**2.1 Evaluation methods**

In the no-choice feeding test, filter paper disks treated with 0.005, 0.05, 0.5 and 5.0% (w/w) of bistrifluron were air-dried and weighed. Each disk was placed in a small plastic cup (ca. 14 ml) with small entry holes to allow termite access. Plastic cups were then put into separate plastic containers (200 ml) and each container retaining 100 of *C. formosanus* workers. The bottom of this larger container was covered with 2-3 mm of plaster and had several small holes made in the base. Assembled units were placed on a damp cotton pad in an incubation chamber so that termites could uptake water through the plaster. Five units were prepared for toxic baits and untreated controls. The units were maintained under appropriate condition for 12 weeks. Dead or moribund termites were counted at given interval to determine the change in mortality over time.

In the two-choice feeding tests, Filter paper disks were treated with 0.5% (w/w) of bistrifluron. The test container was a plastic Petri dish (140 mm in diameter) with approximately 5 mmthick agar [4% (w/w)] on the bottom. Four wells (35 mm in diameter) were made through the agar and plugged by two treated and two untreated disks. One hundred termites were introduced before the dish was covered with a lid and sealed with parafilm. Units were kept under appropriate condition. Mortality and weight of disks consumed were checked at given intervals after termites were released. Five replications were made.

#### **2.2 Speed of action**

In the no-choice test, mortality increases significantly faster as bistrifluron concentration were higher (Fig. 3). When termites were exposed to 0.5% bistrifluron (possible concentration in a commercial product), there had been no significant increase in mortality

Colony Elimination of Subterranean Termites by Bait Application

on performance of a bait product.

**2.3 Allogrooming inhibition effect** 

replications were made for each evaluation time.

**3. Colony elimination in the field cases** 

**3.1 Field trial against** *Coptotermes formosanus* **in Japan** 

a colony.

Using Benzoylphenylurea Compounds, with Special Reference to Bistrifluron 351

In the two-choice test, fed amounts of baits treated with 0.5% bistrifluron seemed less than untreated baits and significant effect on termite survival was not shown during 8-week test period. These results also indicate that feeding preference of termites will greatly influence

Some subterranean termites build their nest in the ground, form tunnel networks and extend their territories. Workers and soldiers are patrolling every time and everywhere in their territories because they are always under threat of attacks from various kinds of enemies. Various microbes are living very next to termites' territory in the ground and trying to take every opportunity to invade it. Some of them are pathogenetic to termites. It is considered that termites have developed behaviours to protect their territories from attacks of many invaders. Allogrooming, the behaviour that a worker grooms other colony member's body, is one of the most important behaviour since microbes attached onto termite's body are effectively removed through the behaviour (Thorne & Traniello, 2003). It is considered natural that incompetence of the above described behaviour should be involved in the mechanism of colony elimination by bait application. That is, once large part of workers are intoxicated with bait toxicant and fail in colony protecting behaviours like allogrooming their colony will collapse by intruding enemies shortly. To examine such a effect of bait toxicant, allogrooming inhibition effect of bistrifluron was evaluated by the no-choice feeding test similarly designed as the test described in the section 2.1 (Kubota et al., 2006). In the no-choice feeding test *C. frmosanus* workers were exposed to filter paper baits treated with 0.5% (w/w) bistrifluron during the test period (5 weeks). At every week termites fed on the bait were stained with red dye, erythrosin (0.5 g/l), on their tergites. Five red-stained workers were left together on a moistened filter paper in a petri dish for 3 hours then the intensity of the red dye that remained on each termite was rated as follows: (1) red dye unchanged, (2) faded or partly disappeared, and (3) no dye remaining at all. Five

When termites were exposed to untreated filter paper no red dye were remaining on all the termites throughout the study period. On the other hand, when they were exposed to toxic bait there were two termites with red dye partly remaining even at 1st week and 24 termites with dye partly remaining or intact at the 2nd week. These results indicate that workers uptaking more than a critical dose of bistrifluron soon become unable to do their regular works to maintain colony health and that it will then result in acceleration of the collapse of

Colony elimination performance of bistrifluron has been examined in the field. In this section the results of the Japanese and Malaysian cases with *Coptotermes* are described. Aside from these studies, Colony elimination performance of bistrifluron was demonstrated against Australian *C. acinaciformis* (Evans 2010) and Malaysian *Globitermes sulphreus* (Neoh et al. 2011).

A bait system using bistrifluron as an active ingredient was applied to a *C. formosanus*  colony which naturally occurred in Okayama city, Okayama prefecture in Japan (Aki, 2005). The bait system worked in the following procedure: bait stations containing wood blocks

until the 4th week. Even when exposed to overdosing 5.0% bistrifluron, 10 times higher concentration, there was no significant increase in mortality at the 1st week. These results indicate that bistrifluron shows very slow action on termites. In comparison to sulfluramid which is often used for ant bait, *C. formosanus* workers exposed to 0.01% (w/w) sulfluramid would die off within a week (Grace et al., 2000). On the other hand, dose dependency of bistrifluron's speed of action suggests that faster or better effect will be obtained by larger dosing. The no-choice feeding test with various concentrations of bistrifluron baits was also conducted against Japanese *R. speratus* workers and similar dose dependency and very slow action at even high doses were also shown (Kubota et al., 2007).

Fig. 3. Time-course change of mortality of *C. formosanus* workers exposed to 0.005, 0.05, 0.5 and 5.0% bistrifluron baits and blank bait in the no-choice feeding test (data from Kubota et al., 2006)

Fig. 4. The results of two-choice feeding test: A) fed mounts of bistrifluron 0.5% baits vs. blank baits, and also blank baits vs. blank baits in controls; B) time-coarse change of mortality of *C. formosanus* workers exposed to 0.5% bistrifluron baits and blank baits (data from Kubota et al., 2006)

In the two-choice test, fed amounts of baits treated with 0.5% bistrifluron seemed less than untreated baits and significant effect on termite survival was not shown during 8-week test period. These results also indicate that feeding preference of termites will greatly influence on performance of a bait product.

#### **2.3 Allogrooming inhibition effect**

350 Pesticides in the Modern World - Risks and Benefits

until the 4th week. Even when exposed to overdosing 5.0% bistrifluron, 10 times higher concentration, there was no significant increase in mortality at the 1st week. These results indicate that bistrifluron shows very slow action on termites. In comparison to sulfluramid which is often used for ant bait, *C. formosanus* workers exposed to 0.01% (w/w) sulfluramid would die off within a week (Grace et al., 2000). On the other hand, dose dependency of bistrifluron's speed of action suggests that faster or better effect will be obtained by larger dosing. The no-choice feeding test with various concentrations of bistrifluron baits was also conducted against Japanese *R. speratus* workers and similar dose dependency and very slow

Fig. 3. Time-course change of mortality of *C. formosanus* workers exposed to 0.005, 0.05, 0.5 and 5.0% bistrifluron baits and blank bait in the no-choice feeding test (data from Kubota et

Fig. 4. The results of two-choice feeding test: A) fed mounts of bistrifluron 0.5% baits vs. blank baits, and also blank baits vs. blank baits in controls; B) time-coarse change of mortality of *C. formosanus* workers exposed to 0.5% bistrifluron baits and blank baits (data

action at even high doses were also shown (Kubota et al., 2007).

al., 2006)

from Kubota et al., 2006)

Some subterranean termites build their nest in the ground, form tunnel networks and extend their territories. Workers and soldiers are patrolling every time and everywhere in their territories because they are always under threat of attacks from various kinds of enemies. Various microbes are living very next to termites' territory in the ground and trying to take every opportunity to invade it. Some of them are pathogenetic to termites. It is considered that termites have developed behaviours to protect their territories from attacks of many invaders. Allogrooming, the behaviour that a worker grooms other colony member's body, is one of the most important behaviour since microbes attached onto termite's body are effectively removed through the behaviour (Thorne & Traniello, 2003).

It is considered natural that incompetence of the above described behaviour should be involved in the mechanism of colony elimination by bait application. That is, once large part of workers are intoxicated with bait toxicant and fail in colony protecting behaviours like allogrooming their colony will collapse by intruding enemies shortly. To examine such a effect of bait toxicant, allogrooming inhibition effect of bistrifluron was evaluated by the no-choice feeding test similarly designed as the test described in the section 2.1 (Kubota et al., 2006).

In the no-choice feeding test *C. frmosanus* workers were exposed to filter paper baits treated with 0.5% (w/w) bistrifluron during the test period (5 weeks). At every week termites fed on the bait were stained with red dye, erythrosin (0.5 g/l), on their tergites. Five red-stained workers were left together on a moistened filter paper in a petri dish for 3 hours then the intensity of the red dye that remained on each termite was rated as follows: (1) red dye unchanged, (2) faded or partly disappeared, and (3) no dye remaining at all. Five replications were made for each evaluation time.

When termites were exposed to untreated filter paper no red dye were remaining on all the termites throughout the study period. On the other hand, when they were exposed to toxic bait there were two termites with red dye partly remaining even at 1st week and 24 termites with dye partly remaining or intact at the 2nd week. These results indicate that workers uptaking more than a critical dose of bistrifluron soon become unable to do their regular works to maintain colony health and that it will then result in acceleration of the collapse of a colony.
