Colchicum Autumnale) Bulb Powder on Red Imported Fire Ants (Solenopsis Invicta

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Article Toxicity and Sublethal Eﬀects of Autumn Crocus (Colchicum autumnale) Bulb Powder on Red Imported Fire Ants (Solenopsis invicta)

Sukun Lin 1, Deqiang Qin 1, Yue Zhang 1, Qun Zheng 1, Liupeng Yang 1, Dongmei Cheng 2, Suqing Huang 3, Jianjun Chen 4,* and Zhixiang Zhang 1,*

1 Key Laboratory of Natural Pesticide and Chemical Biology of the Ministry of Education, South China Agricultural University, Guangzhou 510642, China; [email protected] (S.L.); [email protected] (D.Q.); [email protected] (Y.Z.); [email protected] (Q.Z.); [email protected] (L.Y.) 2 Department of plant protection, Zhongkai University of Agricultural and Engineering, Guangzhou 510225, China; [email protected] 3 College of Chemistry and Chemical Engineering, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; [email protected] 4 Department of Environmental Horticulture and Mid-Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Apopka, FL 32703, USA * Correspondence: jjchen@uﬂ.edu (J.C.); [email protected] (Z.Z.)

Received: 18 September 2020; Accepted: 17 November 2020; Published: 21 November 2020

Abstract: Autumn crocus (Colchicum autumnale L.) is a medicinal plant as it contains high concentrations of colchicine. In this study, we reported that the ground powder of autumn crocus bulb is highly toxic to invasive Solenopsis invicta Buren, commonly referred to as red imported fire ants (RIFAs). Ants fed with sugar water containing 5000 mg/L of bulb powder showed 54.67% mortality in three days compared to 45.33% mortality when fed with sugar water containing 50 mg/L of colchicine. Additionally, the effects of short-term feeding with sugar water containing 1 mg/L of colchicine and 100 mg/L of autumn crocus bulb powder were evaluated for RIFAs’ colony weight, food consumption, and aggressiveness, i.e., aggregation, grasping ability, and walking speed. After 15 days of feeding, the cumulative colony weight loss reached 44.63% and 58.73% due to the sublethal concentrations of colchicine and autumn crocus bulb powder, respectively. The consumption of sugar water and mealworm (Tenebrio molitor L.) was substantially reduced. The aggregation rates decreased 48.67% and 34.67%, grasping rates were reduced to 38.67% and 16.67%, and walking speed decreased 1.13 cm/s and 0.67 cm/s as a result of the feeding of the two sublethal concentrations of colchicine and autumn crocus bulb powder, respectively. Our results for the first time show that powder derived from autumn crocus bulbs could potentially be a botanical pesticide for controlling RIFAs, and application of such a product could be ecologically benign due to its rapid biodegradation in the environment.

Keywords: autumn crocus; Colchicum autumnale; colchicine; invasive species; pest control; red imported ﬁre ant (RIFA)

Key Contribution: This study found that colchicine in autumn crocus (Colchicum autumnale L.) bulb powder was 0.3%, and solutions made from the bulb powder or commercial colchicine were toxic to the red imported ﬁre ant (Solenopsis invicta Buren), a notorious invasive pest. Our results suggest that autumn crocus bulb powder could be potentially used as a botanical pesticide for control of red imported ﬁre ants.

Toxins 2020, 12, 731; doi:10.3390/toxins12110731 www.mdpi.com/journal/toxins Toxins 2020, 12, 731 2 of 12

1. Introduction Solenopsis invicta Buren, commonly known as the red imported fire ant (RIFA), is a dominant and detrimental invasive species [1–3]. RIFA is native to South America [4], and was discovered in southern Alabama in the 1930s [5] and subsequently in Australia [6,7], New Zealand [8], Malaysia [9], Taiwan [10,11], and mainland China [12]. A mature RIFA colony contains a single queen or multiple queens with a large number of workers ranging from 200,000 to 400,000 [13]. They are extremely aggressive andToxins voracious 2020, 12, x FOR feedersPEER REVIEW and can quickly disrupt ground fauna, causing2 the of 12 imbalance of specific natural ecosystems [14]. Fire ant workers sting relentlessly when their mound is disturbed. 1. Introduction To control RIFAs, many contact insecticides, including chlorpyrifos and pyrethroid, have been widely Solenopsis invicta Buren, commonly known as the red imported fire ant (RIFA), is a dominant used [15]. However,and detrimental the use invasive of synthetic species [1–3]. insecticides RIFA is native may to South cause America environmental [4], and was discovered contamination in and are also prone to insecticidesouthern Alabama resistance. in the 1930s Thus, [5] and naturally subsequently occurring, in Australialow-resistant, [6,7], New Zealand and [8], ecologicallyMalaysia friendly control methods[9], haveTaiwan been [10,11], explored and mainland for RIFAChina [12]. management A mature RIFA [16 colony,17]. Amongcontains a them,single queen biological or pesticides multiple queens with a large number of workers ranging from 200,000 to 400,000 [13]. They are or biopesticidesextremely are valuable aggressive alternatives and voracious tofeeders synthetic and canpesticides quickly disrupt [18 ground]. fauna, causing the Biopesticidesimbalance are of natural specific natural products ecosystems derived [14].from Fire ant living workers organisms sting relentlessly including when their microbes, mound nematodes, is disturbed. To control RIFAs, many contact insecticides, including chlorpyrifos and pyrethroid, and plants thathave canbeen limitwidely orused reduce [15]. However, pest the populations use of synthetic [19 insecticides,20]. Biopesticides may cause environmental are usually unique and complexcontamination and have diandff erentare also modesprone to insecticide of action; resistance. thus, Thus, pests naturally are less occurring, likely low-resistant, to develop resistance. Biopesticides areand generallyecologically friendly safer than control synthetic methods have ones been to explored the environment for RIFA management as they [16,17]. can be Among quickly degraded, them, biological pesticides or biopesticides are valuable alternatives to synthetic pesticides [18]. and they are alsoBiopesticides more friendly are natural to nontarget products derived organisms from living [21,22 organisms]. The useincluding of biopesticides microbes, has been increasing globallynematodes, each and year. plants North that can America limit or reduce is the pest largest populations biopesticide [19,20]. Biopesticides market, are accounting usually for 44% of the market valueunique [23 and]. complex and have different modes of action; thus, pests are less likely to develop resistance. Biopesticides are generally safer than synthetic ones to the environment as they can be Autumnquickly crocus degraded, (Colchicum and they autumnale are also moreL.), friendly also knownto nontarget as organisms meadow [21,22]. saff ron,The use is of an herbaceous perennial plantbiopesticides in the familyhas been Colchicaceaeincreasing globally and each isyear. cultivated North America as anis the ornamental largest biopesticide plant [24,25] and market, accounting for 44% of the market value [23]. medicinal herb [26Autumn]. Its crocus flowers (Colchicum arise autumnale from underground L.), also known bulbsas meadow without saffron, leavesis an herbaceous and often achieve a height of 20 toperennial 25 cm plant (Figure in the1 A).family All Colchicaceae organsof and the is cultivated plant are as toxican ornamental and contain plant [24,25] alkaloids, and of which colchicine contributesmedicinal herb 50–70% [26]. Its offlowers the arise total from alkaloid underground content bulbs [ 25without]. These leaves alkaloidsand often achieve withstand a storage, height of 20 to 25 cm (Figure 1A). All organs of the plant are toxic and contain alkaloids, of which drying, and heatcolchicine treatment. contributes Colchicine 50–70% of the is total a medication alkaloid content used [25]. to These treat alkaloids gout andwithstand Bechet’s storage, disease [27,28]. It can inhibit microtubuledrying, and heat treatment. formation Colchici in cellne is mitosis,a medication thus used inducing to treat gout polyploid and Bechet’s disease plants [27,28]. [29,30 ]. However, autumn crocusIt can and inhibit colchicine microtubule have formation been in rarely cell mitosis, reported thus inducing to have polyploid any insecticidalplants [29,30]. However, activities. autumn crocus and colchicine have been rarely reported to have any insecticidal activities.

Figure 1. Autumn crocus (Colchicum autumnale) plants at the time of ﬂowering (A), underground bulbs (B), sliced bulb (C), ground bulb powder (D), HPLC chromatogram of colchicine standard at 10 mg/L (E) and autumn crocus bulb powder sample (F). The fourth (far right) peak in (F) had the same UV-Vis spectra as the peak of colchicine (E). Toxins 2020, 12, 731 3 of 12 Toxins 2020, 12, x FOR PEER REVIEW 3 of 12

In ourFigure studies 1. Autumn with RIFAs,crocus (Colchicum we found autumnale that) autumnplants at the crocus time of had flowering toxic (A e),ff undergroundects on RIFAs. Thus, this study wasbulbs intended (B), sliced bulb toanalyze (C), ground colchicine bulb powder levels (D), HPLC in autumn chromatogram crocus of colchicine bulb and standard determine at 10 toxic and sublethal effmg/Lects (E of) and colchicine autumn crocus and bulb autumn powder crocussample (F bulb). The fourth powder (far right) on RIFAs, peak in (F including) had the same the mortality, UV-Vis spectra as the peak of colchicine (E). colony weight, food consumption, i.e., sugar water and mealworm (Tenebrio molitor), and aggressiveness (aggregation,In our grasping studies ability,with RIFAs, and we walking found that speed). autumn Our crocus study had fortoxic the effects first on time RIFAs. demonstrated Thus, this that autumnstudy crocus was bulbs intended could to beanalyze considered colchicine as levels a biopesticide in autumnfor crocus effectively bulb and controlling determine toxic RIFAs. and sublethal effects of colchicine and autumn crocus bulb powder on RIFAs, including the mortality, 2. Resultscolony weight, food consumption, i.e., sugar water and mealworm (Tenebrio molitor), and aggressiveness (aggregation, grasping ability, and walking speed). Our study for the first time 2.1. Colchicinedemonstrated Content that in autumn Autumn crocus Crocus bulbs Bulb could be considered as a biopesticide for effectively controlling RIFAs. Standard colchicine solutions (0.1 to 10 mg/L) were analyzed by HPLC, which resulted in an equation2. ofResultsy = 186,423 x + 8971.9 (R2 = 0.9989) for determination of colchicine concentrations in autumn crocus bulb powder. The HPLC prolife showed that colchicine standard presented a single peak at the 2.1. Colchicine Content in Autumn Crocus Bulb retention time of 10 min (Figure1E), while four peaks appeared in analysis of the crocus bulb powder, and the firstStandard two were colchicine joined, solutions the third (0.1 and to the10 mg/L) fourth were were analyzed independent by HPLC, (Figure which1 resultedF). The in fourth an peak equation of y = 186,423x + 8971.9 (R2 = 0.9989) for determination of colchicine concentrations in had the same retention time as the colchicine standard solution (Figure1E,F). To further confirm if the autumn crocus bulb powder. The HPLC prolife showed that colchicine standard presented a single fourth peakpeak at was the colchicine,retention time HPLC-UV-Vis of 10 min (Figure analysis 1E), while was four performed. peaks appeared The resultin analysis showed of the that crocus the fourth peak hadbulb the powder, same UV-Vis and the spectrafirst two aswere the joined, peak ofthe colchicine, third and the suggesting fourth were that independent it was colchicine. (Figure 1F). The third peak alsoThe hadfourth the peak similar had the UV-Vis same retention spectra time to colchicine as the colchicine (data standard not shown), solution it (Figure could be1E,F). colchicoside. To The firstfurther twopeaks confirm were if the not fourth determined. peak was colchicine, Based on HPLC-UV-Vis the equation analysis mentioned was performed. above, colchicine The result content in autumnshowed crocus that bulbthe fourth powder peak washad the found same to UV-Vis be 0.30% spectra (n =as3). the peak of colchicine, suggesting that it was colchicine. The third peak also had the similar UV-Vis spectra to colchicine (data not shown), 2.2. Toxicityit could of Colchicinebe colchicoside. and The Autumn first two Crocus peaks Bulb were Powder not determined. to S. invicta Based on the equation mentioned above, colchicine content in autumn crocus bulb powder was found to be 0.30% (n = 3). The death of RIFAs occurred after three days of feeding with sugar water containing different concentrations2.2. Toxicity of of colchicineColchicine and (1 Autumn to 50 Crocus mg/L) Bulb or Powder autumn to S. crocus invicta bulb powder (100 to 5000 mg/L), and mortalityThe rates death varied of RIFAs significantly occurred after with three respective days of feeding concentrations with sugar water (Figure containing2). A mortality different rate of 45.33% wasconcentrations evident when of colchicine RIFAs (1 were to 50 fed mg/L) with or a au solutiontumn crocus containing bulb powder colchicine (100 to at 5000 50 mg mg/L),/L; the and mortality mortality rates varied significantly with respective concentrations (Figure 2). A mortality rate of rates gradually declined with the concentration decrease and became only 5.33% at the concentration 45.33% was evident when RIFAs were fed with a solution containing colchicine at 50 mg/L; the of 1 mgmortality/L (Figure rates2A). gradually When RIFAs declined were with fed the with concentration a solution decrease containing and became autumn only crocus 5.33% bulbat the powder at 5000concentration mg/L, the mortalityof 1 mg/L (Figure rate was2A). When 54.67%. RIFAs With were thefed with reduction a solution of containing bulb powder autumn content crocus in the solutions,bulb the powder mortality at 5000 rates mg/L, decreased. the mortality The rate mortality was 54.67%. was With 6.67% the reduction at the bulb of bulb powder powder concentration content of 100 mg/inL (Figurethe solutions,2B). On the the mortality contrary, rates the decreased. mortality The rate mortality of the RIFAswas 6.67% fed withat the sugar bulb waterpowder solution only wasconcentration 3.33%. of 100 mg/L (Figure 2B). On the contrary, the mortality rate of the RIFAs fed with sugar water solution only was 3.33%.

Figure 2. Mortality rates of S. invicta ants after three days of feeding with 10% sugar water containing

different concentrations of colchicine (A) and autumn crocus bulb powder (B). The ‘00 on the x-axis was the control, i.e., 10% of sugar water solution without any addition. Data are presented as mean standard error (S.E.). Different letters above bars indicate significant differences in mortality ± due to concentration effects within a treatment (A or B) at p < 0.05 level based on Tukey’s honestly significant difference (HSD) test (n = 3). Toxins 2020, 12, 731 4 of 12

A sublethal concentration of colchicine (1 mg/L) and autumn crocus bulb powder (100 mg/L) in 10% sugar water was selected for testing sublethal effects on RIFAs over 15 days of observation. There were no significant differences in mortality rate among treatments on day 3, but significant difference occurred between the control and colchicine and crocus bulb powder solution treatments Toxinson day 2020 6, (Figure12, x FOR3 PEER). Subsequently, REVIEW mortality rates of RIFAs fed with colchicine and crocus powder4 of 12 solutions became significantly higher than control after 6 days of feeding. Furthermore, the mortality ratesFigure of RIFAs 2. Mortality fed with rates crocus of S. bulb invicta powder ants after solution three days became of feeding significantly with 10% greater sugar water than containing those fed with colchicinedifferent from concentrations days 9 to 15. of Oncolchicine day 15, (A the) and mortality autumn ofcrocus ants bulb receiving powder 100 (B mg). The/L autumn‘0′ on the crocus x-axis bulb powderwas solutionthe control, increased i.e., 10% of to sugar 52.67%, water which solution was without significantly any addition. higher Data than are thosepresented receiving as mean 1 ± mg /L colchicinestandard in sugarerror (S.E.). water Different (33.33%) lett anders theabove control bars indicate group (9.33%)significant (Figure differences3). in mortality due to concentration effects within a treatment (A or B) at p < 0.05 level based on Tukey’s honestly significant 2.3. Edifferenceffect of Colchicine (HSD) test and (n Autumn= 3). Crocus on Colony Growth of S. invicta

ASolutions sublethal made concentration from either of colchicine colchicine or (1 autumn mg/L) and crocus autumn bulb powdercrocus bulb at sublethal powder concentrations (100 mg/L) in 10%had sugar significantly water was negative selected impacts for testing on the sublethal survival effe ofctsS. invictaon RIFAscolonies. over 15 The days weight of observation. loss of colonies There werereceiving no significant 10% sugar differences water containing in mortality 100 mg rate/L among autumn treatments crocus bulb on powder day 3, but or 1significant mg/L colchicine difference was occurredsignificantly between greater the than control that ofand the colchicine control (10% and sugar crocus water bulb only) powder on day solution 3 (Figure treatments4). From dayson day 6 to 6 (Figure15, theweight 3). Subsequently, loss of colonies mortality fed with rates 1 mgof /RIFAsL colchicine fed with and colchicine 100 mg/L and crocus crocus bulb powder powder solutions solutions becamequickly significantly increased. Colonies higher than fed with control 100 after mg/L 6 crocusdays of bulb feeding. powder Furthermore, lost significantly the mortality greater rates weight of RIFAsthan those fed fedwith with crocus 1 mg bulb/L colchicine, powder whereassolution thebecame colchicine significantly treatment greater caused than significantly those fed higher with colchicineweight loss from than days the 9 control. to 15. On After day 15, 15 daysthe mortality of feeding, of ants the receiving weight loss 100 percentage mg/L autumn of coloniescrocus bulb fed powderwith 100 solution mg/L autumn increased crocus to bulb52.67%, powder which and was 1 mgsignificantly/L colchicine higher solutions than werethose 58.73% receiving and 1 44.63%, mg/L colchicinerespectively, in comparedsugar water to (33.33%) 9.57% of and the control.the control group (9.33%) (Figure 3).

Figure 3. 3. ToxicityToxicity of of a a sublethal sublethal concentration concentration of of colchi colchicinecine at at 1 1 mg/L mg/L and and autumn autumn crocus crocus bulb bulb powder powder at 100 100 mg/L mg/L in in 10% 10% sugar sugar water water to toS. invictaS. invicta antsants over over 15 days 15 days of feeding. of feeding. Data are Data presented are presented as mean as ±mean S.E. DifferentS.E. Diff letterserent lettersat each at observation each observation day in daydicate indicate significant significant differenc differenceses among among treatments treatments at p ±

2.3.2.4. Effect Effects of of Colchicine Colchicine and and Autumn Autumn Cr Crocusocus on Bulb Colony Powder Growth on Food of S. Consumption invicta of S. invicta SolutionsThe consumption made from of sugareither watercolchicine and T.or molitorautumn(mealworm) crocus bulb bypowder colonies at sublethal of RIFAs concentrations was examined. hadAt the significantly beginning ofnegative the experiment, impacts on per the gram survival of ants of in S. respective invicta colonies. colonies The consumed weight 178.67loss ofmg colonies sugar receivingwater, 164.33 10% mg sugar sugar water water containing containing 100 100 mg/L mg/L autumn autumn crocus crocus bulb bulb powder powder, or and 1 mg/L 171.67 colchicine mg sugar waswater significantly containing 1greater mg/L colchicinethan that of (Figure the control5A). There (10% were sugar no water significant only) on di ffdayerences 3 (Figure in the 4). amount From daysof sugar 6 to water15, the consumptionweight loss of among colonies the fed three with treatments 1 mg/L colchicine on day and 1. On 100 day mg/L 3, thecrocus amount bulb ofpowder sugar solutionswater consumed quickly byincreased. ants in theColonies colonies fed fed with with 100 colchicine mg/L crocus and bulb autumn powder crocus lost bulbsignificantly was similar greater but weightsignificantly than those less than fed thatwith consumed 1 mg/L colchicine, by ants in wher the controleas the colonies.colchicine Starting treatment from caused day 6, significantly the amount higherof sugar weight water loss consumed than the by control. ants fed After with 15 three days food of feeding, sources the became weight significantly loss percentage different, of colonies and the feddiff erenceswith 100 continued mg/L autumn to day crocus 15. The bulb sugar powder water and consumption 1 mg/L colchicine by ants solutions in the control were colonies 58.73% wasand 44.63%, respectively, compared to 9.57% of the control.

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Toxins 2020, 12, 731 5 of 12

103.67 mg on day 15, which was signiﬁcantly higher than that consumed by ants in the other two treatments colonies. The sugar water consumption by the ants receiving 100 mg/L autumn crocus bulbs was 14.67 mg on day 15, which was signiﬁcantly lower than that consumed by ants fed with Figure 4. Accumulated colony weight loss (mean ± S.E.) of S. invicta colonies over 15 days of feeding Toxins1 mg /2020L colchicine, 12, x FOR PEER (45.67) REVIEW (Figure 5A). 5 of 12 with 10% sugar water, the sugar water containing 1 mg/L colchicine, and 100 mg/L autumn crocus bulb, respectively. Different letters at each recording day indicate significant differences among treatments at p < 0.05 level based on Tukey’s HSD test (n = 3).

2.4. Effects of Colchicine and Autumn Crocus Bulb Powder on Food Consumption of S. invicta The consumption of sugar water and T. molitor (mealworm) by colonies of RIFAs was examined. At the beginning of the experiment, per gram of ants in respective colonies consumed 178.67 mg sugar water, 164.33 mg sugar water containing 100 mg/L autumn crocus bulb powder, and 171.67 mg sugar water containing 1 mg/L colchicine (Figure 5A). There were no significant differences in the amount of sugar water consumption among the three treatments on day 1. On day 3, the amount of sugar water consumed by ants in the colonies fed with colchicine and autumn crocus bulb was similar but significantly less than that consumed by ants in the control colonies. Starting from day 6, the amount of sugar water consumed by ants fed with three food sources became significantly different, and the differences continued to day 15. The sugar water consumption by ants in the control colonies wasFigure 103.67 4. mgAccumulated on day 15, colonywhich was weight significantly loss (mean higherS.E.) than of S. invictathat consumedcolonies overby ants 15 daysin the of other feeding two Figure 4. Accumulated colony weight loss (mean ±± S.E.) of S. invicta colonies over 15 days of feeding treatmentswith 10% colonies. sugarsugar water,water, The the thesugar sugar sugar water water water consumption containing containing 1 mg1 by mg/L/L the colchicine, colchicine,ants receiving and and 100 100 mg100/ Lmg/L mg/L autumn autumn autumn crocus crocus crocus bulb, respectively. Different letters at each recording day indicate significant differences among treatments at bulbsbulb, was respectively. 14.67 mg on Different day 15, lettewhichrs wasat each significantl recordingy lowerday indicate than that significant consumed differences by ants fed among with 1 p < 0.05 level based on Tukey’s HSD test (n = 3). mg/Ltreatments colchicine at p (45.67) < 0.05 level(Figure based 5A). on Tukey’s HSD test (n = 3).

2.4. Effects of Colchicine and Autumn Crocus Bulb Powder on Food Consumption of S. invicta The consumption of sugar water and T. molitor (mealworm) by colonies of RIFAs was examined. At the beginning of the experiment, per gram of ants in respective colonies consumed 178.67 mg sugar water, 164.33 mg sugar water containing 100 mg/L autumn crocus bulb powder, and 171.67 mg sugar water containing 1 mg/L colchicine (Figure 5A). There were no significant differences in the amount of sugar water consumption among the three treatments on day 1. On day 3, the amount of sugar water consumed by ants in the colonies fed with colchicine and autumn crocus bulb was similar but significantly less than that consumed by ants in the control colonies. Starting from day 6, the amount of sugar water consumed by ants fed with three food sources became significantly different, and the differences continued to day 15. The sugar water consumption by ants in the control colonies was 103.67 mg on day 15, which was significantly higher than that consumed by ants in the other two treatments colonies. The sugar water consumption by the ants receiving 100 mg/L autumn crocus FigureFigure 5. 5.The The amount amount of of sugar sugar water water (A (A) and) andT. T. molitor molitor(B ()B consumed) consumed by byS. S. invicta invictaworkers workers over over 15 15 days bulbs was 14.67 mg on day 15, which was significantly lower than that consumed by ants fed with 1 ofdays feeding. of feeding. Data are Data presented are presented as mean as meanS.E. Di ± ffS.E.erent Different letters atletters each samplingat each sampling day indicate day indicate significant ± mg/Ldi colchicinesignificantfferences among differences(45.67) treatments (Figure among 5A). attreatmentsp < 0.05 levelat p < based0.05 level on Tukey’sbased on HSD Tukey’s test (HSDn = 3).test (n = 3).

AA di differentfferent pattern pattern was was observed observed in in food food consumption consumption when when colonies colonies were were fed fedT. T. molitor molitor(Figure (Figure5 B). Ants5B). in Ants the coloniesin the colonies treated treated with 100 with mg 100/L autumnmg/L autumn crocus crocus bulb powder bulb powder and 1 mgand/L 1 colchicine mg/L colchicine solutions consumedsolutions 47.67consumed mg and 47.67 55.33 mg mgand of 55.33T. molitor mg of, T. respectively, molitor, respectively, and ants and in the ants control in the colonies control colonies consumed 57.33 mg of T. molitor on the first day. There were no significant differences in consumption of T. molitor among the three treatments on day 1. On the third day, the consumption of T. molitor increased greatly. Ants in the colonies treated with 100 mg/L autumn crocus bulb powder and 1 mg/L colchicine solutions consumed 99.33 mg and 108.67 mg of T. molitor, respectively, and ants in the control treatment consumed 137.33 mg of T. molito. The amount of T. molitor consumed by ants in the colonies treated with colchicine and autumn crocus bulb did not differ significantly, but ants in the control group consumed significantly more T. molitor than the two other treatments. Ants in the control colonies continuously consumed more T. molitor until day 6, and then their consumption gradually decreased until day 15. The consumption of T. molitor by ants in colonies treated with colchicine and autumn crocus bulb powder decreased starting from day 3 until day 15. On day 15, per gram of ants in the Figure 5. The amount of sugar water (A) and T. molitor (B) consumed by S. invicta workers over 15 days of feeding. Data are presented as mean ± S.E. Different letters at each sampling day indicate significant differences among treatments at p < 0.05 level based on Tukey’s HSD test (n = 3).

A different pattern was observed in food consumption when colonies were fed T. molitor (Figure 5B). Ants in the colonies treated with 100 mg/L autumn crocus bulb powder and 1 mg/L colchicine solutions consumed 47.67 mg and 55.33 mg of T. molitor, respectively, and ants in the control colonies

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consumed 57.33 mg of T. molitor on the first day. There were no significant differences in consumption of T. molitor among the three treatments on day 1. On the third day, the consumption of T. molitor increased greatly. Ants in the colonies treated with 100 mg/L autumn crocus bulb powder and 1 mg/L colchicine solutions consumed 99.33 mg and 108.67 mg of T. molitor, respectively, and ants in the control treatment consumed 137.33 mg of T. molito. The amount of T. molitor consumed by ants in the colonies treated with colchicine and autumn crocus bulb did not differ significantly, but ants in the Toxinscontrol2020, 12group, 731 consumed significantly more T. molitor than the two other treatments. Ants in the6 of 12 control colonies continuously consumed more T. molitor until day 6, and then their consumption gradually decreased until day 15. The consumption of T. molitor by ants in colonies treated with coloniescolchicine treated and withautumn 100 crocus mg/L autumn bulb powder crocus decrea bulb powdersed starting and from 1 mg /dayL colchicine 3 until day consumed 15. On day 39.33 15, mg andper 65.67 gram mg of ofantsT. molitorin the ,colonies respectively, treated which with was100 mg/L significantly autumn lower crocus than bulb 104.33 powder mg and consumed 1 mg/L by antscolchicine in the control consumed colonies 39.33 (Figuremg and5 65.67B). mg of T. molitor, respectively, which was significantly lower than 104.33 mg consumed by ants in the control colonies (Figure 5B). 2.5. Effects of Colchicine and Autumn Crocus on the Aggressiveness of S. invicta 2.5. Effects of Colchicine and Autumn Crocus on the Aggressiveness of S. invicta The aggressiveness of RIFAs was evaluated in terms of their aggregation, grasping ability, and walkingThe aggressiveness speed (Figure of6). RIFAs There was were evaluated no di fferences in term ins of aggregation their aggregation, among workersgrasping fedability, with and sugar waterwalking and speed sugar (Figure water containing6). There were 1 mg no/L differences colchicine in and aggregation 100 mg/L among autumn workers crocus fed bulb with powder sugar on daywater 1 of and the sugar experiment water containing (Figure6A). 1 Frommg/L colchicine day 3, the and aggregation 100 mg/L rateautumn of the crocus control bulb treatment powder on was significantlyday 1 of the higher experiment than the(Figure other 6A). two From treatments. day 3, the Starting aggregation from the rate day of 6the to control day 15, treatment the aggregation was ratessignificantly of small workershigher than treated the other with two autumn treatments. crocus Starting bulb powder from the became day 6 to the day lowest, 15, the followed aggregation by the treatmentrates of small of colchicine workers treated and then with the autumn control. crocus On day bulb 15, powder the aggregation became the ratelowest, of workersfollowed receivingby the sugartreatment water of containing colchicine 100 and mg then/L autumnthe control. crocus On bulbday 15, powder the aggregation was only 34.67%rate of workers compared receiving to 48.67% sugar water containing 100 mg/L autumn crocus bulb powder was only 34.67% compared to 48.67% for the colchicine treatment and 88.67% for the control. for the colchicine treatment and 88.67% for the control.

FigureFigure 6. 6.The The mean mean aggregating aggregating rate rate ( A(A),), grasping grasping raterate ((BB),), andand walkingwalking speedspeed ((C) of small workers of S.of invicta S. invictafed with fed with sugar sugar water water and sugarand sugar water water containing containing 1 mg 1/ Lmg/L colchicine colchicine or 100 or mg100/ Lmg/L autumn autumn crocus bulb.crocus Data bulb. are Data presented are presented as mean as S.E.mean Di ±ff erentS.E. Different letters at letters each samplingat each sampling day indicate day significantindicate ± diffsignificanterences per differences parameter per among parameter treatments among treatments at p < 0.05 at level p < 0.05 based level on based Tukey’s on HSDTukey’s test HSD (n = test3). (n = 3). The grasping rates of workers fed with the three food sources exhibited the same pattern as the aggregationThe grasping rates during rates theof workers 15 days fed of evaluationwith the three (Figure food6 B).sources No di exhibitedfferences the were same recorded pattern onas the day 1, butaggregation differences rates occurred during on the day 15 3days where of evaluation the control (Figure treatment 6B). No showed differences significantly were recorded greater on grasping day rate1, thanbut differences the rest of treatments.occurred on Significantday 3 where di fftheerences control among treatment the three showed treatment significantly groups greater occurred fromgrasping day 6 rate to 15. than On the day rest 15, of the treatments. grasping rateSignif oficant workers differences fed 100 among mg/L the autumn three crocustreatment bulb groups powder droppedoccurred to from 16.67%, day which6 to 15. was On significantlyday 15, the grasping lower than rate 38.67%of workers of the fed 1100 mg mg/L/L colchicine autumn treatmentcrocus bulb and powder dropped to 16.67%, which was significantly lower than 38.67% of the 1 mg/L colchicine 89.33% of the control treatment groups. treatment and 89.33% of the control treatment groups. The walking speed of small workers is presented in Figure6C. There were no significant di fferences The walking speed of small workers is presented in Figure 6C. There were no significant in walking speed among small ants fed with the three food sources at the beginning of the experiment. differences in walking speed among small ants fed with the three food sources at the beginning of On day 3, the walking speed of small workers fed with autumn crocus bulb powder solution was the experiment. On day 3, the walking speed of small workers fed with autumn crocus bulb powder significantlysolution was slower significantly than those slower fed withthan thethose two fed other with sources. the two However, other sources. starting However, on day 6,starting the walking on speedday of6, smallthe walking workers speed fed withof small sugar workers water onlyfed with was significantlysugar water only faster was than significantly those fed with faster colchicine than andthose autumn fed with crocus colchicine bulb powder and autumn solutions. crocus From bulb day powder 9 to day solutions. 15, the walking From day speed 9 to of smallday 15, workers the receiving 100 mg/L autumn crocus bulb powder solution was the lowest, followed by those receiving the treatment of 1 mg/L colchicine treatment, and then the control. On day 15, the walking speed of small workers fed with 100 mg/L autumn crocus bulb powder was reduced to 0.67 cm/s, which was significantly lower than that of small workers receiving 1 mg/L colchicine sugar water (1.13 cm/s) and the control treatment (1.97 cm/s).

3. Discussion As an aggressive invasive species, S. invicta causes major problems, including displacement or elimination of native ground fauna and negative eﬀects on human health and public safety [31,32]. Toxins 2020, 12, 731 7 of 12

Lard et al. [33] estimated that the cost for control of RIFAs in the United States alone was greater than USD 6 billion annually. S. invicta specimen was first identified in Wuchuan, Guangdong Province, China on September 23, 2004 [34]. It was reported that S. invicta forage activities occur year-round, peaking in the summer and fall in South China. The expansion rate of S. invicta in China ranged from 26.5 to 48.1 km/yr [35]. Currently, S. invicta has been found in more than 10 provinces in China [35]. Lin et al. [36] predicted S. invicta could potentially threaten 41 species on the China National List of Protected Wildlife. Due to its invasion, the abundance of native ant species decreased more than 30% in China [37]. Common methods for control of RIFAs include the use of insecticides, such as chlorpyrifos and pyrethroid or biological control agents, such as the decapitating phorid fly, Pseudacteon tricuspis and a pathogen of fire ants, Thelohania solenopsae [38]. Additionally, plant-based chemicals have also been explored. For example, essential oil extracted from leaves of cinnamon (Cinnamomum osmophloeum) and trans-cinnamaldehyde were found to have an inhibitory effect on RIFAs [39]. As far as is known, there has been no report on the use of autumn crocus bulb powder for RIFA control. Our results showed that when RIFAs were fed a sugar water solution containing autumn crocus bulb powder at 5000 mg/L, the mortality rate reached 54.67% in three days, which was higher than 45.33% when fed 50 mg/L colchicine (Figure2). Since the autumn crocus bulbs are easy to produce, and powder can be quickly ground, the use of autumn crocus bulb powder could be a simple and convenient way for effective control of RIFAs. Autumn crocus has been used as an approved medical plant for over 3000 years even though it is poisonous [40]. Approximately 30 tropolone alkaloids have been identified in C. autumnale, of which colchicine, demecolcine, and colchicoside are the most abundant [41]. The concentration of colchicine ranged from 0.12 to 1.9% in bulb, 0.02 to 1.42% in leaves, 0.15 to 0.85% in flowers, and 0.14 to 1.2% in seeds. Demecolcine varied from 0.18% to 0.37% in bulb, and about 0.08% in leaves; however, colchicoside occurs mainly in seeds ranging from 0.48 to 0.1% [41]. In the present study, colchicine content in crocus bulb powder was 0.3%. Using solutions made from the powder, along with colchicine solution as a positive control, RIFAs exhibited almost identical patterns in response to both colchicine and autumn crocus bulb powder solutions. Our results suggest that the mortality of RIFAs is caused by the action of colchicine in autumn crocus bulb powder. The sublethal concentrations of colchicine and autumn crocus powder solutions resulted in serious chronic effects, including the increased weight loss of colonies, reduced consumption of sugar water and T. molitor, and reduced aggressiveness, such as aggregation, grasping ability, and walking speed. RIFAs are well known for their aggressiveness and strong food resource competitiveness [42,43]. The reduced consumption of food and colony expansion as well as their reduced aggregation and grasping ability and walking speed may suggest that another way of controlling RIFAs is to reduce their aggressiveness. Due to the chronic effects, their ability to attack other organisms will be severely handicapped, and their feeding and survival will become difficult, thus affecting the survival of the colony [44]. We did observe an interesting behavior in our experiment. When 10 healthy workers were placed in the same beaker with a T. molitor, the workers killed the T. molitor. However, when 20 workers, which were fed with sugar water solution containing 100 mg/L crocus bulb powder for 15 days, and were placed in the same beaker with a T. molitor, T. molitor survived even though the number of workers was doubled in the beaker. This observation suggests that the aggressiveness of the workers fed with sugar water containing 100 mg/L autumn crocus bulb powder were significantly reduced and unable to kill T. molitor. There is a renewed interest in the use of biopesticides for controlling pests [45,46] due to their biodegradability, less harm to nontarget organisms, and minimum effects on pest resistance [47]. Autumn crocus plant has been cultivated for more than 3000 years, and it is easily produced. The whole plant, leaves, flowers and bulbs contain high concentrations of colchicine [41]. Thus, not only bulbs but also the whole plants could be ground to produce powder, and solutions extracted from the plant tissue could be used for control of RIFAs. The application of the extracts will harm RIFAs and be Toxins 2020, 12, 731 8 of 12 benign to non-target organisms and the environment due to their biodegradability [48,49]. However, precautions should be taken when use of crocus plants. The plants should be handled by professionals with protection during extraction and application of the extract for RIFA control.

4. Conclusions This study for the first time documented that solutions made from autumn crocus bulb powder are lethal to RIFAs. The toxicity is mainly attributed to colchicine as RIFAs showed the same response patterns as the commercial colchicine solutions. A sublethal concentration of crocus bulb powder was shown to significantly affect colony growth, food consumption, and aggressiveness of RIFAs, thus reducing their invasiveness. Our study demonstrated that autumn crocus bulb powder could be potentially used as biopesticide for control of RIFAs by either directly killing the RIFAs at a high concentration of crocus bulb powder or by reducing their viability and invasiveness at a lower concentration. Future studies will focus on the mechanisms underlying the toxicity to RIFAs, which may help for the development of valuable botanical pesticides for RIFA control.

5. Materials and Methods

5.1. Insects, Chemicals, and Plant Red imported ﬁre ants (S. invicta) were obtained from nests in Guangzhou city, Guangdong Province, China and raised in the laboratory for bioassays. The ants were fed with 10% (w/w) sugar water and mealworm. All bioassays were conducted in the laboratory with a temperature of 26 2 C and a relative humidity of 70%. ± ◦ Colchicine standard solution was made using a 97% pure product purchased from Guangzhou Yiyang Trading Co., Ltd. (Guangzhou, China) and stored in a 18 C freezer in the dark. − ◦ Autumn crocus plants (C. autumnale) used in this study were grown at the Insecticidal Botanical Garden at South China Agricultural University, Guangzhou, China (Figure1A). The bulbs of autumn crocus were collected, washed (Figure1B), sliced (Figure1C), dried, and ground into powder (Figure1D) for the experiments described below.

5.2. HPLC Analysis of Colchicine in Autumn Crocus Powder In reference to Abidin et al. [50] and our preliminary tests, methanol was used for the extraction of colchicine. The colchicine content in the bulb powder of autumn crocus was determined by HPLC. Briefly, 20 g of bulb powder was mixed with 500 mL of methanol in a beaker. After mixing well, the sample was extracted by ultrasonic extraction for 30 min. An amount of 1 mL of the extract was taken and mixed with 50 mL of methanol. The solution was then filtered through a 0.22 µM filter membrane, and the filtrate was used for HPLC analysis of colchicine. A standard curve was established using diluted colchicine standard solutions ranging from 0.1 to 10 mg/L. There were three independent replicates for the extraction and analysis. HPLC was performed with an Agilent XDB-C column (250 mm 4.6 µm 5 µm) using a 18 × × mobile phase of methanol–water (45:55), with a flow rate of 1 mL/min, column temperature of 30 ◦C, detection wavelength of 245 nm, and injection volume of 10 µL.

5.3. Toxicity of Colchicine and Autumn Crocus Bulb The colchicine standard solution was diluted in 10% (w/w) sugar water to give concentrations of 50, 30, 10, 5, and 1 mg/L, and autumn crocus bulb powder was dissolved in 10% (w/w) sugar water to give doses of 5000, 3000, 1000, 500, and 100 mg/L. Sugar water (10% w/w) solution was used as the control. All solutions were freshly prepared. The experiment was conducted according to the procedures described by Zhang et al. [51]. Ants were starved for 24 h before the experiment. A total of 50 ants were placed in a 1.5 mL test tube and plugged with a cotton ball. The starved ants were subjected to one of the following three treatments: Toxins 2020, 12, 731 9 of 12

(a) each of the ﬁve colchicine concentrations mentioned above, (b) each of the ﬁve autumn crocus bulb powder concentrations, and (c) ants were fed with 10% sugar water as the control. The experiment was a completely randomized design with three replications. The number of dead ants was recorded after 3 days of treatment. Additionally, time-course mortality rates of ants fed with sugar water, sugar water containing 1 mg/L colchicine and 100 mg/L autumn crocus bulb powder were recorded every 3 days until day 15.

5.4. Sublethal Eﬀects of Colchicine and Autumn Crocus Bulb Powder on S. invicta Colony Growth Based on the results of the above toxicity experiment, sugar water solutions containing 1 mg/L colchicine and 100 mg/L autumn crocus bulb powder were selected as sublethal levels for the subsequent experiments. A colony was collected from the ﬁeld (Guangzhou, Guangdong Province, China) and fed as mentioned above in laboratory conditions (26 2 C and 70% relative humidity) for one week. ± ◦ The colony was divided into 3 sub-colonies (>25 g) of the same weight, and each sub-colony had alates, broods, workers and at least one functional queen. Sub-colonies were housed in trays (L W H, × × 41.75 27.5 12 cm) and provided with castone® nests (Diameter Height, 150 25 mm). × × × × The ants in the sub-colonies were starved for 24 h, and then were subjected to three treatments, i.e., fed with (a) 10% (w/w) sugar water as the control, (b) 1 mg/L colchicine solution, and (c) 100 mg/L autumn crocus bulb powder solution. The solution was provided in a 15 mL test tube and stoppered with a cotton ball in which enough T. molitors was provided for the sub-colonies. The experiment was a completely randomized design with three replications. To quantify the impact of colchicine and autumn crocus bulb powder on S. invicta colony growth, each sub-colony was weighed every 3 days up to 15 days after the treatments. The accumulated percentage of weight loss was calculated using the following Equation (1):

Weight loss (%) = [(m m )/m ] 100% (1) 2 − 1 2 × where m1 = the weight of each sub-colony after 0, 3, 6, 9, 12, and 15 days of the treatments and m2 = the initial weight of each sub-colony.

5.5. Eﬀects of Colchicine and Autumn Crocus Bulb Powder on Food Consumption of S. invicta To determine ant consumption of sugar water and T. molitors, sugar water and T. molitors were weighed before and after 1, 3, 6, 9, 12 and 15 days of feeding to the ﬁre ant sub-colony. The average consumption (mg sugar water or T. molitor per g ant) was calculated for each sub-colony at each sampling day. The experiment was a completely randomized design with three replications. The amount of sugar water evaporated was estimated by weighing three tubes containing each dosage of colchicine and autumn crocus bulb powder before and after they had been kept under the same conditions without the presence of the ants [52].

5.6. Effects of Colchicine and Autumn Crocus Bulb Powder on the Aggressiveness of S. invicta The aggressiveness of RIFAs was evaluated by three indexes in the experiment, namely aggregating rate, grasping rate, and walking speed of workers. To reduce experimental error, small workers, about 3 mm in length [39,53] were used for recording the walking speed. These three parameters were measured every 3 days up to 15 days of treatment. There were 50 workers per sub-colony. The experiment was a completely randomized design with three replications. Based on the behavior of RIFAs in our research, the aggregation was defined as the gathering of more than five workers. The aggregation rate was calculated using the Equation (2) below:

Aggregation rate (%) = (p /p ) 100% (2) 1 2 × where p1 = the number of aggregated workers and p2 = the number of total workers measured. Toxins 2020, 12, 731 10 of 12

To test grasping rate, the workers were placed on A4 paper, which was gently rotated 180◦ after 5 s, and the workers were considered to possess grasping ability if they did not fall from the A4 paper. The following Equation (3) was adopted:

Grasping rate (%) = (q /q ) 100% (3) 1 2 × where and q1 = the number of workers possessing grasping ability and q2 = the number of total workers measured. For evaluation of walking speed, workers were placed on a piece of A4 paper, and the walking distance in 3 s was measured. The walking speed and average walking speed were obtained by the following Equations (4) and (5):

Walking speed = Walking distance of workers/3 s (4)

Average walking speed = the sum of all the workers’ walking speed/headcount (5)

5.7. Data Analysis Collected data from the aforementioned experiments were subjected to one-way analysis of variance (ANOVA) using SPSS Statistics, Version 17.0, 2009 (International Business Machines Corporation, Armonk, NY, USA). If significant differences occurred among treatments, means were separated by Tukey’s honestly significant difference (HSD) test at p < 0.05 level. Means were presented in graphs with standard error which were drawn using Microsoft Excel.

Author Contributions: Conceived the experiments: Z.Z.; designed the experiments: D.C. and S.H.; performed the experiments: S.L.; analyzed the data: D.Q., Y.Z., Q.Z., and L.Y.; wrote the manuscript: S.L.; wrote, edited, and reviewed the manuscript: J.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by key-Area Research and Development Program of Guangdong Province (NO. 2020B020224002) and Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (No. 2019KJ122). Acknowledgments: The authors thank Terri A. Mellich for critically reviewing this manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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