Funding for this study was obtained through the Vienna Science and Technology Fund (WWTF) LS13-048 to ISD. Special thanks go to Diane W. Davidson (University of Utah; now retired) for sharing her knowledge about the Bornean COCY ants with us. We appreciate administrations of the Kuala Belalong Field Studies Center (KBFSC) and Universiti Brunei Darussalam (UBD) for project approval as well as Brunei&#39;s Forestry Department and Brunei&#39;s Research and Innovation Centre for permission to collect ants and approval and issuance of export permits. Special thanks go to UBD and KBFSC staff, especially Muhammad Salleh bin Abdullah Bat, Teddy Chua Wee Li, Masnah Mirasan, Rafhiah Kahar, Roshanizah Rosli, Rodzay Wahab, Chan Chin Mei, and Kushan Tennakoon for facilitating our research.

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The authors have nothing to disclose.

In this manuscript, we present a complete protocol for analyzing the volatilome of the content found in the hypertrophied MGRs of C. explodens minor worker ants. Since the ants used here may &#39;explode&#39; and eject their MGR contents in an uncontrolled manner when touched with forceps, it is recommended to use a &#39;soft&#39; collection technique as provided by an insect aspirator (Figure 1). For some ant species including COCY ants, it may be necessary to limit the maximum number of ants to five individuals per 50 mL vial, since otherwise self-poisoning of the ants (e.g., by accumulation of formic acid in the headspace) may occur. For freezing the ants in the field, a cool bag stuffed with deep-frozen cold packs may be used. The samples should be rapidly frozen and stored under cooled conditions (e.g., -20 &#176;C, best at -80 &#176;C), but it is not recommended to kill and store the ants in liquid nitrogen, since increased damage of their glands has been observed with this method.
The presented buffer- and solvent-free dissection methodology is suitable for obtaining the wax-like mandibular gland reservoir contents as well as other glands present in frozen worker ants. For volatilome analysis of the MGR content of C. explodens, major aspects to be considered during dissection are continuous cooling of the ant (step 2.1.4) and minimizing cross-contaminations of the MG samples with other gland contents/fluids present in the ant gaster (step 2.5, Figure 4, and Figure 15). A considerable fraction of the frozen ants may be damaged, either because the ants &#39;exploded&#39; during sampling or were damaged during transport on dry ice from the sampling site to the laboratory. If the gaster region is broken, these ants are not suitable for further analysis (Figure 2C, D). If only antennae and legs are missing or broken, these ants may still be used for extraction of the MGR contents and further analysis. Since the MGR contents of cooled C. explodens ants have a wax-like consistency it is straight-forward to isolate them with the use of dissection needles (step 2.5, Figure 3E, and Figure 14). Additional care needs to be taken while handling the sticky MGR content. It may stick to anything coming into physical contact with it and will also often adhere to the dissection forceps, which increases the risk of contamination of other samples. It is necessary to only touch the MGR content&#160;with the dissection needle and immediately transfer it into the glass vials used for extraction (steps 2.5 and 2.6). Moreover, it is recommended to clean the dissection equipment with a MeOH/H2O mixture when switching between ants or gland-types (step 2.8).
Gland contents of other ant species may be present in a liquid state even during cooling with cold packs. In this case, the whole gland including the membrane may first be isolated and punctured afterwards to obtain the content. Liquid secretions may also be obtained with the help of fine capillary pipettes. With an average body length of about 4-5 mm (without antennae), workers of C. explodens belong to the smaller species of the COCY group. Their often swollen gaster comprises about 2-2.5 mm in length and 1-1.5 mm in width. Workers of the so-far largest known species of the COCY group can reach a size of about 8 mm in body length with a gaster size of about 3 mm in length and 2.5 mm in width. Since the protocol is well suited to dissect and investigate individual ants and gasters of that size of different COCY species, the here used dissection instruments and microscope may have to be adapted to be applicable for smaller ants or smaller organs. Moreover, the number of ants per analytical sample may have to be increased as well.
While dissecting the ant for the MGR content, it is critical not to damage any other glands or the intestines - the contents of which may cross-contaminate the sample (step 2.5, Figure 4 and Figure 15). In the optimum case, after the extraction of the MGR contents, the DG, as well as the VG are visibly kept intact (Figure 4). Contaminations with the contents of the DG, which is located beneath the MGRs, are difficult to avoid completely. Reasons for this may also include the partial disruption of the gland integrity during transport on dry ice from the sampling-site to the laboratory. It is also possible that the DGs become damaged during sampling or in the process of exploding, as we have noticed for the MGR in a number of investigated ants. Since the DG contents can be analyzed the same way as the MGR contents (Sections 3 and 4), the results can be compared to the resulting data after analysis of the MGR content samples (Section 6). In the case of MGR content extracts obtained from C. explodens ants, the compounds also contained in the DG begin to elute late in the chromatogram (Figure 12). To prevent mistaking DG compounds for MGR constituents, the respective metabolites can be excluded from further analysis. From studies on other ant species it is known that DGs can also contain (highly) volatile compounds1,24, which typically elute early in the GC-chromatogram.
In previous studies dealing with the chemical composition of the MGR contents of COCY ants, whole ants or their whole gasters were analyzed16,17,18,23, whereas here the MGR contents themselves were obtained via dissection of the ants.
Analyzing dissected MGR contents allows investigators to perform a broad range of biochemical studies, including identification of metabolites contained therein. Since the performed study here focused on the identification of the volatile constituents contained in the MGR of C. explodens, GC-MS was chosen for its analysis (Section 4). For this, the extracts should ideally be measured immediately after preparation or stored at -80 &#176;C until analysis. It should be noted that (extended) storage may cause chemical alterations of the extracts (see Notes after steps 3.3 and 4.2). Since the concentration of the MGR contents may cover a range of several orders of magnitude, it may be necessary to analyze the MGR extracts at different GC split ratios. Since the two main compounds in the MGR content extract of the ants taken to exemplify the protocol caused column overloading when a split ratio of 2:1 was used, this sample was analyzed a second time at a higher split ratio of 50:1 (step 4.3.1.2and Figure 5). The GC-MS parameter settings presented in Section 4 are suitable for data generated with the GC-MS devices specified in the Table of Materials. Next to the RI calibrants (step 4.1.1), a solvent-blank (step 4.1.2) should also be analyzed to recognize non-biological artifacts and contaminants during subsequent data analysis. For&#160;metabolite identification, it is also important to measure authentic standards of annotated compounds under the same GC-MS conditions as used for analyzing the sample extracts (steps 5.4.7and following). To improve the accuracy and reliability of the final data, it is recommended to analyze all sample categories (solvent-blank, RI calibrants, sample extracts, and standards) within the same measurement sequence. Moreover, the authentic standards should be analyzed at a concentration comparable to that observed for the sample extracts. This helps to enhance the accuracy of RI values and similarity between sample and standard mass spectra, which will finally lead to increased and more meaningful metabolite annotation/identification. To this end, the standards can be repeatedly measured using different split factors.
For&#160;metabolite identification, the sophisticated software, MetaboliteDetector is used for spectra deconvolution and RI and spectra comparison to an implemented NIST-library as well as to an established in-house library (Section 5). It is recommended to run MetaboliteDetector on a 64 bit LINUX-based operating system (e.g., KUBUNTU) and to copy the generated *.CDF data files (step 4.4) from the portable data storage device onto the local hard drive. MetaboliteDetector is capable of importing raw GC-MS data in centroid or profile netCDF format. The software of most GC-MS instruments should be able to convert the recorded raw data into this format19. Before starting the data analysis, it is highly recommended to read the literature available for previous MetaboliteDetector software versions to become familiar with the features and the graphical user interface19,25,26.
For RI calibration and subsequent RI value calculation of the compound peaks present in the sample extracts, a library containing RIs and spectra of RI calibrants (here, n-alkanes) is used. Such a library can either be self-established, or a preexisting one (&#39;CalibrationLibrary_Alkanes&#39;) can be downloaded27. The provided default calibrant library contains RIs and spectra for n-alkanes ranging from C09 to C39 which have been analyzed as described in Section 4. The provided library enables users working with Metabolite Detector to directly start with the calibration process of their data. If needed, this library can also be extended with additional entries for further alkanes. Based on the similarity of reference and experimentally derived RIs and mass spectra (see step 5.3 and sub-steps, Figure 7, Figure 8, Figure 9),&#160;annotation or identification of the compounds can be performed (step 5.4 and sub-steps, Figure 11). It is also crucial that after automated data processing with MetaboliteDetector, the user will manually check for correct peak picking and spectrum deconvolution by inspecting mass spectra underlying the &#39;triangle&#39; for each putative compound of interest. Moreover, depending on the GC-MS instrumentation and the parameter settings used for data generation, adaptions of the presented MetaboliteDetector settings may be necessary. The MetaboliteDetector software is capable of performing many more useful operations than explained in this manuscript, e.g., display of extracted ion current (EIC) chromatograms, export of chromatograms as .csv, automatic batch-quantification of compounds, and many more.
The protocol presented in this manuscript can serve as suggestion for experiments performed by other researchers focusing on isolation of glands or gland contents from insects, volatilome analysis, as well as metabolite identification.

The overall goal of the workflow presented here is the general investigation of chemical compositions in secretions of insects. This is done with the primary aim of elucidating the ecological roles of the secretion as a whole, or of single compounds thereof. Moreover, we are interested in investigating the metabolic pathways underlying the compounds found in the respective secretions. Especially gland contents from ants (Hymenoptera, Formicidae) have gained rising interest in the past years, because they provide sources of hitherto unexplored potentially bioactive compounds (glues, antimicrobials, etc.)1,2. Minor worker ants of some species belonging to the COCY group3,4 may provide such compounds contained in their hypertrophied MGRs which extend from the mouthparts to the gaster5,6. When threatened by putative enemies, minor workers of C. explodens7 and some related species can make use of their MGR contents in an unusual way: They sacrifice themselves by rupturing their gastral wall to eject the sticky content of the MGRs explosively onto the opponent, whereupon the putative enemy is detained and may even die5,6,8,9. The purpose of the development and the use of the herein presented methods was to help improve the understanding of the chemical composition and the nature of tentatively toxic constituents of this ant secretion.
To this end, we present a protocol for the dissection of C. explodens worker ants to obtain the gastral portion of their wax-like MGR contents with subsequent solvent-extraction and analysis by GC-MS.
GC-MS analysis is one of the well-established methods for the profiling and identification of volatile metabolites (volatilome) from insects. Typical analytes of interest in ants include cuticular hydrocarbons10, semiochemicals11, and in general, compounds with biological activity12. Samples can be obtained from whole animals or from body parts and fluids isolated via&#160;dissection of the insect13,14. Sample preparation techniques include extraction of the therein contained metabolites with the use of solvents14 or headspace-solid-phase-microextraction (HS-SPME)15.
For metabolomic studies, it is vital that the samples are frozen rapidly directly after sampling, in order to minimize changes in the chemical composition and quantity of the compounds. The ants used for this study were killed by rapid freezing on-site in a cool bag stuffed with deep-frozen cold packs. The samples were then stored in a -20 &#176;C freezer using generator-driven electricity, before they were transported to the laboratory on dry ice. The dissection procedure presented here offers the possibility to isolate MGR contents without analyzing the whole ant or the gaster as a whole, as it has been done before for different COCY species16,17,18. Moreover, the presented protocol also enables direct access and analysis of the surrounding glands and tissues, like the venom gland (VG)5,8, the Dufour&#39;s gland (DG)8, or the intestines in other biological studies, or to check for possible cross-contaminations introduced during handling or dissection of the ants. To minimize the changes in the chemical composition of the MGR contents during dissection, either by thawing samples or by using chemicals, the dissection process was optimized to be carried out on a cold pack (-20 &#176;C), without the use of any additional buffers, washing solutions, or solvents. The samples obtained via this method are suitable for answering qualitative and quantitative questions.
Data analysis for the purpose of putative metabolite annotation and identification is done via the open-source software MetaboliteDetector19, which was developed for the automatic analysis of GC-MS-based metabolomics data. It detects single ion peaks present in chromatograms, performs a deconvolution step, and extracts the deconvoluted mass spectra of chemical compounds contained in the analyzed samples. Putative identification of compounds with MetaboliteDetector is based on the determined retention index (RI; the Kovats RI can be calculated automatically by the software) as well as similarity of the deconvoluted mass spectra. RI and spectral match factor can be cross-checked against either existing reference libraries (that can be imported, if they are in the common NIST format), or against an established in-house library. This is in accordance to the guidelines for (putative) compound identification suggested, e.g., by the Chemical Analysis Working Group (CAWG) of the Metabolomics Standards Initiative (MSI), where a minimum of two independent and orthogonal data relative to an authentic compound (here retention time (RT)/RI and mass spectrum) analyzed under identical experimental conditions are proposed as necessary to confirm non-novel metabolite identifications20.
The complete experiment is conducted on the MGR content of the COCY model species C. explodens, but the dissection steps can also be adapted to isolate the other glands present in the ant gaster. Moreover, while we present a protocol for the comprehensive analysis of the volatilome of the MGR content, the more generic parts of the workflow describing extraction, GC-MS measurement, and data evaluation can also be used for analysis and (putative) identification of volatile metabolites in general.
Since the experiments described in this manuscript are carried out on insects, no ethical approval is required. Field work, sampling of the ants, as well as their use for publication are in accordance with the guidelines regulating this project through the Universiti Brunei Darussalam, Brunei&#39;s Research and Innovation Centre and Brunei&#39;s Forestry Department, Brunei Darussalam.

<table><tbody><tr><td>Tube 50 ml, 115 x 28 mm, flat/conical base PP</td><td>Sarstedt</td><td>62,559,001</td><td>see Figure 1 in manuscript</td></tr><tr><td>PVC Tubings</td><td>Rehau</td><td>290 4489</td><td>see Figure 1 in manuscript</td></tr><tr><td>Mesh, stainless steel, 0.63 mm mesh size</td><td>Antstore</td><td>1000378</td><td>see Figure 1 in manuscript</td></tr><tr><td>Freezer</td><td>Severin</td><td>KS 9890</td><td>&amp;nbsp;-20 &amp;deg;C or lower</td></tr><tr><td>polystyrene foam box, inner dimensions 155 mm x 100 mm x 45 mm</td><td>Thorsten Koch</td><td>4260308590481</td><td></td></tr><tr><td>Petri dish, glass, 100 mm x 15 mm</td><td>Aldrich</td><td>BR455742</td><td></td></tr><tr><td>Cold pack 150 mm x 100 mm</td><td>Elite Bags</td><td>1998</td><td>freeze to -20 &amp;deg;C</td></tr><tr><td>Bucket with crushed ice</td><td></td><td></td><td></td></tr><tr><td>1.5 mL Short Thread Vials, 32 x 11.6 mm, clear glass, 1st hydrolytic class, wide opening</td><td>La-Pha-Pack</td><td>11090500</td><td></td></tr><tr><td>Screw caps for 1.5 mL Short Thread Vials, closed, Silicon white/PTFE red septum, 55&amp;deg; shore A, 1.0 mm</td><td>La-Pha-Pack</td><td>9151799</td><td></td></tr><tr><td>Stereomicroscope</td><td>Bresser</td><td>5806100</td><td></td></tr><tr><td>Forceps, Superfine Tip curved</td><td>Medizinische Instrumente May, Norman May</td><td>PI-0005B</td><td></td></tr><tr><td>Forceps, Superfine Tip straight</td><td>blueINOX</td><td>BL-3408</td><td></td></tr><tr><td>Dissection needle 140 mm, pointed, straight</td><td>Heinz Herenz Medizinalbedarf GmbH</td><td>1110301</td><td></td></tr><tr><td>Methanol, LC-MS CHROMASOLV, Honeywell Riedel-de Ha&amp;euml;n</td><td>fisher scientific</td><td>15654740</td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td>Rotizell-Tissue-T&amp;uuml;cher</td><td>Carl Roth GmbH + Co.KG</td><td>0087.2</td><td></td></tr><tr><td>Acetic acid ethyl ester ROTISOLV &amp;ge;99,8 %</td><td>Carl Roth GmbH + Co.KG</td><td>4442.1</td><td>freeze to -20 &amp;deg;C</td></tr><tr><td>Vortex Genie 2</td><td>neoLab</td><td>7-0092</td><td></td></tr><tr><td>0.1 mL micro-inserts for 1.5 mL Short Thread Vials, 31 x 6 mm, clear glass, 1st hydrolytic class, 15 mm tip&amp;nbsp;</td><td>La-Pha-Pack</td><td>06090357</td><td></td></tr><tr><td>Screw caps for 1.5 mL Short Thread Vials, with hole, RedRubber/PTFE septum,&amp;nbsp; 45&amp;deg; shore A, 1.0 mm</td><td>La-Pha-Pack</td><td>9151819</td><td></td></tr><tr><td>Alkane standard solution C&lt;sub&gt;8&lt;/sub&gt;-C&lt;sub&gt;20&lt;/sub&gt;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>04070</td><td></td></tr><tr><td>Alkane standard solution C&lt;sub&gt;21&lt;/sub&gt;-C&lt;sub&gt;40&lt;/sub&gt;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>04071</td><td></td></tr><tr><td>n-Hexane SupraSolv</td><td>Merck</td><td>104371</td><td></td></tr><tr><td>GC-autosampler, e.g. MPS2XL-Twister</td><td>Gerstel</td><td></td><td></td></tr><tr><td>Agilent Gas chromatograph 6890 N</td><td>Agilent</td><td></td><td></td></tr><tr><td>Gooseneck splitless Liner</td><td>Restek</td><td>22406</td><td></td></tr><tr><td>Helium (5.0 - F50)</td><td>Messer</td><td>102532501</td><td></td></tr><tr><td>GC capillary column HP-5MS UI 30 m &amp;times; 0.25 mm &amp;times;0.25 &amp;micro;m</td><td>Agilent</td><td>19091S-433UI</td><td></td></tr><tr><td>Agilent Mass Selective Detector 5975B</td><td>Agilent</td><td></td><td></td></tr><tr><td>MSD ChemStation Data Analysis Application software&amp;nbsp;</td><td>Agilent</td><td></td><td></td></tr><tr><td>MetaboliteDetector software (3.1.Lisa20170127Ra-Linux)</td><td>Hiller K</td><td></td><td>download from: http://metabolitedetector.tu-bs.de/node/10</td></tr><tr><td>Calibration Library for MetaboliteDetector</td><td>Hiller K</td><td></td><td>download from: http://metabolitedetector.tu-bs.de/node/10</td></tr><tr><td>MD Conversion Tool for NIST-library</td><td>Hiller K</td><td></td><td>download from: http://metabolitedetector.tu-bs.de/node/10</td></tr></tbody></table>

isolation of mandibular gland reservoir contents from bornean 'exploding ants' (formicidae) for volatilome analysis by gc-ms and metabolitedetector

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis cylindrica (COCY) group. For this purpose, the model species C. explodens is used. Ants belonging to the minor worker caste possess distinctive hypertrophied mandibular glands (MGs). In territorial combat, they use the viscous contents of their enlarged mandibular gland reservoirs (MGRs) to kill rival arthropods in characteristic suicidal &#39;explosions&#39; by voluntary rupture of the gastral integument (autothysis). We show the dissection of worker ants of this species for the isolation of the gastral portion of the wax-like MGR contents as well as listing the necessary steps required for solvent-extraction of the therein contained volatile compounds with subsequent gas chromatography-mass spectrometry (GC-MS) analysis and putative identification of metabolites contained in the extract. The dissection procedure is performed under cooled conditions and without the use of any dissection buffer solution to minimize the changes in the chemical composition of the MGR contents. After solvent-based extraction of volatile metabolites contained therein, the necessary steps for analyzing the samples via liquid-injection-GC-MS are presented. Lastly, data processing and putative metabolite identification with the use of the open-source software MetaboliteDetector is shown. With this approach, the profiling and identification of volatile metabolites in MGRs of ants belonging to the COCY group via GC-MS and the MetaboliteDetector software become possible.

Watch this Scientific Journal Video about Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' Formicidae for Volatilome Analysis by GC-MS and MetaboliteDetector at JoVE.com

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' Formicidae for Volatilome Analysis by GC-MS and MetaboliteDetector

Minor workers of the ant species Colobopsis explodens are dissected to isolate the wax-like content stored in their hypertrophied mandibular gland reservoirs for subsequent solvent-extraction and analysis by gas chromatography-mass spectrometry. The&#160;annotation and identification of volatile constituents using the open-source software MetaboliteDetector is also described.

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis ...

isolation-mandibular-gland-reservoir-contents-from-bornean-exploding

Research

JoVE Journal

Environment

8.8K Views.  University of Natural Resources and Life Sciences Vienna (BOKU), Austria. Minderjarige werknemers van de mier soorten Colobopsis explodens worden ontleed om te isoleren van de inhoud van de wasachtige opgeslagen in hun hypertrophied mandibulaire klier reservoirs voor latere oplosmiddel-extractie en analyse met gaschromatografie-massaspectrometrie. De aantekening en de identificatie van vluchtige bestanddelen met behulp van de open-source software MetaboliteDetector wordt ook beschreven.

Video: Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' Formicidae for Volatilome Analysis by GC-MS and MetaboliteDetector

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Biology

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Neuroscience

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Developmental Biology

Sampling and Analysis of Animal Scent Signals

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.

We have developed an effective methodology for sampling and analysis of odor signals in order to understand how they may be used in animal communication. Particularly, we use headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile components of animal odors and scent-markings.

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas ...

Behavior

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

Biochemistry

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Metabolite Profiling of Hard Coral Samples Using GC-MS

Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the cnidarian-dinoflagellate symbiosis and how coral responds to stress (i.e., during temperature-induced bleaching). Steady-state metabolite profiling of the coral holobiont, which comprises the cnidarian host and its associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi, and viruses), has been successfully applied under ambient and stress conditions to characterize the holistic metabolic status of the coral.
However, to answer questions surrounding the symbiotic interactions, it is necessary to analyze the metabolite profiles of the coral host and its algal symbionts independently, which can only be achieved by physical separation and isolation of the tissues, followed by independent extraction and analysis. While the application of metabolomics is relatively new to the coral field, the sustained efforts of research groups have resulted in the development of robust methods for analyzing metabolites in corals, including the separation of the coral host tissue and algal symbionts.
This paper presents a step-by-step guide for holobiont separation and the extraction of metabolites for GC-MS analysis, including key optimization steps for consideration. We demonstrate how, once analyzed independently, the combined metabolite profile of the two fractions (coral and Symbiodiniaceae) is similar to the profile of the whole (holobiont), but by separating the tissues, we can also obtain key information about the metabolism of and interactions between the two partners that cannot be obtained from the whole alone.

Author Spotlight: Separation of Coral Host Tissues and Algal Symbionts and Analyzing Their Metabolites 

Here, we present the extraction and preparation of polar and semi-polar metabolites from a coral holobiont, as well as separated coral host tissue and Symbiodiniaceae cell fractions, for gas chromatography-mass spectrometry analysis.

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the ...

Optimizing Pinewood Nematode Infection in Pinus pinaster

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) are&#160;free from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

Author Spotlight: In Vitro Co-Culture System of Pine Shoots and Pinewood Nematode for Studying Host Volatile Response

The protocol describes the in vivo and in vitro pinewood nematode infection of Pinus pinaster and their volatilome analysis through Gas Chromatography (GC) and GC coupled to Mass Spectrometry (GC-MS).

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector

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