Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 Sociobiology 60(2): 169-173 (2013) DOI: 10.13102/sociobiology.v60i2.169-173 Effects of Hematoporphyrin Monomethyl Ether on Worker Behavior of Red Imported Fire Ant Solenopsis invicta ZX Zhang 1, 2 , Y Zhou 1, DM Cheng 1, 3, * Introduction The red imported fire ant, a pest newly introduced to mainland China in 2005 (Zhang et al., 2007) and widely distributed in South China, threatens households, agriculture and wildlife. During foraging, worker ants leave a chemical pheromone trail to guide additional worker ants to the food source. These additional worker ants retrieve the food and return to the colony, also marking the pheromone trail laid down by the first group of ants (Xu et al., 2007). The abili- ties to walk, grasp, aggregate and recognize food and water are important for forager ants or other worker ants that leave the nest. If they lose the ability to move and recognize, they will have difficulty living in the complicated external circum- stances, which will directly cause a decrease in the food in their nest. This occurrence will eventually affect their popula- tion, and may even induce nestmates to become aggressive and bite each other. Photosensitizer such as α-Terthienyl (α-T) can affect Abstract The effect of hematoporphyrin monomethyl ether (HMME) activated under visible light on worker behavior of Solenopsis invcita was studied with the potter spray tower method. The results showed that greater than 10 mg/L HMME activated under visible light could reduce the walking, grasping, aggregation, and water and food recognition abilities of red imported fire worker ant significantly, but 100 mg/L HMME in darkness could not affect their abilities or behaviors significantly. Therefore, HMME may be a poten- tial novel insecticide that can be used as a substitute for toxic insecticides for controlling red imported fire ants. Sociobiology An international journal on social insects 1 - Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, China 2 - State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou, China 3 - Department of Plant Protection, Zhongkai University of Agriculture and Engineering, Guangzhou, China RESEARCH ARTICLE - ANTS Article History Edited by: Yi-Juan Xu, SCAU, China Received 13 March 2013 Initial acceptance 24 April 2013 Final acceptance 13 May 2013 Keywords Solenopsis invicta, Red imported fire ant, Hematoporphyrin Monomethyl Ether, Behavior. Corresponding author Dongmei Cheng Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education South China Agricultural University Guangzhou, China, 510642 E-Mail: zdsys@scau.edu.cn the walking behavior of worker ants and even kill them di- rectly (Yan et al., 2012; Liu et al., 2011). However, α-T ac- tivated under UV light is not suitable for controlling worker ants because the worker ants leaving the nest usually wander under visible light. Hematoporphyrin monomethyl ether (HMME), a novel photosensitizer activated under visible light, is the second gen- eration of porphyrin-related photosensitizer; it consists of two monomer porphyrins, namely, 3-(1-methyloxyethyl)-8-(1-hy- droxyethyl) deuteropor-phyrin IX and 8-(1-methyloxyethyl)- 3-(1-hydroxyethyl) deuteropor-phyrin IX, that are mutually locational isomers (Chen et al., 2000). HMME possesses some excellent properties, such as strong photodynamic ef- fect and fast removal from organs or cells. Moreover, HMME has been reported for the treatment of some cancers (Ander- son et al., 2002; Ascencio et al., 2007; Moghissi et al., 2000; Yoshihiro et al., 1993). HMME may be useful for controlling worker ants. To our knowledge, no research has been conducted on ZX Zhang, Y Zhou, DM Cheng - HMME Effects on Worker Behavior of red imported fire ant170 the effect of HMME on red imported fire worker ants. This study aimed to investigate the effects of HMME activated un- der visible light on the behavior of red imported fire ants. Materials and methods Solenopsis invicta colonies were collected from the suburb of Guangzhou and maintained in the laboratory for bioassays. The collected ants were fed with a mixture of 10% honey and live insects (Tenebrio molitor). A test tube (25 mm×200 mm), which was partially filled with water and plugged with cotton, was used as a water source. The ants were maintained in the laboratory at 25±2 °C. Large worker ants used in the test were about 6 mm in length, whereas small worker ants were about 3 mm in length. HMME was provided by the Institute of Red and Green Photosensitizer (Shanghai, China). The stock solution was prepared in ethanol at a concentration of 10 mg/mL and kept in the dark at -20 °C. A 75 W bromine tungsten lamp source (provided by Zolix Instrument Co. Ltd., Beijing, China) provided spectral radiation from 350 nm to over 2500 nm, which falls within the entire visible range of wavelengths. The stock solution was reconstituted at 5, 10, 25, 50 and 100 mg/L in acetone–water mixtures (3/7, v/v). Freshly prepared HMME solutions were kept from light at all times, except during actual measurement. HMME solution was applied to the worker ants with a potter spray tower (Burkard Manufacturing Co. Ltd., UK) using methods similar to those described by Harris et al (1962). Worker ants were placed in a glass Petri dish (120 mm in diameter) whose vertical wall was coated with a Fluon emulsion. The ants were then placed in the spray tower and sprayed with 2 ml HMME solution. The treated worker ants were transferred into a clean 500 ml beaker, whose vertical wall was coated with a Fluon emulsion (the same as below), and then placed in darkness immediately and incubated at 25 °C for 30 h. They were then exposed to visible light emitted by the bromine tungsten lamp (20 cm in height above the 500 ml beaker) for 10 min. The treated worker ants were placed in darkness immediately and incubated at 25 °C for 1h, and then their behaviors were observed. Each treatment was replicated three times, and each replicate included 30 to 40 worker ants. In the following methods used to observe the behav- ior of worker ants regarding water and food recognition, the worker ants were placed in darkness without food and water for 10 h before treatment. Controls were similar to the above steps, except that 10 min of light treatment was replaced with 10 min of dark treatment. Behavior observation on walking ability of worker ants Worker ants were placed on an A4 paper. They were regarded as possessing walking ability if they could walk con- tinuously for 10 cm and did not fall down. The formula was as follows: walking rate = number of worker ants possessing walking ability/number of worker ants per replicate × 100. Behavior observation on worker ants’ aggregation Worker ants were placed in a 500 ml beaker, and 20 min later, worker ant aggregation was observed. Aggregation was considered present if over five worker ants gathered into an aggregate mass. The formula was as follows: aggregation rate = num- ber of worker ants in aggregate mass/number of worker ants per replicate × 100. Behavior observation on grasping ability of worker ants Worker ants were placed on an A4 paper, and 10 s later, the A4 paper was turned over 180 degrees gently. They were regarded as possessing grasping ability if they would not fall down from the A4 paper. The formula was as follows: grasping rate = number of worker ants possessing grasping ability/number of worker ants per replicate × 100. Behavior observation on water recognition of worker ants A water-soaked cotton ball (1 g) and 20 worker ants (the distance between the cotton ball and the ants was 25 cm) were placed on the midcourt line of a porcelain tray (20 cm × 30 cm × 5 cm) whose vertical wall was coated with a Fluon emulsion (the same as below). Then, the water drinking be- havior of worker ant was observed within 30 min. Worker ants were regarded as having water recognition ability if they con- tinuously touched the cotton ball with their mouth for greater than 10 s. A worker ant was removed from the porcelain tray if it had drunk water. The formula was as follows: drinking water rate = number of worker ants drank water/number of worker ants per replicate × 100. Behavior observation on food recognition of worker ants The treatment method was the same as that used for the behavior observation on the water recognition of worker ants, but the cotton ball was replaced with a dead larva of Tenebrio molitor. Worker ants were regarded as possessing food recognition ability if they continuously touched the dead larvae with their mouth for greater than 10 s. The formula was as follows: food recognition rate = number of worker ants could recognize the dead larvae/num- ber of worker ants per replicate × 100. Sociobiology 60(2): 169-173 (2013) 171 Statistical Analysis Data were reported as means ± SE based on three in- dependent experiments. The percentage values were trans- formed into arc sin of square root of the percentages prior to the analysis, and three-factor ANOVA with worker size, light treatment, and concentration as the main effects were conducted. Moreover, the differences between the data were assessed by Duncan’s multiple range test (SAS 1989) with P < 0.05 regarded as statistically significant. The figures were produced using Microsoft Office Excel 2007. Results Three-factor ANOVA with worker size, light treat- ment, and concentration as the main effects, as well as two and three-way interactions between these effects, was con- ducted. Results show that all three main effects and partial in- teractions were significant (Table 1.). Significant differences in walking, aggregation, grasping, and water and food rec- ognition abilities were observed from the different treatment concentrations, light treatment, and size of the worker ants (Table 2.). Small and big worker ants possessed good walking, aggregation, and grasping abilities after treated with HMME in darkness. After treated with 5, 10, 25 and 50 mg/L HMME, the walking rates of small and big worker ants were greater than 96.0%. The walking rates were 86.67% and 98.89% at 100 mg/L, respectively (Fig. 1A, Table 2.). All the aggre- gation rates of small and big worker ants were greater than 92.0% (Fig. 1B, Table 2.), and all their grasping rates were greater than 82.0% at the treatment concentration in darkness (Fig. 1C, Table 2.). HMME activated under visible light could signifi- cantly affect the walking, aggregation, and grasping abilities of red imported worker fire ants. After treated with 10, 25, 50 and 100 mg/L HMME activated under visible light, the walking rates of small worker ants were 82.22%, 53.33%, 14.44% and 0.0%, respectively, and those of big worker ants were 92.22%, 81.11%, 34.44% and 0.0%, respectively, which were significantly different from the values obtained from the treatments in darkness (P < 0.05) (Fig. 1A, Table 2.). The ag- gregation rates of small worker ants were 70.83%, 50.83%, 26.67% and 2.50%, respectively, and those of big worker ants were 92.50%, 80.00%, 52.50% and 8.33%, respectively, which were significantly different from the values obtained from the treatments in darkness (P < 0.05) (Fig. 1B, Table 2.). The climbing rates of small worker ants were 45.56%, 21.11%, 2.22% and 0.0%, and those of big worker ants were 68.89%, 35.56%, 6.67%, and 0.0%, respectively, which were significantly different from the values obtained from the treat- ments in darkness (P < 0.05) (Fig. 1C, Table 2.). Small and big worker ants possessed good water and food recognition abilities after treated with HMME in dark- ness. After treated with HMME at test concentration in dark- ness, the drinking water rates of small and big worker ants were greater than 73.00%, and their food recognition rates were greater than 90.00% (Fig. 2, Table 2.). The visible light-activated HMME could affect the water and food recognition abilities of worker ants signifi- cantly. After treated with 5, 10, 25, 50 and 100 mg/L HMME activated under visible light, the drinking water rates of small worker ants were 51.11%, 43.33%, 34.44%, 13.33% and 5.56%, respectively, and those of big worker ants were 84.44%, 78.89%, 61.11%, 32.22% and 14.44%, respectively, which were significantly different from the values obtained from the treatments in darkness (P < 0.05) (Fig. 2A, Table 2.). The food recognition rates of small worker ants were 80.00%, 56.67%, 44.44%, 20.00% and 12.22%, respectively, and those of big worker ants were 93.33%, 86.67%, 58.89%, 27.78% and 10.00%, respectively, which were significantly different from the values obtained from the treatments in darkness (P < 0.05) (Fig. 2B, Table 2.). Fig. 1 The effect of visible light-activated HMME on walk- ing ability (A), aggregation (B), and grasping ability (C) of worker ants. ZX Zhang, Y Zhou, DM Cheng - HMME Effects on Worker Behavior of red imported fire ant172 Discussion This study showed that HMME concentration greater than 10 mg/L activated under visible light could reduce the walking, grasping, aggregation, and water and food recogni- tion abilities of red imported worker fire ants. However, 100 mg/L HMME in darkness does not affect these abilities or behaviors of worker ants. This finding suggests that HMME could be transmitted successfully in an ant nest, which is the key factor for effectively controlling red imported fire ants. HMME activated under visible light is a novel pho- tosensitizer that has been effectively used for solid tumors, which means that it is relatively safe for humans. HMME is also safe for the environment because of its photodegradation characteristics. As a formulation for controlling red imported fire ants, HMME can be used as bait or as powder applied in the morning or late afternoon. Thousands of worker ants living in one nest will then carry it into the nest, resulting in effectively decreasing the labor and photodegradation of HMME, and thus, producing good controlling effect. In conclusion, this study showed that HMME is a po- tential novel alternative for moderately and highly toxic in- secticides to control red imported fire ant. Reference Anderson, G. S., Miyagi, K., Sampson, R. W. & Sieber, F. (2002). 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Fa ct or s W al ki ng a bi lit y A gg re ga tio n G ra sp in g ab ili ty W at er re co gn iti on a bi lit y Fo od re co gn iti on a bi lit y F va lu es P va lu es F va lu es P va lu es F va lu es P va lu es F va lu es P va lu es F va lu es P va lu es A 23 5. 87 0 0. 00 01 82 .4 73 0. 00 01 20 4. 86 0 0. 00 01 96 .5 51 0. 00 01 88 .7 86 0. 00 01 B 99 0. 86 9 0. 00 01 38 4. 24 2 0. 00 01 15 05 .8 17 0. 00 01 60 7. 76 1 0. 00 01 66 8. 21 7 0. 00 01 C 22 .7 14 0. 00 01 20 .8 20 0. 00 01 30 .0 94 0. 00 01 12 9. 99 0 0. 00 01 18 .0 87 0. 00 01 A ×B 15 9. 55 4 0. 00 01 51 .7 79 0. 00 01 13 4. 49 7 0. 00 01 31 .0 30 0. 00 01 51 .7 49 0. 00 01 A ×C 2. 45 0 0. 04 68 0. 87 9 0. 50 23 2. 14 4 0. 07 61 1. 76 4 0. 13 83 3. 60 6 0. 00 75 B ×C 4. 23 3 0. 04 51 13 .7 27 0. 00 05 2. 49 6 0. 12 07 0. 98 1 0. 32 68 9. 76 6 0. 00 30 A ×B ×C 7. 79 0 0. 00 01 1. 41 8 0. 23 50 7. 29 0 0. 00 01 1. 01 0 0. 42 22 1. 30 1 0. 27 93 A : C on ce nt ra tio n, B : L ig ht tr ea tm en t, C : W or ke r s iz e (P =0 .0 5) Ta bl e 2. In flu en ce s of d if fe re nt tr ea tm en ts o n be ha vi or s of re d im po rt ed fi re a nt s . Tr ea tm en ts Pe rc en ta ge (M ea ns ± SE , % ) C on ce nt ra tio n (m g/ L ) L ig ht W or ke r s iz e W al ki ng a bi lit y A gg re ga tio n G ra sp in g ab ili ty W at er re co gn iti on Fo od re co gn iti on 0 L ig ht Sm al l 10 0. 00 ±0 .9 6a 98 .3 3± 3. 91 ab c 10 0. 00 ±0 .0 0a 95 .5 6± 1. 11 bc d 92 .2 2± 5. 09 bc de B ig 10 0. 00 ±0 .0 0a 99 .1 7± 1. 38 a 10 0. 00 ±0 .0 0a 98 .8 9± 1. 11 ab 96 .6 7± 1. 57 ab c D ar kn es s Sm al l 10 0. 00 ±0 .9 6a 99 .1 7± 0. 72 a 10 0. 00 ±0 .0 0a 96 .6 7± 1. 92 ab c 97 .7 8± 0. 96 ab B ig 10 0. 00 ±0 .0 0a 99 .1 7± 1. 38 a 10 0. 00 ±0 .0 0a 10 0. 00 ±0 .0 0a 97 .7 8± 1. 11 ab 5 L ig ht Sm al l 96 .6 7± 2. 48 b 90 .8 3± 5. 83 cd 84 .4 4± 2. 94 b 51 .1 1± 4. 01 ij 80 .0 0± 5. 98 f B ig 97 .7 8± 1. 57 ab 95 .8 3± 2. 47 ab c 96 .6 7± 1. 92 a 84 .4 4± 4. 01 ef g 93 .3 3± 1. 84 bc de D ar kn es s Sm al l 98 .8 9± 0. 96 ab 99 .1 7± 0. 72 a 10 0. 00 ±0 .0 0a 90 .0 0± 3. 85 cd ef 95 .5 6± 0. 96 ab cd B ig 10 0. 00 ±0 .0 0a 98 .3 3± 1. 38 ab 10 0. 00 ±0 .0 0a 98 .8 9± 1. 11 ab 98 .8 9± 1. 84 a 10 L ig ht Sm al l 82 .2 2± 10 .1 1d 70 .8 3± 4. 77 e 45 .5 6± 4. 01 d 43 .3 3± 1. 92 jk 56 .6 7± 4. 16 gh B ig 92 .2 2± 1. 84 c 92 .5 0± 3. 80 bc 68 .8 9± 5. 77 c 78 .8 9± 4. 84 fg 86 .6 7± 7. 86 ef D ar kn es s Sm al l 98 .8 9± 0. 96 ab 97 .5 0± 1. 18 ab c 98 .8 9± 1. 11 a 88 .8 9± 4. 01 de f 93 .3 3± 2. 48 bc de B ig 96 .6 7± 1. 84 b 96 .6 7± 1. 86 ab c 98 .8 9± 1. 11 a 98 .8 9± 1. 11 ab 95 .5 6± 2. 22 ab cd 25 L ig ht Sm al l 53 .3 3± 8. 80 e 50 .8 3± 6. 28 f 21 .1 1± 4. 84 e 34 .4 4± 4. 84 k 44 .4 4± 8. 37 h B ig 81 .1 1± 12 .1 1d 80 .0 0± 8. 28 de 35 .5 6± 6. 19 d 61 .1 1± 4. 84 hi 58 .8 9± 7. 43 g D ar kn es s Sm al l 10 0. 00 ±0 .0 0a 97 .5 0± 1. 18 ab c 97 .7 8± 2. 22 a 86 .6 7± 1. 92 de f 97 .7 8± 1. 11 ab B ig 98 .8 9± 0. 96 ab 97 .5 0± 1. 18 ab c 98 .8 9± 1. 11 a 96 .6 7± 1. 92 ab c 96 .6 7± 1. 84 ab c 50 L ig ht Sm al l 14 .4 4± 4. 81 g 26 .6 7± 7. 91 g 2. 22 ±4 .8 4g 13 .3 3± 3. 85 lm 20 .0 0± 2. 48 ij B ig 34 .4 4± 10 .2 3f 52 .5 0± 10 .3 7f 6. 67 ±1 .9 2f 32 .2 2± 2. 94 k 27 .7 8± 5. 30 i D ar kn es s Sm al l 98 .8 9± 3. 64 ab 95 .0 0± 1. 18 ab c 98 .8 9± 1. 11 a 85 .5 6± 2. 94 ef g 96 .6 7± 1. 84 ab c B ig 10 0. 00 ±0 .0 0a 95 .0 0± 1. 86 ab c 98 .8 9± 1. 11 a 93 .3 3± 1. 92 cd e 94 .4 4± 1. 57 bc de 10 0 L ig ht Sm al l 0. 00 ±0 .0 0h 2. 50 ±2 .5 0h 0. 00 ±0 .0 0g 5. 56 ±2 .2 2m 12 .2 2± 1. 11 j B ig 0. 00 ±0 .0 0h 8. 33 ±5 .0 7h 0. 00 ±0 .0 0g 14 .4 4± 2. 94 l 10 .0 0± 1. 92 j D ar kn es s Sm al l 86 .6 7± 1. 92 cd 92 .5 0± 2. 50 bc 82 .2 2± 2. 94 b 73 .3 3± 5. 09 gh 90 .0 0± 1. 92 de f B ig 98 .8 9± 1. 11 ab 98 .3 3± 0. 83 ab c 10 0. 00 ±0 .0 0a 94 .4 4± 1. 11 cd e 91 .1 1± 1. 11 cd e a Sh ar in g sa m e le tte rs m ea ns n ot s ig ni fic an tly d iff er en t f ro m e ac h ot he r ( P >0 .0 5, D un ca n’ s M ul tip le R an ge T es t) .