By Fibroporia Radiculosa (Peck) Parmasto 1968

Article Feeding Deterrence to Reticulitermes speratus (Kolbe) by Fibroporia radiculosa (Peck) Parmasto 1968

Nadia Nuraniya Kamaluddin, Shigeru Matsuyama and Akiko Nakagawa-Izumi * Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba, Ibaraki Prefecture 305-8571, Japan; [email protected] (N.N.K.); [email protected] (S.M.) * Correspondence: [email protected]; Tel.: +81-29-853-4578

Academic Editors: Tsuyoshi Yoshimura, Wakako Ohmura, Vernard Lewis and Ryutaro Iwata Received: 27 January 2017; Accepted: 28 February 2017; Published: 6 March 2017

Abstract: Brown rot fungus Fibroporia radiculosa (Peck) Parmasto grown in decayed wood and non-wood material, potato dextrose agar (PDA) media, deterred Reticulitermes speratus (Kolbe) feeding. Decayed wood and PDA media were extracted and tests were performed to assess termite feeding behavior towards the extracts. We found that the extract from PDA media also suppressed termite feeding, although it did not induce mortality. Using gas chromatography and mass spectrometry analysis, two bioactive compounds were detected from the decayed wood extract, and one was detected from the PDA extract. Based on National Institute of Science and Technology (USA) Mass Spectral library match and compound fragmentation, both of the compounds belong to the sesquiterpenes family.

Keywords: Fibroporia radiculosa; feeding behavior; Reticulitermes speratus

1. Introduction Both higher and lower termites interact with fungi [1]. In particular, termites and wood-decay fungi both decompose woody substances; therefore, several forms of interactions likely formed between these two organisms. For example, non-entomopathogenic fungi can influence the palatability of wood [2,3], acting as feeding attractants, stimuli or preference inducers [4,5], or as deterrents [6] towards termites. Thus, fungi-termite interactions are ubiquitous, although they are sometimes provisional in character and cannot be categorized as symbiosis [7]. Factors such as fungal strain, degree of decay, as well as tree and termite species, should be considered in fungi-termite interactions, especially with respect to termite feeding preferences for fungus-decayed wood [1,2]. Wood-decay fungi facilitate termite consumption of lignocellulose [8] through the degradation or modification of wood components. Wood-decay fungi trigger various responses in termite feeding behavior under field and laboratory conditions [1,4,8]. For instance, fungal chelate accumulation during wood decay initiates lignin oxidation, cellulose depolymerization, and lignin degradation reactions, which may act as chemosensory signals to termites that the wood is heavily degraded and therefore nutritionally inadequate [6]. However, wood-decay fungi also appear to detoxify wood extractives, rendering soft wood more suitable for termite consumption [5]. Although interactions between termites and decay fungi are frequently observed on wood as a fungal substrate, it remains unclear whether these interactions are actually mediated by the presence of wood components or solely between the termite and fungus themselves. Therefore, it is important to clarify whether termite feeding is related to fungal secretion or products released during wood degradation. In our previous study, we reported a feeding deterrent effect from stakes decayed by brown rot basidiomycete Fibroporia radiculosa [9]. We determined that the compounds linked to this deterrent effect are also present in n-hexane wood extracts; both field-decayed wood stakes and

Insects 2017, 8, 29; doi:10.3390/insects8010029 www.mdpi.com/journal/insects Insects 2017, 8, 29 2 of 10 laboratory-decayed wood extract yielded the same outcome. This study aimed to further verify the identity and origin of the compound from Fibroporia radiculosa (Peck) Parmasto that is responsible for the feeding deterrent effect on Reticulitermes speratus (Kolbe).

2. Materials and Methods

2.1. Termites Five R. speratus colonies were used in this study: three colonies (Oarai 1, 2, and 3) were obtained from Oarai, Ibaraki Prefecture, Japan, during October 2016. Two colonies (Kagoshima 1 and 2) were obtained from a ﬁeld maintained by the Research Institute for Sustainable Humanosphere (RISH) in Kagoshima Prefecture, Japan, during December 2014. All termite colonies were housed in plastic containers in a temperature- and moisture-controlled laboratory until use. Different colonies were used for each test; no colony was used more than once.

2.2. Pure Cultures of Fibroporia radiculosa Fibroporia radiculosa cultures were previously isolated, identified, and purified from decayed stakes obtained from a RISH field in 2009 [9]. The fungus was maintained in a potato dextrose agar (PDA) slant containing 100 ppm benlate and 50 ppm tetracycline to prevent mold growth, and kept under refrigerated conditions (2 to 3 ◦C) until use.

2.3. Preparation of Decayed Wood and PDA Media Laboratory decayed woods were prepared by drying block samples from sapwood of Pinus densiﬂora (20 mm × 20 mm × 5 mm) at 60 ◦C for 48 h, followed by sterilization by ethylene oxide gas. Three sterilized woods were aseptically placed on a F. radiculosa mycelium grown in a diluted potato dextrose agar medium and kept inside an incubator at 27 ◦C for eight weeks. To prepare diluted Potato Dextrose Agar (PDA) medium for laboratory decayed wood, 1.3 g of PDA powder (Nissui Pharmaceuticals, Tokyo, Japan) and 2.6 g of agar (Nacalai Tesque, Kyoto, Japan) were diluted in 100 mL tap water. The medium was poured into a 500 mL screw-capped bottle and sterilized by autoclaving at 121 ◦C for 15 min. Sterilized medium was inoculated with F. radiculosa pure cultures and incubated at 27 ◦C for seven days or until the mycelium has covered the diluted PDA surface. Meanwhile, decayed PDA medium were prepared via mixing 3.9 g of PDA powder (Nissui Pharmaceuticals, Tokyo, Japan) with 100 mL of tap water, then poured into a 500-mL screw-capped bottle for autoclaving at 121 ◦C and 15 min. Pure culture of F. radiculosa were transferred aseptically to the media surface. Cultured medium were cultivated in a 27 ◦C incubator for eight weeks.

2.4. Extract Preparation of Extract and PDA Media Decayed P. densiflora blocks were removed from the incubation bottle. Blocks surface were carefully cleaned from fungal mycelium. The woods were freeze-dried overnight, cut into smaller pieces, and ground using a mortar and pestle to obtain decayed wood powder. Five grams of the decayed wood powder was inserted into a 200 mL conical flask and subjected to extraction with 100 mL of n-hexane at 18–25 ◦C overnight (10–12 h). The mixture was filtered using filter paper (No. 1, Advantec Toyo Roshi, Tokyo, Japan) and concentrated using a rotary evaporator into a 10 mL solution. For extract preparation from fungal cultures cultivated on growth media, the mycelium-covered medium surface was carefully scraped using a spatula; the collected material was ground with a glass stirrer, and then added to a 300 mL conical flask. The ground medium (100 g) was extracted with 100 mL of n-hexane overnight or up to 24 h. The mixture was filtered using No. 1 filter paper (Advantec Toyo Roshi) and concentrated to 5 mL in a rotary evaporator at 10 ◦C, then refrigerated (2–3 ◦C) for a maximum of 14 days after extraction. Insects 2017, 8, 29 3 of 10

2.5. Puriﬁcation of Extract The decayed wood and PDA extracts were puriﬁed using small-scale column chromatography. Into a cotton wool plugged Pasteur pipette (146 mm, Corning, New York, NY, USA) silica gel (C200, Wako Pure Chemicals Industry, Osaka, Japan) was dry-packed (4 cm long). Then an aliquot of 4 mL of extract was added; n-hexane, diethyl ether, and ethyl acetate were used. Each portion was divided into two equal parts for bioassay and chemical analyses using gas chromatography/mass spectrometry (GC/MS).

2.6. Feeding Deterrent Bioassays (No-Choice and Two-Choice Feeding Tests)

2.6.1. No-Choice Feeding Test of Crude and Purified Extract from Wood and PDA Media Decayed by F. radiculosa No-choice feeding tests were conducted as follows: paper disks (Ø 8 mm, thick type; Advantec Toyo Roshi) were soaked with 100 µL of decayed wood extract (containing 50 mgeq (milligrams equivalent) of decayed wood), 100 µL of decayed PDA extract (containing 500 mgeq of decayed PDA media), or 100 µL of sound wood extract (containing 5 mgeq of sound wood) used as control. The disks were then treated with 100 µL of n-hexane were used as a solvent treatment. Treated paper disks were placed inside a fume hood for 15 min to remove the solvent before being transferred into individual plastic saucer (Ø 1 cm) on top of 15-mm-thick hard plaster (New Plastone Dental Stone, GC Corp., Tokyo, Japan). The top of hard plaster was covered with a thin layer of wet sand on which a sample disk on the saucer was placed. The bottom of each cup was perforated (perforations 10 mm in diameter) to supply moisture through the plaster layer, which was in contact with a damp paper pad at the bottom of the test chamber. Fifty R. speratus workers of Kagoshima 1 colony were introduced to each cup. Test chambers were kept inside an incubator for five days at 25 ◦C and 70%–80% relative humidity (RH). Seven biological replicates were performed (seven cups each with 50 R. speratus) for each treatment. At the end of the test period, paper disks were weighed to assess mass loss from termite feeding and the remaining termites were counted to determine the mortality percentage. Purified extracts bioassay was conducted in a similar manner to crude extracts, but with 300 µL (150 mgeq decayed and sound wood, 1500 mgeq of decayed PDA) of purified extracts. The tests were conducted for four days at 25 ◦C and 70%–80% RH, and replicated five times (five cups of 50 R. speratus for each treatment) using Oarai 3 colony. In no-choice tests, starvation treatment was used as a control towards termite mortality. Fifty termite workers (per cup) were not given any food during test period. The number of replication for starvation treatment is identical to other treatments (seven times for crude extracts and five times for purified extracts) in each feeding test. After the period finished, dead termite were counted to determine the mortality percentage.

2.6.2. Two-Choice Feeding Test Using Extract from F. radiculosa Decayed Wood and PDA Media Two-choice feeding test were performed to assess termite preferences between paper disks treated with decayed wood and PDA media extract. Inside a 200 mL cup (prepared as described in Section 2.6.1), paper disks treated with 100 µL of either decayed wood or PDA extract were placed, 4 cm apart, in a single plastic saucer. One hundred termite workers were introduced into each cup. Test chambers were kept inside an incubator for ﬁve days at 25 ◦C and 70%–80% RH. One R. speratus colony (Kagoshima 2) from Kagoshima and two colonies from Oarai (Oarai 1 and 2) were used in this test. Eight biological replications (eight cups each with 100 R. speratus) were performed for each colony. Paper disks were weighed at the end of the test period to assess the mass loss from termite feeding.

2.7. Gas Chromatography and Mass Spectrometer Analyses Chemical analyses of the bioactive fraction in decayed wood and PDA extract was carried out using GC-MS analysis. JEOL MS-600 (JEOL Ltd., Tokyo, Japan) mass spectrometer was coupled with Hewlett-Packard 6890N (Hewlett-Packard, Palo Alto, CA, USA) gas chromatograph was used Insects 2017, 8, 29 4 of 10 Insects 2017, 8, 29 4 of 10 forinjected GC-MS at 200 and °C helium in the was split-less used as mode the carrier (sampling gas at time: 1 mL/min 0.75 min), in a constantseparated flow with mode. a fused Samples silica werecapillary injected column at 200(DB-5MS,◦C in the25 m split-less × 0.25 mm, mode 0.25 (sampling μm film thickness, time: 0.75 Agilent min), separated Technology with Inc., a fusedSanta silicaClara, capillary CA, USA). column Oven (DB-5MS,temperature 25 mwas× maintained0.25 mm, 0.25 at 50µm °C film for thickness,3 min, increased Agilent to Technology 300 °C at a Inc.,rate Santaof 5 °C/min, Clara, CA,and USA). kept steady Oven temperature for 6 min. The was tran maintainedsfer line at was 50 ◦keptC for at 3 min,280 increased°C. Mass tospectra 300 ◦ Cwere at a rateobtained of 5 ◦ byC/min, electron and ionization kept steady at for70 eV, 6 min. at a The scan transfer speed of line 0.29 was s with kept a at mass 280 ◦ rangeC. Mass of spectra40–600 amu. were obtainedMajor peaks by electronin the chromatogram ionization at were 70 eV, compared at a scan to speed those of of 0.29 compounds s with amass in the range National of 40–600 Institute amu. of MajorScience peaks and Technology in the chromatogram (USA) Mass were Spectral compared (NIST to MS) those library of compounds for identification. in the National Institute of Science and Technology (USA) Mass Spectral (NIST MS) library for identification. 2.8. Statisitical Analysis 2.8. Statisitical Analysis For feeding tests, treatment and control samples were compared using non-parametric test, Kruskal-WallisFor feeding H. tests, The significance treatment and level control was set samples at p = 0.05. were All comparedtests were usingperformed non-parametric using IBM SPSS test, Kruskal-WallisStatistics version H. 22 The software significance (IBM levelCorp., was New set York, at p = NY, 0.05. USA). All tests were performed using IBM SPSS Statistics version 22 software (IBM Corp., New York, NY, USA). 3. Results 3. Results 3.1. Feeding Deterrent Effect, Toxicity, and Bioactivity of Extracts Obtained from Decayed Wood and PDA 3.1. Feeding Deterrent Effect, Toxicity, and Bioactivity of Extracts Obtained from Decayed Wood and Media PDA Media Mass loss differences among the treated paper disks were significantly different (χ2(4) = 16.409, Mass loss differences among the treated paper disks were significantly different (χ2(4) = 16.409, p = 0.03). The extract obtained from F. radiculosa-decayed PDA media was a significantly stronger p = 0.03). The extract obtained from F. radiculosa-decayed PDA media was a significantly stronger feeding deterrent than the untreated paper disk (Figure 1). Although the decayed wood extract did feeding deterrent than the untreated paper disk (Figure1). Although the decayed wood extract did not deter termite feeding to the same extent that the PDA media extract did, the mortality caused by not deter termite feeding to the same extent that the PDA media extract did, the mortality caused by it it was higher. Based on pairwise comparison, feeding on decayed wood extract led to a similar was higher. Based on pairwise comparison, feeding on decayed wood extract led to a similar termite termite mortality percentage as feeding on sound wood extract. In contrast, feeding on decayed PDA mortality percentage as feeding on sound wood extract. In contrast, feeding on decayed PDA media media extract induced a considerably lower mortality that than from feeding on both decayed and extract induced a considerably lower mortality that than from feeding on both decayed and sound sound wood extracts (χ2(5) = 21.797, p = 0.01). These results suggest that mortality was primarily wood extracts (χ2(5) = 21.797, p = 0.01). These results suggest that mortality was primarily caused by caused by original or fungus-modified wood components. original or fungus-modified wood components.

Figure 1.1. BoxBoxplot plot of of (a ()a mass) mass loss loss and and (b) mortality(b) mortality in termites in termites caused caused by feeding by feeding on paper on diskpaper treated disk withtreated extracts with extracts from decayed from decayed wood or wood potato or dextrose potato dextrose agar (PDA) agar (Kruskal-Wallis (PDA) (Kruskal-Wallis H test, p < H 0.05). test, p < 0.05). The two-choice feeding test was performed to compare the bioactivity of the extracts (TableThe1). two-choice There were feeding signiﬁcant test differenceswas performed among to comp the treatmentsare the bioactivity under the of Kruskal-Wallis the extracts (Table H test 1). (Thereχ2(5) =were 27.252, significantp < 0.01). differences Lower mass among loss mediansthe treatments were detected under the for Kruskal-Wallis paper disks treated H test with (χ2 PDA(5) = extract,27.252, p suggesting < 0.01). Lower that it mass hada loss stronger medians deterrent were effectdetected on termite for paper feeding. disks Mortalitytreated with was PDA not recorded extract, insuggesting this test. that it had a stronger deterrent effect on termite feeding. Mortality was not recorded in this test.

Insects 2017, 8, 29 5 of 10

Insects 2017, 8, 29 5 of 10 Table 1. Mass loss medians of two-choice feeding test between crude decayed wood and PDA media extractTable in 1. test Mass colonies. loss medians of two-choice feeding test between crude decayed wood and PDA media extract in test colonies. Colony Decayed Wood Extract (%) PDA Extract (%) Colony Decayed Wood Extract (%) PDA Extract (%) ColonyColony 1 (Kagoshima 1 (Kagoshima 2) 2) 21.19 21.190 0 ColonyColony 2 2 (Oarai (Oarai 1) 1) 34.60 34.6019.02 19.02 Colony 3 (Oarai 2) 59.60 3.42 Colony 3 (Oarai 2) 59.60 3.42 Kruskal-Wallis H test, p < 0.05. Kruskal-Wallis H test, p < 0.05.

3.2.3.2. No-Choice No-Choice Feeding Feeding Test Test Using Using Purified Purified Extract Extract ToTo eliminate eliminate impurities impurities and and determine determine thethe bioactivebioactive fraction, fraction, crude crude decayed decayed wood wood and and PDA PDA extracts were purified using column chromatography. Differences among treatments were detected extracts were purified using column chromatography. Differences among treatments were detected by by mass loss from termite feeding (χ2(6) = 26.575, p < 0.01) and termite mortality caused by mass loss from termite feeding (χ2(6) = 26.575, p < 0.01) and termite mortality caused by consumption of consumption of purified extract (χ2(7) = 31.034, p < 0.01). Analysis of mass loss indicated that χ2 p purifiedconsumption extract (of (7)decayed = 31.034, wood< extract 0.01). Analysispurified with of mass n-hexane loss indicated and diethyl that ether consumption was significantly of decayed woodlower extract than purifiedthat of extracts with n- purifiedhexane using and diethylother treatments ether was (Figure significantly 2). These lower extracts than also that resulted of extracts in purifiedlow survival, using other indicating treatments that (Figurethe compound2). These resp extractsonsible also for resulted feeding indeterrence low survival, or mortality indicating was that thepresent compound in the responsible extracts purified for feeding by n-hexane deterrence and diethyl or mortality ether. Although was present paper disks in the treated extracts with purified the by n-diethylhexane ether and fraction diethyl from ether. the Although decayed paperPDA extrac disksttreated resulted with in low the termite diethyl feeding ether fraction and slightly from the decayedhigher PDA mortality extract compared resulted to in the low other termite treatments feeding, both and the slightly feeding higher deterrence mortality and compared the effect on to the othertermite treatments, survival both were the inconclusive feeding deterrence and not as and clear the as effect those onof termitethe n-hexane survival or diethyl were inconclusiveether fraction and notfrom as clear the asdecayed those ofwood the n- extract.hexane Thus, or diethyl the bioact etherive fraction compound from may the decayedhave been wood lost from extract. the Thus, PDA the bioactiveextract compoundduring the pu mayrification have been process. lost from the PDA extract during the purification process.

FigureFigure 2. (2.a )(a Mass) Mass loss loss and and ( b(b)) mortality mortality causedcaused by feeding feeding of of purified puriﬁed decayed decayed wood wood and and potato potato dextrosedextrose agar agar (PDA) (PDA) extract extract in in a a no-choice no-choice feedingfeeding test (Kruskal-Wallis H H test, test, p p< 0.05).< 0.05).

Insects 2017, 8, 29 6 of 10

Insects3.3. Analysis2017, 8, 29 of Decayed Wood and PDA Extract with GC-MS 6 of 10 Insects 2017, 8, 29 6 of 10 We compared chromatograms from the GC-MS analysis of the sound and decayed wood 3.3.extracts,3.3. Analysis Analysis as ofwell of Decayed Decayed as the Wood Woodn-hexane and andPDA PDA fraction ExtractExtract from with the GC-MS GC-MS decayed wood extract (Figure 3). Comparisons between the chromatograms of the sound and decayed wood extracts revealed two unique peaks in WeWe compared compared chromatograms chromatograms from from the the GC-MS GC-MS analysis analysis of theof soundthe sound and decayedand decayed wood wood extracts, the decayed wood extract at retention times (RTs) of 21.11 and 27.72 min. After silica gel column as wellextracts, as the as nwell-hexane as the fraction n-hexane from fraction the decayed from the wood decayed extract wood (Figure extract3). (Figure Comparisons 3). Comparisons between the chromatography, peak 1 eluted in the n-hexane fraction, suggesting that the compound was chromatogramsbetween the chromatograms of the sound andof the decayed sound and wood decayed extracts wood revealed extracts two revealed unique two peaks unique in thepeaks decayed in hydrocarbon. The mass spectrum of peak 1 (Figure 4) showed an ion at m/z 204 accompanied by an woodthe extractdecayed at wood retention extract times at retention (RTs) of 21.11times and(RTs) 27.72 of 21.11 min. and After 27.72 silica min. gel After column silica chromatography, gel column ion at m/z 189 (204-15), which was indicative of a sesquiterpene hydrocarbon (C15H24). On the other peakchromatography, 1 eluted in the peakn-hexane 1 eluted fraction, in the suggesting n-hexane thatfraction, the compoundsuggesting wasthat hydrocarbon.the compound The was mass hand, peak 2 (Figure 5) was not detected after the chromatography. Based on the analysis of crude spectrumhydrocarbon. of peak The 1 (Figuremass spectrum4) showed of peak an ion1 (Figure at m/z 4)204 showed accompanied an ion at m/z by an204ion accompanied at m/z 189 by (204-15), an extracts,ion at m/z these 189 compounds (204-15), which share was a indicativebase ion at of m/z a sesquiterpene of 161. By comparing hydrocarbon the (C mass15H24 ).spectra On the with other those which was indicative of a sesquiterpene hydrocarbon (C15H24). On the other hand, peak 2 (Figure5) in hand,the NIST peak MS2 (Figure library, 5) was peaks not 1detected and 2 afterwere the indicated chromatography. as longifolene Based on(similarity the analysis index of crude88.9) and was not detected after the chromatography. Based on the analysis of crude extracts, these compounds hinesolextracts, (similarity these compounds index 78.2), share respectively. a base ion at m/zInterestingly, of 161. By comparing peak 2 (RT the 27.72) mass spectrawas detected with those in both share a base ion at m/z of 161. By comparing the mass spectra with those in the NIST MS library, decayedin the NISTwood MS and library, PDA peaks media 1 andcrude 2 wereextracts, indicated indicating as longifolene that the (similarity origin of index peak 88.9)2 is andfrom F. peaks 1 and 2 were indicated as longifolene (similarity index 88.9) and hinesol (similarity index 78.2), radiculosahinesol (similarityrather than index from 78.2), the respectively. wood metabolism, Interestingly, because peak 2the (RT compound27.72) was detectedwas also in foundboth in respectively. Interestingly, peak 2 (RT 27.72) was detected in both decayed wood and PDA media crude non-wooddecayed woodmedia andinoculated PDA media with thecrude fungus extracts, (Figure indicating 6). that the origin of peak 2 is from F. extracts,radiculosa indicating rather thatthan the from origin theof wood peak 2metabolism, is from F. radiculosa because ratherthe compound than from was the woodalso found metabolism, in becausenon-wood the compound media inoculated was also with found the fungus in non-wood (Figure 6). media inoculated with the fungus (Figure6).

FigureFigureFigure 3.3. 3. ChromatogramChromatogram Chromatogram ofof extractsextractsextracts fromfrom so soundsoundund wood, wood,wood, crude crudecrude decayed decayeddecayed wood, wood,wood, and and andthe then-thehexanen- n-hexanehexane fractionfractionfraction from from from decayed decayed decayed wood woodwood extract. extract. PeakPeak Peak 1 1 (retention (retention (retention time time time (RT) (RT) (RT) 21.11) 21.11) 21.11) eluted eluted eluted in the in in the n-thehexanen- n-hexanehexane fraction fraction fraction afterafterafter chromatography. chromatography. chromatography.

Figure 4. Mass spectra of peak 1 (retention time (RT) 21.11) from (a) crude decayed wood extract and FigureFigure(b) from 4. 4.Mass Mass n-hexane spectra spectra fraction of of peak peak after1 1 (retentionchromatography. (retention time time (RT) (RT) 21.11) 21.11) from from ( a(a)) crude crude decayed decayed wood wood extract extract and and (b(b)) from fromn- n-hexanehexane fraction fraction after after chromatography. chromatography.

Insects 2017, 8, 29 7 of 10 InsectsInsects 2017 2017, ,8 8, ,29 29 77 of of 10 10

FigureFigure 5. 5. Mass MassMass spectra spectraspectra of ofof peak peakpeak 2 22 (retention (retention(retention time timetime [RT] [RT][RT] 27.72) 27.72) from fromfrom ( (aa))) crude crudecrude decayed decayeddecayed wood woodwood extract extractextract and andand ((bb)) crude crude PDA PDA extract. extract.

Figure 6. Chromatogram of blank, crude decayed, and diethyl ether fraction from decayed PDA extract. FigureFigure 6.6. ChromatogramChromatogram ofof blank,blank, crudecrude decayed,decayed, anandd diethyldiethyl etherether fractionfraction fromfrom decayeddecayed PDAPDA Peak 2 (retention time (RT) 27.72) was also present in the decayed wood extract. extract.extract. Peak Peak 2 2 (retention (retention time time (RT] (RT] 27.72) 27.72) wa wass also also present present in in the the decayed decayed wood wood extract. extract. 4. Discussion 4.4. Discussion Discussion We compared the feeding deterrence and mortality of R. speratus fed with crude extracts and WeWe comparedcompared thethe feedingfeeding deterrencedeterrence andand mortalitymortality ofof R.R. speratus speratus fedfed withwith crudecrude extractsextracts andand fractions from wood and PDA medium decayed by F. radiculosa growth, with a view to determine the fractionsfractions fromfrom woodwood andand PDAPDA mediummedium decayeddecayed byby F.F. radiculosa radiculosa growth,growth, withwith aa viewview toto determinedetermine chemical structure and the origin of the compounds responsible for these effects on the termites. thethe chemical chemical structure structure and and the the origin origin of of the the compou compoundsnds responsible responsible for for these these effects effects on on the the termites. termites. 4.1. No- and Two-Choice Feeding of Extract Obtained from Growth Medium Infected with F. radiculosa 4.1.4.1. No- No- and and Two-Choice Two-Choice Feeding Feeding of of Extract Extract Obtained Obtained from from Growth Growth Medium Medium Infect Infecteded with with F. F. radiculosa radiculosa Paper disks soaked with both decayed wood and decayed PDA extract had a lower mass loss than PaperPaper disksdisks soakedsoaked withwith bothboth decayeddecayed woodwood andand decayeddecayed PDAPDA extractextract hadhad aa lowerlower massmass lossloss did the untreated paper disks, consistent with results from our previous study [9] showing the feeding thanthan did did the the untreated untreated paper paper disks, disks, consistent consistent with with results results from from our our previous previous study study [9] [9] showing showing the the deterrent in decayed wood was successfully extracted. The PDA extract had a stronger deterrent effect, feedingfeeding deterrentdeterrent inin decayeddecayed woodwood waswas successfullysuccessfully extracted.extracted. ThThee PDAPDA extractextract hadhad aa strongerstronger wherein the feeding is clearly suppressed. deterrentdeterrent effect, effect, wherein wherein the the fe feedingeding is is clearly clearly suppressed. suppressed. In contrast, the decayed PDA extract resulted in a lower mortality compared with that of the InIn contrast,contrast, thethe decayeddecayed PDAPDA extractextract resultedresulted inin aa lowerlower mortalitymortality comparedcompared withwith thatthat ofof thethe decayed wood extract in a no-choice feeding test (Figure1); this outcome might be due to the absence decayeddecayed woodwood extractextract inin aa no-choiceno-choice feedingfeeding testtest (Figure(Figure 1);1); thisthis outcomeoutcome mightmight bebe duedue toto thethe of wood extractives. The fact that the mortality did not signiﬁcantly differ between extracts from absenceabsence of of wood wood extractives. extractives. The The fact fact that that the the mo mortalityrtality did did not not significantly significantly differ differ between between extracts extracts fromfrom sound sound wood wood and and decayed decayed wood wood implies implies the the role role of of the the wood wood material material in in lowering lowering the the termite termite

Insects 2017, 8, 29 8 of 10 sound wood and decayed wood implies the role of the wood material in lowering the termite survival. Although a mortality effect was not apparent for the PDA extract, the deterrent effect of this extract may act as a mortality cofactor. Reduced consumption caused by a feeding deterrent effect can reduce fitness, thereby exposing termites to a variety of factors that affect mortality [10]. Fungal secondary metabolites, such as volatile organic compounds (VOCs), can modulate insect feeding behavior. In certain conditions, fungal secondary metabolites can adversely affect termite feeding on rotting wood. Similar to allelopathy mechanisms in plants, the export of toxic secondary metabolites might drive competition between insects and fungi [11] that feed within the same niche. Deterrents released by F. radiculosa initially affected the pre-ingestion stage in termite feeding, specifically during orientation or host selection. In the pre-ingestion stage (food recognition followed by acceptance or rejection), feeding deterrents interfere with sensors located in the mouthparts, food canal, and antennae of the insects [12]. The deterrent interferes with the termite’s gustatory and olfactory sensation via blocking impulse transmission towards neural control circuits. In the absence of a feeding stimulant or the presence of a deterrent, termites will perceive the interference as a sign that the compound is inedible or nutritionally inadequate, leading to the rejection of decayed wood as a food source. Interestingly, in the two-choice feeding test (Figure2), two colonies displayed distinctive preferences towards decayed wood extract over decayed PDA. The data suggest that there may be a stronger feeding stimulant, masking the detection of a deterrent and/or toxic compound. This possibility is not unexpected; termites were found to prefer a cellulosic disk treated with Pinus densiflora extract and exhibited continuous feeding stimulation in the presence of this extract [13].

4.2. Mass Loss and Mortality Caused by the Feeding of Extract Obtained from Chromatographed Decayed wood and PDA and Analysis by GC-MS The n-hexane and diethyl ether fractions deterred termite feeding and induced termite mortality. The fact that bioactivity of the extract was retained after chromatography indicates the non-polar nature of the feeding deterrent. Purified extract with the more polar ethyl acetate did not show any notable feeding deterrence or mortality compared with the controls. Multiple components were involved in the deterrence of termite feeding from the decayed wood extract. Peak 1 (RT 21.11) remained in the n-hexane fraction of decayed wood and was undetected in decayed PDA media (Figure3); thus, this compound is suspected to be a wood modification product synthesized during fungal decay. Meanwhile, peak 2 (RT 27.72) was detected in the chromatogram from crude decayed samples (Figure6), indicating that peak 2 is a compound released by F. radiculosa rather than a metabolized wood product. The mass spectra of peaks 1 and 2 indicated that these compounds are sesquiterpenes. Peak 1 (Figure4), which the NIST MS library indicated as longifolene, plays an important role in wood resistance to termite attacks. Xu et al. [14] suggested that this is one of the key components of Pinus massoniana resistance to C. formosanus attacks. Studies examining the function of hinesol (match of peak 2, RT 27.72) shown in Figure5, as a termite feeding deterrent have not been conducted to date to the best of our knowledge, although several studies have already documented the association of other sesquiterpenes with insect behavior. The differences in the amount and type of feeding deterrents produced by decayed wood and PDA media may be strongly linked with carbon and nitrogen as nutrient sources for F. radiculosa. Unlike fungi growing on soft wood, fungi grown in PDA medium are nourished with only a single sugar source. Secondary metabolite production depends on the composition of the culture medium and culture conditions. For example, sesquiterpene production by the cultured basidiomycete Lentinus lepideus varied depending on the type of sugar-enriched standard culture media. The sugar type influenced both the sesquiterpene increase and the decrease of volatile cinnamic acid derivatives; thus, the production of secondary metabolites in the form of VOCs is dependent on the fungal nutrient source [15]. Insects 2017, 8, 29 9 of 10

5. Conclusions Extracts from fungi-decayed PDA media inhibited R. speratus feeding, indicating that the deterrent substance originated from F. radiculosa rather than the wood decay process. Despite the stronger feeding deterrent effect of the crude PDA extract, its effects differed from those of decayed wood extract. Conceivably, decayed wood extract may contain two bioactive compounds that deter feeding and are toxic to termites. Chromatography easily removed the decayed PDA media–derived bioactive compound, causing the puriﬁed extract to lose its deterrent characteristics and weakly suppress termite feeding. According to the NIST MS match, the bioactive compounds were indicated to be sesquiterpenes—the decayed wood extract contained two bioactive suspect compounds, longifolene (similarity index 88.9) and hinesol (similarity index 78.2), while the PDA extract contained only hinesol. The F. radiculosa growth medium appears to be primarily responsible for the differences in the bioactive compound produced. Future studies on termite-fungi interactions should aim to further investigate the effects of F. radiculosa growth media and variations in feeding deterrents produced.

Acknowledgments: We would like to thank Tsuyoshi Yoshimura (Kyoto University) for providing the termite collection permit in RISH Field. Nadia N. Kamaluddin would like to thank for the funding provided by Indonesian Endowment Fund of Education (LPDP). Author Contributions: Nadia N. Kamaluddin, Shigeru Matsuyama and Akiko Nakagawa-Izumi conceived of and designed the experiments; Nadia N. Kamaluddin performed the experiments and analyzed the data. Nadia N. Kamaluddin, Shigeru Matsuyama and Akiko Nakagawa-Izumi wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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