Application of Direct Immersion Solid-Phase Microextraction (DI-SPME) for Understanding Biological Changes of Mediterranean Fruit Fly (Ceratitis capitata) During Mating Procedures

Samples from three different mating stages (before, during and after mating) of the Mediterranean fruit fly Ceratitis capitata were used in this experiment. Samples obtained from whole insects were subjected to extraction with the two mixtures of solvents (acetonitrile/water (A) and methanol/acetonitrile/water (B)) and a comparative study of the extractions using the different solvents was performed. Direct immersion-solid phase microextraction (DI-SPME) was employed, followed by gas chromatographic-mass spectrometry analyses (GC/MS) for the collection, separation and identification of compounds. The method was validated by testing its sensitivity, linearity and reproducibility. The main compounds identified in the three different mating stages were ethyl glycolate, α-farnesene, decanoic acid octyl ester, 2,6,10,15-tetramethylheptadecane, 11-tricosene, 9,12-(Z,Z)-octadecadienoic acid, methyl stearate, 9-(Z)-tricosene, 9,11-didehydro-lumisterol acetate; 1,54-dibromotetrapentacontane, 9-(Z)-hexadecenoic acid hexadecyl ester, 9-(E)-octadecenoic acid and 9-(Z)-hexadecenoic acid octadecyl ester. The novel findings indicated that compound compositions were not significantly different before and during mating. However, new chemical compounds were generated after mating, such as 1-iodododecane, 9-(Z)-tricosene and 11,13-dimethyl-12-tetradecen-1-acetate which were extracted with both (A) and (B) and dodecanoic acid, (Z)-oleic acid, octadecanoic acid and hentriacontane which were extracted with (A) and ethyl glycolate, 9-hexadecenoic acid hexadecyl ester, palmitoleic acid and 9-(E)-octadecenoic acid, which were extracted with solvent (B). This study has demonstrated that DI-SPME is useful in quantitative insect metabolomics by determining changes in the metabolic compounds in response to mating periods. DI-SPME chemical extraction technology might offer analysis of metabolites that could potentially enhance our understanding on the evolution of the medfly.


Introduction
The developed analytical methods for the analysis of volatile and non-volatile compounds are increasingly being used as tools for the study of plant chemistry and the evolution of insect-plant stages. In this experiment, the DI-SPME samples of mature medfly adults at three different mating stages (before, during and after), were analyzed using a GC-MS. To examine the effectiveness of DI-SPME, two different solvent extractions were used to compare the DI-SPME, which indicated quantitative and qualitative differences between these solvents in the type and peak areas of compounds. For further testing of DI-SPME, a GC-MS was used to compare the composition of two extracts solvents after directly immersing the SPME fiber in the extract. Comparison of these compound profiles revealed that DI-SPME had higher levels of the lighter chemicals and lower levels of ponderous chemicals. Firstly, the choice of the sealing and desorption time was carried out by fixing the time (2, 4, 8 and 16 h of sealing times). The best results were obtained with the recently developed 50/30 µm Carboxen/DVB/PDMS and, thus, 16 h sealing time was selected for further method development.
The example used is a GC-coupled MS-based metabolomics study in extracted samples where two mating time lines are compared between extract solvents. The two principal components were plotted: the first solvent extraction had 56% and 11.1%, and the second extraction had 39.3% and 28.3% ( Figure 1). The heat map showed a clear difference between the samples, particularly during and after mating stage (Figures 2 and 3).
Molecules 2018, 23, x 9 of 13 acids, such as oleic, palmitic, stearic, linoleic, and palmitoleic acids have been found in the exocrine secretions and cuticular extracts of many insects [45]. These compounds are important in intermediates and metabolites of biological pathways, and analytical techniques to study these compounds are of interest [46]. Filho et al. [47] showed that DI-SPME is more sensitive than HS-SPME, and it is thus the method of choice for the analysis of clean aqueous samples. The two extraction modes were evaluated and, despite being less sensitive than HS-SPME in the case of the more volatile compounds, DI-SPME mode successfully extracted 16 pesticides, while HS-SPME was able to extract only 12 compounds.

Insect Rearing
A medfly colony was obtained from the Department of Primary Industries and Regional Development (DPIRD), and flies were reared in the Post-harvest Biosecurity and Food Safety Laboratory at Murdoch University (Perth, Western Australia). All the flies were reared under the  Identifications are based on comparisons of both mass spectral data and GC retention indices with those of authentic reference compounds. Several components remain unidentified. Most of the unidentified run components were present at low concentrations, and were therefore thought to be contaminants. Some compounds were presented before mating, but they were missing during the mating stage. Some chemicals, were increased and some decreased within the mating stages (Tables 1 and 2). McDonald [27] reported that medfly males are stimulated to more frequent episodes of calling activity, when they are able to detect the presence of other medfly males. However, this interaction to visual and acoustic cues rather than to chemical communication. Jacobson et al. [25] and Ohinata et al. [28] studied which components are necessary to trigger an attractive response from female flies. This has been addressed to varying degrees except the present one, which is primarily a qualitative and semi-quantitative examination of the male emission complex. Ongoing laboratory evaluations of the major pheromone components identified indicate that many compounds contribute differentially, but synergistically to the pheromone's attractiveness for the female medflies. Other intermediate to low-concentration components may also be required to attain full parity with calling males. Flath et al. [29]; Al-khshemawee et al. [30] reported that three different medfly ages (5-6, 11-12, and 20-21 days old), and early-, mid-, and late-morning samples were used to collect volatiles. Thirty-two components were identified. However, propan-2-ol, hexanal, phenol, (Z,E)-α-farnesene, prop-2-yl-(E)-3-octenoate, ethyl (E)-2-octenoate, and propyl (E)-3-octenoate had been only partially identified in an earlier study. Quantitatively, ethyl acetate, 1-pyrroline, ethyl (E)-3-octenoate, geranyl acetate, and α-farnesene were the most abundant emission components from 5-6-and 11-12-day from old flies. The major compound for al fly ages was (2S)-2-hexenoic acid. Shelly [31] investigated the influence of α-copaene-containing plants on the mating system of C. capitata and the possibility of using attractants in prerelease exposure of males to increase the effectiveness of sterile insect release programs. Mature males were exposed to 20 µL of the attractant over a 6-h period and then were held for 2 d before testing. In field-cage trials, treated males (exposed to attractants) obtained significantly more matings than control males (no exposure) for all three substances. The potential exists for the development of an effective and useful female attractant, especially if essential components and their optimum release rates can be pinpointed and reproduced.
DI-SPME-GC-FID was first reported in an analysis of 13 commonly known benzodiazepines in urine [14]. The same group reported a modification of the method to analyze the hydrolysis of benzodiazepines from benzophenones extraction [32]. DI-SPME has been reported for quantitative analysis of biological samples including plant tissues [33], pesticides [4,34], milk [35], pharmaceuticals [36], wine [37] and water [38]. Myung et al. [39] optimized the DI extraction in blood samples for sorption of 1-octanol. Frérot et al. [40] used an organic solvent to soak or wash SPME in detected pheromones from the female abdominal tip of the Lepidopteran Sesamia nonagrioides. The pheromones of Metamasius hemipterus (Coleoptera) were sampled using SPME and compared to typical analytical methodologies. The SPME technique was shown to be cheaper, easier, faster and more reproducible [41]. SPME has been used to analyze cuticular hydrocarbons from ants [42]. DI-SPME has been used with pentane or hexane to analyze signaling chemicals and long-chain hydrocarbons from different parts of wasps' bodies [43]. The SPME technique has also been used to detect long-chain free fatty acids from insect exocrine glands, using a GC-MS [44]. Long chain fatty acids, such as oleic, palmitic, stearic, linoleic, and palmitoleic acids have been found in the exocrine secretions and cuticular extracts of many insects [45]. These compounds are important in intermediates and metabolites of biological pathways, and analytical techniques to study these compounds are of interest [46]. Filho et al. [47] showed that DI-SPME is more sensitive than HS-SPME, and it is thus the method of choice for the analysis of clean aqueous samples. The two extraction modes were evaluated and, despite being less sensitive than HS-SPME in the case of the more volatile compounds, DI-SPME mode successfully extracted 16 pesticides, while HS-SPME was able to extract only 12 compounds.

Insect Rearing
A medfly colony was obtained from the Department of Primary Industries and Regional Development (DPIRD), and flies were reared in the Post-harvest Biosecurity and Food Safety Laboratory at Murdoch University (Perth, Western Australia). All the flies were reared under the following conditions: temperature = 23 ± 2 • C, relative humidity = 75 ± 5%, and light: dark cycle = 12:12-h [48]. Adults were placed in screen cages (40 cm cubes), each containing medfly food made from crystaline sugar (Bidvest, Sydney, Australia) and yeast hydrolysate (Australian Biosearch, Sydney, Australia) at a ratio of 4:1, and 50 mL water. Approximately 10-12 days after adult emergence from pupae and mating, eggs were collected each day. These were deposited on a mesh side of the cage and fell into a water tray kept adjacent to the cage.

DI-SPME Conditions
A GC-MS 7890B gas chromatograph equipped with a 5977B MSD mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), with an Agilent HP-5MS column (30 m, 0.25 µm, 0.25 µm film thickness) was used in the experiments. The carrier gas used was helium at 99.999% (BOC, Sydney, Australia). The conditions for the GC-MS were as follows: injector port temperature of 270 • C; initial oven temperature of 60 • C, which increased to 320 • C (at 5 • C/min); MS Quad at 150 • C; MS source at 230 • C; pressure at 10.629 psi. The flow rate was 1.2 mL/min; the splitless was 30 mL/min at 1.0 min. The total run time was 45.40 min.
3.3. DI-SPME Procedure and Sampling Setup SPME fiber 50/30 µm with Carboxen/DVB/PDMS (Sigma-Aldrich, Bellefonte, PA, USA) coating was inserted into extracted samples. SPME in the samples was conditioned at room temperature (25 ± 5 • C) for 16 h with a sampling depth of 3 cm. The DI-SPME extraction was carried out by immersing the fiber (length: 1.3 cm) into the extracted solution. After extraction for 16 h sealing time, the fiber was withdrawn into the needle, removed from the vial and immediately introduced into the GC injector port for thermal desorption. Samples in triplicate were used for extraction. For sample preparation, adult medflies (0.05 g) were taken before, during and after mating stages. Insects were grinded using tissuelyser at 270 rpm for 2 min. Two extraction solvents, acetonitrile/water (1:1) and methanol/acetonitrile/water (2:2:1) (CAS: 67-56-1, UN1230, Thermo Fisher Scientific, Perth, Australia), were used to extract the samples. Extraction solvent (1 mL) was added to the samples, and centrifuged at 2000 rpm for 5 min. The extraction samples were transferred to a 2 mL analytical vial. SPME was inserted directly into the vial for 16 h at room temperature. Then, the DI-SPME was analyzed using a GC-MS for 15 min desorption time. The samples were analyzed in biological triplicates.

Statistical Analysis
To observe the impact of observations, principal component analysis (PCA) with the correlation matrix method was used for statistical analysis using the online MetaboAnalyst 3.0 (2017) (Bellevue, Quebec, USA) tool, a comprehensive online tool for metabolomics analysis and interpretation. PCA was used to transfer the original data onto new axes where principal components corresponded to significant information represented by the original data. Three principal components are chosen from the result of PCA and sPLS-DA analysis based on Xia and Wishart [49]. The plots classifier was used to integrate the two components obtained from PCA and produce a segmented image. Since the heatmap centers were chosen randomly in the original means and the obtained results can be different for every run of the algorithm, the overall classification accuracies were averaged over different data.

Conclusions
In this study, two DI-SPME extraction solvents for were used at three different stages of the medfly adult life. The first extraction solvent was acetonitrile/water, and the second solvent was methanol/acetonitrile/water. Samples were collected before, during and after mating. This study compared these extraction solvents based on the metabolites extracted. The GC-MS analytical data showed a wide spectrum of compounds and DI-SPME sampling was developed to identify these compounds from medfly extracts. These results indicate that DI-SPME coupled with the GC-MS could be performed successfully on medfly extracts. Using DI-SPME with GC analysis of extracts, high sensitivity and good repeatability were obtained. This work is an example of the application of DI-SPME-GC in the analysis of complex samples and provides a way in which to prepare the samples of SPME coatings. Further development of DI-SPME is promising, and may provide an efficient extraction technique for biological samples.