RESEARCH ARTICLE Proximate mechanism of behavioral manipulation of an orb-weaver spider host by a parasitoid wasp Thiago Gechel Kloss1,2*, Marcelo Oliveira Gonzaga3, Leandro Licursi de Oliveira4, Carlos Frankl Sperber4 1 Programa de PoÂs-GraduacËão em Entomologia, Universidade Federal de VicËosa, VicËosa, Minas Gerais, Brazil, 2 Universidade Federal do EspõÂrito Santo, Centro de Ciências Exatas, Naturais e da SauÂde, Departamento de Biologia, Alegre, EspõÂrito Santo, Brazil, 3 Instituto de Biologia, Universidade Federal de a1111111111 UberlaÃndia, UberlaÃndia, Minas Gerais, Brazil, 4 Departamento de Biologia Geral, Universidade Federal de a1111111111 VicËosa, VicËosa, Minas Gerais, Brazil a1111111111 a1111111111 * [email protected] a1111111111 Abstract Some ichneumonid wasps induce modifications in the web building behavior of their spider OPEN ACCESS hosts to produce resistant ªcocoonº webs. These structures hold and protect the wasp's Citation: Kloss TG, Gonzaga MO, de Oliveira LL, cocoon during pupa development. The mechanism responsible for host manipulation proba- Sperber CF (2017) Proximate mechanism of bly involves the inoculation of psychotropic chemicals by the parasitoid larva during a spe- behavioral manipulation of an orb-weaver spider host by a parasitoid wasp. PLoS ONE 12(2): cific developmental period. Recent studies indicate that some spiders build cocoon webs e0171336. doi:10.1371/journal.pone.0171336 similar to those normally built immediately before ecdysis, suggesting that this substance Editor: Peter Schausberger, University of Vienna, might be a molting hormone or a precursor chemical of this hormone. Here, we report that AUSTRIA Cyclosa spider species exhibiting modified behavior presented higher 20-OH-ecdysone lev- Received: August 14, 2016 els than parasitized spiders acting normally or unparasitized individuals. We suggest that the lack of control that spiders have when constructing modified webs can be triggered by Accepted: January 19, 2017 anachronic activation of ecdysis. Published: February 3, 2017 Copyright: © 2017 Kloss et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Data Availability Statement: All relevant data are Parasites and parasitoids often alter specific behaviors of their hosts. Examples of changes within the paper and its Supporting Information range from subtle shifts in one aspect of the host's behavior to the performance of completely files. novel and complex behavioral patterns [1]. Some of these changes might be primarily due to Funding: This work was supported by MOGÐ the host's own defensive responses to infection [1,2] or to exploitation of the host's nutritional FundacËão de Amparo à Pesquisa do Estado de resources by the parasites [3]. Others changes in behavior are classified as host manipulation Minas Gerais (Grant number APQ-02104-14, CRA- to acquire some benefit in survivorship or dispersal ability [1]. In the majority of manipulation 30058/12) (http://www.fapemig.br/pt-br/); cases, however, little is known about the proximal mechanisms responsible for behavioral CoordenacËão de AperfeicËoamento de Pessoal de alterations [4]. For example, the cricket Nemobius sylvestris (Bosc, 1792) performs the unusual NõÂvel Superior (http://www.capes.gov.br/); TGK, MOG, CFS: Conselho Nacional de Desenvolvimento behavior of searching for water and jumping into it after being attacked by mature individuals CientõÂfico e TecnoloÂgico (CNPq) (Grant numbers of the hairworm Paragordius tricuspidatus (Dufour, 1828). This behavior is induced by the par- 140508/2012-0, 300295/2016-2,309787/2012-2, asite producing protein from the Wnt family, which act on the central nervous system of the PLOS ONE | DOI:10.1371/journal.pone.0171336 February 3, 2017 1 / 11 Proximate mechanism of behavioral manipulation of orb-weaver spiders 306157/2014-4, 403733/2012-0; 445832/2014-2, host [5]. Another reported case of host manipulation involves the injection of venom contain- 573802/2008-4) (http://www.cnpq.br/); ing a dopamine-like substance into the cerebral ganglia of the cockroach Periplaneta ameri- Financiadora de Estudos e Projetos (www.finep. cana (Linnaeus, 1758) by the parasitoid wasp Ampulex compressa (Fabricius, 1781). This gov.br); and Sistema Nacional de LaboratoÂrios em Nanotecnologias (SisNANO)/MinisteÂrio da Ciência, action directly stimulates the grooming-releasing circuits within the host's cerebral ganglia, Tecnologia e InformacËão (www.mcti.gov.br/ which enters into a subsequent nonparalytical hypokinetic state [6,7]. sisnano). All funders had no role in study design, Behavioral modifications of spiders by parasitoid wasps that form part of the tribe Ephial- data collection and analysis, decision to publish, or tini [8] (Hymenoptera: Ichneumonidae, Pimplinae) have been observed in several host fami- preparation of the manuscript. All funders offer lies. The wasps lay one egg on the spider's abdomen, where the ectoparasitic larva develops by research grants or money for laboratory equipment sucking the host's hemolymph through perforated holes in the spider's cuticle. Usually, the maintenance. host spider builds a modified web just before the wasp larva enters the final stage of develop- Competing Interests: The authors have declared ment [9±18], but, in some cases, the number of radii and spirals of the orb web gradually that no competing interests exist. decrease over several days before the spider dies [19,20]. After the spider builds this modified structure, the larva kills its host, and builds its cocoon attached to the silk threads spun by the host. Cocoon webs appear to be more resistant to rupture [18,20,21] and have fewer compo- nents (e.g., radii and sticky spirals in orb webs) that are utilized for prey interception and retention. Variations of this general pattern include the addition of barrier threads [22], shorter radii, doubled number of lines in each radius, construction of a reinforced frame [19], reinforcement of the retreat by addition of more threads [14] or a veil sheltering the cavity containing spider and larva [23]. These alterations are not a by-product of infection [20,21], but appear to be directly induced by the parasitoid larva. This hypothesis is supported by the fact that removing the larva after the construction of the modified web results in the spider gradually building webs that progressively resembles normal webs [19]. The exact proximate mechanism of spider manipulation is unknown, however recent evi- dence has showed that it might be related to the process of ecdysis. Modified webs of Cyclosa argenteoalba BoÈsenberg & Strand, 1906 [18], Allocyclosa bifurca (McCook, 1887) [19], and Nephila clavipes (Linnaeus, 1767) [20] resemble the molting webs spun by unparasitized indi- viduals just before molting. During the molting period, spiders have increasing levels of the hor- mone 20-OH-ecdysone (20E), which is responsible for renewing their old exoskeletons [24]. This fact resulted in us hypothesizing that the presence of parasitoid wasp larvae would induce the increase of 20E levels in their spider hosts just before pupation. These higher 20E levels would elicit a behavioral response of the parasitized spiders, to build molting webs. In the present study, we evaluated this hypothesis, by testing whether the that 20E levels in parasitized spiders that already present modified web-building behavior are higher than those in unparasitized spiders, and whether they are higher than parasitized spiders performing unmodified web-building behav- ior. We also tested a second hypothesis, that 20E levels would be higher in the third stage of the parasitoid larvae, which is one stage that induces the behavioral modification in the spider host, than in the second stage larvae. In addition, we described the structure of molting webs and com- pared these to normal webs and cocoon webs. We utilized two orb-weaver spiders C. morretes Levi, 1999 and C. fililineata Hingston, 1932 (Araneidae) parasitized by the wasps Polysphincta jan- zeni Gauld, 1991 and P. sp. nr. purcelli (Ichneumonidae), respectively, as models. Materials and methods Study species Cyclosa fililineata is a small spider (total length of 4.7 mm) that is found from Panama to Argentina. C. morretes is slightly larger (total length of 7.2 mm) and has only been reported from Brazil [25,26]. The parasitoid of C. morretes, P. janzeni, has been previously reported from Costa Rica [27] and the Brazilian Atlantic rainforest [21,28]. The parasitoid of C. filili- neata, P. sp. nr. purcelli, is probably a new species, with the closely related Polysphincta purcelli PLOS ONE | DOI:10.1371/journal.pone.0171336 February 3, 2017 2 / 11 Proximate mechanism of behavioral manipulation of orb-weaver spiders Gauld, 1991 being reported from Belize and Costa Rica [27,29]. Web modifications of C. mor- retes parasitized by P. janzeni and C. fililineata parasitized by P. nr. sp. purcelli have been recently described (see [21]). Study area All spiders/larvae were collected during July 2014 and January 2015 from two Atlantic rainfor- est reserves located in the Santa Teresa municipality, in EspõÂrito Santo state, Brazil; specifically, EstacËão BioloÂgica de Santa LuÂcia (19Ê57056@S, 40Ê32024@W) and Reserva BioloÂgica Augusto Ruschi (19Ê54026@S, 40Ê33011@W). Spider and wasp collection We collected 10 and 6 unparasitized