Insects

The termites, C. formosanus, used in this study were collected from one colony in Fukuoka, Japan and were maintained in plastic boxes (49 × 36 × 32 cm) in a dark chamber at 25° C; they were fed on seasoned pinewoods (kuromatsu; Pinus thunbergii). Workers were transferred from the above colonies into 90 × 15 mm Petri dishes containing a wet paper disc (No 2. Qualitative Filter Paper (90 mm dia.), Toyo Roshi Kaisha Ltd.,  www.advantec.co.jp/) and placed in a dark chamber at 25° C for one to three weeks before use.

Preparation of conidial suspensions

Entomopathogenic fungi, Beauveria brongniartii 782, Paecilomyces fumosoroseus K3, and Metarhizium anisopliae 455 were maintained, and 1.0 × 10⁷ conidia/ml conidia suspensions were prepared as described in Yanagawa et al. (2009). FITC-labeled conidia suspensions were described as A-series suspensions, and unlabeled conidia suspensions were described as B-series suspensions.

Detection of fungal conidia on cuticle of C. formosanus

In order to know the removal pattern of three fungal isolates, conidia attached to the cuticle after inoculation were detected in the same way as in the previous study (Yanagawa et al. 2008). Termites were submerged in an FITC-labeled conidial suspension (A-series) for inoculation and dried on filter paper. After inoculation, they were partitioned into groups of 10 in 90 × 15 mm Petri dishes containing a wet paper disc and incubated in a dark chamber at 25° C. At 0, 3, 6, and 24 h post-treatment, they were carefully mounted in a drop of Vectashield (Vector Laboratories,  www.vectorlabs.com) to stabilize the fluorescence, and the conidia attached on their cuticle were counted with an epifluorescent microscope (Axioplan 2, Carl Zeiss,  www.zeiss.com) at 200 x. The sum of five sites (head, thorax, and 2nd, 4th, and 6th abdominal segments) on each C. formosanus surface were calculated for the number in a square millimeter to estimate the amount on the whole body.

Grooming behavior of C. formosanus with/without antennae

To clarify how antennae affect grooming behavior, this behavior was examined for termites with or without antennae that had been inoculated with fungal conidia. To prepare termites without antennae, both antennae were cut off at the scape after termites had been cold anesthetized on ice for 30 min. Termites with no antennae were reared in 90 × 15 mm Petri dishes containing a wet paper disc in a dark chamber at 25° C for over one week to allow the antennal cut end to heal. Termites with intact antennae were also anesthetized on ice for 30 min and reared for the same period as those without antennae in order to obtain control data. Thereafter, they were inoculated with M. anisopliae 455 conidial suspension (B-series) as described above. After inoculation, five termites were each put into Petri dishes (35 × 15 mm) and covered with a cardboard box during the experiment to reduce a room light effect on movements. Termites were left for 15 min to reduce the impact of treatment. Since it is impractical to observe and estimate the level of the grooming behavior for its whole duration, the frequency of touching each other was counted on photographs taken every 30 s for 30 min. Thus, total sixty photographs were taken per dish. Only the C. formosanus whose mouthparts touched their nestmates were counted. The number was estimated for each treatment with and without antennae inoculated with M. anisopliae 455 conidial suspensions or with a Tween 20 solution (control). Data obtained from 30 replicates and overall 600 termites were examined. The number of contacting termites was converted to the touching rate (%); total number of nestmate-touching termites/60 (all photographs).

Feeding preference

Since the conidia removal due to grooming behavior in C. formosanus occurs by eating them (Yanagawa and Shimizu 2007), the preference for fungi was estimated by food intake rate (%) of the fungal-flavored filter paper disc, modifying the method of Ohmura et al. (2006). Though feeding rates could be affected by factors other than olfactory, as the discs used in this test had the same texture, the termites could not discriminate them by touch easily. Thus, especially at first, they should have discriminated between discs by utilizing antennal chemosensory receptors, which detected chemical cues emitted from conidial suspensions. To estimate the effects of the feeding activity on grooming behavior, the application time was decided as first 24 h in which the grooming behavior showed its benefit (Yanagawa and Shimizu 2005). Six quadrangular filter paper discs (7 × 7 mm) were placed on the bottom of Petri dishes (35 × 15 mm). The amount of food ingested was estimated by the following procedure. Three of six discs were treated with 150 µl test solution (B-series) containing 2 mg/ml of a blue food color (Blue Food Color No. 1, TCIFC: FO147, Tokyo Kasei Kogyo Co., LTD.  www.tokyokasei.co.jp), and the others were treated with 150 µl colorless 0.025% Tween 20 solution (Treatment I). To avoid the influences of the food color material, the experiments were also conducted with opposite coloring (Treatment II) (Figure 2). Five workers were placed on the dish in each trial and allowed to feed on discs for 24 h. Thereafter, the C. formosanus in each dish were subjected to homogenization in 500 µl of 50% EtOH. After centrifuging twice at 15,000 rpm for 10 min, 150 µl of supernatant liquid was collected and the absorbance (Abs) of each was measured at 630 nm by an absorptiometer (DU-640E, Spectrophotometer, Beckman Coulter_TM,  www.beckmancoulter.com). The feeding preference of conidial suspension was calculated by the following equation: Feeding preference (Absorbance Index, AI) = (Abs Treatment I - Abs _{Treatment II}) / Abs _{Treatment II}. Data of 10 replicates, 100 C. formosanus per fungus, were pooled and analyzed per fungal isolate.

Single sensillum recording

C. formosanus were fixed in a specimen holder made of a standard blue pipette tip (100–1000 µl) with wet cotton, and their antennae were arranged to stick out of the end of the tip, the orifice diameter of which had been fitted to the size of the termite. The cotton was moistened with physiological saline for cockroaches (200 mM KCl, 1.55%; 200 mM CaCl₂-2H₂O, 0.9%; 200 mM Na₂HPO₄-2H₂O, 0.1%; 200 mM NaH₂PO₄-H₂O, 0.9%) (Yamazaki and Narahashi 1959). An antennal platform was made of small piece of acrylic plate, and an antennal pillar was made of an electrolytically sharpened tungsten rod (0.5 mm in diameter) as protectors to prevent the antennae from moving during recording of electrical activities. The specimen was set on a research microscope (BX51WI, OLYMPUS) with the protectors. The active electrode, which was also an electrolytically sharpened tungsten rod, was inserted into the base of a sensillum, enough to make an electrical contact. The indifferent electrode, a silver wire (0.2 mm in diameter), was set in the specimen holder to contact the wet cotton. Electrical events were recorded using standard equipment.

The stimulus and control air were prepared as described in Yanagawa et al. (2009). Briefly, fresh air from the pure air source was divided to the two stream routes, a control stream and a stimulus stream, which were switched by two electric valves for stimulation, and the small glass bottles (30 ml) were set between the two routes. Each bottle contained a different kind of odor substance. One bottle contained 1 ml of 0.025% Tween 20 solution without any suspension. Each of the others contained 1 ml B-series conidial suspensions; B. brongniartii 782, P. fumosoroseus K3, or M. anisopliae 455. The air passing through these bottles was separately fed to glass tubes for stimulation. The specimen was exposed to the control clean air during the interstimulus time.

Figure 1.

Persistence of conidia on the Coptotermes formosanus cuticle. (black): Metarhizium anisopliae 455 conidia remaining rate on the cuticle, (white): Paecilomyces formosanus K3 conidia remaining rate on the cuticle, (red): Beauveria brongniartii 782 conidia remaining rate on the cuticle. Y bar expresses the conidia remaining rate (%) on the cuticle; number of conidia on the cuticle at each time interval/number of conidia just after inoculation. Bars at top of columns represent standard errors. High quality figures are available online.

Identification of sensillum

After impulse activities to a given odor were successfully recorded from sensilla, the regions of the sensilla were sketched in order to help locate the same sensilla on scanning electron microscope (SEM) observation. Samples were prepared by ultrasonic cleaning in 70% acetone, fixed in 4% (v/v) osminium acid for 2 h and dehydrated through an acetone series. They were then dried and coated with platinum-palladium using an ion spatter. Observations were carried out with a Hitachi S-4100 scanning electron microscope ( www.hitachi-hta.com) equipped with a field emission gun.

Statistical analysis

To examine the time-depended conidial reduction of each fungal species on the C. formosanus cuticle, analysis of variance (ANOVA) was applied to the mean number of conidia. The relationships expressed by linear regression (y: mean number of conidia attached to cuticle, x: time (h) postinoculation, r²: Peason's correlation coefficient) were obtained. Then, to compare the differences in conidia attachment and persistence on the C. formosanus surface among the three fungi, Poisson regression was applied to the counted data, and the KruskalWallis test was applied to assay the reduction rate. Grooming frequency data were analyzed by the Mann-Whitney test. Feeding preference was analyzed by Wilcoxon signed rank test to examine the fungal effects on C. formosanus feeding preference in each B-series conidial suspension . Then, multiple regression was conducted to compare the difference of those conidial suspensions by assuming AI = β₀ + β_IT_II+ β₂×1 + β3X₂, where TII indicates the food color material; (X₁, X₂) is (0, 0) for B. brongniartii 782, (1, 0) for M. anisopliae 455, and (0, 1) for P. fumosoroseus K3. Thus β₂ shows the effects of TII on AI, and β₂ and β₃ represents, respectively, the effect of M. anisopliae 455 and P. fumosoroseus K3 on AI when measured from B. brongniartii 782.

Detection of fungal conidia on cuticle of C.formosanus

The binding of FITC-labeled conidia to the cuticle was quantified at 0, 3, 6, and 24 h after inoculation. The number of FITC-labeled conidia decreased markedly during the first 3 h after inoculation, and thereafter, the average number of conidia was less than 3/mm² 24 h after inoculation in all genera of entomopathogenic fungi (Table 1). Attachment and persistence patterns among three genera of fungal conidia were statistically different (Poisson regression, p < .0001). Interestingly, the amount of conidia remaining on the cuticle was similar among three fungi when presented as a percentage of the amount just after inoculation (KruskalWallis test, p = 0.93). This suggests that the more conidia attached, the more disposed they are to remove them. However, the slope of linear regression was sharper for the conidia of P. fumosoroseus K3 and B. brongniartii 782 than for those of M. anisopliae 455 conidia: approximately 3.5% of M. anisopliae 455 conidia remained on the surface at 24 h post inoculation, but less than 1% of P. fumosoroseus K3 and B. brongniartii 782 (Figure 1).

Figure 2.

Grooming frequency of Coptotermes formosanus termites with/without antennae.

A) Comparison of touching frequency of termite groups. AT (black): Termite group with intact antennae treated with Tween 20 solution. AF (white): Termite group with intact antennae treated with 1.0 × 10⁷/ml M. anisopliae 455 conidial suspension. NAT (blue): Termite group without antennae treated with Tween 20 solution. NAF (green): Termite group with intact antennae treated with 1.0 × 10⁷/ml M. anisopliae 455 conidial suspension. Bars on top of the columns represent standard errors. B) Details of touching condition in a dish. × bar expresses the number of termites, which touch a nestmate per dish. One means that one termite touches their nestmates in a dish. Y bar expresses the touching frequency (%); the % of touching termites in / all photographs. Bars on top of the columns represent standard errors. High quality figures are available online.

Grooming behavior of C. formosanus with/without antennae

The touching frequency was similar between C. formosanus treated with Tween 20 solution and with conidia suspension of M. anisopliae 455 in the group of termites that had intact antennae (Mann-Whitney test, p = 0.212) and in the group that had no antennae (Mann-Whitney test, p = 0.796) (Figure 2A). The behavioral change was initiated by the presence of antennae, but not by the presence of entomopathogenic microbe. Termites without antennae touched their nestmates significantly more often than the group with antennae (Mann-Whitney test, p < .0001) (Figure 2A), and more termites in a dish participated in the mutual grooming behavior in the group without antennae (Figure 2B).

Feeding preference

To estimate the feeding preference of C. formosanus, the ingested amounts of filter paper discs were compared between those given a disc containing a 0.025% Tween 20 solution only (control discs) and those given a disc containing B-series conidial suspensions of M. anisopliae 455, P. fumosoroseus K3, and B. brongniartii 782 (Figure 3).

C. formosanus consumed the M. anisopliae 455 disc significantly less than the control disc (Wilcoxon signed rank test: p = 0.002, Table 2a). In contrast, they consumed the P. fumosoroseus K3 disc significantly more than the control disc (Table 2a, Wilcoxon signed rank test: p = 0.055). Paper discs including B. brongniartii 782 were consumed at the same rate as control discs (Wilcoxon signed rank test: p = 0.688, Table 2a). The results of multiple regression analysis showed no influences of the food color material (p = 0.267 in TII parameter, Table 2b) and the isolate P. fumosoroseus K3 (p = 0.160 in X₂ parameter, Table 2b). The significant difference appeared in the result of the isolate M. anisopliae 455 (p = 0.014 in X1 parameter, Table 2b). This means that termites stop eating significantly more when food material contained the conidia of M. anisopliae 455 compared with B. brongniartii 782 (estimate = -0.121 in X₁ parameter, Table 2b). Moreover, in the previous study (Yanagawa et al. 2009), feeding behavior of C. formosanus, was enhanced when food material contained the conidia of P. fumosoroseus K3 compared with B. brongniartii 782, and the results in this study confirm this, although the change is not significant (estimate = 0.067 in X₂ parameter, Table 2b). Therefore, C. formosanus rejected the fungal substances in the order of M. anisopliae 455 > B. brongniartii 782 > P. fumosoroseus K3. This tendency agrees with the conidia-remaining numbers and the rate on the surface at 24 h after inoculation (Table 1, Figure 2).

Table 1.

Attachment and persistence of conidia on termite surface

Single sensillum recording

Electrical activities were recorded from the sensilla responding to odors originating from these three kinds of fungi in order to examine whether C. formosanus recognize fungal odors and whether they distinguish the three genera of entomopathogenic fungi by antennal olfactory sensilla. C. formosanus antennal sensilla were randomly probed with the recording electrode to obtain stable recordings. Figure 4 shows examples of impulses responses to fungal odors recorded from the same single sensillum. The impulses seen in Figure 4A are small, and those in Figure 4B are large. The impulses in Figure 4D differ somewhat in shape from Figure 4A and 4B, indicating that at least three different receptor cells belong to the sensillum, each of which responds to one of the odors of M. anisopliae 455, P. fumosoroseus K3, and Tween 20 (control odor) but not to the odor of B. brongniartii 782. However, the sensilla from which responses were recorded did not necessarily show the same combination of receptor cells as described below.

The response profiles of the antennal sensilla to the odors of three entomopathogenic fungi and one control were investigated. Overall 327 sensilla from which responses to at least one of these four kinds of odors were successfully recorded are listed in Table 3. Whether one receptor cell responded to plural kinds of odors or whether receptor cells in a single sensillum each responded to different odors is not clear (see Table 3).

Figure 3.

Feeding preference test. Treatment I,(orange): Paper disc containing 50 µl of 1.0 × 10⁷/ml conidia and 2 mg/ml blue food coloring suspended in Tween 20 solution, (white): Paper disc containing 50 µl of Tween 20 solution. Treatment II, (green): Paper disc containing 50 µl of 2 mg / ml blue food coloring suspended in a Tween 20 solution. (purple): Paper disc containing 50 µl of 1.0 × 10⁷/ ml conidia suspended in a Tween 20 solution. High quality figures are available online.

Identification of sensillum

About 50 sensilla from which responses had been successfully recorded were observed with scanning electron microscopy and identified (Figure 5). The hole at the base of the sensillum indicates the recording site in Figure 4B. All identified sensilla was chaetica in shape (Figure 5A). The sensilla had several longitudinal grooves on their cuticular surface, a single blunt tip, and a broad base (Figures 5B, 5C, 5D). The length of the cuticular apparatuses varied between 10 and 40 µm.