Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v67i1.4438Sociobiology 67(1): 59-64 (March, 2020) Introduction Ants comprise 5% of the world’s one hundred worst invasive alien species and of the seventeen land invertebrates listed, five (28%) are ants (Vander Meer & Milne, 2017). Among the most problematic invasive species, the Asian needle ant, Monomorium chinensis, Santschi and the Yellow crazy ant, Anoplolepis gracilipes, Smith, cause devastating environmental and urban problems all over the world and area threat to local biodiversity (Zheng et al., 2008). Current control measures of these species include perimeter spraying or direct application to nest sites for successful control. However, this can result in insecticidal run-off and environmental contamination often poisons other non-target organisms (Welzel & Choe, 2016). Kafle et al. (2010) stated that the solid bait design was not ideal due to a tendency to become excessively hydrated or dehydrated and Abstract The current control measures used against common household ants in urban and agricultural settings include perimeter insecticide applications. These often have the potential to cause problems of poisoning non-target organisms, due to the insecticidal runoff and environmental contamination. A gel-baiting technique is the most effective tool to control ants with less insecticides released into the environment. In this study two commercial gel baits; the Boric acid (2.5% boric acid) and the Fipronil (0.01% fipronil) baits, were evaluated against laboratory made baits (lab baits). The lab baits, consisted of: 1.5% boric + fructose (F1.5), 1.5% boric acid + fructose + molasses (M1.5), 3% boric acid + fructose (F3), and 3% boric + fructose + molasses (M3) were evaluated based on preference and mortality rates of the common household ant species: the Asian needle ant, Monomorium chinensis, Santschi, and the Yellow crazy ant, Anoplolepis gracilipes, Smith, under laboratory conditions. An inconsistent preference was observed between species and different baits; however, the fipronil bait and the lab bait M3, were preferred more by both ant species compared to the other baits tested. Lab bait M3 also had a faster killing speed than the boric acid bait and the lab bait F3.Based on the results it was concluded that lab bait M3 was a more efficient ant bait and is a potential alternative control measure to the current commercial baits. Sociobiology An international journal on social insects LN Kafle, AC Neupane, YM Wang, SR Gangai Article History Edited by Evandro Nascimento Silva, UEFS, Brazil Received 26 March 2019 Initial acceptance 07 January 2020 Final acceptance 31 January 2020 Publication date 18 April 2020 Keywords Bait, boric acid, Monomorium chinensis, Anoplolepis gracilipes, preference. Corresponding author Samantha Rose Gangai Department of Tropical Agriculture and International Cooperation National Pingtung University of Science and Technology 1, Sheufu Rd, Neipu, Pingtung 91201, Taiwan. E-Mail: chamandah@gmail.com needing frequent maintenance. Considering that most baits contain toxic substances, they are therefore environmentally hazardous and arising concern to world wide health agencies and environment authorities (Kafle & Shih, 2013). Fipronil is a broad use insecticide used to control ants and other insect pests. It is highly toxic to sea and freshwater vertebrates and invertebrates. Furthermore, Fipronil has been found to be highly toxic to some birds, although non-toxic to ducks. It has also been found as highly toxic to honey bees, but non-toxic to earthworms (Jackson et al., 2009). Fibronil is frequently found in urban waterways and aquatic systems in amounts exceeding the LC50 values and therefore there is concern for its potential impact on non-target organisms, as has been reported in California (Welzel & Choe, 2016). Similarly, Boric acid is a pesticide that can also be found in nature and has been used for ant control since the early 1900’s. Boric acid is slow acting and non-repellent Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung, Taiwan RESEARCH ARTICLE - ANTS Development of New Boric Acid Gel Baits for Use on Invasive Ants (Hymenoptera: Formicidae) LN Kafle, AC Neupane, YM Wang, SR Gangai – Development of new boric acid gel baits 60 thereby enhancing long-term ingestion (Klotz & Moss, 1996: Klotz & Williams, 1996). Boric acid and sodium salts can be used to control insects, spiders, mites, algae, moulds, fungi, and weeds. The delayed activity of boric acid promotes a thorough distribution of the active ingredient within the nest, leading to the death of the entire colony (Klotz & Williams, 1996). Commercial ant baits with boric acid as an active ingredient typically use concentrations of > 5% (Klotz et al., 2000). During this study, we formulated a new composition of ant bait with boric acid as an active ingredient (AI). In order to determine an effective range of boric acid, we compared different lab baits with two concentrations of boric acid (1.5% and 3%) against two commercial ant baits; being them fipronil (0.014%) and boric acid (2.5%). The study focused on (1) evaluating the feeding preferences for lab baits against commercial baits, and (2) comparing the efficacy of the most preferred lab bait against commercial baits. The results of this study aim to develop new gel baits with a specific percentage of boric acid as the active ingredient (AI) for effective control of common household ants; M. chinensis and A. gracilipes. Materials and Methods The source of ants Polygyne colonies of the Asian needle ant, Monomorium chinensis, Santschi and the Yellow crazy ant, Anoplolepis gracilipes, Smith, were obtained by excavation from field populations located around the National Pingtung University of Science and Technology campus, Pingtung, Taiwan. The ants were separated from the soil using the water drip method, as described by Kafle et al. (2008, 2009), and Chen (2007), and reared under ambient laboratory conditions (27±1 °C and 50±3% RH), photoperiod of 14:10 h (L:D) and placed on a standard diet of water, meal worm larvae, peanut butter, and 10% sugar water at least one week before the tests. The test population, usually made up of the major workers, were isolated from the main population and starved for 24 hours before each test was conducted. Preparation of the laboratory baits A stock solution made up of 25% boric acid was prepared by heating25g of boric acid powder (99%) (Shimada Chemical Works, Taipei, Taiwan) and75gof fructose (SunRight Foods Corporation, Taipei, Taiwan) at a temperature of 140ºC for 15 minutes, in order to completely dissolve the boric acid in the fructose. After cooling the mixture, 6g of the stock solution was added to 19g of either fructose or molasses to make up1.5% boric baits. Similarly, to make up 3% boric baits, 13g of fructose or molasses was added to 12g of stock solution for 25g in each lab bait. The specific ratios of each component contained in the lab baits are shown in Table 1. Commercial Baits Two popular commercial ant baits used in Taiwan were compared for efficacy against the laboratory baits: the boric acid bait (2.5% boric acid, Chung Tai Sing Chemical Industry Company Limited, Hsinchu, Taiwan) and the fipronil bait (0.01% fipronil, Family Consumer Products Company Limited, Miaoli, Taiwan). The baits were bought at the local market. Preference Tests In order to evaluate the feeding preferencesof the M. chinensis and A. gracilipes on the four laboratory baits (Table 1) and two commercial baits, similar studies undertaken by Kafle et al. (2010) and Klotz (2000) were adapted for this test. Acrylic rectangular foraging stations (18.5cm x 10.5cm x 4cm (L x W x H) were set up and modified to suit the purpose of the experiment. Fluon was applied to the inner vertical surfaces to prevent ants from escaping the container. The test baits were arranged side by side on parafilm squares (1 cm x 1 cm) along one end of the setup and water was not provided during the test period. An artificial cardboard nest (2 cm x 2 cm) setup, as an artificial ant nest, were placed 1cm away from the inner wall and10cm away from the baits. The feeding preferences were recorded on 60-minute videos. These observations were reviewed and interval counts were done at 5 minute frames. Ant-to-bait counts were determined by the number of ants actually feeding on the baits or foraging on the individual parafilm squares for at least 10 seconds. During the test period any dead ant was removed and replaced with live ants from the isolated population. This was to maintain the test population during the study period. A hundred M. chinensis and fifty A. gracilipes workers from the test populations were used in each individual test. Each test was replicated four times under the laboratory conditions at temperatures of 25 ± 3ºC and RH 52 ± 3% and 14:10h L:D photoperiods. Mortality Tests According to the results of the preference tests, the lab baits F3 and M3 had the highest preferences used in the mortality test. Rectangular foraging stations similar to those used in the preference tests were set as described by Klotz et al. (2000) and Kafle et al. (2010), and modified for this experiment. Table 1. Ratio of each constituent used in new laboratory bait prepa- rations. Constituent (%) Lab baits* F1.5 F3 M1.5 M3 Fructose 94 88 0 0 Molasses 0 0 94 88 Stock solution (25% Boric acid) 6 12 6 12 Total vol. (%) 100 100 100 100 *F = Fructose, M = Molasses, 1.5 = 1.5% boric acid and 3 = 3% boric acid Sociobiology 67(1): 59-64 (March, 2020) 61 Fluon was applied to the inner vertical surfaces to prevent ants from escaping. A single test bait per feeding arena was placed at one end of the foraging arena on a parafilm strip 1cm away from the vertical wall. An artificial cardboard nest was placed 10cm away from the bait at the opposite end of the foraging arena 1cm away from the inner wall. Water was provided during the mortality tests and mealworm larvae were provided as food in the control sets and in all mortality tests. Efficacies were compared across the baits; the boric acid, fipronil, lab bait F3, and lab bait M3, in tests similar to those described by Kafle et al. (2010). Observations of the test were recorded at 3-hour intervals for the first 24 hours and every 6 hours thereafter, until a hundred percent mortality was reached. Thirty worker ants from the test populations of both species were used and each test was replicated four times under laboratory conditions;at temperatures of 25 ± 3ºC and RH 52 ± 3% and 14:10h L:D photoperiods. Data collection and analysis Each test setup was randomized and the data was sorted and compared on an Excel spreadsheet using the procedures described by Vander Meer (2017) and Kafle et al. (2010). The lethal time (LT50) was calculated through probit analysis using StatPlus (2017) and the means were compared using SNK of SAS (2017). Results Preference Tests Two fructose based lab baits, the F1.5 and F3 (Table 1), were compared with the two commercial baits, fipronil and boric acid, to determine the preference of M. chinensis and A. gracilipes. The percentage of M. chinensis observed foraging on the Fipronil bait was significantly higher than those observed on the rest of tested baits; however, the number of ants observed on the lab bait F1.5, the boric acid bait and the lab bait F3 were not significantly different (F = 6.2, p < 0.01) (Table 2). The number of A. gracilipes observed foraging on the lab bait F3 was significantly higher than the other baits tested. The boric acid bait had the next highest preference. However, the number of A. gracilipes observed foraging on the two commercial baits and the lab bait F1.5 were not significantly different (F = 1.49, p < 0.27) (Table 2). Similarly, the number of M. chinensis observed foraging on the fipronil bait was significantly higher than the other three baits. However, the number of ants observed on the lab bait M1.5 and fipronil bait or lab bait M3 and the boric acid bait were not significantly different (F = 12.84, p < 0.01) (Table 3). The number of M. chinensis observed foraging on the Fipronil bait was statistically higher than the other three baits tested. However, there was no significant difference observed on the percentage of ants on all the baits tested (F = 0.14, p < 0.93) (Table 3). The preference tests showed that lab bait M3 was more preferred by M. chinensis than the fructose based baits, while A. gracilipes preferred both the lab F3 and lab M3 baits. Therefore, only the lab baits F3 and M3 were evaluated against the two commercial baits in the mortality tests. Baits tested No. of ants foraging (Mean ± SE)* Monomorium chinensis Anoplolepis gracilipes Boric acid bait (2.5% Boric acid) 8.26 ± 0.37b 12 ± 3.72a Fipronil bait (0.01% Fipronil) 12.25 ± 1.12a 8.25 ± 1.38a Lab bait 3F (5% Boric acid) 7.00 ± 0.93b 8.25 ± 1.11a Lab bait 4F (10% Boric acid) 5.25 ± 0.8b 15.25 ± 3.68b *Means within the same column followed by the same letter are not significantly different (p<0.05) (SNK test, SAS, 2017). Table 2. Preference of Monomorium chinensis and Anoplolepis gracilipes on new fructose and commercial baits under laboratory conditions. Table 3. Preference of Monomorium chinensis and Anoplolepis gracili- pes on new molasses and commercial baits under laboratory conditions. Baits tested No. of ants foraging (Mean ± SE)* Monomorium chinensis Anoplolepis gracilipes Boric acid bait (2.5% Boric acid) 5.74 ± 0.97b 10.75 ± 4.25a Fipronil bait (0.01% Fipronil) 13.09 ± 2.88a 13 ± 3.24a Lab bait 3M (5% Boric acid) 7.82 ± 3.39b 9.75 ± 4.11a Lab bait 4M (10% Boric acid) 12.19 ± 4.77a 12.25 ± 3.97a *Means within the same column followed by the same letter are not signifi- cantly different (p<0.05) (SNK test, SAS, 2017). Mortality Tests Mortality of M. chinensis by lab baits F3 and M3 against two commercialwere compared with the two commercial baits, fipronil and boric acid under the laboratory conditions. The percentage of M. chinensis killed by the lab baits(F3 and M3) fipronil and boric acid baits were not significantly different at 6 HAT and 12 HAT (6 HAT: F= 1.07, p < 0.41; 12 HAT: F = 2.61, p < 0.08) (Table 4). At 24 HAT, the percentage of ants killed by the fipronil bait was significantly higher than the lab bait M3 and the control. However, there was no significant difference in the percentage of ants killed by the boric acid, fipronil baits and the M3 or the boric acid bait, and the lab baits F3 and M3 (F = 7.81, p < 0.01) (Table 4). The mortality rate of the M. chinensis by the lab bait M3, fipronil and boric acid baits were significantly higher than the control at 48, 72 and 84 HAT. However, the percentage of ants killed by all three baits was not significantly different from each other. It was observed that all baits could kill up to 100% of M. chinensis within 84 HAT. At 72 HAT, the lab bait F3 killed 100% ants (48 HAT: F = 8.83, p < 0.01; 72 HAT: F = LN Kafle, AC Neupane, YM Wang, SR Gangai – Development of new boric acid gel baits 62 194.62, p < 0.01; 84 HAT: F = 2255.53, p < 0.01). Based on the LT50 results, fipronil was the fastest killing bait, followed by the boric acid bait the lab bait M3 and then the lab bait F3 (Table 4). Mortality of A. gracilipes by lab baits F3 and M3 against two commercial were compared with the two commercial baits, fipronil and boric acid under the laboratory conditions. The percentage of A. gracilipes killed by the fipronil bait was significantly higher than the F3, M3 and boric acid baits at 6 HAT and 12 HAT. However, the percentage of ants killed by the boric acid bait and the lab baits F3 and M3 were not significantly different (6 HAT: F = 136.08, p < 0.01; 12 HAT: F = 29.76, p < 0.01) (Table 5). Observation timeα Ant mortality (%) (Mean ± SE)* Boric acid bait Fipronil bait Lab bait F3 Lab bait M3 Control 6 HAT 2.62±1.02a 5.96±2.57a 0±0a 3.33±3.33a 2.50±1.60a 12 HAT 12.82±4.40a 26.94±5.51a 0.81±0.81a 25.61±15.48a 2.50±1.60a 24 HAT 46.38±11.86ab 73.86±9.96a 5.63±2.63b 44.96±17.25ab 6.67±1.36b 48 HAT 68.11±13.19a 86.10±7.60a 59.73±13.58a 62.83±13.02a 20.84±2.50b 72 HAT 93.58±4.05a 96.75±1.90a 100±0a 97.80±2.21a 24.17±1.60b 84 HAT 100±0a 100±0a 100±0a 100±0a 24.17±1.60b LT50 β (h) 27.67 18.23 39.01 27.92 - *Means within the same row followed by the same letter (lower case) are not significantly different (p < 0.05) (SNK test, SAS, 2017) α HAT = Hours after treatment β LT50 values (h) were determined by Probit analysis (StatPlus, 2017) F3 = 3% boric acid M3 = 3% boric acid Observation timeα Ant mortality (%) (Mean ± SE)* Boric acid bait Fipronil bait Lab bait F3 Lab bait M3 Control 6 HAT 3.70±0.80c 78.60±5.57a 1.62±0.93c 14.93±1.59b 6.67±1.36bc 12 HAT 13.17±1.95c 90.32±4.62a 1.62±0.93c 43.56±13.87b 10.83±1.56c 24 HAT 98.49±1.51a 100±0a 1.62±0.93c 97.14±2.86a 20.83±2.85b 48 HAT 100±0a 100±0a 76.15±3.50b 98.57±1.43a 25.83±3.44c 72 HAT 100.0±0a 100.0±0a 100.0±0a 100.0±0a 26.67±3.04b LT50 β 16.63 3.49 36.25 12.44 - * Means within the same row followed by the same letter (lower case) are not significantly different (p < 0.05) (SNK test, SAS, 2017) α HAT = Hours after treatment β LT50 values (h) were determined by probit analysis (StatPlus, 2017) F3 = 3% boric acid M3 = 3% boric acid Table 4. Mortality of M. chinensis by lab baits F3 and M3 against two commercial baits under laboratory conditions. At 24 HAT, the percentage of ants killed by the fipronil bait was significantly higher than the other three baits tested. However, the percentage of ants killed by the boric acid, fipronil and the lab bait F3 were not significant different (F = 600.52, p < 0.01) (Table 5). Similarly, at 48 HAT, the percentage of A. gracilipes killed by the fipronil and boric acid baits reached 100% and was significantly higher than lab baits F3 and M3. However, the percentage of ants killed by the boric acid and fipronil baits and lab bait M3 was not significantly different (F = 296.33, p < 0.01) (Table 5). Table 5. Mortality of A. gracilipes by lab baits F3and M3 against two commercial baits under laboratory conditions. The percentage of A. gracilipes killed by the lab baits F3 and M3, and fipronil and boric acid baits reached 100% of ants at 72 HAT and the percentage of ants killed by all tested baits was significantly higher than the control (F = 580.94, p < 0.01). Based on the LT50 value the fipronil bait was the fastest killing bait of M. chinensis and A. gracilipes, followed by the lab bait M3, the boric acid bait and the lab bait F3 (Table 5). Discussion Molasses and fructose are two of the most popular natural sugars used in insect control. A similar study undertaken by Ulloa-Chacon and Jaramillo (2003) emphasized that toxic baits prepared with insecticides added to a sugar solution were attractive to the Ghost ant and appropriate for use when Sociobiology 67(1): 59-64 (March, 2020) 63 implemented in trials for the control of various species, similar to the Argentine ant (Iridomyrmex humilis Mayr). During the preference tests on the molasses baits, a higher percentage of ants were observed to be actively foraging on the lab baits M1.5 and M3. The ants were attracted to the molasses over the fructose mainly due to the sucrose content, and thus both the M. chinensis and A. gracilipes were lured when it was used in these baits (Binkley & Wolfrom, 1953; Curtin, 1983; Saric et al., 2016). Both M. chinensis and A. gracilipes are generalist feeders that seek out sources of carbohydrates, lipids and proteins as food sources (Vanderwoude et al., 2006). Therefore, it was observed how both species in this study actively fed on the sugar based baits. They were able to exploit their food resources by communicating with odour trails and rapid recruitment (Sparks, 2015) in a similar way. They also may have been able to communicate danger when workers were stuck in the baits. During the study, it was observed that when workers became stuck in the bait and were left there for a while, other ants also ceased feeding. This behavior was observed in both M. chinensis and A. gracilipes. Bluthgen and Fiedler (2004a) observed the preference for sugars and amino acids of the Nectarivorous ants in an Australian tropical rain forest. Using artificial nectar solutions, the feeding behavior of fifty-one ant species on these solutions was recorded. The results stated that preferences among carbohydrates were principally consistent between ant species. A very significant observation was that many of the ant species preferred: sugar, glucose, fructose or sucrose solutions containing mixtures of amino acids, such as: alanine; arginine, asparagine, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and other substances in natural nectar over pure sugar solutions. These results correspond with previous studies thus confirming the variability in amino acids and carbohydrates proposes to play a key role in ant preferences and nutrition (Bluthgen & Fiedler, 2004b; Bluthgen et al., 2004;Vander Meer et al., 1995; Lanza, 1993). In a study on the bait preferences and toxicity of insecticides to White-footed ants, Technomyrmex albipes, Warner (2003) noted that fructose was found to be more significant than the nectar of Brownea sp., or other sugars, while both the nectar and fructose of Clerodendrum myricoides were significantly favored more than glucose and sucrose. This is evidence that different species of ants have a specific preference for sugar types. Therefore, the results varied in the toxicity tests. All baits achieved 100% mortality by 72 HAT. The killing speed of lab bait M3 was faster than the boric acid bait and the lab bait F3. A main reason for this may be the ingredients used in the baits. The lab bait F3 contained just fructose and boric acid. However, lab bait M3 contained fructose, molasses and boric acid, which may have had an impact on the feeding preference and thus the results. The killing speed of lab baits F3 and M3 against M. chinensis and A. gracilipes were faster than the boric acid bait and slower than the fipronil bait. It is assumed this is due to the different active ingredients used in each of the baits. The fipronil bait used in this study contained 0.01% active ingredient. Fipronil is a sweet smelling poison that also aided it as a preference. It is toxic to insects by contact or ingestion (Jackson et al., 2009). Thus, is used in many products when a delayed kill is desired. Fipronil disrupts the insect central nervous system and causes the hyperexcitation of nerves and muscles resulting in death. Klotz et al. (2000) presumes that although there is little information available concerning the physiological mode of action of boric acid on insects, it has been shown that borate ions form strong complexes with sugar alcohols, and other organic functional groups. Harper et al. (2012) also suggest that boron may be involved in the disruption of intercellular adhesion because saturated boric acid solutions can be used to dissociate cells. Some advantages of using boric acid as an active ingredient in ant baits are the delayed activity and solubility in water. At a low concentration, boric acid is slow- acting and less likely to be repellent (Klotz & Williams, 1996). As federal and state laws become more stringent on the use of residual insecticide spray, these baiting techniques may provide an effective control strategy (Welzel & Choe, 2016). The test formulations proved that the boric acid based molasses bait (M3) was an efficient formula. The results of this study showed, i) the new Lab bait M3 is a potential alternative to current commercial gel baits, and ii) boric acid can be used as an alternative for the hazardous pesticide like fipronil, as the active ingredient in ant gel baits. Acknowledgement This study was supported by the Ministry of Science and Technology (MOST 106-2313-B-020-002 Project), Taiwan. The authors are grateful to the Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology for providing the laboratory facilities. Author’s contribution The study concept and design: L. Kafle and Y. M. Wang, the acquisition of data: SR Gangai, Analysis and Interpretation of data: A.C. Neupane, S.R Gangai and L. Kafle, Manuscript preparation: A.C. Neupane and L. Kafle, and Critical revision: L. Kafle, A. C. Neupane and Y. M. Wang. References Binkley, W.W. & Wolfrom, M.L. (1953). Composition of cane juice and cane final molasses. Advances in Carbohydrate Chemistry: Scientific Report Series No. 15 (8):1 -25. Bluthgen, N. & Fiedler, K. (2004a). Preferences for sugars and amino acids and their conditionality in a diverse nectar- LN Kafle, AC Neupane, YM Wang, SR Gangai – Development of new boric acid gel baits 64 feeding ant community. Journal of Animal Ecology, 73:155– 166. doi: 10.1111/j.1365-2656.2004.00789.x Bluthgen, N. & Fiedler, K. (2004b). Competition for composition: lessons from nectar-feeding ant communities. Ecology, 85: 1479-1485. doi: 10.1890/03-0430 Bluthgen, N., Gottsberger, G. & Fiedler, K. (2004). Sugar and amino acid composition of ant-attended nectar and honeydew sources from an Australian rainforest. Austral Ecology, 29: 418-429. doi: 10.1111/j.1442-9993.2004.01380.x Chen, J. (2007). Advancement on techniques for the separation and maintenance of the red imported fire ant colonies. Insect Science, 14: 1- 4. doi: 10.1111/j.1744-7917.2007.00120.x Curtin, L.V. (1983). Molasses – general considerations. Molasses in Animal Nutrition, National Feed Ingredients Association, West Des Moines, Iowa. Pp.1- 11. Harper, B., Gervais, J.A., Buhl, K. & Stone, D. (2012). Boric acid - technical fact sheet. National Pesticide Information Center, Oregon State University Extension Services. http:// npic.orst.edu/factsheets/archive/borictech.html. (accessed date: 28 December, 2017). Jackson, D., Cornell, C.B., Luukinen, B., Buhl, B. & Stone, D. (2009). Fipronil - general fact sheet. National Pesticide Information Center, Oregon State University Extension Services. http://npic.orst.edu/factsheets/archive/fiptech.html. (accessed date: 28 December, 2017). Kafle, L., Wu, W.J., VanderMeer R.K. & Shih, C.J. (2008). Simplified approaches to determine the attractant preference of Solenopsis invicta (Hymenoptera: Formicidae). Applied Entomology and Zoology, 43 (3): 383-390. doi: 10.1303/ aez.2008.383 Kafle, L., Wu, W.J., VanderMeer R.K., Huang, Y.Y. & Shih, C.J. (2009). Microencapsulated bait: does it work with red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae)? Sociobiology, 53(3): 729-737. Kafle, L., Wu, W.J. & Shih C.J. (2010). A new fire ant (Hym.: Formicidae) bait base carrier for moist conditions. Pest Management Science, 66(10): 1082- 8. doi: 10.1002/ps.1981 Kafle, L. & Shih, C.J. (2012). Determining the most effective concentration of cypermethrin and the appropriate carrier particle size for fire ant (Hymenoptera: Formicidae) bait. Pest Management Science, 68(3) 394-398. doi: 10.1002/ps.2275 Kafle, L. & Shih, C.J. (2013). Toxicity and repellency of compounds from Clove (Syzygium aromaticum) to red imported fire ants Solenopsis invicta (Hymenoptera: Formicidae). Journal of Economic Entomology, 106 (1):131-135. doi: 10.1603/EC12230 Klotz, J.H. & Moss, J.I. (1996). Oral Toxicity of a boric acid - sucrose water bait to Florida carpenter ants (Hymenoptera: Formicidae). Journal of Entomological Science, 31 (1): 9–12. doi: 10.18474/0749-8004-46.2.89 Klotz, J.H. & Williams, D.F. (1996). New approach to boric acid ant baits. The IPM Practitioner, (8): 1-4. Klotz, J.H., Greenberg, L., Amrhein, C. & Rust, M.K. (2000). Toxicity and repellency of borate-sucrose water baits to argentine ants (Hym.: Formicidae). Journal of Economic Entomology, 93 (4): 1256-1258. doi: 10.1603/0022-0493-93.4.1256 Lanza, J., Vargo, E.L., Pulim, S. & Chang, Y.Z. (1993). Preferences of the fire ants Solenopsis invicta and S. geminate (Hymenoptera. Formicidae) for amino acid and sugar components of extrafloral nectars. Environmental Entomology, 22: 411-417. doi: 10.1093/ee/22.2.411 Saric, L.C., Filipcev, B.V., Simurina, O.D., Plavsic, D.V., Saric, B.M., Lazarevic, J.M. & Milovanovic, I.L. (2016). Sugar beet molasses: property and applications in osmotic dehydration of fruits and vegetables. Food and Feed Research, 43 (2): 135-144. doi: 10.5937/FFR1602135Š SAS Institute. (2017). SAS User’s Guide: Statistics, Version 8. SAS Institute, Inc., Cary, NC. Sparks, K. (2015). Molecular phylogenetics of Australian Monomorium. A thesis for degree of Doctor of Philosophy. School of Biological Sciences, UA, Australia. StatPlus. (2017). Analysist Soft.Version 5.4. StatPlus House Inc. CA, USA. Ulloa-Chacon, P. & Jaramillo, G. (2003). Effects of boric acid, fipronil, hydramethylnon, and diflubenzuron baits on colonies of ghost ants (Hymenoptera: Formicidae). Journal of Economic Entomology, 96 (3): 856-862. doi: 10.1093/jee/96.3.856 VanderMeer, R.K., Lofgren, C.S. & Seawright, J.A. (1995). Specificity of the red imported fire ant (Hymenoptera: Formicidae) phagostimulant response to carbohydrates. Florida Entomologist, 78: 144-154. doi: 10.2307/3495679 VanderMeer, R.K. & Milne, D.E. (2017). Enhanced pest ant Control with hydrophobic bait. Journal of Economic Entomology, 110 (2): 567-574. doi: 10.1093/jee/tow300 Vanderwoude, C., Siolo, S., Sio, F. & Tupufia, S. (2006). Assessment of yellow crazy ants (Anoplolepis gracilipes) on Nuulua Island, Aleipata. A status assessment report, pp 1-30. Warner, J.R. (2003). Bait preferences and toxicity of insecticides to white-footed ants Technomyrmex albipes (Hymenoptera: Formicidae). A thesis for degree of Master of Science. Graduate School of the University of Florida, USA, 59 p. Zheng, J.H., Mao, R. Q. & Zhang, R. J. (2008). Competitive interactions between the yellow crazy ant and the red imported fire ant (Hymenoptera: Formicidae). Journal of Entomological Science, 43 (3): 331-336. doi: 10.18474/0749-8004-43.3.331