DOI: 10.15482/usda.adc/1528422
title: Data from: Attraction, mobility, and preference by Lasioderma serricorne (F.) (Coleoptera: Ptinidae) to microbially-mediated volatile emissions by two species of fungi in stored grain

description: Our goals were to 1) isolate, and culture two fungal morphotypes, 2) characterize the volatile emissions from grain inoculated by each fungal morphotype (Aspergillus flavus or Fusarium spp.) compared to uninoculated and sanitized grain, and 3) understand how MVOCs from each morphotype affects mobility, attraction, and preference by L. serricorne. Headspace collection revealed that the Fusarium- and A. flavus-inoculated grain produced significantly different volatiles compared to sanitized grain or the positive control. Changes in MVOC emissions affected close-range foraging during an Ethovision assay, with a greater frequency of entering and spending time in a small zone with kernels inoculated with A. flavus compared to other treatments. In the release-recapture assay, MVOCs were found to be attractive to L. serricorne at a longer distances in commercial pitfall traps. While there was no preference shown among semiochemical stimuli in a still-air, four-way olfactometer, it is possible that methodological limitations prevented robust interpretation from this assay. Overall, our study suggests that MVOCs are important for close- and long-range orientation of L.serricorne during foraging, and that MVOCs may have the potential for inclusion in behaviorally-based tactics for this species.
4-way olfactometer assay - Sierra_2021_olfactometer_Ag_Data_commons.csv
In order to assess preference by L. serricorne among the stimuli, a still-air four-way olfactometer was used. This device (Sigma Scientific, LLC., Micanopy, FL, USA) consisted of a cylindrical central release glass chamber (9.0 × 12.1 cm D: H) with exit ports (2.5 × 3.5 cm D:L) at each cardinal direction connecting the central chamber to four identical glass chambers (7.0 × 11.5 cm D:H) where 10 g of each odor source was placed (Table 1). The bottom of the release arena was acid-etched to provide a surface over which insects could easily crawl. Each of the four outer chambers were spaced at a 90° angle from each other. Inert septa buffered and sealed each component. A removable glass lid with a hole was used to cover the top of the main central glass chamber to allow for airflow, but also prevent insects from exiting. A single L. serricorne was released into the middle of the central arena for each bioassay and given a maximum of 4 min to make a decision to choose one of the stimuli. A choice was considered to have been made when the individual traveled 2.54 cm into the adjoining 1-way exit port that branched off to the odor chamber. After every 5 replicates, the entire olfactometer was rotated 90° to control for positional effects. After every 15 replicates, the whole apparatus was washed first with methanol followed by hexane, and a new, independent set of each treatment was used, with the grain replaced. The time to decision and choice of odor treatment were recorded and an individual insect was never tested more than once. Non-responsive (NR) insects were also recorded, but excluded from the final statistical analysis. The assay was conducted in a biosafety cabinet to maintain a constant flow of air and odor. A total of n = 200 replicate individuals were tested.
Release-recapture Assay - sierra_release_recapture_exp_2021_fungal_volatiles_agdata_commons.csv
A trapping assay was utilized to determine how microbial fungal volatiles impact the capture by L. serricorne in monitoring traps at a distance. In a large, walk-in environmental chamber (5 × 6 × 2 m, L:W:H), plastic bins (86.3 × 30.5 × 39.4 cm L:H:W) were set up to perform the experiment under constant environmental conditions (28°C, 59% RH, 14:10 L:D). A ring of polytetrafluoroethylene (Millipore Sigma, Burlington, MA, USA) around the top and bottom of the plastic bins was added to prevent insects from climbing out of the bins. The bottom of the bins was roughened with sandpaper to provide traction. The traps consisted of commercially-available pitfall traps (Storgard, Trece, Adair, OK, USA) with two connectible pieces (Doud and Phillips 2020; Doud et al. 2021), containing a central well where the treatments were added as bait. Traps were baited with treatments in Table 1 and placed in one corner inside the bins. A total of 20 adult, mixed-sex L. serricorne were released in the corner opposite from where the trap was placed. The position of traps were randomized in a different corner inside the bin to reduce any positional bias between each replicate. The insects were given 24 h to respond to stimuli. Afterwards, traps were collected, and the number of insects recaptured was recorded. A total of n = 8 replicate releases were performed per treatment.
Ethovision Movement Assay - ethovision_sierra_2021_microbial_volatiles_agdatacommons.csv
A movement assay was used to assess if fungal inoculated grain impacted close-range foraging behavior of L. serricorne. This was accomplished through the use of video-tracking with a network camera (GigE Network Camera, Basler AG, Germany), suspended using a pole and clamp 80 cm above the test arenas, coupled with Ethovision Software (Noldus Inc., Leesburg, VA, USA). The test arenas consisted of five petri dishes (100 × 15 mm) with 85 mm diameter filter paper adhered to the bottom of the petri dish with double sided tape, on an artist’s lightbox, each containing a single L. serricorne and a single treatment kernel (Table 1). Each test arena was halved vertically, with the left half designated for the control half (e.g., no kernel) and the right half for the treatment half. Embedded within each half of the arenas were 1.16 cm diameter control kernel zones the treatment kernel zones, located midway and halfway on each respective half (Supplemental Figure 1). These were coded as hidden zones in Ethovision and the smaller treatment kernel zones were where the grain treatments were placed, consisting of a single kernel of each treatment listed in Table 1. A single L. serricorne was released at the midpoint in the center of each arena. Trials lasted for a period of 30 min. The Ethovison software automatically measured several variables including the total distance moved, the cumulative duration of time spent in each half/kernel zone, the frequency of entry to each half/kernel zone, and latency to entering the half/kernel zone. The location of each treatment was randomized between each replicate to control any positional bias. There was a total of n = 20 replicates per treatment. No petri dish, filter paper, or adult was ever used more than once.
Headspace volatile collection assay - headspace_compounds_sierra_2021_fungal_volatiles_final_agdatacommons.csv
To determine how microbial colonization impacted volatile emissions from grain, headspace collections were performed on fungal-inoculated, uninoculated, and UV-treated grain. The headspace volatile collection apparatus consisted of four 500-ml glass chambers, each with an air inlet and outlet. Central air was first purified, then regulated with a flow meter to 1 L/m, after which it entered the headspace chamber via PTFE tubing and was collected onto a volatile collection trap (VCT). Each glass chamber was secured during headspace volatile collection with a plastic lid and a PTFE-faced butyl septum. After each use, chambers were washed with methanol first followed by hexane inside a laminar fume hood. The VCT consisted of an angled drip-tip collection point borosilicate glass tube with a mesh (Stainless Steel #316 screen), packed with 20 mg of PoraPak-Q™ chemical absorbent held in place with a borosilicate glass wool plug, and followed by a PTFE Teflon™ compression seal, which was used to collect and concentrate MVOCs over 3 h periods from 20 g of each treatment (Table 1). Background volatiles were also collected from empty glass chambers as a negative control. Volatiles were eluted from VCTs inside the fume hood with 150 µl of dichloromethane, which was pushed through with N2 gas into labeled 2 mL GC vials with 150-µl glass inserts with PTFE polymer feet and magnetic seal caps. Using a microsyringe (2-μl capacity, Hamilton Co., Reno, NV, USA), 1 µl of internal standard (190.5 ng tetradecane) was added to each sample. Between each use, the syringe was washed with 2 µl of dichloromethane in triplicate to avoid cross contamination between samples. All headspace samples were sealed with Teflon tape and stored at -13°C until GC-MS analysis below. For reuse of VCT, traps were washed in triplicate with 700 µl of dichloromethane, which was pushed through with N2 gas.
All headspace collection sample extracts were run on an Agilent 7890B gas chromatograph (GC) equipped with an Agilent Durabond HP-5 column (30 m length, 0.250 mm diameter and 0.25 μm film thickness) with He as the carrier gas at a constant 1.2 mL/min flow and 40 cm/s velocity, which was coupled with a single-quadrupole Agilent 5997B mass spectrometer (MS). The compounds were separated by auto-injecting 1 μl of each sample under split mode into the GC-MS at room temperature (approximately 23°C). The flow was split in a 15:1 ratio with a split flow rate of 18 ml/min. The GC program consisted of 40°C for 1 min followed by 10°C/min increases to 300°C and then held for 26.5 min. After a solvent delay of 3 min, mass ranges between 50 and 550 atomic mass units were scanned. Compounds were tentatively identified by comparison of spectral data with those from the NIST 17 library and by GC retention index (Adams 2007). Using the ratio of the peak area for the internal standard to the peak area for the other compounds in the headspace, the emission rates of samples was normalized in ng of volatile per g of grain, per μl of solvent, and per h of collection were calculated.
Sequencing Data - sequencing_data.zip
2.2 Fungal Isolation
To initially isolate the two fungal morphotypes, L. serricorne were allowed to disperse on agar. A total of 32 g of potato dextrose agar (Merck, Darmstadt, Germany) was mixed with 900 ml of deionized water in a 1000-mL glass media bottle with a magnetic stirring rod placed inside the bottle. The agar solution was autoclaved (533LS, Getinge, Rochester, NY, USA) for 30 min and then stirred on a hot plate to cool down for 20 min. Before pouring the potato agar solution into petri dishes (100 × 15), the biosafety cabinet (75 × 73 × 95 cm L:H:W, #302381101, Labconco, Kansas City, MO, USA) was sanitized with 70% ethanol and exposed to UV light for 10 min. A total of 36 petri dishes with potato dextrose agar solution were left in the biosafety cabinet to solidify overnight.
A single L. serricorne was introduced into to a single dish and microbial growth was visualized after 3 and 5 d. Each agar petri dish was sealed with parafilm, and stored in an environmental chamber under constant conditions of 30°C, 60% RH, and 14:10 L:D photoperiod. Transfer of L. serricorne from containers to agar was performed inside the biosafety cabinet to prevent contamination. Pictures of the agar dishes and corresponding microbial growth using a DSLR camera (EOS 7D Mark II, Canon, Tokyo, Japan) mounted to 3D imaging StackShot (CogniSys, Inc., Traverse City, MI, USA) equipped with a dual flash (MT-26EX-RT, Canon, Tokyo, Japan). Light was diffused using a partially cut occluded plastic jar (15.2 × 7.6 cm D:H) making a total of 12 replicates. This was used to generate the two primary morphotypes for the rest of the assays in the experiments below, and to generate sequences for the morphotypes.
Microbial Community Isolation & Sequencing
To identify microbes associated with the cuticle of L. serricorne, fungal morphotypes were isolated from cuticles and cultured for the purpose of sequencing. From the fungal isolation (above), two fungal morphotypes were selected by close examination of microbial characteristics such as spore shape, size, and color. Pure isolations from each morphotypes were made by excising a 1 × 1 cm agar plug of fungi and subculturing it onto a new potato dextrose agar dish, sealed with parafilm to obtain a pure culture. DNA was extracted from pure cultures after 7 d of growth in an environmental chamber set to 30°C, 60% RH, and 14:10 L:D photoperiod using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (D6010, Zymo Research Corp, Irvine, CA, USA). Concentration of DNA and quality were assessed using the Take 3 Assay on a microplate reader (Gen5™, BioTek Instruments, Winooski, VT, USA) before performing the PCR.
Polymerase chain reaction (PCR) was used to amplify the ITS region with the ITS5/4 primer set: ITS4 5’-TCCTCCGCTTATTGATATGC-3’ and ITS5 5'-GGAAGTAAAAGTCGTAACAAGG-3'. Each reaction contained 1.0 μl extracted DNA, 1.0 uL of each primer (10 μM), 9.5 μl of nuclease free water, and 12.5 μl of master mix containing 50 units/ml of Taq DNA polymerase master mix (Hot Start Taq 2X Master Mix, Promega, Madison, WI, USA). Briefly, the PCR program consisted of 2 min of initial denaturation at 95°C, followed by 30 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1.5 min, and a final extension at 72°C for 5 min (S1000 Thermal Cycler, Bio-Rad, Hercules, CA, USA). PCR products were treated with ExoSAP-it prior to sequencing following the manufacturer’s protocol (ThermoFisher Scientific, Waltham, MA, USA) Amplicons were sequenced bidirectionally on an ABI 3730XL instrument (Eurofins Scientific, Brussels, Belgium), and the resulting sequences were quality-filtered and aligned using Sequencher (v. 5.4.6, Gene Codes, Ann Arbor, MI, USA). The consensus sequences were searched against NCBI’s nucleotide database (nt) using the BLASTn algorithm. In order to circumvent taxonomic misassignments, the consensus sequences were also checked against the UNITE Database using the Ribosomal Database Project Classifier algorithm.