1475 2016 The suc- 14 – 12 , 9 15 , 9 Regarding the insecticide, the use of Terezinha MC Della 16 , 15 a , 12 , 4 (Forel, 1893). Correspondence to: RNC Guedes, Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa,[email protected] MG 36570–000, Brazil. E-mail: [email protected] or Departamento de Entomologia, Universidade FederalBrazil de Viçosa, Viçosa, MG, Departamento de Biologia Animal, Universidade Federal de Viçosa, Viçosa,Brazil MG, The limited number of active ingredients used in toxic baits is ∗ a b four distinct practices of insecticide application are usedapplication, – fumigation, powder thermal fogginguse and of toxic the baiting latter – is the widespread in planted forests. due to the atypical requirement for thesethe compounds colony to but suppress without compromising worker foraging, allowing organosynthetic compounds isvery pervasive, limited albeit number of restricted compounds. to a cess of a toxicattractant, bait the against mass leaf-cutting and antsingredient size is used. of dependent Citrus the on extract bait and its granulessize the and are current the optimised granule active mass for and forest targeting the plantations. main pest species attacking in Joel C Couceiro, a Eucalyptus a* Acromyrmex subterraneus subterraneus 11 – 9 , 4 Karina D Amaral, a This scenario is further aggravated by the They are dominant species in natural as 9 , 4 : 1475–1481 www.soci.org © 2016 Society of Chemical Industry 5 , – 4 1 72 8 , 7 , 4 2016; and Raul NC Guedes colony suppression; ant control; leaf-cutting ant management; insecticide activity; behavioural activity; insecticide-induced The concerns with leaf-cutting ants are particularly acute b 6

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Insecticides are the prevailing tools used for controlling As truly social insects, leaf-cutting ants exhibit a number of pecu- leaf-cutting ants throughout their range of occurrence. Although liarities that include social organisation,tion, foraging, hygiene and fungus complex nest cultiva- structure, which make theirnotoriously control difficult. Pest Manag Sci Leaf-cutting ants have been broadly recognised asspecies important pest in Neotropical Americaof since the the New European colonisation World. Keywords: impairment 1 INTRODUCTION Supporting information may be found in the online version of this article. RESULTS: Although forager activity was not directly(auto- impaired and by allogrooming) fipronil, and the waste insecticide removalties affected and, of forager more fungus nestmate importantly, garden interactions greatly cultivation affected and the progenythe minor handling. colonies, The workers, ultimately fast impairing leading decay their to of activi- their the demise fungus within garden 8 compromised days. the sustainabilityCONCLUSION: of The behavioural effects ofinsecticide sublethal activity insecticide as exposure ant towards baits and minor© should workers 2016 be Society are targeted of the in Chemical main developing Industry such determinants compounds. of Abstract BACKGROUND: Attractive toxic baits are thefor prevailing the method production for of managing pulp,circumvent leaf-cutting the paper, ants typical timber in defences and the of the charcoal. eucalyptdirectly eusocial For forests harm leaf-cutting planted and/or successful ants. compromise use The foraging workers, challenge in but isare that these to will poorly baits, have eventually known, suppress an the the and insecticide insecticidal colony. here These incolonies underlying compounds the the of mechanisms bait need potential the that mechanism representative to will Neotropical of not fipronil activity in toxic baits for leaf-cutting ants was assessed using baits Lailla C Gandra, suppression by fipronil used in attractive toxic (wileyonlinelibrary.com) DOI 10.1002/ps.4239 Mechanism of leaf-cutting ant colony Research Article Received: 25 October 2015 Revised: 20 January 2016 Accepted article published: 27 January 2016 Published online in Wiley Online Library: 29 February wide monoculture expanses aimed at pulp, paper, woodcoal and production. char- in planted forest systems with the prevailing use of well as in human-altered environments (e.g. agricultural fields, pas- tures, forests), where they aretion, a with recognised conservative as loss adollars. threat estimates to in produc- the range of billions of current problems faced by forest companies,ant making management leaf-cutting evenrestrictive more requirements imposed challenging by – forest the certificationand initiatives ever-increasing the lack of control agentsspecies and below economic techniques thresholds. to keep these pest Lucia ) et al. : 1475–1481 A. wilkesiana 72 , 0.003% fipronil; ® 2016; Pest Manag Sci Worker ant mortality (mass of dead 30 10 cm). A 14 cm mark was made on each tray, × 30 cm × The mass of bait given to each colony was based on the com- The minor workers remain for much longer in the fungus garden (30 cm extending from its edgewas to considered the the entrance of totalcolony. Each the extent tray ant arena of was nest, further the14 which cm subdivided foraging halfway mark along made arena the (i.e. at ofplaced a at each 7 the cm 14 cm distance). mark Theconsidered at bait the as granules edge being were of harvested the when tray,(i.e. and carried half the over bait the the was extent 7 of cmnest. Either the mark baits foraging or arena) placebo incolony. baits the were offered direction only of once the to each mercial label rate and amounted to 3 g for1 L each colony volume for of about fungus a gardenBiocarv (Isca Ind. Attack Quím., Verde Curitiba, PR, Brazil).taining The fipronil commercial baits were con- given justreplicates), while once placebo to baits without each insecticide of wereto delivered five the colonies other (i.e. fiveing colonies. up The for baits 24 h, remained after which availableand the for remaining weighed. pick- granules were A retrieved provision of freshly cut leaves ( started 24 h afterdaily bait to provision, each and colonylasted 5 for g until the of eventual duration suppression leaves(i.e. of of 8 was days). the the offered experiment, fipronil-baited which colonies 2.3 Experimental measurements 2.3.1 Colony-based determinations The mass of bait harvestof after freshly the allocation, cut asdetermined leaves well for each as collected colony. the daily mass by the foraging ants, was workers) was also assessed dailyin by means which of the midden disposed inspection, deadan ants analytical scale were (AUW220D; retrieved Shimadzu, and Kyoto, Japan). weighedcounting Individual on of the deadnumbers ants and entanglement was with one not another. possible owing to the large 2.3.2 Worker-based determinations The behaviour ofdaily foraging for and eachand colony minor 10:30 a.m. for workers using a 30 minJapan). was digital These camera for recorded (HDR-XR520V; recordings each Sony, were Tokyo, placement caste performed to the from between end the of 8:00 time thetual experiment, of suppression which bait lasted of until the even- number fipronil-baited of colonies waste (i.e. removal 8 activitiesants, days). transferring fungus waste The fragments, (e.g. etc.) dead toaging the workers waste was recorded pile between performed 9:00the by and number for- 11:00 of a.m., autogrooming as (i.e. was ing self-grooming) (i.e. and grooming allogroom- of an individual byactivities another performed of during the the same observation species) period. and are involved in the tasksferring of mycelium hyphal manipulation (i.e. fragments trans- fromfungus one garden), place mycelium hygienisation to (groomingof another and old within removal fungus fragments) the and larvaltion transport. of For these better tasks, visualisa- the fungusrecipient, garden was and placed the in workers a were transparent nifiying observed glass with during the task aid performance.workers; of There a the was mag- order no marking ofwas of the observed colony once was per random,which day each and activity for each was 30 performed colony min; wascolonies the recorded and for treatments. number the different of times in 28 29 16 , , 9 5% W), , 15 4 , ± ′′ 13 www.soci.org LC Gandra 54 This is so ′ 4 52 © 2016 Society of Chemical Industry ∘ Acromyrmex sub- S, 42 ′′ C temperature, 75 14 ∘ ′ 5 was used in the experiments with a mixed assortment of Fipronil and sulfluramid suc- 45 ± ∘ 15 , 27 13 , – 12 23 ad libitum Müll. Arg. (Euphorbiacea). and none of them was commercially fea- 11 , 22 (Forel, 1893) were used for the experiments 10 – , 4 18 A. s. subterraneus Very few compounds were subjected to such 18 , 17 14 Therefore, the direct mortality of foraging workers is , 6 ant bait, was the insecticide of choice in the early use 16 , 15 Acalipha wilkesiana ® The aim of the present study was to improve our understanding The organochlorine dodecachlor, the active ingredient of the an undesirable traitstandard for focus of insecticidal insecticide research baits, rapidlypest killing contrasting species. an arthropod with the terraneus subterraneus performed under laboratorycan conditions. be achieved As either colony by directlyers, suppression compromising the the fungus minor garden work- or the antformer queen, is we more likely hypothesised because that fipronil the is notnor reported as as a a fungicidal sterilant compound. of the mechanism of fipronil activityof in leaf-cutting achieving ant the colonies suppression when usedof in the attractive representative baits. Neotropical leaf-cutting Colonies ant Mirex of toxic baits against leaf-cutting ants until its prohibition. wileyonlinelibrary.com/journal/ps Each colony produced about 1foraging L activity; of all fungus of andprogeny the exhibited at each colonies similar of were the developmental monogynic, stagespupae) exhibiting (i.e. eggs, and larvae and an estimatedreceived worker weekly number bait ofbaits) granules 8000. to allow without conditioning Each of the colony insecticide foraging workersand to (i.e. to accept prevent baits bait placebo rejection during the experiments.were The also colonies daily provided freshly cut leaves, with the prevalence of leaves fromshrub the evergreen Ten colonies of this species werestate field collected of in Viçosa Minas County, Gerais, Brazil (20 22.1 MATERIALS AND METHODS Colony collectionThe and leaf-cutting laboratory ant establishment because it attacks eucalypt forests in Neotropical America. This compound set a highpersistence standard and for efficacy low owing acute tothe toxicity its baits to high to foraging contaminate workers, the nest, allowing minor causing workers wholesale within mortality 5 of days. the baits to be harvested and brought to the nest. because leaf-cutting ant baitsnear are the applied ant by trailsto and distributing the nest them nest openings size, inand and quantities introduce the proportional foraging them workersdeath. into pick the up the colony, granules eventually leading to its mechanistic studies, sible. Thus, the understanding of such mechanismspression of in colony sup- currently usedremains insecticidal a baits knowledge gap for impairing leaf-cuttingtive the insecticides ants development against of leaf-cutting alterna- ants atavailable a compounds time are when facing currently pointed restrictionstification by companies. forest cer- cessfully followed the useunderlying of dodechlaclor mechanisms in to antunknown. baits, achieve but colony their suppression remain and established in theunder laboratory controlled where conditions theyrelative of were humidity and 25 maintained 12 h scotophase, as described elsewhere. 2.2 Bait application The teneach colonies individual were colony split was into placed two on groups a of white five, plastic and tray

1476 1477 25.50, 59.18, = = 0.56 auto- 1,34 1,43 ± F F = 0.006] P = 8.68, –x > 0.05] Acromyrmex subterraneus 1,27 F P [ Placebo-baited colonies Placebo-baited 1.70e y = 2.60 + 5678 = 0.010] = 0.24, P 2 R [ Fipronil-baited colonies y = 0.49 Time (days) Time (days) = 3.03, wileyonlinelibrary.com/journal/ps x 1,30 0.01) (Fig. 2). The number of auto- F = P = 0.09, 2 R Fipronil-baited colonies y = 1.20 – 0.12 [ 7.17, 0.05) (Fig. 3a). The allogrooming interactions = > > 0.05] 1,34 P P F provided either with fipronil or with placebo bait granules. 1234 y = 0.026 [ Placebo-baited colonies 12345678 Mass of freshly cut leaves harvested (a) and mass of dead ants

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

Daily harvest of freshly cut leaves Daily harvest Mass of dead ants removed daily from nest

Mass of dead ants (g) ants dead of Mass Mass of freshly-cut leaves (g) leaves freshly-cut of Mass 0.001); in the placebo-baited colonies such activity was main- 0.001) than in the placebo-baited colonies (19.27 (a) < < (b) exhibited a distinctbetween treatments diminishing 24 h trend, after with bait provision small and rapid differences increase groomings; tained until theprovision) end ( of the experiment (i.e. 8 days after bait and allogrooming interactions amongvary foraging with workers time did among not declined the with placebo-baited colonies, time24 but h in quickly after the baitcantly provision, fipronil-baited less the colonies frequent autogrooming in (Fig. was the already 3). fipronil-baited signifi- At colonies ( P P Figure 1. (b) in coloniessubterraneus of the leaf-cuttingEach ant symbol represents the species mean of five biologicaland replicates (i.e. the colonies), vertical barsfrom daily represent 30 min the observation associated for eight standard consecutive days. errors obtained 3.2 Worker-level effect ofThe fipronil number of wasteworkers removal in activities the performedHowever, such colony by decline was foraging declined steeper for with the fipronil-baitednearly colonies, time stopping in after 4 both days treatments. of the bait provision ( ). 4 HP picric 2 0.51 g 0.09 g; + ± ± 0.05) (Sys- < P 0.01) (Fig. 1b). = 0.14 g dead ants P ± ,20mMofNa test ( 0.25). The foraging 4 t- = PO 3.03, P 2 0.006), which remained with model complexity. = 2 = R P 1,30 1.24, F = ; fipronil: 4.69 8 8.68, t 1 − = 0.01). The colonies provided with 0.05), the amount of dead ants was = > 1,27 P F P 0.004), particularly 24 h after bait provi- 0.25 g of placebo baits and 1.80 = : 1475–1481 © 2016 Society of Chemical Industry ± P 3.08, 72 3.41 g) than in the fipronil-provided colonies = 0.07 g; 8 ± ± t 3.99, 2016; = 8 t ; 1 0.86 g) ( − 0.05 g dead ants colony -values and steep increases in F ± ± 0.05) (Fig. 1a). The mass of dead ants observed in the colony C) in Zamboni’s fixative solution (paraformaldehyde ∘ > waste piles0.15 was also distinct between treatments (placebo: (3.85 ants collected 1.09 of fipronil-containing baitsing in by the 24 h.with ant In significantly colonies contrast,colonies more greatly leaf (14.69 differed leaves harvest- between cut treatments, on the placebo-provided P relatively constant throughout the experiment (2.60 Pest Manag Sci 3RESULTS 3.1 Colony-level effectThe of fipronil mass of baitdiffer granules between collected treatments, by with the fipronilpick-up ant not by interfering colonies with did foraging bait not workers ( Leaf-cutting ant colony suppression by fipronil2.3.3 Queen-based reproductiveAt assessment the end of thebaits, experiment, when the 8 fipronil-treated days colonies were from virtually inactive, the the queen offering of of each the ant colony wastion for removed insects and (0.1 M dissected of NaCl, in 20 mM saline of KH solu- www.soci.org very high at thecolonies start and declined of thereafter the until ittrol assessments matched 8 for that days of the after the the fipronil-baited con- bait provision ( sion (Fig. 1b). While thenumber of placebo-baited dead colonies ants exhibited in the awith colony low waste time piles, which (0.26 did not vary 2.4 Statistical analyses The mass of bait granulesor collected without by fipronil, the as foraging well workers asdead with the workers, total mass was of compared harvest leaves by and Student’s acid) for subsequent morphological inspection undercroscope a (Stemi stereomi- 2000; Zeiss, Göttingen, Germany)a digital equipped camera with (Canon Power A640; Canon, Lake Success, NY). The reproductive tract of each queen was isolated, fixed(4 and stored tat v.13; Systat, San Jose,recorded CA). (i.e. All number of of the workersties other involved recorded) response in followed variables the an different approximateleast-squares normal activi- regression analyses distribution, were and performed overbait time introduction, after using the curve-fitting procedure2D of v.5.01 TableCurve (Systat). The regression models to(i.e. describe dependent) the variables response were selected on thehigh basis of parsimony, fipronil-containing baits exhibited significant reduction 24the h bait after was offered,when and compared with with the colonies further provided with steady placebonot baits decline (i.e. containing thereafter, fipronil) ( Although ants from differentpiles, castes were the observed amount in ofthe the dead fipronil-baited waste colonies, minor particularly workersprovision. at largely All the dominated of onset in the ofbait fipronil-baited provision, the unlike colonies the bait died placebo-baited colonies. after 8 days of The regression responses for treated anddistinguished untreated on colonies were the basis of thedistinct equation different parameters models and also obtained, the their non-overlapping means and respective standard errors of the observedregression data curve. points of each colony et al. < 0.001] P : 1475–1481 –x 72 = 59.18, 1,34 F 16.35e + 2016; = 0.63, 2 = 1.55 R y Fipronil-baited colonies [ , an opportunistic parasitic provided either with fipronil or Pest Manag Sci Time (days) Time (days) Escovopsis > 0.05] = 19.27 P < 0.001] Placebo-baited colonies Placebo-baited [ y P 2345678 = 79.98, > 0.05] –x = 2.03 Indeed, the results obtained provided support P y [ Placebo-baited colonies 1,34 F 26 1 1 , 25 , 18 23 , , = 0.70, 2 17 17 = 0.99 –1.99e , 0 2345678 0 , R y Fipronil-baited colonies [ Number of autogrooming (a) and allogrooming (b) interactions 4 4

5 4 3 2 1 5 0 0

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20 15 10 Autogrooming Autogrooming (number) interactions Autogrooming Autogrooming activites (number) activites Autogrooming We hypothesised that the direct effect of fipronil on minor (a) (b) fungus in ant–fungus symbiosis. workers was likely moreant important than queen its and directthe effects fungus foraging on workers. garden the This without expectationreported was directly fungicide based compromising activity on the ofthan lack fipronil sterilant of and effect itsbaits on to neurotoxic be insects, picked rather up besides by thethe the foraging nest. workers need and introduced for into the toxic Figure 3. performed by foraging workers inAcromyrmex colonies of subterraneus the subterraneus leaf-cuttingwith ant species placebo baitbiological granules. replicates Each (i.e. symbolassociated colonies), standard represents and errors the theeight obtained mean consecutive vertical days. from of bars daily five represent 30 min the observation for achieves ant colony suppression,(i) directly which impairing could the bedirectly ant by compromising queen means either reproductive of outputminor (ii) and/or the workers fungus handlingprogeny. garden or the (iii) fungus the garden and the queen for our hypothesis of acompromising prevailing the minor-worker-targeted care activity of theallowing fungus it garden to be and overrun with thus ultimately 40.55, 0.001) = 0.01] www.soci.org LC Gandra = < P P 1,34 F –x = 7.17, © 2016 Society of Chemical Industry 1,34 40.55, 0.001) (Fig. 3b). F 2.33e = < + P 1,34 The underlying mech- Acromyrmex subterraneus = 0.18, F 9 2 , = 3.43 4 R Placebo-baited colonies Placebo-baited y [ 79.98, 0.44 mycelium hygienisation = ± 1,34 F Time (days) 0.50 mycelium manipulation tasks ± < 0.001] P 2345678 0.05), while mycelium hygienisation in the > –x = 25.50, P 0.001) (Fig. 5). 1,34 0.05), while such activity decreased 24 h after F 1 < > P P provided either with fipronil or with placebo bait granules. = 0.35, 2 0 = 1.02 – 4.81e Number of waste removal tasks performed by foraging workers R y Fipronil-baited colonies [ 42.72,

7 6 5 4 3 2 1 0 = Waste removal tasks performed (number) performed tasks removal Waste 0.001) (Fig. 4a). Mycelium hygienisation also differed between 1,34 The common behavioural activities of minor ants in the fun- < F P treatments. The placebo-baitedlevel colonies of activity maintained through time the (4.11 same fipronil-baited colonies declined, with grooming stoppingcompletely almost 6 and 7 days after baiting ( tasks performed; 3.3 Queen-level effectThe of fipronil reproductive tractstereomicroscope of to assess the reproductive queensinspected impairment. queens, was from All either of inspected thecolonies, the placebo- exhibited under healthy or reproductive the tracts a without fipronil-baited anyent appar- differences (supporting information Fig. S1).not The atrophied, ovaries and were the eggsproduction were of visible apparently in healthy the eggs oviducts and young with larvae. the wileyonlinelibrary.com/journal/ps 4Fipronil DISCUSSION is one ofattractive the baits few successful foreucalypt insecticides forests managing in in use Neotropical leaf-cutting America. in ants, toxic particularly in thereafter until allogrooming interactionsdays completely after stopped fipronil bait 3 offering ( subterraneus Each symbol represents the mean of five biologicaland replicates (i.e. the colonies), vertical barsfrom daily represent 30 min the observation associated for eight standard consecutive days. errors obtained Figure 2. from colonies of the leaf-cutting ant species gus garden are themycelium manipulation hygienisation of (grooming mycelium andfragments), hyphae along removal with and larval of the transport.manipulation old The number fungus tasks of mycelium performedtime, but by remained minor higherout workers in the placebo-baited varied experiment colonies with through- (3.78 performed; anism of colonywe suppression attempted to was shed unknown light on for the fipronil. mechanism Here of how fipronil (Fig. 4b). Larval transport alsocantly declined more with compromised in time, fipronil-baited but colonies, which was exhib- ited signifi- only negligible activity as early( as 4 days after the bait supply fipronil bait access to negligible levels after 4 days (

1478 1479 ,a = 0.02] P Escovopsis –x = 6.04, ), no fungicidal 1,34 19 , F 4.20e 18 , + 9 , 4 = 0.15, 2 = 2.63 R y [ Placebo-baited colonies Placebo-baited Escovopsis Curiously, the fipronil-baited Acromyrmex subterraneus subterra- , but the said suppression took 26 Such an effect of abamectin led to – wileyonlinelibrary.com/journal/ps Time (days) 23 19 , Thus, as fipronil did not directly affect 18 32 , < 0.001] 31 P 2345678 –x = 42.76, contamination and eventual suppression of the A. s. subterraneus 1,34 F 1 2.441e + = 0.55, 2 Larva transportation activity performed by minor workers within = 0.19 0 R y Fipronil-baited colonies [ Escovopsis

provided either with fipronil or with placebo bait granules. Each sym- 8 6 4 2 0 Larva transportation tasks (number) tasks transportation Larva While both forager and minor workers were affected by fipronil, Fungicidal activity has never been associated with fipronil, activity by fipronilfavour was apparent. However,fungus the garden. This insecticide is explained did bybait the over the insecticidal minor activity workers of that the handlediffer the fungus in garden, which sizemay and behaviour account from forMinor the their workers, foraging differences for workers, instance,baits, in which allowing remain higher susceptibility longer levels of to inworkers. exposure contact fipronil. to fipronil with than the foraging the insecticide exhibiteddid mild not effects hinderfipronil on affected the both auto- harvesting the and allogrooming, and of foragers,minor interfered worker with freshly activities which within cut the nest,The noticeably leaves. waste higher removal. However, mortality ofchanges minor in workers their likely behaviour contributedworkers recorded from to colonies in of the our similar density study,also from different but exhibited treatments as altered behaviour, minor fipronilmediator was of probably such the changes. main Regardless,tion nest were compromised hygiene owing and to protec- and eventual removing failure fipronil-intoxicated in ants recognising tion and of allowing the fungus contamina- garden. the suppression of colonies of the sameour leaf-cutting experiment, ant species in a relatively long timepotential to use of occur this (over compound in 9 toxic baits. weeks), thus limiting the thus the perception thatleaf-cutting it directly ants affects the is fungus unlikely. garden of Therefore, this insecticide does not seemreproduction, directly to unlike impair the queen sterilantqueens effect of caused leaf-cutting ants. by abamectin in Figure 5. colonies of the leaf-cutting antneus species bol represents the mean of fivevertical biological bars replicates represent (i.e. the colonies), and associated30 the standard min errors observation obtained for from eight daily consecutive days. either fungus (i.e. ant garden fungus or colonies exhibited a quick debilitationgarden, and which decline of became the heavily fungus contaminated with specialised and occasionally virulenteliminate the fungus ant fungus. able to attack and 13 , 9 . < 0.002] P Acromyrmex –x = 10.74, 1,34 F 2.60e + A. s. subterraneus = 0.24, 2 = 0.79 R y Fipronil-baited colonies [ > 0.05] = 4.11 P y Placebo-baited colonies [ Time (days) Time (days) > 0.05] = 3.78 < 0.001] P y [ Placebo-baited colonies Placebo-baited P 2345678 provided either with fipronil or with placebo bait : 1475–1481 © 2016 Society of Chemical Industry –x = 40.55, 72 1,34 F 1 1 3.30e + 2016; = 0.54, 2 = 0.25 0 0 2345678 R y Fipronil-baited colonies [ Number of mycelium hypha manipulation tasks (a) and mycelium

6

6 4 2 0 4 2 0 Mycelium grooming tasks performed (number) performed tasks grooming Mycelium Mycelium manipulating (number) performed tasks manipulating Mycelium Mycelium grooming Fipronil did not interfere with bait pick-up by the foraging The queens of the leaf-cutting ants baited with fipronil did not (a) (b) workers, which wereules able into the to nest introduce atTherefore, the the no same fipronil rate repellence aswas bait or the detected gran- placebo direct bait in granules. into short-term the the foraging acute colony, ants, mortality aing which pivotal colonies were bait of well trait leaf-cutting for accepted ants its such success in as suppress- Pest Manag Sci The fipronil-baited ant colonies3–4 were quickly days, again suppressed reinforcing within leaf-cutting the ants. high efficacy of fipronil baits for exhibit any signs of reproductive impairment as theirtract reproductive remained healthy,ovarioles. with The produced marks eggs ofstill visible egg full within release the spermatheca. werepresence of Another also new brood visible, clue until the suppression as to of the was fungus queen garden. a fertility was the subterraneus subterraneus granules. Each symbol represents the mean ofcolonies), five biological and replicates (i.e. the verticalobtained from bars daily represent 30 min the observation associated for eight standard consecutive errors days. hygienisation (mycelium grooming) tasks (b) performedin by the minor fungus garden workers of colonies of the leaf-cutting ant species Figure 4. Leaf-cutting ant colony suppression by fipronil www.soci.org , Rev Bull ,ed. DOI: et al. :99–117 Eucalyptus 58 Atta sexdens : 1475–1481 72 Forel (Hymenoptera: 2016; , 2nd edition. Ceres, São (Hymenoptera: Formici- Forest Stewardship Coun- Annu Rev Entomol Formigas-Cortadeiras – da Formigas-Cortadeiras – da -eudesmol (Hymenoptera: As Formigas Cortadeiras 𝛽 :68–102 (2015). :31–46 (1997). -eudesmol in nestmate recog- Modern Crop Protection Com- Annu Rev Entomol :89–93 (2005). 𝛽 22 :1529–1535 (1998). 11 (Hymenoptera: Formicidae). 11 , ed. by Della Lucia TMC. Folha de 117 :208–221 (1999). Pest Manag Sci :445–454 (2006). , ABRAF, Brasília, Brazil (2013). :1–66 (2003). 23 , ed. by Della Lucia TMC. Editora UFV, , ed. by Della Lucia TMC. Editora UFV, Recommendations Regarding Derogations 47 :5–6 (1962). 176 18 Ser Tecn IPEF :163–172 (2005). no Novo Mundo, in 45 , Effects of neonicotinoids and fipronil on non-target Chem Res Toxicol Atta :467–473 (2008). Environ Sci Pollut Res Acromyrmex laticeps nigrosetosus , ed. by Krämer W, Schirmer U, Jeschke P and Witschel e Ciênc. Agrotec Sociobiology 98 Manual de Entomologia Agrícola Bol Campo Entomol Exp Applic Manejo Integrado – Doenças, Pragas e Plantas Daninhas , . et al. por Atta sexdens rubropilosa As Formigas Cortadeiras et al. Acromyrmex subterraneus subterraneus. Pest Manag Sci Sociobiology Acromyrmex subterraneus subterraneus Bonn, Germany (2010). :1059–1064 (2000). , -Eudesmol induced aggression in the leaf-cutting ant 10.1146/annurev-ent-010715-023646 (2016). effects ofant abamectin suppressing56 colonies of the leaf-cutting Abamectin-driven alterations on queen ovariesant of the leaf-cutting ed. by Zambolim L.(2000). Editora UFV, Viçosa, MG, Brazil, pp.Pesticide-induced 245–273 stress ingrated arthropod pest pests for management optimized programs. inte- dae). Biologia ao Manejo, Vol.Viçosa, MG, 1 Brazil, pp. 80–101 (2011). formigas-cortadeiras em laboratório, in by Della Lucia TMC. Folha de Viçosa,(1993). Viçosa, MG, Brazil, pp. 151–162 𝛽 rubropilosa urophylla Formicidae). Viçosa, Viçosa, MG, Brazil, pp. 163–190 (1993). formigas-cortadeiras. Filho E Paulo, Brazil (1988). combate, in RNC and Jham GN, Interferencenition of in Entomol Res sive response of pest ant species to Environ Contam Toxicol channel antagonists (fiproles),pounds, vol. 3 in M. Wiley-VCH, Weinheim, Germany, pp. 1283–1326 (2012). resistance, and secondary(2013). effects. Goulson D invertebrates. Nascimento IC, Distribuição das formigas-cortadeirasAcromyrmex dos gêneros Formicidae). of fipronil insecticidephotoproduct. and its sulfone metabolite andenvironmental fate, desulfinyl ecotoxicology, and human health concerns. TMC, Manejo de formigas-cortadeiras, in Biologia ao Manejo, Vol.Viçosa, MG, 1 Brazil, pp. 400–419 (2011). FSC-GUI-30-001 Version 1-O EN, ForestGermany Stewardship (2010). Council, Bonn, to Use Alpha-Cypermethrin, Deltamethrin,SulfluramidinFSCCertifiedForestsinBrazil. 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Harvard University Press, Cam- , 33 , 4 :225–247 (2012). Formigas-Cortadeiras – da Biologia ao 53 The Ants Eucalypt Cultivation in Brazil Springer-Verlag, Berlin, Germany (2003). , ed. by Della Lucia TMC. Editora UFV, Viçosa, MG, Brazil, Int J Pest Manag Thus, the findings reported here suggest alterna- :671–675 (1989). 36 13 , 35 fungus. This supplemented the reported behavioural , 4 24 h) and extensive mortality of minor workers took place < Leaf-Cutting Ants. Brazil (2000). Vasconcelos HL, Aleaf-cutting pest ants: is keystoneManag a taxa pest? of The natural dilemma of ecosystems. Neotropical Manejo, Vol. 1 pp. 13–26 (2011). ants: peculiarities, trends and challenges. (2014). das formigas-cortadeiras, in bridge, MS (1990). Calle Z, Leaf-cutting antsand revisited: control. towards rational management Fast ( The fipronil activity that primarily targeted minor workers of the 1 Fowler HG, Pagani MI, Silva AO, Forti LC, Pereira-da-Silva V and 2 Wirth R, Herx H, Ryel RJ, Beyschlag W and Hölldobler B, 4 Della Lucia TMC, Gandra LC and Guedes RNC, Managing leaf-cutting 5 Hölldobler B and Wilson EO, 3 Della Lucia TMC and de Souza DJ, Importância e história de vida 6 Montoya-Lerma J, Giraldo-Echeverri C, Armbrecht I, Farji-Brener A and 7MoraALandGarciaCH, under fipronil exposure viain attractive our baits, assessments which and was reportedhas observed here. been Mortality resembled reported what forof the which organochlorine in dodecachlor, insecticidal themon baits use until against leaf-cutting its ants eventual was prohibition com- in the early 1990s. wileyonlinelibrary.com/journal/ps REFERENCES SUPPORTING INFORMATION Supporting information may be found inarticle. the online version of this ACKNOWLEDGEMENTS The financial support provided bytific the and National Technological Development Council (CNPq), oftion the Scien- (Brazilian CAPES Founda- Ministry ofFoundation Education) of Research and Aid the (FAPEMIG) is Minas greatly appreciated. Gerais State tive venues in pursuing management ofplantations leaf-cutting that ants in are forest consistent with currenttion agencies demands and of product certifica- marketability. the former canagainst impair the pathogen ants’ contamination,of naturally which the protective little behaviour is successagainst the so leaf-cutting ants, far alleged despite obtainedthe cause the with tactic. high entomopathogen laboratory use efficacy of our study we(minor) were workers were unable able to perform their further taskswith within to the nest, fipronil observe affecting their thatfungus handling the garden, of allowing surviving the the progenyEscovopsis contamination and of of the the effects latter by observed the intated the the foraging impact workers,suppression. of which fipronil further in facili- the colony, leading toleaf-cutting ants, its probably aided quick by theand impairment hygienic of behaviours, led protective to aand fast likely progeny decay mishandling. of This compromised the colony sustain- fungusability garden and resulted inare colony therefore demise worthy in targetsdal 4–8 in compounds developing and days. other alternative Such control insectici- The effects tactics association of for neurotoxic insecticide the and pathogen leaf-cutting also ant. promising, seems as recently suggested.

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