Electrophysiological and Alarm Responses of Solenopsis Invicta Buren (Hymenoptera: Formicidae) to 2-Ethyl-3,5-Dimethylpyrazine
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insects Article Electrophysiological and Alarm Responses of Solenopsis invicta Buren (Hymenoptera: Formicidae) to 2-Ethyl-3,5-dimethylpyrazine 1,2,3 1,3, 2, , Ya-Ya Li , Deguang Liu * and Li Chen * y 1 College of Plant Protection, Northwest A & F University, Yangling, Xianyang 712100, China; [email protected] 2 State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 3 State Key Laboratory of Crop Stress Biology for Arid Areas (Northwest A & F University), Yangling, Xianyang 712100, China * Correspondence: [email protected] (D.L.); [email protected] (L.C.); Tel.: +86-29-8709-2697 (D.L.); +86-10-6480-7780 (L.C.) Present address: College of Life Science, Hebei University, Baoding 071001, China. y Received: 23 November 2019; Accepted: 10 December 2019; Published: 13 December 2019 Abstract: 2-Ethyl-3,5-dimethylpyrazine is an isomer of 2-ethyl-3,6-dimethylpyrazine, the alarm pheromone component of the red imported fire ant, Solenopsis invicta Buren. The pyrazine was synthesized and its alarm activity was investigated under laboratory conditions. It elicited significant electroantennogram (EAG) activities, and released characteristic alarm behaviors in fire ant workers. The EAG and alarm responses were both dose-dependent. Two doses of the pyrazine, 1 and 100 ng, were further subjected to bait discovery bioassays. Fire ant workers excited by the pyrazine were attracted to food baits, and their numbers increased over time. Ants displayed very similar response patterns to both low and high doses of the pyrazine. The pyrazine impregnated onto filter paper disc attracted significantly more fire ant workers than the hexane control for all observation time intervals at the low dose, and in the first 15 min period at the high dose. The pyrazine loaded onto food bait directly tended to attract more fire ant workers than the hexane control. These results support the potential use of 2-ethyl-3,5-dimethylpyrazine to enhance bait attractiveness for the control of S. invicta in invaded regions. Keywords: red imported fire ant; bioassay; antennal response; myrmecology; social behavior; alarm pheromone 1. Introduction Chemical communication is of great importance in social organizations, which can have large impacts on survival and maintenance of integrity of ant colonies. When facing potential dangers, ants release alarm pheromones to alert nestmates for colony defense [1]. In response to alarm pheromones, ants show characteristic behaviors, such as attacking and biting the odor source or moving rapidly away from the odor source [2,3]. Furthermore, alarm pheromones may act as attractants, thus recruiting more workers for intensified attacks on intruders. Alarm responses can vary among species [3]. Generally, ants of those species with large and dense colonies often run toward the pheromone source with mandibles opened [4]. Ants have evolved a wide variety of compounds in communicating threats [5]. Pyrazines are a major group of pheromonal compounds isolated from mandibular glands and venom reservoir in Insects 2019, 10, 451; doi:10.3390/insects10120451 www.mdpi.com/journal/insects Insects 2019, 10, 451 2 of 9 Insects 2019, 10, x FOR PEER REVIEW 2 of 8 ants [6–8]. Pyrazines originating from mandibular glands typically trigger alarm responses, whereas thoseants [6–8]. from Pyrazines poison glands originating often serve from a mandibular trail following glands function typically [7]. trigger alarm responses, whereas thosePyrazines from poison seem glands to be often widespread serve a trail among following the two function subfamilies, [7]. Ponerinae and Myrmicinae, of the Formicidae.Pyrazines seem Few to species be widespread in Dolichoderinae, among the Ecitoninae, two subfamilies, Ectatomminae, Ponerinae and and Formicinae Myrmicinae, produce of the pyrazinesFormicidae. as Few alarm species and trail in Dolichoderinae, pheromones [8,9 Ecit]. Mostoninae, ant Ectatomminae, pyrazines are tri-substituted and Formicinae derivatives. produce Mono-,pyrazines di-, as and alarm tetra-substituted and trail pheromones pyrazines are[8,9]. much Most less ant common. pyrazines Numerous are tri-substituted dimethylalkylpyrazines, derivatives. whichMono-, show di-, 2,5-dimethyl-3-alkyl and tetra-substituted or 2,6-dimethyl-3-alkyl pyrazines are substitution much less patterns, common. have been Numerous detected fromdimethylalkylpyrazines, mandibular gland secretions which show of a variety 2,5-dimethyl-3-alkyl of ant species [7 ,8or]. 2,6-dimethyl-3-alkyl substitution patterns,2-Ethyl-3,6-dimethylpyrazine have been detected from mandibular (Figure1) is glan a componentd secretions of a poison variety gland of ant secretionsspecies [7,8]. of ant species2-Ethyl-3,6-dimethylpyrazine in Myrmicinae (Acromyrmex (Figure, Atta 1), Dacetonis a component, Manica of, Messorpoison, glandMetapone secretions, Myrmica of ant, Pheidole species, Pogonomyrmexin Myrmicinae, and (AcromyrmexTetramorium, )[Atta8,10, ,11Daceton] and, Ectatomminae Manica, Messor (Eciton, Metapone burchelli, )[Myrmica12], and, primarilyPheidole, functionsPogonomyrmex as a trail-following, and Tetramorium pheromone.) [8,10,11] Thereand Ectatomminae are several reports (Eciton of 2-ethyl-3,6-dimethylpyrazineburchelli) [12], and primarily infunctions mandibular as a trail-following glands of dolichoderine pheromone. (Iridomyrmex There are several purpureus reports), ectatommine of 2-ethyl-3,6-dimethylpyrazine (Ectatomma sp.) and ponerinein mandibular (Odontomachus glands of bauri dolichoderine, Pachycondyla (Iridomyrmex indica, Pachycondyla purpureus obscuricornis), ectatommine, and (EctatommaPachycondyla sp.) striata and) antsponerine [9,13 ,14(Odontomachus]. No functions bauri had, beenPachycondyla attributed indica to this, compoundPachycondyla in antsobscuricornis until Vander, and Meer Pachycondyla et al. [15] reportedstriata) ants its [9,13,14]. isolation No and functions identification had been from attributed the mandibular to this compound gland of thein ants red until imported Vander fire Meer ant, Solenopsiset al. [15] reported invicta. 2-Ethyl-3,6-dimethylpyrazine its isolation and identification elicited from rapidthe mandibular and erratic gland movement of the of red fire imported ant workers, fire apparentlyant, Solenopsis functioning invicta. 2-Ethyl-3,6-dimethylpyrazine as an alarm pheromone. elicited rapid and erratic movement of fire ant workers, apparently functioning as an alarm pheromone. Figure 1. Chemical structures of pyrazines. 2-Ethyl-3,5-dimethylpyrazine (Figure (Figure 1),1), anan isomerisomer ofof 2-ethyl-3,6-dimethylpyrazine,2-ethyl-3,6-dimethylpyrazine, isis aa mandibular gland componentcomponent of ponerine ants, Odontomachus brunneus [16],[16], O.O. chelifer [8],[8], O. troglodytes [17[17].]. Unlike Unlike other other pyrazine pyrazine components components from fromO. troglodytes O. troglodytesworkers, workers, however, however, this pyrazine this failedpyrazine to release failed significantto release alarmsignificant behaviors alarm [17 behaviors]. Because [17]. 2-ethyl-3,6-dimethylpyrazine Because 2-ethyl-3,6-dimethylpyrazine is commercially- is availablecommercially-available as a mixture with as itsa isomermixture of with 2-ethyl-3,5-dimethylpyrazine its isomer of 2-ethyl-3,5-dimethylpyrazine (ca. 60% 3,5-dimethyl (ca. isomer 60% and3,5- 40%dimethyl 3,6-dimethyl isomer isomer),and 40% the 3,6-dimethyl synthetic mixture isomer), of 2-ethyl-3,6(or the synthetic 5)-dimethylpyrazine mixture of 2-ethyl-3,6(or has been used 5)- fordimethylpyrazine most electrophysiological has been used and for behavioral most electrophysiological studies [15,18–23]. Bioassaysand behavioral with isomers studies purified[15,18–23]. by preparativeBioassays with GC orisomers HPLC demonstratedpurified by thatpreparative 2-ethyl-3,6-dimethylpyrazine GC or HPLC demonstrated had a relatively that lower2-ethyl-3,6- active responsedimethylpyrazine threshold had than a relatively 2-ethyl-3,5-dimethylpyrazine lower active response and threshold the mixture than of2-ethyl-3,5-dimethylpyrazine the two isomers [15,18]. and theA previous mixture of study the two has isomers reported [15,18]. the electroantennogram (EAG) and behavioral responses of S. invictaA workersprevious to study synthetic has 2-ethyl-3,6-dimethylpyrazinereported the electroantennogram [24]. (EAG) However, and very behavioral limited responses information of onS. alarminvicta activityworkers of to 2-ethyl-3,5-dimethylpyrazine synthetic 2-ethyl-3,6-dimethylpyrazine is available [24]. for fire However, ants in thevery literature. limited information In the present on study,alarm activity we aimed of 2-ethyl-3,5-dimethylpyrazine to examine both EAG and behavioral is available responses for fire ants of fire in antthe workersliterature. to In the the synthetic present pyrazine,study, we andaimed further to examine test for both attractiveness EAG and ofbehavioral the synthetic responses pyrazine-enhanced of fire ant workers baits. to the synthetic pyrazine, and further test for attractiveness of the synthetic pyrazine-enhanced baits. 2. Materials and Methods 2. Materials and Methods 2.1. Ant Sources and Colony Maintenance 2.1. AntMature SourcesS. invicta and Colonycolonies Maintenance were collected from the campus of South China Agricultural University (Guangzhou,Mature S. China). invicta Each colonies colony were with workers,collected broods, from the and campus queens wasof maintainedSouth China in fluon-coatedAgricultural plasticUniversity trays