueous solution containing 100 mM NaOH and 1.0 M CH3COONa (solvent C), and aqueous solution containing 100 mM NaOH and 150 mM CH3COONa (solvent D). The gradient conditions for monomers, dimers, and uronic acids were as follows: for monomers, with a gradient of B 0.5% C 0% 45 min, C 100% 10 min, B 100% 10 min, B 0.5% C 0% 20 min; for dimers, with a gradient of B 50% C 0% 50 min, C 100% 10 min, B 100% 10 min, B 50% C 0% 15 min; for uronic acids, with a gradient of D 100% 10 min. These extraction, hydrolysis, and measurement procedures were conducted using n = 5 samples. For the structural sugars, their yield was calculated as the dehydrated state. The values of other extractives % were calculated by the subtraction of total extractable sugars % from total extractives %.
Elemental analysis (carbon, hydrogen, nitrogen) was conducted by 2400 CHNS Organic Elemental Analyzer (PerkinElmer Japan, Yokohama, Japan). About 1-mg dried samples were burned completely and the produced CO2, H2O, and N2 (after reduction of NOx species) gasses were quantified by a thermal conductivity detector.
Means of components of bamboo tissues were compared among tissue types using the Steel–Dwass test after the Kruskal–Wallis test. Calculations were performed using R 3.5.133.
Carbon assimilation test
The yeast W. anomalus (DBL05Kawaminami) was cultured aerobically in 20 mL of yeast nitrogen base (YNB) (Difco) containing 0.5% glucose at 25 °C in the dark for 2 days with shaking at 85 rpm. The culture media were centrifuged and cell pellets were suspended in sterile water, in which the OD600 was adjusted to 0.10. Fifty μL of the cell suspension was added into a tube (2 mL) with 1 mL of each of 14 different media containing YNB and one of the following carbon sources: d-glucose, d-galactose, d-mannose, d-xylose, l-arabinose, d-fructose, d-galacturonic acid, d-glucuronic acid, sucrose, cellobiose, starch from corn, xylan from corn, carboxymethyl cellulose, and no carbon source (n = 5 to 6). The concentration of each carbon source was 0.5 g L−1, except for xylan at 1.5 g L−1. The tubes were shaken at 85 rpm and incubated at 25 °C in the dark for 7 days. Afterwards, the presence of visible pellets of yeasts and OD600 were recorded to determine the growth of the strain. The degree of assimilation was scored according to the presence of the pellets and the difference in the turbidity increase (ΔOD600) between culture media containing no and a given carbon source as follows: no growth (without a pellet, ΔOD600 < 0.03), weak growth (with a pellet, 0.03 ≤ ΔOD600 < 0.10), moderate growth (with a pellet, 0.10 ≤ ΔOD600 < 0.40), and strong growth (with a pellet, 0.40 ≤ ΔOD600 < 1.00)8.
Source: Ecology - nature.com
