Journal of Economic Entomology, 110(2), 2017, 593–597

doi: 10.1093/jee/tox038
Advance Access Publication Date: 8 March 2017
Household and Structural Insects Research article

Effect of Sweeteners on the Survival of Solenopsis invicta

(Hymenoptera: Formicidae)
Xiaoqiang Zhang,1,* Siqi Chen,1,* Zhiqiang Li,2,3 and Yijuan Xu1,3
1
Red Imported Fire Ant Research Center, South China Agricultural University, Guangzhou 510642, China (719855828@qq.com;
xuyijuan@scau.edu.cn), 2Shenzhen Agricultural Science and Technology Promotion Center, Shenzhen 518057, China

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(179558953@qq.com), and 3Corresponding authors, e-mail: 179558953@qq.com; xuyijuan@scau.edu.cn
*These authors contributed equally to this work.
Subject Editor: Arthur Appel
Received 23 September 2016; Editorial decision 5 January 2017

Abstract
The red imported fire ant, Solenopsis invicta Buren, is a serious agricultural, ecological, and public health pest
in its invaded range. Chemical insecticides have been widely used for the prevention and control of this notori-
ous pest. To search for novel, cost-effective, and environmentally friendly materials for fire ant control, we
tested various sweeteners for their toxicity to S. invicta. The S. invicta that were fed erythritol, aspartame, and
saccharin exhibited significantly higher mortality rates than those fed other sweeteners. After 72 h, the mortality
of the workers that were fed 0.1 or 0.2 g/ml erythritol was above 80%. For males, females, and larvae, mortality
was close to 100% after 9 d at high concentrations. Dose-dependent effects of erythritol were observed for work-
ers, males, females, and larvae at the concentrations tested. Rapid transfer of toxicity among the fire ant colony
was also observed. Our results suggest that erythritol can be developed as an additive ingredient in baits.

Key words: Solenopsis invicta, sweetener, bait, toxicity transfer

The red imported fire ant, Solenopsis invicta Buren, is an enor- also toxic to the oriental fruit fly, Bactrocera dorsalis Hendel
mously successful invasive ant species that is found throughout (Tephritidae) (Zheng et al. 2016). However, the toxicity of sweet-
much of the southern United States (Drees et al. 2010). Solenopsis eners to ants and the effects of sweeteners on behavior have not
invicta has spread worldwide via commerce and is the most preva- been characterized.
lent pest species in parts of the Caribbean. Additionally, new infest- In this study, we examined the effects of six commonly used
ations have been detected and exterminated in Arizona, California, sweeteners on the survival of S. invicta. We then investigated the
Australia, New Zealand, and southern China in past decades dose-dependent effects of erythritol on the mortality of different
(Ascunce et al. 2011). castes. Finally, we assessed the transfer of erythritol among different
In its invaded area, S. invicta poses a serious threat to human castes of S. invicta. The results of these experiments indicate that
health, public safety, agriculture, and forestry production and to the sweeteners have negative effects on the survival of S. invicta and
ecological environment (Wang et al. 2012). Chemical control of this have the potential to be developed into additive ingredients in baits.
pest has proven effective and is becoming the main method for the
prevention and control of ant populations (Drees and Gold 2003).
However, long-term use of highly toxic pesticides is likely to cause
Materials and Methods
serious problems, such as biodiversity reduction and ecological im- Materials
balance (Wojcik et al. 2001). Thus, there is a substantial need for Erythritol (BR, state  99.0%), sucrose (AR, purity ¼ 99.5%), D-
novel, cost-effective, and environmentally friendly methods and in- fructose (BR, purity  99.0%), D-glucose anhydrous (EP, state-
secticides for controlling populations of this pest (Williams and  99.0%), saccharin (BR, 99% purity), and aspartame (BR,
Deshazo 2004, Zheng et al. 2016). state  99.0%) were provided by SePol Laboratory Equipment
Sweeteners are a common flavoring agent. Both natural and arti- Co. Ltd (Guangzhou, China).
ficial sweeteners are widely used in the food industry as food addi- Brilliant blue (Zhengxing Food Additive Co. Ltd, Henan, China)
tives (Shankar et al. 2013). Previous reports have shown that was mixed with 0.01 M PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM
sweeteners exert toxic effects on insects. For example, the mortality Na2HPO4, and 1.4 mM KH2PO4, pH 7.3) to obtain a 0.5% blue
of fruit flies and bees was found to increase rapidly after feeding on dye solution. A 50-ml aliquot of brilliant blue was added to the treat-
erythritol, a natural sweetener (Baudier et al. 2014). Erythritol is ment liquid and water control to enable visual confirmation of

C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.
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594 Journal of Economic Entomology, 2017, Vol. 110, No. 2

consumption of the liquid by S. invicta. Brilliant blue was proved to Ten larvae were randomly selected and placed in a plastic box
have no toxicity effect on the ants and don’t change the palatability (length 14 cm by width 9 cm by height 6 cm) with the inner walls
of the tested solutions in our preliminary test. coated with Fluon. Next, 120 workers of various sizes were intro-
duced into the box to attend the larvae. A 6-ml aliquot of the eryth-
Insect Colonies ritol solution was then placed in a 10-ml centrifuge tube, which was
Solenopsis invicta colonies were collected from the experimental introduced into the box. The mortality of the larvae was scored
field of South China Agricultural University and reared in plastic every 24 h until all of the larvae were dead. Five replicates were per-
boxes (40 by 28 by 10 cm3). All colonies were separated from the formed per treatment. The dead workers were removed from the
soil by adding water to the plastic boxes until the colonies floated box every day, and healthy nestmate workers were added to retain
(Jouvenaz and Wojcik 1977). The ants were fed a mixture of 10% the same number of workers. Additionally, erythritol solution was
honey and live insects (Tenebrio molitor L. [Coleoptera: constantly provided. The larvae were considered dead if their bodies
Tenebrionidae]). A test tube (25 by 200 mm2) that was partially became dry and the workers showed no interest in them. It is diffi-
filled with water and plugged with cotton was used as a water cult to distinguish between the first instar of alate larvae and worker
source. The ants were placed in a plastic box (25 by 17 by 8 cm3), larvae; therefore, the larvae tested in this experiment included larvae

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which served as artificial nest. The fire ants were given a 10% solu- from both workers and the first instars of alates.
tion of honey mixed with water (50 ml) weekly. The inner edge of
each box was painted with Fluon to prevent the ants from escaping. Transfer of Erythritol Among Different S. invicta Castes
Ten male or female alates were randomly selected and placed into
Effects of the Tested Sweeteners on the Survival of different plastic boxes (length 14 cm by width 9 cm by height 6 cm),
S. invicta Workers and 50 fire ant workers and 50 larvae of various sizes were then
The experiments were performed under laboratory conditions with introduced into each box to attend the alates. After 6 h of starvation
a photoperiod of 14:10 (L:D) h, at 24–26  C and 60–70% relative treatment, a 0.1 g/ml erythritol solution mixed with blue dye was
humidity. Thirty medium-sized workers (head width 0.65–0.9 mm) added to a 10-ml centrifuge tube, which was introduced into the
were randomly selected and transferred to a plastic box (length box. Fresh erythritol solution was constantly provided. Blue dye was
40 cm by width 26 cm by height 10 cm) and maintained for 6 h with- added to the treatment liquids and water solution to enable visual
out food and water. A 10-ml centrifuge tube plugged with cotton confirmation of the consumption of the liquid by S. invicta. The ab-
with 6 ml of sweetener solution was then introduced into the plastic domens of the larva, worker, male and female ants were crushed by
box. The mortality of the workers was recorded at 72 h. Workers hand, and if blue liquid was found in the abdomens, then the fire
were considered dead if they showed no reaction to gentle touching
ants were confirmed to have consumed the erythritol and water so-
(also used to assess alates in the following experiment). The sweet-
lution. The rate of dye consumption was scored at 12, 24, and 48 h,
eners were dissolved at concentration of 0.2 g/ml (except 0.01 g/ml
and three replicates were performed per treatment.
for aspartame). Five replicates were performed for each treatment,
and water was utilized as a control.
Statistics
Analyses were conducted using SPSS software v.13.0 (SPSS 2004).
Effects of Erythritol on the Survival of Different
The resultant toxicity and the differences in the concentrations of
S. invicta Castes the sweeteners were compared. The mortality data from the screen-
Erythritol, the only natural sweetener among the three most toxic
ing experiment, mortality data on the toxicity of the sweeteners at
chemicals identified in the previous experiment, was selected for fur-
the different tested concentrations for different developmental stage/
ther exploration. Specifically, the toxicity of erythritol was tested on
cast, and the rate of dye consumption for different developmental
S invicta workers, alates, and larvae. Concentrations of 0.001, 0.01,
stage/cast were subjected to angular transformation (arcsine trans-
0.05, 0.1, and 0.2 g/ml of erythritol were tested in the following
formation) to meet the requirements for a normal distribution. One-
experiment.
way analysis of variance (ANOVA) was performed to evaluate the
Thirty fire ant workers of varying sizes were randomly selected
effects of the different sweeteners on the mortality of S. invicta and
and transferred to a plastic box (length 14 cm by width
the differences in toxicity transfer (rate of dye consumption) at vari-
9 cm by height 6 cm), where they were maintained for 6 h without
ous treatment times. Repeated-measures ANOVA with main effect
food and water. The inner walls of the box were coated with Fluon
(concentrations) was performed to assess mortality during the differ-
to prevent the fire ants from escaping. Then, 6 ml of the erythritol
solution was placed in a 10-ml centrifuge tube, which was intro- ent durations of exposure to various concentrations of the investi-
duced into the box. Mortality was recorded at 12, 24, 36, 48, 60, gated sweeteners.
and 72 h. Five replicates were performed for each treatment.
Ten male or female alates were randomly selected and placed in
Results
different plastic boxes (length 14 cm by width 9 cm by height 6 cm),
and 120 fire ant workers of various sizes were then introduced into Comparison of the Sweetener Toxicities
each box to attend the alates. Next, 6 ml of the erythritol solution We compared the effects of the sweeteners (erythritol, sucrose, D-
was placed in a 10-ml centrifuge tube, which was introduced into fructose, D-glucose anhydrous, saccharin, and aspartame) on the
the box. The mortality of the alates was recorded every 24 h until all survival of fire ant workers. The effects of the sweeteners on S.
of the alates were dead. The dead workers were removed from the invicta differed significantly (one-way ANOVA, F5,24 ¼ 20.024,
plastic box every day, and healthy nestmate workers were added to P < 0.0001) at 72 h. The mortality rates of workers fed erythritol,
maintain the same number of workers. Additionally, erythritol solu- aspartame, and saccharin were higher than 80% and were signifi-
tion was constantly provided. Three replicates were performed for cantly higher than for those fed D-glucose anhydrous and water
each treatment. (control; Fig. 1).
Journal of Economic Entomology, 2017, Vol. 110, No. 2 595

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Fig. 1. Effects of several sweeteners on the survival of S. invicta workers after 72 h. The graph shows the mortality rates of workers fed different sweeteners.
Erythritol, aspartame, and saccharin induced a significant increase in mortality rates compared with the other sweeteners. Bars (6SE) with the same letter do not
differ significantly (Tukey’s test) at the 0.05 level.

Fig. 4. Analysis of the dose-dependent responses of S. invicta females to

erythritol at 24-h intervals.

Fig. 2. Analysis of the dose-dependent responses of S. invicta workers to

Dose-Dependent Effects of the Sweeteners
erythritol at 12-h intervals.
To determine the dose-dependent effects of erythritol, we measured
the mortality of different S. invicta castes fed with five different con-
centrations of erythritol. The mortality rates of the workers, males,
females, and larvae fed erythritol differed significantly under the dif-
ferent concentrations (repeated-measures ANOVA, workers,
F4,20 ¼ 24.582, P < 0.0001; males, F4,10 ¼ 5.78, P ¼ 0.011; females,
F4,10 ¼ 5.338, P ¼ 0.015; larvae, F4,20 ¼ 14.283, P < 0.0001). The
mortality rates of the fire ants increased with an increasing duration
of processing (time) at the same dose concentration (repeated-meas-
ures ANOVA, all P < 0.001). After 72 h, the mortality of the work-
ers fed erythritol was greater than 80% at 0.1 or 0.2 g/ml. For the
males, females, and larvae, mortality was close to 100% after 9 d at
high concentrations (Figs. 2–5).

Toxicity Transfer Among the Fire Ant Colony

According to the rate of dye uptake, we observed that 50% of the
workers showed toxicity transfer at 12 h, which rapidly increased to
100% at 24 h (one-way ANOVA, F2,6 ¼ 196, P < 0.0001, Fig. 6A).
Erythritol could be rapidly transferred to reproductive males, and
Fig. 3. Analysis of the dose-dependent responses of S. invicta males to no significant difference in the rate of dye uptake was observed at
erythritol at 24-h intervals. three time points (one-way ANOVA, F2,6 ¼ 1.857, P ¼ 0.236,
596 Journal of Economic Entomology, 2017, Vol. 110, No. 2

Fig. 6B). There were significant differences in the rate of dye uptake Discussion
in reproductive females at different time points (one-way ANOVA,
Sweeteners are widely used as food additives, and research shows
F2,6 ¼ 28, P ¼ 0.001, Fig. 6C), and the rate was 100% at 48 h. The
that humans exhibit good adaptability to sweeteners (Bornet et al.
rate of dye uptake in the larvae was only 26.67% at 12 h, which rap-
1996, Tetzloff et al. 1996, Storey et al. 2007). Indeed, there are
idly increased to 73.33% at 48 h (one-way ANOVA, F2,6 ¼ 9.867,
many sweeteners that are consumed by humans every day (Hinson
P ¼ 0.013, Fig. 6D).
and Nicol 1992). In addition to their roles as food additives, the
other known functions of sweeteners are limited. The present study
evaluated the effect of sweeteners on the survival and behavior of S.
invicta based on toxicity measurements and behavior assays. The
natural sweetener erythritol and the artificial sweeteners aspartame
and saccharin were identified as the three types of sweeteners with
the greatest effects on S. invicta survival in our screening.
Erythritol exerted dose-dependent effects on workers, males, fe-
males, and larvae. This natural sweetener exhibited toxicity against the

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fire ants at concentrations higher than 0.1 g/ml, which may be a higher
dose compared with pesticides. Erythritol is generally recognized as
safe and can be used as a tabletop sweetener (Lin et al. 2010), which
suggests that erythritol presents potential for use as a novel and effect-
ive insect pest control bait that is safe to humans (Zheng et al. 2016).
Our results showed that erythritol exhibited slow toxicity in the fire
ants, which is helpful for achieving transmission among an entire col-
ony. Generally, insecticides with slow toxicity are considered more ap-
propriate for development into baits (Hooper-B ui and Rust 2015). In
polygyne colonies, it is especially important that the bait achieves
Fig. 5. Analysis of the dose-dependent responses of S. invicta larvae to eryth- the maximum distribution within the colony, so that reproductive
ritol at 24-h intervals. individuals throughout the colony can be effectively controlled

Fig. 6. Toxicity transfer of erythritol in workers (A), males (B), females (C), and larvae (D).
Journal of Economic Entomology, 2017, Vol. 110, No. 2 597

(Jordan et al. 2013). Although erythritol was rapidly transferred to Drees, B. M., K. Schofield, and B. Summerlin. 2010. Worker ant foraging re-
reproductive ants and larvae, the mortality of the reproductive ants sponse on and near mounds of red imported fire ants, Solenopsis invicta
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Hinson, A. L., and W. M. Nicol. 1992. Monitoring sweetener consumption in
in our experiments, which may be due to a relatively poor capacity
Great Britain. Food Addit. Contam. 9: 669–680.
of the workers to endure the toxicity of the sweetener (Hooper-B ui
Hooper-B ui, L. M., and M. K. Rust. 2001. An oral bioassay for the toxicity of
and Rust 2001). Although most or all of the workers were killed,
hydramethylnon to individual workers and queens of Argentine ants,
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In this study, we only evaluated the toxicity and transfer of erythri- Jordan, B. W., B. E. Bayer, P. G. Koehler, and R. M. Pereira. 2013. Bait evalu-
tol, a natural sweetener, among fire ant colonies. Determination of the ation methods for urban pest management, pp. 445–469. Insecticides -
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