Journal of https://doi.org/10.1007/s10886-020-01178-2

Courtship Behavior Confusion in Two Subterranean Species that Evolved in Allopatry (, Rhinotermitidae, Coptotermes)

Thomas Chouvenc1 & David Sillam-Dussès2 & Alain Robert3,4

Received: 20 January 2020 /Revised: 19 March 2020 /Accepted: 30 March 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract Congeneric species that live in sympatry may have evolved various mechanisms that maintain reproductive isolation among species. However, with the spread of invasive organisms owing to increased global human activity, some species that evolved in allopatry can now be found outside their native range and may have the opportunity to interact, in the absence of mechanisms for reproductive isolation. In South Florida, where the Asian subterranean termite, Coptotermes gestroi (Wamann), and the Formosan subterranean termite, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae) are invasive, the two species can engage in heterospecific behavior as their distribution range and their dispersal flight season both overlap. rely on semiochemicals for many of their activities, including finding a mate after a dispersal flight. In this study, we showed that of both species produce (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (DTE) from their tergal glands as a shared sex . We suggest that both species primarily rely on an inundative dispersal flight strategy to find a mate, and that DTE is used as a short distance pheromone or contact pheromone to initiate and maintain the tandem between males and females. The preference of C. gestroi males for C. formosanus females during tandem resulted from the relatively high amount of DTE produced by tergal glands of C. formosanus females, when compared with those of C. gestroi females. This results in confusion of mating in the field during simultaneous dispersal flights, with a potential for hybridization. Such observations imply that no prezygotic barriers emerged while the two species evolved in allopatry for ~18 Ma.

Keywords Coptotermes . Sex pheromone . Reproductive isolation . Cuticular hydrocarbons . Pest species . (3Z,6Z,8E)-Dodeca-3,6,8-trien-1-ol

Introduction ultimately prevent, the exchange of genetic material among distinct populations (Nosil et al. 2017). Although the diver- Evolutionary processes that result in speciation often involve gence between species can emerge from geographic isolation reproductive isolation mechanisms that can reduce, and (allopatric speciation), it may also occur when the gene flow between two populations ceases over time owing to the estab- lishment of reproductive barriers (behavioral, physiological or ecological) while remaining within the same geographical * Thomas Chouvenc [email protected] range (sympatric speciation) (Fitzpatrick et al. 2008; Hoskin et al. 2005;Koppetal.2018;Malletetal.2009; Naisbit et al. 2001). However, occasional gene flow between two distinct 1 Department of Entomology and Nematology, Fort Lauderdale Research and Education Center, Institute of Food and Agricultural species may occur through hybridization events, bringing flu- Sciences, University of Florida, 3205 College Avenue, Fort idity to the concept of species (Mendelson and Shaw 2012; Lauderdale, FL 33314, USA Nosil et al. 2017). With the spread of various organisms be- 2 Laboratory of Experimental and Comparative Ethology UR 4443, yond their native range resulting from global human activity, University Sorbonne Paris Nord, 99 avenue Jean-Baptiste Clément, either as commercial commodities or as invasive pest species, 93430 Villetaneuse, France there is an increasing potential for congeneric species that 3 UPEC, SU, CNRS, IRD, INRA, Institut d’Ecologie et des Sciences initially evolved in allopatry over a geological time scale, to ’ de l Environnement de Paris, iEES Paris, 94010 Créteil now interact in human-mediated sympatry (Crispo et al. Cedex, France 2011). Therefore, there is an increasing possibility for two 4 ’ ’ Institut d Ecologie et des Sciences de l Environnement de Paris, distinct biological species that evolved in allopatry to now iEES Paris, Université de Paris, 94010 Créteil Cedex, France JChemEcol engage in mating interactions owing to a potential absence of conserved within a termite genus and sometimes across fam- prezygotic barriers. In such context, we here propose to inves- ilies (Bordereau and Pasteels 2011; Sillam-Dussès 2010). This tigate the causes of the potential hybridization between two observation reveals that, beyond the use of sex invasive termite species that evolved in allopatry (Chouvenc for mate-finding, there may be a potential lack of species et al. 2015), taking into account the complexity of their life recognition if two sympatric congeneric species rely on the cycle, which relies on brief but intense dispersal flight events same semiochemical cues for the formation of mating pairs. It (Chouvenc and Su 2014;Nutting1969). was suggested that termite speciation primarily resulted from Eusocial such as termites have a reproductive divi- oceanic dispersal events and vicariance (Bourguignon et al. sion of labor with the majority of individuals in the colony as 2016). However, in cases of sympatric termite speciation sterile workers and soldiers. However, as a colony reaches which resulted from the emergence of mating barriers over maturity, it invests in the next generation of functionally re- evolutionary times, reproductive isolation may have main- productive individuals which seasonally engage in dispersal tained termite species boundaries, preventing potential gene flights. Large numbers of winged imagoes (alates) fly away flow among populations (Bordereau et al. 2011;Dieckmann from their parental nests, and each individual tries to locate a and Doebeli 1999; Noor 1999), with some exceptions mate and establish a new colony as new primary king and (Aldrich and Kambhampati 2009; Lefebvre et al. 2008). queen (Nutting 1969). The dispersal flight event is often a Such barriers can be mechanisms that limit or prevent major survival bottleneck in the life cycle of termites, as only heterospecific mating, which often relies on a difference in a small fraction of all alates usually survives the process and the timing of dispersal flight events (Bordereau et al. 2010; successfully establishes an incipient colony (Chouvenc 2019). Connétable et al. 2012). Alternatively, sympatric termite spe- The phenology of termite dispersal flight can cover a wide cies may use a species-specific sex pheromone blend, made of spectrum between two extremes, from many small events a shared common major compound and some minor specific across the mating season (Ferreira 2008; Sugio et al. 2018), compounds, as found in three sympatric Cornitermes species to few inundative events with millions of flying alates that are (Bordereau et al. 2011). highly synchronized among colonies within a geographic area However, with the emergence and spread of invasive ter- (Chouvenc et al. 2017;Connétableetal.2012; Neoh and Lee mite pest species resulting from human activity (Bourguignon 2009). Mate finding strategies may also vary across termite et al. 2016;Chouvencetal.2016b;Evansetal.2013; species in terms of how males and females optimize their Hochmair and Scheffrahn 2010), some congeneric species chance to find each other during chaotic dispersal events that evolved through allopatric divergence may not have had (Mizumoto and Dobata 2019; Sillam-Dussès 2010). the selective pressure to evolve and maintain mating barriers. The alates flight is commonly followed by dealation and They may now have the opportunity to engage in mating courtship, which consists in calling and tandem behaviors behavior in their newly overlapping invasive geographic (Sillam-Dussès 2010). Although there is a high variability in range. Such heterospecific mating behavior was recently ob- secretory glands involved, semiochemicals used, and behav- served between Coptotermes gestroi (Wasmann) and iors displayed across termite taxa (Bordereau and Pasteels Coptotermes formosanus Shiraki(Chouvencetal.2015). 2011; Hanus et al. 2009; Leuthold and Bruinsma 1977), the Coptotermes (Rhinotermitidae) is one of the most important calling behavior is often characterized by the raising termite pest genera in the tropics and subtropics, and her abdomen and exposing some glands secreting a sex pher- C. formosanus and C. gestroi are also two successful invaders omone in order to attract a male. After pairing, the two (Chouvenc et al. 2016a; Evans et al. 2013;RustandSu2012). imagoes exhibit a tandem behavior in which the male follows Both species have evolved in allopatry for ~18 Ma the female in search of a suitable nesting site, and a combina- (Bourguignon et al. 2016), as C. formosanus is native to tion of secretory glands can be involved in the formation and Taiwan and Mainland China, and C. gestroi originates from the maintenance of the tandem pair (Sillam-Dussès 2010). As Southeast Asia (Li et al. 2010; Zhang and Evans 2017). the male approaches the female, it engages and maintains However, as a result of the increase of global human activity, contact by palpating the female abdominal extremity with and despite some differences in their ecological requirements antennae and mouthparts (Nutting 1969; Raina et al. 2003). (Patel et al. 2019a), they have spread and are now established In addition, both males and females usually possess a sternal in human-mediated sympatry in four distinct geographic re- gland that secretes a trail pheromone, which can then be in- gions: Taiwan, Hainan, Hawaii and Southeast Florida (Su volved in reinforcing the tandem or helping to reconnect the et al. 2017). During simultaneous dispersal flight events in tandem in case of a brief loss of contact (Leuthold 1975; south Florida, heterospecific were observed in the Sillam-Dussès 2010). field and such pairs were later able to establish viable hybrid A group of semiochemicals has been identified as trail and/ colonies in laboratory conditions, although the production of or sex pheromones across termite taxa, but some of these are fertile alates from such colonies remains to be observed used widely among diverse termite groups and are usually (Chouvenc et al. 2015, 2017; Patel et al. 2019b). Behavioral JChemEcol assays showed that males of both species would preferentially known as the trail pheromone in the Coptotermes worker caste initiate tandem behavior with C. formosanus females rather (Bland et al. 2007; Sillam-Dussès et al. 2006; Tokoro et al. than C. gestroi females in choice tests (Chouvenc et al. 2015). 1989) and potentially the reproductive caste (Bland et al. This observation was confirmed repeatedly (Fig. 1)suggest- 2007). Females also possess a pair of tergal glands (dorsal, ing that there is courtship behavior confusion in C. gestroi 9th and 10th abdominal segments), which were suggested to males, and semiochemical cues may be involved in such produce a contact sex pheromone that helps males for initiate a confusion. tandem pair (Bland et al. 2004;Rainaetal.2003). Male However, despite their major economic impact and wide Coptotermes do not possess tergal glands (Raina et al. distribution (Evans et al. 2019;RustandSu2012) and being 2003). However, DTE was not detected from female tergal among the most studied termite species (Chouvenc et al. glands and instead, a higher molecular weight 2016a; Krishna et al. 2013), the chemical communication in- compound, trilinolein, was isolated from female tergal glands volved in mating behavior of these two Coptotermes species (Bland et al. 2004), but was never confirmed as a contact sex remains unclear (Bland et al. 2007). According to laboratory pheromone in Coptotermes. Its function remains speculative, observations, Coptotermes females display no or minimal potentially as a nuptial gift from the female to the male (Park calling behavior, unlike most other termite species, and it et al. 2004). was suggested that, during their large dispersal flight events, As the role of tergal glands and the functional chemistry the high density of potential mates in the direct surrounding involved in Coptotermes mating behavior remain unclear, we rendered the need for a long distance volatile sex pheromone here propose that heterospecific mating behavior between two unnecessary, as males would randomly run into females and Coptotermes species may provide an opportunity to answer a engage in tandem behavior through the use of a putative, series of questions regarding the specificity of chemical cues, unidentified contact pheromone (Raina et al. 2003). Such hy- or absence thereof, involved in the formation of tandem pairs potheses were substantiated by Chouvenc et al. (2017), as in Coptotermes. Although DTE was previously not detected alates were observed to aggregate on trees or around artificial in C. formosanus female tergal glands (Raina et al. 2003), we lights, and then chaotically and randomly run into a potential here suggest that it may have been overlooked. Therefore, mate. DTE detection and quantification from Coptotermes female Both females and males possess a sternal gland (ventral, tergal glands were determined through an extensive gland 5th abdominal segment) that produces (3Z,6Z,8E)-dodeca- dissection protocol. In addition, analyses using gas chroma- 3,6,8-trien-1-ol (hereinafter referred to as DTE), which is tography coupled to mass spectrometry (GC-MS), microsco- py and behavioral assays were used, and the implications of the results are then discussed in the broader context of allo- patric speciation.

Methods and Material

Collection of Alate Termites

Alates of C. gestroi and C. formosanus were collected in Broward County (Florida, USA) between March and May of 2018 and 2019 using a light trap (Chouvenc et al. 2015, modified from Peppuy 1999)duringsimultaneousdispersal flight events at sunset, when environmental conditions were conducive (Chouvenc et al. 2017). Alates were temporarily placed in containers with moist corrugated cardboard and brought back to the laboratory. As termites dealated, individ- uals were sorted by species and by sex based on morpholog- ical traits (Scheffrahn and Su 2000), kept in independent Petri dishes with moistened sand, and used for experimentation within 24 h after collection. Because of the reliance on “fresh” alates for experimentation, the availability of termites for be- Fig. 1 Tandem behavior between one female imago in Coptotermes formosanus and one male imago in C. gestroi in a choice test. The havioral assay and dissections was variable over time and tandem was performed after the termite imagoes dropped their wings resulted in a variable number of replicates in assays. JChemEcol

Chemical Extractions and Analysis Cuticular Hydrocarbons of Females

In this study, all samples were prepared with hexane as sol- The cuticular hydrocarbon (CHC) profiles of females of vent. Dealates were placed on ice for at least 10 min before C. gestroi and C. formosanus were compared to investigate dissection. Tergal and sternal glands were independently dis- if the confusion of C. gestroi males to mate with sected from 30 females of each termite species and placed in C. formosanus female could be influenced by a similar com- hexane for 24 h at −4 °C, resulting in extract solutions of 30 position of CHC. Three females of each species were placed in individual-equivalent glands per 200 μl (3 to 5 replicates per individual 1.5 ml vials containing 200 μl of hexane and stirred sample, depending on alate availability). At the end of the for 30 min. The hexane extracts were transferred to new vials, extractions, hexane extracts were separated from the glands, concentrated down to 5 μl under nitrogen flow, and injected in transferred to new vials, and the samples were concentrated the GC-MS with the following specifications: HP-5MS Ultra underslownitrogenflowdownto5μl and injected into a gas Inert column (Agilent technologies 19091S-433UI, 30 m × chromatograph (Agilent Technologies 7890B) coupled to a 250 μm × 0.25 μm), splitless inlet set initially at 290 °C with mass spectrometer (Agilent Technologies 5977A) (GC-MS). 2 ml/min flow; oven temperature programmed from 60 °C to The GC-MS was setup with the following parameters: inlet 195 °C at 20 °C/min, 195 °C to 290 °C at 3 °C/min, and 250 °C splitless with 3 ml/min helium flow, column Agilent 290 °C to 325 °C at 15 °C/min with a 25 min hold time. Technologies DBWAX ultra inert (122-7032UI, 30 m × CHCs were identified, quantified, and compared to the worker 250 μm × 0.25 μm), oven 40 °C for 3 min, then at 5 °C/min profiles of the two respective species, using the worker dataset increase to 250 °C and held for 20 min, MS transfer line at previously obtained by Chouvenc and Su (2017). 250 °C. Histology

Identification and Volatility of (3Z,6Z,8E) The sizes of tergal glands in C. gestroi and C. formosanus -dodeca-3,6,8-trien-1-ol (DTE) females were determined through histology. Female abdo- mens were fixed in Bouin’s aqueous fixing solution for To confirm the presence of DTE and to determine its amount 24 h, and dehydrated in successive baths of ethanol, butanol, in gland extracts, a 1 ng / μl solution of a synthetic DTE and finally paraffin at 60 °C. Specimens were embedded in standard (kindly provided by the Nitto Denko Japanese paraffin blocks and cut on a microtome with 6 μmparasagittal Company) was used. The retention time of this DTE standard, and transversal sections. Sections were mounted and stained its relative abundance (integrated surface area of its peak) and with the Heidenhain’sAzanprotocol(Gabe1968). its MS ion profile were established and used as a reference for Observations and measurements were done with a DP70 dig- female gland extracts. In order to take into account the vola- ital camera mounted on an Olympus BX51 microscope. On tility of DTE and the potential loss of the pheromone during parasagittal sections, the width and the thickness of the tergal the concentration process of the gland extracts under nitrogen glands on the 9th and 10th abdominal segments were mea- flow, 4 ng DTE / 5 μland4ngDTE/200μlhexanesolutions sured every 5 sections, when possible. Lengths of the tergal were prepared and the latter concentrated down to 5 μlunder glands were estimated with transverse sections. The volume of nitrogen flow. The solutions were analyzed by GC-MS to each gland was estimated by simplifying the two independent determine the loss during concentration. This was then used glands as ellipsoids (4/3π xlengthxwidthxthickness)for to estimate the initial DTE amount from the gland extracts females of both species, using the average values for the three before it was concentrated. variables of each gland. A t-test was used to compare each In addition, we hypothesized that DTE, when in presence variable between females of the two species. of trilinolein (a viscous triglyceride secreted by female ter- gal glands (Bland et al. 2004)), may have a reduced volatil- Contact Behavioral Assays ity. To determine if the presence of trilinolein can reduce the volatility of DTE in vitro, two types of solutions were pre- It was already shown that both C. gestroi and C. formosanus pared in addition to the two 4 ng DTE standards described males would prefer to engage in a tandem run behavior with above: a solution with 4 ng DTE + 10 μg trilinolein / 5 μlof C. formosanus females rather than with C. gestroi females in a hexaneandasolutionwith4ngDTE+10μg trilinolein / choice test assay (Fig. 1,Chouvencetal.2015). Thus, most of 200 μl of hexane concentrated to 5 μl under nitrogen flow. the behavioral assays described below focused on C. gestroi Each solution was replicated five times, and injected in the males to determine the source of mating confusion. The bio- GC-MS. All relative abundances of DTE were compared assays tested for the preference of C. gestroi males for various with the 4 ng DTE / 5 μl hexane standard solution, and concentrations of DTE, and between different female tergal compared with an ANOVA. gland extracts from both species. The experiments aimed at JChemEcol determining the amount of DTE from the female tergal glands determine if the males spent significantly more time in contact that would influence the male decision to engage in a with one of the lures in each pairwise assay. heterospecific mating behavior. The behavioral assay was adapted from a choice test protocol (Fig. 2a) widely used to Distance Olfactory Assays detect sex pheromones in termites (Sillam-Dussès 2010). Three C. gestroi males were placed in a 9-cm Petri dish with The experiment aimed at revisiting the concept of “contact a filter paper at the bottom, and two lures positioned 4 cm pheromone” in Coptotermes, as suggested by Raina et al. apart. Each lure was a 2 × 1 cm piece of filter paper folded (2003). In order to test if C. gestroi males can detect DTE in a roof-like shape, and impregnated with 10 μltreatment from a distance, an olfactory assay (Fig. 2b) was adapted from prior to being placed in the Petri dish. Treatment comparisons Wen et al. (2012). Three cylindrical chambers (55 mm diam., included a serial dilution of a standard DTE solution, gland 50 mm high) were connected by a glass Y tube (8 mm i.d.) extracts and controls. The Petri dishes were observed for 300 s with a 50 mm distance for each branch of the Y tube. The two and the total times for which at least one male was in contact test chambers connected at the top of the Y tube were sepa- with the lure were recorded manually for each of the two lures. rated from the Y tube by a metallic mesh to prevent any imago Between 15 to 25 replicates were performed for each assay from escaping the chamber. The introduction chamber at the depending on termite availability. A paired t-test was used to base of the Y was connected to allow a male to enter the Y tube, but the chamber also had an additional hole on the op- posite side, protected by a metallic mesh. This hole was con- nected by a tube to a low pressure vacuum to create a gentle negative air flow from the two test chambers to the introduc- tion chamber. Each chamber contained a thin layer of moist sand and was closed with a punched lid. For each replicate, a C. gestroi male was gently placed in the introduction chamber and then was free to move into the Y tube. The choice for the test chamber was taken into account when the males first touched the metallic mesh of one of these chambers. Replicates in which the termite did not respond within 5 min were discarded. The test chambers contained either live ter- mites, or a paper lure impregnated with DTE dilutions or hexane as control, placed in the center of the treatment cham- ber. The average distance between the split of the Y tube and the two lures or live samples was 7.5 cm. Each assay was replicated 15 to 40 times depending on termite availability and tubes were washed with alcohol, rinsed with water and dried between replicates. Differences in the numbers of visits by males to the two test chambers were tested for significance by a binomial test.

Results

Detection of (3Z,6Z,8E)-Dodeca-3,6,8-trien-1-ol (DTE) in Coptotermes spp. Tergal Glands

Chemical analysis of gland dissections showed that DTE is produced in tergal glands of female Coptotermes (Fig. 3). We estimated by GC-MS that 14.9 ± 4.8 pg of DTE was present in Fig. 2 Behavioral assays were of two kinds: a Contact behavioral assay the tergal glands of one female of C. gestroi, and 128.6 ± in a 9-cm Petri dish where 3 Coptotermes gestroi males may be in contact 8.6 pg in the tergal glands of one female in C. formosanus. with two impregnated paper lures. b Distance olfactory assay with a: Females from each respective species secreted DTE from both introduction chamber, b: treatment chambers, c:Ytube,d:metalmesh, their tergal glands and their sternal gland, but overall e: pin hole for air flow, f: tube connected to vacuum, g: C. gestroi male, h: paper lure with treatment (or live individuals). Distance between c and C. formosanus females produced significantly more DTE than h =7.5cm C. gestroi females with an average of 8.6 fold the amount of JChemEcol

Fig. 3 Partial GC-MS profiles for (a)a1ng(3Z,6Z,8E)-dodeca- 3,6,8-trien-1-ol (DTE) standard, b mirrored extracts of male and fe- male Coptotermes gestroi termi- nal tergites (N =30),c mirrored extracts of male and female Coptotermes formosanus terminal tergites (N =30).d Mass- spectrum of DTE standard and chemical structure. Relative abundances at the same scale for all chromatograms. Arrows indi- cate an identical retention time and a > 98% mass spectrum match to the profile of (3Z,6Z,8E)-dodeca-3,6,8-trien-1- ol

DTE in C. formosanus females when compared to C. gestroi amount of DTE in C. formosanus females when compared females for tergal glands, and an average of 3.8 fold the to C. gestroi females for sternal glands (Table 1). JChemEcol

Table 1 Quantification of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (pg) in GC-MS analysis (mean ± SE, N = 3 to 5 replicates per category; means the tergal glands and the sternal gland of female imagoes (30 individuals with the same letter are not significantly different after ANOVA and dissected per sample) in Coptotermes gestroi and C. formosanus from Tukey HSD test)

Mean quantity (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol ± SE (pg)

Female dissections Coptotermes formosanus Coptotermes gestroi

Tergal gland 128.6 ± 8.6 a 14.9 ± 4.9 b Sternal gland 140.2 ± 25.7 a 36.0 ± 17.04 b

Volatility of (3Z,6Z,8E)-Dodeca-3,6,8-trien-1-ol (DTE) same level of difference of CHC profiles in female imagoes did not prevent C. gestroi males engaging in heterospecific The ratio of relative abundances of the diluted-then- mating behavior with C. formosanus females. concentrated DTE standard and the non-concentrated DTE standard was 0.39 ± 0.06 (N = 5) revealing that approximately Tergal Gland Histology and Measurements 61% of DTE was lost during the concentration process from 200 μlto5μl. However, when trilinolein was added to the The tergal gland sections from females of the two solutions, the DTE loss (14%) was reduced and the amount of Coptotermes species revealed that there are several morpho- DTE remaining after the concentration process was not sig- logical distinctions between the two species. Measurements of nificantly different from that in the reference DTE solution the average width, length and thickness using parasagittal and (Table 2). transversal sections showed that both tergal glands (9th and 10th abdominal segments) in C. formosanus were approxi- Cuticular Hydrocarbon (CHC) Profiles of Female mately three times bigger than in C. gestroi (Table 3). Coptotermes Additional observations showed that the cuticle from the seg- ment previous to a gland partially overlapped with the gland The CHC profiles of C. gestroi and C. formosanus females position in C. formosanus, exposing the location of the gland shared, with minor exceptions, the same alkanes, from (Fig. 5a-c). In comparison, locations of the tergal glands in tetracosane (C24) to heptacosane (C27), and their respective C. gestroi were fully overlapped by the cuticle from previous methyl-branched derivatives at positions 2, 3 and 11 (Fig. 4a). segments, preventing direct exposure of the glands to the air The profiles were qualitatively were very similar apart from (Fig. 5d-f). Therefore, not only tergal glands in C. gestroi were some lower molecular weight CHC’s found in trace amount in smaller than in C. formosanus, they were also less exposed C. formosanus, but they were quantitatively different as the when individuals were at rest. Observations in the field during major CHC was 11Me-C25 in C. gestroi but was 11Me-C27 dispersal flight also suggested that exposure of tergal glands in in C. formosanus (Fig. 4a). When compared to the CHC pro- Coptotermes females was minimal and short-lived (Fig. 6), files obtained from workers of each species using an identical when compared to other termite species (Bordereau and extraction and GC-MS protocol, the specific CHC profiles of Pasteels 2011). females from the two Coptotermes species were within their respective intraspecies variability (Fig. 4b), showing a rela- Contact Behavioral Assays tively high conservation of CHC profiles among castes within a species. Although the difference of CHC profiles between As C. gestroi males were released in the Petri dish with two the two species was correlated with agonism in workers, the treated lures, they readily initiated contact with the lure treated

Table 2 Determination of the relative volatility of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (DTE) during the process of concentrating samples under gentle nitrogen flow from 200 μlto5μlin15min(N = 5; means followed by same letter are not significantly different after ANOVA and Tukey HSD test)

Sample preparation Mean DTE detected ± SE (ng) DTE loss (%)

4 ng DTE in 5 μl hexane (standard) 4.00 ± 0.35 a N/A 4 ng DTE in 200 μl hexane evaporated to 5 μl 1.56 ± 0.26 b 61% 4 ng DTE + 10 μg trilinolein in 5 μl hexane 3.54 ± 0.34 a 11% 4 ng DTE + 10 μg trilinolein in 200 μl hexane evaporated to 5 μl 3.46 ± 0.43 a 14% JChemEcol

Fig. 4 a GC-MS profiles of cu- ticular hydrocarbons of Coptotermes gestroi and C. formosanus female imagoes (1: 2-Me-C24, 2: C25, 3: 11-Me- C25, 4: 2-Me-C25, 5: 3-Me-C25, 6: C26, 7: 11-Me-C26, 8: 2-Me- C26, 9: C27, 10: 11-Me-C27, 11: 2-Me-C27, 12: 3-Me-C27) and (b) compared by PCA with workers GC-MS profiles of both species (from Chouvenc and Su 2017)

with DTE, and maintained the contact for most of the duration on the DTE lure than the C. gestroi TGE (Fig. 8). Such obser- of the experiment. This observation was especially visible vations suggested that the quantity of DTE in tergal glands of with DTE at 1000 pg, 100 pg, 10 pg and to some extent, even C. gestroi was in the range of 10 pg, and corroborated the at 1 pg, when tested against control treated lures (paired t-tests, quantity estimation made by chemical analysis (Table 1). P < 0.05). However, males did not differentiate between lures When testing with C. formosanus gland extracts, C. gestroi treated with 0.1 pg DTE and 0.01 pg DTE when compared to males showed preference for 1 C. formosanus TGE when the controls (Fig. 7). tested against 1 C. gestroi TGE. However, when 1 C. gestroi When using extracts of tergal glands of C. gestroi females, TGE was spiked with 100 pg DTE, C. gestroi males did not C. gestroi males aggregated more on the tergal gland equiva- discriminate when tested against 1 C. formosanus TGE. lent extract (TGE) than on controls or to 1 pg DTE (paired t- Finally, C. gestroi males aggregated significantly more on a tests, P <0.05).However, C. gestroi males were not able to lure treated with 1 C. gestroi TGE + 200 pg of DTE, when discriminate between 10 pg DTE and 1 C. gestroi TGE, but compared with 1 C. formosanus TGE (Fig. 8). These last when increasing DTE to 100 pg, the males aggregated more experiments suggested that there was approximately 100 pg more of DTE in C. formosanus tergal glands than in C. gestroi tergal glands, and again corroborated the quantity estimation by chemical analysis (Table 1). Table 3 Measurements of tergal gland thickness, width and length, obtained from parasagittal and transversal histological sections in Coptotermes spp. females Distance Olfactory Assays Mean ± SE (μm) Measurement Coptotermes formosanus Coptotermes gestroi This experiment showed that C. gestroi males could discrim- Tergal gland, 9th segmenta inate between a group of 5 live C. gestroi females and a group Thickness 37.56 ± 1.90 a 34.39 ± 1.18 a of 5 live C. gestroi males at short distance (7.5 cm) and pref- Length 192.75 ± 4.31 a 117.08 ± 1.91 b erentially walked toward the group of females, but could not Width 1026.78 ± 16.53 a 609.06 ± 31.77 b differentiate between a single male and single female Tergal gland, 10th segmenta C. gestroi (Table 4) implying that the amount of DTE emitted Thickness 45.60 ± 1.12 a 33.58 ± 1.17 b byasinglefemaleC. gestroi in such testing conditions may Length 143.43 ± 4.42 a 112.59 ± 3.31 b not be enough to attract a male at such distance. However, Width 672.34 ± 16.24 a 325.03 ± 34.10 b C. gestroi males could detect 1 tergal gland extract equivalent Extrapolated volumesb of C. gestroi females at a distance when compared with a 9th Tergal gland 31.1 nl 10.3 nl control and were also able to detect DTE as low as 100 pg 10th tergal gland 18.4 nl 5.1 nl (threshold) in the same experimental conditions (Table 4). Finally, C. gestroi males preferentially walked toward a group a The t-test compared two values within a line; N = 3 individuals for each of 5 live C. formosanus females rather than a group of 5 live type of measurement with between 3 to 10 sub-replicates per individual; means followed by the same letter are not significantly different at C. gestroi females (Table 4). P <0.05 When using C. formosanus males for behavioral assays, b Gland volumes were extrapolated from an ellipsoid formula using the they were able to detect DTE as low as 1 pg (threshold), and mean measurements also preferentially walked toward a group of 5 live JChemEcol

Fig. 5 Histological sections of the female tergal glands at the 9th tergite (TG-9) or at the 10th ter- gite (TG-10) in Coptotermes formosanus (left side) or in C. gestroi (right side) stained with Azan (a, d:sagittalsections;b, e: transversal section of the 9th ter- gite; c, f: transversal section of the 10th tergite). Scale bar = 100 μm

C. formosanus females rather than a group of 5 live C. gestroi DTE, released by the tergal glands, is the sex pheromone that females (Table 5). C. gestroi females use to attract males, and also most likely used as sex pheromone in C. formosanus.WhileC. gestroi males were able to detect as low as 1 pg DTE when in contact, Discussion they were only able to detect the pheromone over short dis- tances (7.5 cm distance) at 100 pg DTE and above. In This study was able to demonstrate that (3Z,6Z,8E)-dodeca- 3,6,8-trien-1-ol (DTE) was present in the tergal glands of both Coptotermes formosanus and C. gestroi females, contradic- ting the initial findings by Raina et al. (2003) and Bland et al. (2007)thatfemaleC. formosanus tergal glands do not produce DTE. Our experimental bioassays demonstrated that

Fig. 7 Choice-test preference in contact behavioral assay of Coptotermes gestroi males between two samples, hexane (as control) or a range of dilutions of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (DTE). Box-plots repre- sent the difference in times (sec) that at least one of 3 C. gestroi males Fig. 6 Female of Coptotermes gestroi during a dispersal flight. This spent in contact with each samples over the experimental period of 300 s. female that just landed on an oak tree and just dealated. Tergal glands * indicates a significant difference in time spent (paired t-test, P <0.05) from distal tergites (arrow) are marginally exposed on one of the two samples (N =15) JChemEcol

males, it was not considered as a sex pheromone but rather as a nuptial gift transferred from the females to the males (Bland et al. 2004;Parketal.2004). Instead, we showed that when in presence of trilinolein, a high molecular weight tri- glyceride, the volatility of DTE was drastically reduced in vitro. The relatively high viscosity of trilinolein (Rabelo et al. 2000) secreted by female tergal glands and coating the surface of the terminal abdominal tergites (Park et al. 2004) may therefore reduce the volatility of DTE, converting a vol- atile sex pheromone into a functional contact sex pheromone. The transfer of trilinolein to males observed by Park et al. (2004) may therefore simply have been a passive transfer through recurrent contacts during tandem behavior. The fact that Coptotermes has the ability to display major synchronous dispersal events (Chouvenc et al. 2017) could partially explain why it may only rely on a sex pheromone that primarily functions as a contact pheromone or as a short- distance pheromone. Alates aggregate on large trees, marked- Fig. 8 Choice-test preference in contact behavioral assay of Coptotermes ly so in C. gestroi, but also visible in C. formosansus, and gestroi males between two samples: hexane (as control), tergal glands extracts (TGE) of 1 female-equivalent of Coptotermes gestroi or artificial light sources, when several thousands of individuals C. formosanus,or(3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (DTE). Box-plots run frantically on the bark and chaotically run into a potential represent the difference in times (sec) that at least one of 3 C. gestroi mate. Yet, we cannot fully exclude that DTE could still work males spent in contact with each samples over the experimental period of as a long-range sex pheromone in Coptotermes. It is still pos- 300 s. N = 15 to 25, depending on the availability of C. gestroi males at the time. * indicates a significant difference in time spent (paired t-test, sible that during large dispersal flights, when thousands of P < 0.05) on one of the two samples female alates aggregate on a tree, they could indirectly create a DTE plume resulting from the cumulative DTE secretory comparison, C. formosanus males were able to detect as low activity from their sternal and tergal glands, potentially as 1 pg DTE at short distances. Our results therefore support impacting the ability of males to alter their flight toward such the concept initially suggested by Raina et al. (2003)thatthe trees. This hypothesis remains to be tested, but our results and sex pheromone in Coptotermes primarily functions as a con- field observations suggest that males of both species are rela- tact pheromone, but potentially also as a short-distance sex tively inefficient in locating females during swarms, especial- pheromone. However, we argue that this characteristic did ly for C. gestroi males. It was showed that Coptotermes and not emerge from the volatile properties of DTE, but instead Reticulitermes have a distinct mate finding behavior in case is a consequence of the co-secretion of the involatile trilinolein the tandem was interrupted. Reticulitermes females would by tergal glands. Indeed, trilinolein has been identified as a stop for a long period of time for the male to find her and sex-specific component of the females of Coptotermes reconnect, while Coptotermes females would resume walking formosanus, but because it did not elicit sex attraction in in a much shorter time (Mizumoto and Dobata 2019). This

Table 4 Distance olfactory assays allowing male Sample Coptotermes gestroi male choice Coptotermes gestroi to detect a paper lure impregnated with DTE Chamber 1 Chamber 2 N Chamber 1 Chamber 2 dilutions, tergal gland extract equivalent (TGE), or hexane (as 10,000 pg DTE Hexane 30 26 4 * control), or groups of live females 1000 pg DTE Hexane 30 22 8 * or males from different species, 100 pg DTE Hexane 30 24 6 * placed in two different chambers in test-choice (* indicates signifi- 10 pg DTE Hexane 30 18 12 cant difference using a binomial 1 pg DTE Hexane 30 19 11 test, P <0.05) 5 C. gestroi females 5 C. gestroi males 30 26 4 * 1 C. gestroi female 1 C. gestroi male 30 18 12 5 C. formosanus females 5 C. gestroi females 40 25 15 * 1 C. gestroi TGE Hexane 30 22 8 * JChemEcol

Table 5 Distance olfactory assays allowing male Sample Coptotermes formosanus male choice Coptotermes formosanus to detect a paper lure impregnated Chamber 1 Chamber 2 N Chamber 1 Chamber 2 with DTE dilutions, or hexane (as control), or groups of live females 10,000 pg DTE Hexane 15 12 3 * from different species, placed in 1000 pg DTE Hexane 30 27 3 * two different chambers in test- 100 pg DTE Hexane 30 27 3 * choice (* indicates significant difference using a binomial test at 10 pg DTE Hexane 15 13 2 * P < 0.05) 1 pg DTE Hexane 15 13 2 * 0.1 pg DTE Hexan 15 10 5 5 C. formosanus females 5 C. gestroi females 40 36 4 *

distinct behavior may reflect a peculiar mate finding strategy behavior has not previously been observed in C. formosanus in Coptotermes that relies on short distance cues, when com- which questioned the occurrence of a long-range sex phero- pared to other species (Bordereau and Pasteels 2011), as they mone (Raina et al. 2003), we were able to observe a short- may primarily rely on an inundative method. In this context, lived calling behavior, with marginal exposure of the tergal the sex pheromone may serve to initiate and to maintain the glands by females (Fig. 6). It is likely that this behavior was tandem, but not to create the encounter in the first place, as it not previously noticed because of its ephemeral occurrence would not be necessary during large synchronized dispersal during the dispersal event in the field, and its absence of dis- events. play during later observations in the laboratory. In addition, DTE was already identified in the sternal gland of the color of the dorsal cuticle in C. formosanus alates is much C. formosanus males and females but its role when secreted lighter than in C. gestroi, which potentially makes it challeng- ventrally was unclear and it was not considered as a sex pher- ing to see the tergal gland exposure during the dispersal flight omone (Bland et al. 2007). According to these authors, the events. amount of DTE was about 220 pg in the female sternal gland, We here suggest that the inherently low reliance on a dis- with a high variability in the colonies studied, while we esti- tance sex pheromone and the high reliance on the enhanced mated in the current study that the quantity of DTE was closer encounter rate in Coptotermes is an important factor for the to 140 pg per sternal gland. This minor difference of DTE emergence of mating confusion between the two termite spe- estimation could emerge from a regional difference in cies, in addition to a shared sex pheromone and difference of C. formosanus, or in small differences of the calibration in tergal glands size between the two species. Coptotermes the DTE standard used and the GC-MS column used. We here formosanus tergal glands are larger and produced significantly suggest that males and females secrete DTE from their sternal more DTE than C. gestroi tergal glands, and C. formosanus gland as a trail pheromone to create a path in order to ensure males can detect lower concentrations of DTE at a short dis- the tandem cohesion by reducing an accidental tandem sepa- tance than C. gestroi males. When given the choice, males ration and improve tandem reformation in case of separation from both species preferentially engaged in tandem behavior (Bordereau and Pasteels 2011; Mizumoto and Dobata 2019; with C. formosanus females (Chouvenc et al. 2015)because Nutting 1969). Interestingly, workers of C. formosanus and of the higher amount of DTE they perceive. Besides, by ma- C. gestroi secrete the same compound, DTE, from their sternal nipulating the content of DTE from tergal gland extracts, we gland and use it as a trail pheromone (Bland et al. 2007; were able to show that when C. gestroi males have the choice Sillam-Dussès et al. 2006; Tokoro et al. 1989). Such use of between two gland extracts, they would preferentially main- compounds as trail pheromone or as sex pheromone according tain contact with the lure containing the highest absolute to the caste secreting the compound has been already reported amount of DTE, regardless of the origin of the extract. Such in termites (e.g. Bordereau et al. 1993, 2010;Laduguieetal. observation therefore confirms that DTE is a semiochemical 1994; Sillam-Dussès et al. 2011; Robert et al. 2004). This secreted by female tergal glands in both C. gestroi and study underlines once again the high conservative nature of C. formosanus with no species specificity. This phenomenon releaser pheromones in termites. has already been detected in the sympatric fungus-growing In termites, the swarming is followed by the courtship be- termites, Pseudacanthotermes spiniger and P. militaris.In havior, which consists of calling behavior, i.e. the female rais- the laboratory, males of P. militaris are preferentially attracted ing its abdomen to expose its glands and to secrete the sex by female sternal gland extracts of P. spiniger which contain pheromone, and then the tandem, i.e. the female, followed by more DTE than their own glandular extracts. The species the male, searching for a nesting site. Although the calling specificity of the mating behavior in these two sympatric JChemEcol species is ensured by dispersal flights occurring at different find a mate without the use of long distance semiochem- times of the season (Bordereau et al. 1993; Bordereau and icals, as live trees may be default points of rendezvous. Pasteels 2011). DTE then becomes important to initiate and maintain the The situation is different in Coptotermes gestroi and tandem between a male and a female. Such strategy could C. formosanus since they have evolved in allopatry for be a numbers game highly biased in favor of mature col- ~18 Ma, and only recently have become sympatric in onies producing the most alates in the area (Chouvenc few restricted geographic areas, including South Florida 2019). This might imply that Coptotermes,knowntobe (Bourguignon et al. 2016; Chouvenc et al. 2015;Suetal. one of the rare termite genera to infest live trees, would 2017). Their dispersal flights now overlap both in location primarily rely on first finding a tree to increase the poten- and in time and heterospecific mating behavior was ob- tial to find a mate, instead of relying on semiochemicals served since 2013 (Chouvenc et al. 2017). Therefore, if directly produced by such potential mates. Such strategy minor specific compounds are secreted with the sex pher- would increase the chance for a tandem pair to form at a omone (DTE), they may not have much relevance to proper initial nesting site and could partially explain the C. gestroi males for exclusive conspecific mating prefer- ecological success of this genus, and the invasive success ence, as observed in some sympatric termite species. For of these two species, considered as two of the most eco- example, DTE is the only compound of the sex phero- nomically important invasive in the world mone in Cornitermes bequaerti, whereas it is associated (Bradshaw et al. 2016; Chouvenc et al. 2016b; Krishna with (E)-nerolidol in the sympatric Cornitermes et al. 2013). cumulans, and with (E)-nerolidol and (Z)-dodec-3-en-1- In conclusion, with the emergence of heterospecific ol in the sympatric Cornitermes silvestrii (Bordereau mating opportunities between the two termite species in et al. 2011). Another mechanism used in nestmate recog- Florida owing to a shared chemical cue, an absence of nition to prevent heterospecific interaction is the occur- species recognition, and an overlap of dispersal flight rence of different cuticular hydrocarbons (CHC) coating events, our study therefore suggests that it is unlikely the cuticle of termites (Clément and Bagnères 1998; that prezygotic barriers were established between the Howard and Blomquist 2005).Here,wewereabletocon- two Coptotermes speciesoverthe~18Maofevolution- firm that imagoes of the two Coptotermes species share ary times in allopatry. The sex pheromone DTE is widely qualitatively similar CHC profiles, although the relative used in Rhinotermitidae (Sillam-Dussès 2010), and it is quantities of the different components differ. When com- possible that DTE was conserved as the primary sex pared with the CHC profiles obtained from workers of pheromone throughout the Coptotermes genus. If this both species, females displayed a CHC profile similar to statement holds true, the existence of multiple sympatric the one from workers of their own species. Such CHC Coptotermes species in Australia and Southeast Asia profile differences were associated with agonistic behav- (Chouvenc et al. 2016a) raises the question if reproduc- ior between foraging workers of both species (Chouvenc tive isolation mechanisms were established among such and Su 2017), but in the current study, such quantitative species or if potential gene flows may have casually oc- CHC difference did not deter C. gestroi males from en- curred. A further investigation of Coptotermes species gaging in tandem behavior with C. formosanus females that may have gone through sympatric speciation, with (Chouvenc et al. 2015). It is likely that during the dispers- a focus on their flight phenology, their chemical ecology al flight, the males are so excited and focused on finding a and their mating behavior may therefore provide a female that they may not be able to detect the quantitative unique insight on evolutionary pressures that may have differences between the two different CHC profiles. The shaped reproductive isolation mechanisms, or lack there- relative similarity of female CHC between the two of, during termite diversification. Coptotermes species may therefore render these differ- ences in chemical cues secondary to the sex pheromone Acknowledgements Thanks to Ronald Pepin, Reynaldo Moscat, Alvin emitted by females. Puzio, Aaron Mullins, Joseph Velenovsky, Sang-Bin Lee for their help in collecting alates and performing some of the behavioral assays, Andy Fu Therefore, our study showed that both C. gestroi and and Johnalyn Gordon for their technical assistance with the chemical data C. formosanus share DTE as sex pheromone from tergal acquisition, Nan-Yao Su for the use of research space and his undeterred glands but C. formosanus females produce more DTE support for termite research, and two anonymous reviewers for their con- than C. gestroi females, which would explain why both structive comments. C. formosanus and C. gestroi males would preferentially Funding Information Thisstudywassupportedinpartbyagrant mate with C. formosanus females, as observed in the from the USDA National Institute of Food and Agriculture, Hatch field, raising the possibility for the establishment of hy- project number FLAFLT-005660/, by NSF-DEB grant agreement brid colonies (Chouvenc et al. 2015). Both species pri- No. 1754083, and a University of Florida Early Career Seed marily rely on an inundative dispersal flight strategy to Grant No. REA1801100. JChemEcol

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