An antifungal compound secreted by termite workers, phenylacetic acid, inhibits the growth of both termite egg-mimicking fungus and entomopathogenic fungi

Social insects live in closely related family groups but face risks of intrusion and infection by pathogenic and parasitic microbes. To cope with the microbes invading their nests and feeding sites, social insects produce various types of antimicrobial substances. Subterranean termites occupy microbe-rich decaying wood and soil at high density, expanding their nest area by exploring and feeding on wood outward from the royal chamber (room for kings and queens). Although antimicrobial agents have been identi�ed in many termite species, few studies have investigated those used by foraging workers in unexplored decaying wood, which is richer in microbes than the well-sterilized royal chamber and its surroundings. Here we report that phenylacetic acid, an antifungal aromatic compound, is secreted by foraging workers of the Japanese subterranean termite Reticulitermes speratus. The compound was detected by gas chromatography-mass spectrometry analysis of ethyl acetate extracts of shelter papers infested with the workers, and antimicrobial tests demonstrated that it inhibits the mycelial growth of the entomopathogenic fungus Metarhizium anisopliae and the termite egg-mimicking fungus Athelia termitephila. Our study provides new insights into the antimicrobial defense mechanisms of termites, including by combining different types of antimicrobial substances secreted by different castes, and thus the survival strategy of entomopathogenic and parasitic fungi in termite nests.


Introduction
The remarkable diversity and ecological success of social insects have been attributed to their ability to cope with infectious microbes invading their nests and feeding sites (Traniello et al. 2002;Cremer et al. 2018).Because social insects live in dense clusters with closely related family members susceptible to infections by the same pathogen (Cremer et al. 2007), infectious diseases can easily spread between group members, in contrast to solitary insects (Meunier 2015).Because larger social groups tend to have higher disease burdens, social insects have evolved prophylaxis and control measures to maintain hygiene in response to the potentially intense selection pressures posed by parasites and pathogens (Anderson and May 1979;Fefferman et al. 2007;Cremer and Sixt 2008).
Termites are social insects that form colonies of hundreds of thousands of close relatives, maintaining dense nests in microbe-rich wood and soil (Tsunoda et al. 1999).Maintaining a protected environment in their nests, by preventing pathogen invasion and growth, is thus a critical task of termites.This is achieved by the production of antimicrobial agents, such as α-pinene and limonene from Nasutitermes species (Zhao et al. 2004), trinervitane and n-hexanoic acid from Zootermopsis species (Rosengaus et al. 2004), and naphthalene, an antiseptic and an anthelmintic (Middleditch et al. 1981) found in the inner wall of the nest of Coptotermes formosanus Shiraki (Chen et al. 1998).Antifungal and antibacterial proteins (lysozymes, termicin, spingerin, and Gram-negative-binding proteins) have also been identi ed in many termite species (Bulmer and Crozier 2006; Terrapon et al. 2014;Mitaka et al. 2017a).However, as none these substances is effective against all pathogenic microorganisms, termites must have multiple antimicrobial substances with different antimicrobial spectra to combat the various types of pathogenic microorganisms in the environment.
Reticulitermes termites including R. speratus are classi ed as multiple-site nesters, whereby nests of a single colony are often interconnected by belowground tunnels and aboveground shelter tubes (Yanagihara et al. 2018).In a colony of R. speratus, a royal chamber, i.e., a room of kings and queens, is located deep in one of the nest trees (Yanagihara et al. 2018) and this chamber is kept at low risk of pathogen infection due to the antifungal protection provided by the antifungal volatile queen pheromone (mixture of n-butyl-n-butyrate and 2-mehtyl-1-butanol) (Matsuura and Matsunaga 2015) and antibacterial egg recognition pheromone (lysozyme) (Matsuura et al. 2007).On the other hand, the foraging area, i.e., the area away from the royal chamber and where soldiers protect nest entrance (Yanagihara et al. 2018) and workers often forage for new wood food (Crosland et al. 1997), are considered to be microbe-rich environment compared with the sanitized royal chamber.Therefore, workers and soldiers in the foraging area are at higher risk of disease transmission than those around the royal chamber.Previous studies revealed that soldier pheromone (β-elemene) secreted from soldiers suppress the growth of entomopathogenic fungi, Metarhizium anisopliae and Beauberia bassiana (Mitaka et al. 2017b), and mellein contained in bodies of workers and soldiers in the foraging area also suppress the growth of these fungi (Mitaka et al. 2019).The antifungal activity of these compounds is not so strong and only retards mycelial growth of entomopathogenic fungi.Nevertheless, foraging areas where workers come and go are usually clean and few areas of microbial growth are found (Cremer et al. 2007).This suggests that some antimicrobial substance may be present not only in the body of individual termite but also in the inside of the nest.
In addition to pathogenic microorganisms, termite-egg-mimicking fungus 'termite ball' are also parasitic in egg piles in the nests of Reticulitermes termites (Yashiro and Matsuura 2007).Termite balls are the sclerotia of Athelia termitephila (Maekawa et al. 2020).They mimic termite eggs both morphologically (Matsuura 2005) and chemically (Matsuura et al. 2009) and thereby be protected from desiccation and other microorganisms by egg-caring behavior of workers.Once the care of workers becomes absent, termite balls start to germinate (Matsuura et al. 2000;Komagata et al. 2022) and can consume surrounding eggs (Matsuura and Matsunaga 2015).Since the interaction is parasitic, in that it is bene cial for the fungus but costly for the host termites, the germination and growth of termite balls are usually inhibited throughout the nests in the led colonies (Matsuura 2006).The volatile queen pheromone, which is emitted by queens and eggs (Matsuura et al. 2010), is known to be able to inhibit germination and mycelial growth of termite balls (Matsuura and Matsunaga 2015).However, the queen pheromone should be absent or in very low concentration in foraging area far from the royal chamber, and β-elemene and mellein emitted by workers and soldiers have no inhibitory effect against termite balls (Mitaka et al. 2017b(Mitaka et al. , 2019)).Therefore, it is expected that compounds other than the queen pheromone inhibit growth of entomopathogenic microorganisms and termite balls in the interior surface of foraging areas.
In this study, ethyl acetate extracts of secretions on shelter papers infested with R. speratus workers were analyzed by gas chromatography-mass spectrometry (GC-MS), which led to the identi cation of phenylacetic acid.Tests of its activity against the growth of the egg-parasitic fungus A. termitephila and the entomopathogenic fungi M. anisopliae and B. bassiana were conducted.The antibacterial activities of this compound against several species of entomopathogenic (Bacillus subtilis, Bacillus thuringensis, and Serratia marcescens) and opportunistic (Micrococcus luteus, and Pseudomonas aeruginosa) bacteria were examined as well, based on previous laboratory bioassays demonstrating the pathogenicity of these entomopathogen to termites (Shimizu and

Ethics
No speci c permits were required for the described eld activities.Speci c permission was also not required to access or sample the termite colonies, as they were collected from unprotected public lands.This study did not involve endangered or protected species.However, in Japan, phenylacetic acid is classi ed as a raw material for stimulant production; we obtained the necessary designation certi cate by the governor of Kyoto Prefecture to allow its purchase from FUJIFILM Wako Pure Chemical Corp., Osaka, Japan.

Preparation of shelter papers
Substances secreted by termite workers into the inner walls of their nest were trapped using shelter papers (Fig. 1A).Two hundred workers from a colony were placed in a 35 mm dish lined with a lter paper (30 mm diameter, 0.26 mm thickness; Advantec No. 2; Toyo Roshi Kaisha, Ltd, Tokyo, Japan), with 20 dishes prepared from each of the ve colonies (TI003, 190124C, 190409B, 200622A, and 200622B).
The papers were moistened with 150 µL distilled water and the dishes were incubated at 25°C.Shelter papers were collected from each dish every 24 h for 7 days and preserved in a freezer at − 20°C for use in the analyses.
To extract the chemical compounds released by the termites, the shelter papers (n = 300) from each colony were placed in 200 mL glass jars and extracted with 200 mL ethyl acetate (FUJIFILM Wako Pure Chemical Corp.) for 24 h.The resulting crude extracts were concentrated to 1 mL using a rotary evaporator and then subjected to GC-MS analyses for chemical identi cation.

GC-MS analysis
GC-MS analyses were performed on a JMS-Q1500GC (JEOL Ltd., Tokyo, Japan) combined with an Agilent Technologies 7890B GC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a DB-1MS column (30 m × 250 µm × 0.25 µm, Agilent Technologies).The column temperature was held at 50°C for 5 min, then increased from 50 to 300°C at 20°C/min, and held for 5 min.Then a 1 µL sample was injected using an injector in splitless mode, helium as the carrier gas (1 mL/min), and an injection port temperature maintained at 250°C.MS data were obtained under the following conditions: 50 µA ionization current, 70 eV ionization energy, 2 kV accelerating voltage, and a 40-500 m/z scan range.Both GC and MS systems were controlled using an msPrimo system controller ver.1.06 (JEOL Ltd.).The data were analyzed using the software Escrime ver.2.04 (JEOL Ltd., https://www.jeol.co.jp/en/products/detail/JMS-Q1500GC.html).Candidate compounds were predicted from the mass spectral library (NIST11).Phenylacetic acid was identi ed according to its retention time and mass spectrum, based on a comparison with the commercial standard.

Antifungal tests
The antifungal activity of phenylacetic acid was tested against A. termitephila, M. anisopliae, and B. bassiana.The strain of A. termitephila used in this bioassay was isolated from the nest of R. speratus as described in a previous study (Mitaka et al. 2019).M. anisopliae (NBRC31961) and B. bassiana (NBRC103721) strains were provided by the Biological Resource Center (NBRC, National Institute of Technology and Evaluation, Tokyo, Japan) and cultured on potato-dextrose agar (PDA) plates at 28°C.
The assay was performed by placing a 5 mm diameter plug of growing mycelia from each fungal culture in the center a Petri dish (90 × 15 mm) containing phenylacetic acid at concentrations of 0, 50, 500, and 5,000 ng/µL (Fig. 2A).The Petri dishes were wrapped with two layers of Para lm and incubated at 28°C for 14 days.Five replicates were prepared for each treatment of each fungus.The size of the mycelia was measured by taking vertical photographs of each dish every 7 days after inoculation using a digital camera (tg-6; Olympus).The colony area in cm 2 was determined by counting the total number of pixels in the fungal colony area using ImageJ software (US National Institutes of Health, Bethesda, MD, USA) (Schneider et al. 2012).

Antibacterial tests
The antibacterial activity of phenylacetic acid against B. subtilis, B. thuringensis, M. luteus, P. aeruginosa, and S. marcescens was tested as described previously (Zhu et al. 2011).Strains of B. subtilis (NBRC3009), B. thuringensis (NBRC13865), M. luteus (NBRC16250), and P. aeruginosa (NBRC3080) were provided by the NBRC.The S. marcescens strain used in the bioassay was isolated from the nest of R. speratus following a procedure developed in a previous study (Inagaki and Matsuura 2018) and cultured on lysogeny broth (LB; NakaraiTesque) agar plates at 30°C.The agar diffusion assay was performed by spreading 100 µL bacterial inoculum (1-2 × 10 8 CFUs/mL) on an LB agar plate.Then 40 µL aqueous phenylacetic acid at concentrations of 0, 50, 500, and 5,000 ng/ µL (equivalent to 0, 2,000, 20,000, and 200,000 ng of phenylacetic acid) was added to a 7 mm (diameter) well punched in the center of the plate.As the negative control, distilled water was added to the well.Ten replicates were prepared for each treatment of each bacterium, except the 5,000 ng/µL treatment (20 replicates).The plates were incubated at 30°C for 24 h, after which antimicrobial activity was assessed by measuring the diameter of the inhibition zone using a digital caliper and then calculating the apparent area.Because the latter included the area of the well, the practical area of the inhibition zone was calculated by subtracting the area of the well from the apparent area of the inhibition zone.

Statistical analysis
All statistical analyses were performed using R software v. 4.2.2 (R Core Team 2016).Analysis of variance (ANOVA) followed by Tukey's HSD test was used in the antifungal tests, based on measurements of fungal colony area (the size of mycelia), and in the antibacterial tests, based on measurements of the area of the inhibition zone, on agar plates containing different concentrations of phenylacetic acid.

Estimation of the phenylacetic acid concentration in the foraging area
Field colonies of R. speratus contain a median and maximum of 24,500 and 451,800 workers, respectively (Takata et al. 2023b).In the subterranean termite Coptotermes formosanus, 20% of the workers are estimated to be foragers (Lee et al. 2022).Assuming the same proportion for R. speratus, the median and maximum numbers of workers in eld colonies of R. speratus are 4,900 and 90,360, respectively.If all foraging workers pass through an area of the same size as the shelter paper (706.858mm 2 [≈ 15 mm × 15 mm × π]) where they secrete phenylacetic acid for 24 h, the median and maximum concentrations of phenylacetic acid soaked into the spot would be 48,794.200ng (= 9.958 ng per worker × 4,900 workers) and 899,804.900ng (= 9.958 ng per worker × 90,360 workers), respectively.

Antifungal activity of phenylacetic acid
To test the inhibitory effects of phenylacetic acid on fungal mycelial growth, colonies of typical entomopathogenic fungi (M.anisopliae and B. bassiana) and the egg-mimicking termite ball fungus (A.termitephila) were exposed to authentic phenylacetic acid at different concentrations (Fig. 2).Inhibitory effects against M. anisopliae were observed at 50-5,000 ng/µL, against A. termitephila at 500-5,000 ng/µL, and against B. bassiana at only 5,000 ng/µL.

Antibacterial activity
To determine whether phenylacetic acid inhibits bacterial growth, colonies of entomopathogenic bacteria (B.subtilis, B. thuringensis and S. marcescens), a typical Gram-positive bacterium (M.luteus), and a typical Gram-negative bacterium (P.aeruginosa) were exposed to authentic phenylacetic acid at different concentrations.The compound inhibited the growth of all bacteria only at the highest tested concentration of 5,000 ng/µL (Fig. S1).

Discussion
In R. speratus colonies, foraging areas are a more microbe-rich environment than the royal chamber, such that foraging workers were predicted to secrete substances that inhibit microbial growth.Our GC-MS analysis revealed that foraging workers of R. speratus secrete phenylacetic acid (Fig. 1), initially identi ed as a growth-promoting substance in the 1930s but later shown to possess substantial antimicrobial activity in bacteria, fungi, algae, land plants, and insects (Fernández-Marín et al. 2015; Cook 2019).However, its antimicrobial effects against the entomopathogenic microorganisms of termites had not been investigated.The amount of phenylacetic acid secreted by R. speratus is su cient to suppress the growth of M. anisopliae and A. termitephila in their colonies, as according to the GC analysis the amount secreted per worker per 24 h was 9.958 ng.Assuming that all foraging workers in the eld continually secrete it over a foraging area equivalent to the area of one shelter paper (706.858mm 2 ), then after 24 h the cumulative median and maximum amounts of phenylacetic acid will be 48,794 ng and 899,805 ng, respectively.In PDA media with phenylacetic acid in our antifungal tests (Fig. 2A), the amount of this compound in an area equivalent to one shelter paper (assuming a thickness of 1 mm, that is, a volume of 706.858 mm 3 = 706.858µL) is 35,343 ng for the 50 ng/µL treatment, 353,429 ng for the 500 ng/µL treatment, and 3,534,290 ng for the 5,000 ng/µL treatment, respectively.The results of antifungal tests demonstrated that phenylacetic acid inhibited mycelial growth of M. anisopliae at the 50-5,000 ng/µL treatments, A. termitephila at the 500-5,000 ng/µL treatments, and B. bassiana at the 5,000 ng/µL treatment in antifungal tests (Fig. 2B).These mean that eld termites would suppress the mycelial growth of M. anisopliae if the number of foraging workers were above the median, and the growth of both M. anisopliae and A. termitephila if the number of foraging workers reaches the maximum.The growth of B. bassiana, however, would not be suppressed even at the maximum number of foraging workers.On the other hand, in the antibacterial tests, phenylacetic acid resulted in inhibition zones for all tested strains at the 5,000 ng/µL treatment, although the size of the zone was very small (≤ 0.8 cm 2 , Fig. S1).Even with the maximum number of foraging workers secreting the compound in the eld, bacterial growth would barely be suppressed.R. speratus releases a variety of antimicrobial substances with different effects on microbial growth.Mellein, secreted by foraging workers and soldiers, inhibits the mycelial growth of B. bassiana but not that of A. termitephila (Mitaka et al. 2019).Several termite pheromones also include antimicrobial compounds.For example, lysozyme, a component of termite egg recognition pheromone, has broad antibacterial activity against Gram-positive bacteria including Bacillus species (Matsuura et al. 2007), while queen pheromone, a mixture of n-butyl-n-butylate and 2-methyl-1-butanol, inhibits the germination and mycelial growth of M. anisopliae, B. bassiana, Isaria farinose, Sclerotium tuliparum, Athelia rolfsii, and A. termitephila (Matsuura and Matsunaga 2015).However, queen pheromone is secreted only by queens and eggs (Matsuura 2012).The internal structure of Reticulitermes termite nests is multi-layered (Yanagihara et al. 2018) and the radius of the foraging area can range from one meter to tens or even hundreds of meters (Vargo and Husseneder 2009).Therefore, the effective range of the antifungal activity of queen pheromone would be limited to the area around the royal chamber and its side egg chamber.In this work, phenylacetic acid secreted from foraging workers was shown to inhibit the mycelial growth of M. anisopliae and A. termitephila, with suppression involving the entire termite nest where colony members are active.Our results suggest that each caste of R. speratus makes use of multiple antimicrobial substances in combination to inhibit the growth of pathogenic microorganisms.
Antimicrobial activity of phenylacetic acid was also reported in Atta leaf-cutting ants, and these ants secrete this compound from the metapleural gland to inhibit the spore germination and growth of pathogenic fungi (Fernández-Marín et al. 2015).In that study, B. bassiana and Metarhizium brunneum were isolated from the ant nests but only the mycelial growth of B. bassiana was inhibited by phenylacetic acid.This contrasts with our bioassays, in which the growth of M. anisopliae but not B. bassiana was suppressed (Fig. 2).Thus, susceptibility to phenylacetic acid likely varies among fungal species within the same genus and within strains of the same species.Parallel to the competition between pathogens and termites, parasites and their host termites seem to be engaged in a coevolutionary arms race, in which parasites eventually gain resistance to antimicrobial substances.In open eld environments, the lower relatedness among parasites within infected hosts leads to higher levels of within-host competition, which selects for higher parasite virulence (Frank 1994).The coexistence of multiple strains of parasitic fungi within the nests of social insects gives parasitic fungi a distinct advantage in the coevolutionary arms race between hosts and parasites (Yashiro et al. 2011).In response, termites are likely to use multiple antimicrobial substances, alone, or in combination, against parasitic microorganisms that may be costly to the colony.The process of acquiring resistance to termite-secreted antimicrobial substances in entomopathogenic and parasitic fungi by species or strain should be investigated in the future.
Although the site of the biosynthesis of phenylacetic acid in the termite body is still unknown, it is probably in the gut tissues.A previous study reported that Reticulitermes avipes fed high amounts of xylan, a hemicellulose contained in wood, increased the production of phenylacetic acid in the gut (Brasseur et al. 2016).In the phenylalanine biosynthesis pathway of many organisms, phenethylamine is produced and subsequently metabolized to phenylacetic acid (Ramos and Filloux 2007).Thus, amino acid metabolism by gut symbionts of R. speratus may include the production of phenylacetic acid, which is then excreted.Its presence in termite feces may help maintain nest hygiene.In Zootermopsis nevadensis, acetate produced by gut symbionts and excreted by the termite suppresses the growth of S. marcescens (Inagaki and Matsuura 2018).The mechanism of action of acetate and other weak acids is thought to involve cytoplasmic acidi cation of pathogenic microorganisms, in turn inhibiting enzyme activity and amino acid transport (Hillenga et al. 1995;Lambert and Stratford 1999;Weber et al. 2012).Phenylacetic acid is also a weak acid such that it presumably inhibits pathogen activity by a similar mechanism.Further studies are needed to determine the site of phenylacetic acid biosynthesis and the mechanism underlying its inhibition of fungal growth.
In summary, R. speratus secrete phenylacetic acid into their nest materials, where it inhibits the mycelial growth of the parasitic termite egg-mimicking fungus A. termitephila and the entomopathogenic fungus M. anisopliae.Thus, this compound is one of several antimicrobial compounds, such as the antimicrobial components of pheromones (Matsuura et al. 2007;Mitaka et al. 2017b) and other antimicrobial agents (Mitaka et al. 2019), released by termites to protect their colonies.The simultaneous use of multiple antimicrobial substances enables termites to cope with a wide variety of parasitic microorganisms.Our work contributes to a more in-depth understanding of the defense mechanisms of termites against pathogenic and parasitic microorganisms.

Figure 1 Chemical
Figure 1