Developmental strategy of the endoparasite Xenos vesparum (strepsiptera, Insecta): Host invasion and elusion of its defense reactions

Developmental Strategy of the EndoparasiteXenos vesparum (Strepsiptera, Insecta): Host Invasionand Elusion of Its Defense Reactions

Fabio Manfredini,1 Fabiola Giusti,1 Laura Beani,2 and Romano Dallai1*

1Dipartimento di Biologia Evolutiva, University of Siena, Siena 53100, Italy2Dipartimento di Biologia Animale e Genetica ‘‘Leo Pardi’’, University of Florence, Florence 50125, Italy

ABSTRACT To successfully complete its endoparasiticdevelopment, the strepsipteran Xenos vesparum needs toelude the defense mechanisms of its host, the waspPolistes dominulus. SEM and TEM observations afterartificial infections allow us to outline the steps of thisintimate host–parasite association. Triungulins, the mo-bile 1st instar larvae of this parasite, are able to ‘‘softly’’overcome structural barriers of the larval wasp (cuticleand epidermis) without any traumatic reaction at theentry site, to reach the hemocoel where they settle. Theparasite molts 48 h later to a 2nd instar larva, whichmoves away from the 1st instar exuvium, molts twicemore without ecdysis (a feature unique to Strepsiptera)and pupates, if male, or develops into a neotenic female.Host encapsulation involves the abandoned 1st larvalexuvium, but not the living parasite. In contrast to theusual process of encapsulation, it occurs only 48 h afterhost invasion or later, and without any melanization. Infurther experiments, first, we verified Xenos vesparum’sability to reinfect an already parasitized wasp larva.Second, 2nd instar larvae implanted in a new host didnot evoke any response by hemocytes. Third, we testedthe efficiency of host defense mechanisms by implantingnylon filaments in control larval wasps, excluding anyeffect due the dynamic behavior of a living parasite;within a few minutes, we observed the beginning of atypical melanotic encapsulation plus an initial melaniza-tion in the wound site. We conclude that the immuneresponse of the wasp is manipulated by the parasite,which is able to delay and redirect encapsulationtowards a pseudo-target, the exuvia of triungulins, andto elude hemocyte attack through an active suppressionof the immune defense and/or a passive avoidance ofencapsulation by peculiar surface chemical properties.J. Morphol. 268:588–601, 2007 � 2007 Wiley-Liss, Inc.

KEY WORDS: endoparasitic development; host defenseelusion; encapsulation; melanization

Host–parasite relationships are important exper-imental models for understanding how the insect‘‘innate’’ immune system (Schmidt et al., 2001) rec-ognizes a variety of invaders (e.g., endoparasiticwasps) and how parasites—more or less closelyrelated to the host—overcome the insect immuneresponse. Hemocytes, the cellular component ofthe insect immune system (Lavine and Strand,2002), play a major role in defense against meta-

zoan parasites. Small pathogens are phagocytizedby hemocytes, whereas parasitoids and other mac-roparasites are encapsulated: hemocytes recognizethe foreign invader as nonself and change fromfreely circulating cells to adhesive cells, forming amultilayered sheath of cells. From an evolutionaryperspective, relatively few studies have focused onthe behavioral ecology of immature parasites: ‘‘ex-ploitative organisms that live in continuous, inti-mate association with their host,’’ forced to adaptthemselves to a changing environment (Brodeurand Boivin, 2004). In this chain of events both cel-lular and humoral components of host immuneresponse might be compromised, and profoundchanges in life-history parameters are likely tooccur.

Xenos vesparum belongs to Strepsiptera(Insecta), a cosmopolitan order of obligate endopar-asites of other insects. Strepsiptera have a directlife cycle; therefore, the first organism infected isthe final host, which may be infected at differentdevelopmental stages by the 1st instar larva of theparasite, the so-called triungulin (the infectivemotile stage). The primary host of X. vesparum(Stylopidae) is Polistes dominulus (Vespidae), aprimitively eusocial paper wasp. Like other mem-bers of the order (except Mengenillidae), X. vespa-rum shows a striking sexual dimorphism. Males,after the 4th larval instar, follow a complete holo-metabolous development: they pupate and emergeas free-living winged adults from the puparium,which extrudes through the host abdominal ter-gites. During their short life-span (4–6 h), theyactively search for a receptive female. Neoteniclarviform females are the result of an involutiveprocess. At the end of the 4th instar, they extrude

Contract grant sponsor: PAR.

*Correspondence to: Prof. Romano Dallai, Department of Evolu-tionary Biology, University of Siena, Via A. Moro 2, Siena I-53100,Italy. E-mail: [email protected]

Published online 16 April 2007 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10540

JOURNAL OF MORPHOLOGY 268:588–601 (2007)

� 2007 WILEY-LISS, INC.

with their anterior region, the cephalothorax,through the host cuticle. They are larviparous per-manent endoparasites, which at maturity fill mostof the wasp hemocoelic cavity, and lack distinctantennae, mouthparts, eyes, wings, legs, andexternal genitalia. The opening of the ventralcanal in the cephalothorax allows both the extra-genital insemination and the escape of the triun-gulins (Beani et al., 2005a).

This characteristic protrusion of the cephalo-theca of the puparium or the sclerotized anteriorportion of cephalothorax is called ‘‘stylopization’’ ofthe host (from the family Stylopidae). Stylopizedwasps, regardless of their putative caste, neitherwork nor reproduce. Instead they desert the colonyearly on to form, first, summer extra-nidal aggre-gations, where parasite mating is likely to occur(Hughes et al., 2004b), then autumn-winter dia-pausing groups, where the fertilized Xenos vespa-rum females overwinter inside their hosts to infectnew colonies the next spring (Beani, 2006). Thesefemales may be used as long-term dispensers oftriungulins to infect wasp larvae in the laboratory.

We have monitored the endoparasitic develop-ment of Xenos vesparum inside the body of Polistesdominulus by means of artificial infections. First,we focus on how the triungulin manages to pene-trate the wasp larva, which is protected by com-pact overlapping cuticle layers, and to evade thewound healing reaction at the entry site (Rowleyand Ratcliffe, 1981). Second, we describe step-by-step the developmental stages of the parasite inthe hemocoel, up to its 4th instar which is wellsynchronized with the pupal-imaginal stages ofthe wasp. Third, we discuss the implications of thecompromised defense reaction of the host: thedelayed encapsulation of a pseudo-target (the exu-via of triungulins) and the absence of melaniza-tion. Through various experimental trials (reinfec-tions, subcuticle applications of 2nd instar larvaeand nylon implants, a nonliving triungulin-sizedstimulus) we have tested the immune response of

P. dominulus larvae and the ability of X. vesparumto elude this response.

Recently, an unusual immune defense has beendescribed in Strepsiptera (Kathirithamby et al.,2003): the parasite Stichotrema dallatorreanummanipulates the tissues of the hosts Segestidaenovaeguineae and S. defoliaria defoliaria (Orthop-tera, Tettigonidae) to build a ‘‘host-derived epider-mal bag,’’ a sort of ‘‘camouflage’’ against the hostdefense reaction. The immune elusion mechanismsof Xenos vesparum could exploit similar compo-nents of the host’s defense repertoire; alternativelythey may represent a different adaptive strategywhich has evolved during an ancient and deepassociation with its host.

MATERIALS AND METHODSStudy Animals

Nests of Polistes dominulus Christ were collected during latespring from various locations in Tuscany (Italy). Each colonywas housed in a 20 3 20 3 20 cm Plexiglas cage (with sugar,water and Sarcophaga sp. larvae ad libitum) for 1 month, toverify that nests were not yet infected by strepsipterans in thefield. In all, nine colonies were artificially infected in laboratorywith 1st instar larvae (triungulins) of Xenos vesparum Rossi.Our sources of triungulins were 13 wasps, collected from twohibernating groups (Impruneta and Volterra), parasitized by asingle female of X. vesparum. These females, extruding theircephalothorax from wasp abdomen, released triungulins after2–3 weeks at 12L/12D and 288C.

Artificial Infections and Dissections

Infections (15 in all) were started at mid-June and extendeduntil the beginning of August. After the temporary removal ofadult wasps from the experimental colony, the nest was placedunder a stereo microscope. Using a needle, two to four triungu-lins were transferred (Fig. 2A–D) from the abdomen of stylop-ized wasps to ‘‘late’’ larval stages of Polistes dominulus (4th,5th and sometimes 3rd instars, Fig. 2A), without removingthem from the nest and marking the cells with a distinct color.Triungulins were generally placed in the anterior portion of thewasp body, dorsally and posterior to the mandibles. After 10min the nest was then tied in its cage, so the wasps would notabandon the colony.

The majority of stylopized adult wasps in the field present asingle extruded parasite (Hughes et al., 2004a, b), although af-ter dissections higher numbers of immature parasites wererecorded (personal observations); moreover the parasite loadper brood in naturally infected nests is higher and variable(Hughes et al., 2003). We tried to simulate a natural infectionpattern, in which several triungulins are likely to reach onelarva. For this reason, and in order to increase the probabilityof successful infections (at least two parasites per host), weplaced from two to five triungulins in each wasp larva. For thepurpose of this study, we selected hosts with a maximum ofthree parasites although the pattern was quite variable (Fig. 1).We omitted the cases of superparasitism (more than three)because competition for nutrients could interfere with normalparasitic development. Although every immature stage of thehost was susceptible (Hughes et al., 2003), we focused on the‘‘late’’ larval stages.

We dissected wasp specimens after 24 h, 30 h, 48 h, 3 days,1 week, 2 weeks, 3 weeks, and 1 month, to monitor Xenos ves-parum endoparasitic development, under a stereo microscope,

Fig. 1. Distribution of 235 living Xenos vesparum parasites(or exuvia in absence of living parasites) in 89 Polistes dominu-lus wasps (late larvae, pupae and adults).

ENDOPARASITIC DEVELOPMENT OF XENOS VESPARUM 589

Journal of Morphology DOI 10.1002/jmor

in 0.1 M phosphate buffer pH 7.2 containing 3% sucrose (PB).All measurements are means 6 SD.

Reinfection Trials

To determine whether Xenos vesparum can parasitize an al-ready infected host, about 2 days after the first infection, a rein-fection of five Polistes dominulus larvae and one pupa was donefollowing the same procedure described above. We chose thistime span to be sure that the wasp defense reaction had alreadybeen activated by the first entry of triungulins at the momentof the second infection.

Polistes dominulus dissections were performed at the follow-ing times: one larva was dissected about 3 days after the firstinfection (24 h from the second infection); four larvae and onepupa were dissected 5 days after the first infection (3 days fromthe second infection).

Implantation of 2nd Instar Larvae

To verify whether Xenos vesparum 2nd instar larvae evokean attack from Polistes dominulus hemocytes when implantedin naive hosts, two preliminary trials were carried out. Weobtained 2nd instar X. vesparum larvae by dissecting specimenswhich had been infected 3 days previously. Using a glass capil-lary, 2nd instar larvae were implanted into naive specimens,previously extracted from their nest cells. We infected two 5thinstar wasp larvae with one parasite, seven with two and twopupae with three. To prevent dehydration, the organisms wereplaced in glass pipes, which were wrapped in aluminum foil,and kept at room temperature. Dissections were done 24 h(three larvae and two pupae) and 3 days (six larvae) after im-plantation.

Nylon Implantation Trials

To test the normal Polistes dominulus defense against for-eign objects, we implanted 1 3 0.25 mm nylon filaments in five‘‘late’’ larvae and two pupae. The alcohol-sterilized filamentswere inserted into the hemocoel of P. dominulus specimens pre-viously extracted from their nest cells, and organisms were pre-vented from dehydration as described above. We dissected thewasps at 24 and 48 h after implantation. Presence or absence ofcapsules and melanization was observed by light stereo micros-copy. These specimens were used as control animals for the effi-ciency of the normal immune system and compared with parasi-tized wasps.

Scanning Electron Microscopy

For SEM observations, samples were fixed in 2.5% glutaral-dehyde in PB for 2 days at 48C, postfixed in 1% osmium tetrox-ide in PB for 2 h at 48C, rinsed in PB several times, and dehy-drated in a graded series of ethanol to 100% ethanol. The mate-rial was processed by critical point drying in a Balzer’s CPD030. Samples were gold-evaporated in a Balzer’s MED 010 sput-terer and observed in a Philips XL20 scanning electron micro-scope operating at 10 kV.

Transmission Electron Microscopy

After dissection, the material was fixed and rinsed as de-scribed above. Ethanol dehydration was followed by a step inpropylene oxide and embedding in Epon-Araldite. Ultrathin sec-tions and serial sections, obtained with a Reichert Ultracut II Eand a LKB Ultratome III, were routinely stained and observedwith a TEM Philips CM 10 at 80 kV. Semithin sections were

stained with 1% toluidine blue and observed with a LeicaDMRB light microscope.

RESULTSThe Parasite’s Perspective

Artificial infections with 1st instar larvae.In Xenos vesparum development, four larval in-stars can be distinguished, based on changes inshape, size and cuticles. Our data are in agreementwith histo-morphological studies carried out inother Strepsiptera: Elenchus tenuicornis (Kathiri-thamby et al., 1984), Stichotrema dallatorreanum(Kathirithamby, 2000) and Pseudoxenos iwatai(Maeta et al., 2001). The 1st instar larva (Fig. 2D)has the general appearance of the triungulin pre-viously described for this order (Kathirithamby,1989), although SEM data show some species-spe-cific features (Pohl, 1998). It is a free-living orga-nism (length about 270 lm), with antennae, eyesand mouthparts (mandibles and labium) on thehead, slender legs terminating in filiform tarsi,serrated sternites, and tarsal expansions on pro-and meso-thoracic legs, used for clinging to thehost or the substrate. Their jumping ability (per-sonal observations) is probably due to abdominalcaudal appendages (setae).

The timing of emergence of 1st instar larvae isquite variable, depending on environmental condi-tions (principally light and temperature). EveryXenos vesparum female of our group was able togive rise to hundreds of triungulins between Mayand July (in line with 3,000–4,000 oocytes in thehemocoel). Triungulins, being extremely motileorganisms, were difficult to follow after theirtransfer onto the larva, because they could movefrom one nest cell to another. At dissection, wesometimes discovered a higher number of parasitesthan we had placed onto the larva.

Generally, laboratory infections were successfuland stylopized wasps emerged from each artifi-cially infected nest. Figure 1 shows the trend ofparasite distribution. We dissected a total of 89infected Polistes dominulus specimens: 69 larvae,9 pupae, and 12 adults. We counted 235 Xenos ves-parum within the wasps: 181 living parasites withtheir 1st instar exuvia; 46 living parasites withouttheir exuvia; 8 exuvia in absence of visible livingparasites. Only rarely did we find a dead triungu-lin on the wasp larva cuticle or outside in its nestcell (6 cases).

Entry into the host. When the triungulinsmade contact with the cuticle of the wasp larva,they moved randomly for a while. Then theystopped and with their heads repeatedly hit theexternal coating of the wasp at an oblique angle.There was no evidence of a specific entry target: inthe head, not far from the mandibles (Fig. 2A,B),but also in the middle of the body or at its poste-rior extremity. Usually they avoided the sclerotized

590 F. MANFREDINI ET AL.


buccal apparatus of our experimental ‘‘late’’ larvae;in a few cases, we observed wasp larvae chewingtriungulins.

First instar larvae needed about 6–24 h to crossboth exocuticle and endocuticle (Fig. 2C). This pic-ture was taken 6 h after infection, when the triun-gulin has not fully entered: its anterior portion isalready under the wasp cuticle, opaque and withmany microtubercles; its posterior region is stilloutside and appears bright and smooth (Fig. 2D).Triungulins created a small opening in the cuticle(Fig. 2E), which we were unable to distinguishunder microscope after a few minutes, and crossedexocuticle and then endocuticle (Fig. 3). Weobserved a mechanical detachment in the outerlayers of the host cuticle and a not-yet-defined sub-stance, probably secreted by the anterior region oftriungulins (Fig. 4A,B). Noticeably, the entry holein the larval coating did not melanize.

Once triungulins reached the epidermis (Fig. 4C),they began to sink into the hemocoel by passingthrough the epidermic layer (Fig. 5A), which com-pletely surrounded them (Fig. 5B,C), until theywere finally released in the wasp hemocoel. Atransient and incomplete host epidermal infolding

surrounds the parasite at this stage. We did notfind any preferred region of the wasp hemocoelwhere triungulins settled, e.g., near the epidermisas well as deeper inside, in the fat tissues or closeto nervous ganglia. Either head or dorsal/ventralbody regions were susceptible.

Fig. 2. A–C: Infection of the host. A: Polistes dominulus 4th instar larva in the nest, with a Xenos vesparum triungulin (tr)near the wasp mandibles, on the left. B: Triungulin (tr) making contact with the cuticle (ct) of the wasp larva. C: Triungulin 6 h af-ter the beginning of the infection. Note that it has not yet fully entered: the anterior portion is already under the wasp cuticle(arrowhead) while the posterior region is still outside. D, E: The triungulin. SEM. D: Dorsal view. E: Triungulin partially pene-trated into the host with its anterior region. Note the small opening (op) at the level of the wasp’s cuticle.

Fig. 3. Semithin sagittal section through the body of a triungulinwhich has not fully entered into the wasp larva. Note themechanicaldetachment of both the exocuticle (ex) and the endocuticle (en).



First molt 48 h post-infection. Both 24- and30-h postinfection dissections revealed intact tri-ungulins inside the body of the host. Some werestill moving in the hemocoel, as could be seen withthe stereoscope through the thin cuticle of the

wasp larva. Neither 1st instar exuvia nor 2ndinstar larvae of the parasite was present: moltinghad not occurred yet. By 2 days after infection,young 2nd instar larvae were easily detectable

Fig. 4. Semithin serial cross sections through the anterior por-tion of a Xenos vesparum 1st instar larva infecting a new host:early steps. A, B: Triungulin probably secreting a substance(arrowhead) while making contact with the wasp cuticle (ct). C:Triungulin reaching the epidermis (ep) of the wasp larva.

Fig. 5. Semithin serial cross sections through the anteriorportion of a Xenos vesparum 1st instar larva infecting a newhost: later events. A: The triungulin has crossed the cuticle (ct)and it starts breaking the epidermic layer (ep). B: Formation ofan incomplete host epidermal infolding (ep) around the parasitewhile it is sinking into the wasp hemocoel (wh). C: Triungulinfully surrounded by the epidermal infolding.



near the exuvia (Fig. 6A,D). In a few cases, wewere able to observe the motile larva abandoningthe 1st instar exuvium through a dorsal opening,just behind the head (Figs. 6E, 7A,B).

Second instar larvae (Fig. 8A) lack body appen-dages (they keep only short protrusions of thethree pairs of legs) and are unpigmented, exceptfor two black spots on the dorsal side of the head(the eyes) that facilitate their identification. Thisdevelopmental stage (183 6 8 lm in length) ischaracterized by a soft thin cuticle (0.6 6 0.4 lm).Many minute folds of the epicuticle (Fig. 8B)increase its surface. In a space of 7.5 lm, we havecounted �30-folds (max length: 0.75 lm) of differ-ent shape and size.

We usually found a correspondence between thenumber of endoparasitic larvae and the number oftriungulin exuvia inside the wasp body, althoughsometimes there were more exuvia, probablybecause we were unable to localize the small livingparasites when they were encompassed by the hostfatty tissues. Exuvia were often free in the hemocoelat 48 h post-infection, but some were already encap-sulated. Generally, we have not seen capsulesaround the larvae nor signs of melanization.

Third instar larva and later developmentalstages. Third instar larvae appeared about 3 daysafter infection. At this stage the parasite appeared

very similar to 2nd instar larvae (Fig. 8C), but thesecond molt was revealed by the presence of a dou-ble cuticle layer (Fig. 8D), easily recognizablearound the posterior portion of early 3rd instarlarva. The outer layer is due to a unique moltingprocess, labeled as ‘‘apolysis without ecdysis’’ byKathirirthamby et al. (1984), who first described itin Elenchus tenuicornis (Strepsiptera, Elenchidae).

Fig. 6. First molt of Xenos vesparum. A–D: Young 2nd instar larvae (arrowheads) are visible near the exuvia (ev) of the triungu-lins. Note the black eyes of the molted parasites. A: 2nd instar larva and triungulin exuvium near the head (he) of the larval wasp.B: Two molted parasites settled in the fat tissues of the host. C: Polistes dominulus 3rd instar larva with a X. vesparum specimeninside its posterior region. D: Detail. E: Ventral view of P. dominulus 4th instar larva with a X. vesparum molting triungulin(arrowhead) under the cuticle.

Fig. 7. A,B: Molting Xenos vesparum 1st instar larva. Theparasite is abandoning the triungulin’s exuvium through a dor-sal opening (arrowhead), just behind the head. A: The shortprotrusions (arrows) of the three pairs of legs are visible in theyoung 2nd instar larva. SEM. B: Light microscope view of thesame stage.



The parasite makes a new cuticle but it does notabandon the previous exuvium, which becomes theouter layer (Fig. 8E,F). As a consequence of thisprocess, the parasite is progressively surroundedby a growing number of cuticle layers, but the onecorresponding to the 2nd larval instar will alwaysremain outside as well as in the neotenic female.

During their continued development, 3rd instarlarvae were characterized by visible body growth(early 3rd instar larvae 324 6 41 lm in length),the loss of all appendages, and the depigmentationof the two dark spots, now light red. In addition,the intestinal tract became more and more evidentand gonads started to differentiate. As describedearlier by Maeta et al. (2001) in Pseudoxenos iwa-tai, when transferred into buffer late 3rd instarlarvae of Xenos vesparum settled with the ventralpart of the body upward; they twisted their bodiesup and downwards, until they closed like a ring.In P. iwatai parasite larvae migrated to the finalposition of future extrusion from the host eumenidwasp at just this stage.

The early 4th instar larva appeared when thewasp was in the pupal stage or in adulthood, 1–2weeks after wasp emergence. At this stage, the twosexes were clearly recognizable. Females kept a lar-viform body (7.5 6 1.5 mm in length). Male pupae,which were smaller (5.25 6 0.75 mm), usuallyextruded a few days before female cephalothoraxes.

The Host ResponseEncapsulation. The total number of capsules

observed in wasps’ hemocoel was 113, but encapsu-lation, which is common in parasite–insect interac-tions, presented some unusual features in ourstudy.

First, capsules were formed only around emptyexuvia (Fig. 9A): neither the living triungulins,nor the developing endoparasitic instars were tar-gets of encapsulation. Second, the temporal dy-namic of the process was peculiar, because encap-sulation of exuvia was delayed. Wasp hemocytesbegan to aggregate around the triungulin exuvium

Fig. 8. Endoparasitic larval stages. A, B: 2nd instar larva. A: Whole parasite. Note the black eyes (arrowheads). B: Detail of theepicuticle with many minute folds (arrows). TEM. C–F: 3rd instar larva. C: Just molted parasite. This stage is very similar to theprevious one except for the presence of a double cuticle layer (arrows). D: Semithin cross section: the double cuticle layer (arrows)and the differentiating gonads (g) are visible. E, F: Two overlapping cuticles (2nd ct and 3rd ct) soon after (E) and few days (F)from molting. TEM.



48 h after infection, without any specific adhesionsite: they attacked the head as well as the back ofthe exuvia (Fig. 9B,D). At the beginning, the pro-cess involved medium- and large-sized (8–15 lmdiameter) hemocytes, round to ovoid cells with a

central nucleus accounting for �40–60% of the cellvolume (Fig. 9E). Capsules were not tightly packedat this stage. During the first week we observedthe growing recruitment of large hemocytes (10–20lm). These irregularly shaped cells (Fig. 9F–G)

Fig. 9. The host defense reaction. A: Semithin section of a capsule forming around the empty exuvium (arrowhead) of a triungu-lin, between the epidermis (ep) and two nerves (nv) of a Polistes dominulus larva. B–D: Polistes dominulus hemocytes aggregatingaround the triungulin exuvium (ev). SEM. C, D: Detail showing the hemocytes pseudopodia contacting the exuvium. E–G: Firststeps in the formation of a capsule: round hemocytes approach the target (E), they change in shape and protrude pseudopodia(arrowheads) while aggregating around the target (F) and finally form the capsule (G). TEM. H, I: Mature capsules (8–10 dayspostinfection). H: A round milky capsule (arrowhead) visible through the cuticle of a P. dominulus 4th instar larva. I: Sectionthrough a mature compact capsule: note the extremely flattened hemocytes. TEM.



were characterized by a few to many variableextensions (pseudopodia) and a central nucleusoccupying �40% of the cell volume.

About 8–10 days postinfection, 700-lm diametermature capsules were visible, with a round shapeand milky color (Fig. 9H). At this stage, hemocyteswere flattened and capsules more compact (Fig. 9I).We found this kind of capsule deep inside the bodyof the wasp larva, embedded in the fatty tissue.More superficially, partial capsules and not yetencapsulated exuvia occurred. We never observedcapsules in adult wasps, but sporadically free exu-via close to living parasites.

Melanization. After infection of Polistes domi-nulus larvae with Xenos vesparum specimens, themelanization response, a common feature of hu-moral insect immunity (Lavine and Strand, 2002),was absent at both the entering point and aroundthe encapsulated exuvia (in 64 cases), while in 3cases we recorded an apparent beginning of mela-nization. In 2 further cases we noticed the depositof a melanin-like substance on the host cuticle atthe entry of the triungulin, but this could be dueto the contact with the forceps, as Hughes andKathirithamby mentioned (2005) when using asimilar technique.

Reinfection trials. A second infection, 55 h af-ter the first one, confirmed the pattern describedabove. Dissections at either 1 or 3 days post-rein-fection, revealed that triungulins were able toenter an already parasitized host, molt and giverise to the usual developmental stages. As in thefirst infections, capsules were found only aroundtriungulins’ exuvia and there was no evidence ofmelanization.

Implantation with 2nd instar larvae. Subcu-ticle applications of Xenos vesparum 2nd instarlarvae (3 days) in naive hosts did not elicit anydefense reactions during the first 24 h. The para-sites (5 specimens) were still alive after dissectionof the wasps and not encapsulated; no signs of

melanization were visible. Three days after im-plantation, we recorded a high mortality of thePolistes dominulus larvae removed from theirnests.

Nylon implantation trials. The filament-inser-tion trials gave variable host-response patterns,regardless of the wasp developmental stage.Twenty-four hours after implantation of nylon fila-ments in the host hemocoel, full capsules wereobserved in five of seven trials; in one case theencapsulation was incomplete and in another thefilament did not elicit any reaction.

We also recorded melanization both at the site ofimplantation (Fig. 10A) and around the capsule(Fig. 10B) in five of seven trials.

Failure of parasite development. We foundthree exceptions to the general rules of immuneresponse above outlined. SEM observations showedtwo young larvae partially encapsulated close tothe triungulin exuvium (Fig. 11A,B). TEM obser-vations revealed another case of abnormal molting,i.e., a young specimen encapsulated in the sameway and partially melanized (Fig. 11C): this orga-nism showed two additional coating layers (exuvia)overlapping the larval cuticle. Wasp hemocytes werevisible close to the outer exuvium (Fig. 11C, the topof the micrograph). Noticeably, a secretion of unknownorigin was found between the two outer layers.Due to its similarity to the midgut contents of thesame specimen (Fig. 11D), we hypothesize it couldbe an outpouring of such contents.

DISCUSSION

To better understand the life-history of the sys-tem Xenos vesparum-Polistes dominulus, we havelooked at it from two angles: the way in whichX. vesparum 1st instar larvae infect a new host(the parasite’s perspective) and the consequentreaction of the wasp (the host’s perspective). TheP. dominulus larva, the target of infection, is a

Fig. 10. Nylon implants in Polistes dominulus 4th instar larvae. A: The implant site of thenylon filament is melanized (arrowhead). B: Three capsules around nylon filaments, 24 h afterimplantation in three different specimens: capsules were not melanized (a), partially (b) ortotally melanized (c).



changing environment and goes through greatphysiological and morphological reorganization (i.e.,pupation). The endoparasitic development ofX. vesparum has to be finely tuned to the develop-ment of its holometabolous host, similarly to koino-biont parasitoids (Vinson and Iwantsch, 1980). Itis therefore not surprising that profound altera-tions occur in the endocrine equilibrium of the hostenvironment: castration, the most relevant conse-quence of stylopization, is well known (Strambiand Strambi, 1973), and further studies on moresubtle physical changes, i.e., epicuticular hydrocar-bons and fat bodies, are in progress (Beani et al.,2005b; Beani, 2006). In our system, we suspectthat the physiological damage to the host is lessdramatic than in organisms affected by parasitoids(Pennacchio and Strand, 2006). Xenos vesparum—an obligate endoparasite, permanent if female—would gain no advantage in killing the host to sat-isfy its nutritional needs. On the contrary, itsfitness comes from the exploitation of a livingwasp, first as a substrate for its growth, then tofacilitate its mating inside abnormal aggregationsof stylopized wasps (Hughes et al., 2004b), and

later as a vector for releasing infective 1st instarlarvae. Consequently, one would expect the effectsof the parasite on the defensive responses of thehost to be limited.

From an evolutionary perspective, the refinedand reciprocal adaptation of both Xenos vesparumand Polistes dominulus to this forced long-termcoexistence (1 year, if a wasp infected by a Xenosfemale overwinters and survives to the nextsummer), suggests an ancient host–parasite inter-action. Moreover, the large variety of organismsparasitized by Strepsiptera (Kathirithamby, 1989)highlights the plasticity of this group, which hadto evolve many different modes of infection. Thisprovides further support for the antiquity of thisinsect order, in agreement with recent findings ofspecimens in Cretaceous amber (Grimaldi et al.,2005).

The Parasite’s Perspective: A ‘‘Soft’’ EntryInto the Host

According to the studies on the interactionsbetween immature parasitoids and their hosts

Fig. 11. Unsuccessful parasite development. A, B: Both micrographs show a young Xenos vesparum larva (arrowheads), prob-ably a 2nd instar, close to the triungulin exuvium (ev) and partially encapsulated. Part of the capsule (cp) was removed. SEM. C:Detail of the two additional coating layers (a, b) overlapping the larval cuticle (ct) from a young X. vesparum specimen encapsu-lated and partially melanized. Wasp hemocytes (hm) are visible close to the outer exuvium, at the top of the micrograph. Note thepresence of a secretion (sc) of unknown origin between the two outer layers. D: Cross section of the midgut (mg) of the same X. ves-parum larva showing the midgut lumen filled with a secretion (sc) similar to that mentioned above.



(Brodeur and Boivin, 2004), the host-seeking stageof Xenos vesparum, the free-living mobile triungu-lin, undergoes a chain of ‘‘hierarchical steps’’ toachieve successful parasitism: it must locate, eval-uate and penetrate the host, evade or overcomethe host immune response and adapt to (or regu-late) the constantly changing host environment.

Polistes dominulus larvae are typically aggregated,so in this case host selection may be a relevant step,whereas parasitoids are unlikely to have a choice ofseveral hosts (Feener and Brown, 1997). Once a triun-gulin has reached a wasp nest, either on a foragingwasp (phoretic transport) or by means of a stylopizedwasp which directly visits a nest (Hughes et al.,2003), a wide range of potential new hosts is avail-able. The mechanism of host discrimination cannot beinvestigated by artificial infections, although a cer-tain motility of the triungulins in the nest, even whenthey were put directly on a wasp larva, suggests anonrandom choice, possibly influenced by the devel-opmental stage, the nutritional state or even the sexof the larval wasp. We were able to successfully infecthosts of different developmental stages, naive or al-ready parasitized, and even some wasp males, whichare less parasitized than females in nature (Hugheset al., 2004a,b). The process of successful infectionrelies on a perfect tuning between the life cycles ofhost and parasite, and in the field host discriminationmay be constrained by many factors: first of all, bythe presence of adult wasps which actively remove tri-ungulins moving on the larvae. In the laboratory,according to Schmid-Hempel and Ebert (2003), the‘‘behavioral defenses,’’ a first line of the defense cas-cade, are ‘‘largely eliminated.’’

The second step, the evasion of the host immuneresponse, takes place as soon as the triungulinbegins to penetrate the coating layers of the wasplarva, the cuticle and the epidermis. We expectedthat this process would evoke a reaction of melani-zation in the entry point, as a consequence of coagu-lum formation during wound-healing. In Galleriamellonella (Lepidoptera, Pyralidae) melanization inthe wound-site was recorded 6–12 h after wounding(Rowley and Ratcliffe, 1981); it occurred after 24 hin our nylon implants. Contrary to expectations, theentry point of Xenos vesparum does not melanizeeven several days after infection; we infer that theparasite does not produce any wound.

The process of entry into the host has alreadybeen described in other Strepsiptera by Maeta et al.(2001) and Kathirithamby (1989). According to theformer, the triungulins of Pseudoxenos iwatai(Strepsiptera, Stylopidae) ‘‘gnawed the chorion withtheir mandibles’’ while invading the eggs of a eume-nid wasp. Kathirithamby instead, quoting Riek (per-sonal communications), describes a considerableamount of liquid secreted by Pseudoxenos sp., andsuggests an enzymatic softening of the cuticle of thehost Sceliphron laetrum (Sphecidae, Hymenoptera).In turn, what we have observed for the Xenos vespa-

rum penetration of the Polistes dominulus larva is amechanical detachment of the outer layers in thehost cuticle and a thin layer of a substance (its func-tion is unknown) between the larva and the wasp’scuticle, which could be a product of the triungulin’ssalivary glands or the contents of its foregut.

Since the parasite does not produce a wound,there is no ‘‘injury factor’’ (Gupta, 1985) to triggerthe coagulation and melanization of the hemo-lymph, and no hemocytes migrate to the entrysite. To avoid the rupture of the wasp epidermallayer, which would represent the ‘‘injury factor’’mentioned above, the triungulin initially pene-trates the host epidermis without breaking it,inducing the formation of a transient and incom-plete epidermal infolding. This process, observedin transverse serial semithin sections at themoment of entry into the host cuticle (Figs. 4A–C,5A–C), is similar to the recently described abilityof another strepsipteran to wrap itself in ‘‘a host-derived epidermal bag’’ (Kathirithamby et al.,2003): the epidermal cells of the hosts Segestideanovaeguineae and S. defoliaria defoliaria (Orthop-tera, Tettigonidae), when detached from the endo-cuticle, lengthen and surround the 1st instar larvaof Stichotrema dallatorreanum (Strepsiptera, Myr-mecolacidae). This bag protects the parasite, ‘‘mas-queraded as self,’’ from the attack of the hemocytesduring its entire endoparasitic development and,at the same time, allows the passage of nutrientsfrom host hemolymph. This mechanism couldexplain another peculiar feature of Strepsiptera,‘‘ecdysless molting’’: the parasite, while growing,cannot abandon the exuvia, because it is ‘‘closelyfitted in its bag’’ (Kathirithamby et al., 2003).

In the case of Xenos vesparum, this process stopsearlier and leads to an incomplete and transient‘‘bag,’’ or better, an epidermal infolding. This struc-ture doesn’t surround the parasite during itswhole endoparasitic development, but only at itsnontraumatic entry into the host, when it allowsthe triungulin to cross the cuticle and to reach thehemocoel without breaking the epithelial cell layerand evoking the defense reaction of the host(Schmid-Hempel, 2005). In the absence of any per-manent and constraining bag wrapping the para-site, ‘‘apolysis without ecdysis’’ (Kathirithambyet al., 1984) has to be differently explained.

The Host’s Response: Encapsulationof a Pseudo-Target, the Exuviumof the Triungulin

Hosts and parasites coexist in a ‘‘labile equilib-rium’’ which is the result of evolutionary optimiza-tion mechanisms. This equilibrium balances itselfbetween two extremes: ineffective parasites, unableto produce enough offspring to survive, and too-effective parasites, which reduce the survivalchance of the host, thus compromising their own



existence (Gotz and Boman, 1985). In this context,resistance is all-or-nothing, because if the hostfails to actuate an effective immune response, itwill die (Rolff and Siva-Jothy, 2003).

In the case of metazoan parasites, eggs andimmature parasitoids, or xenobiotics (e.g., nylonfilaments), the typical defense reaction in insectsis a melanotic encapsulation which takes place intwo distinct phases. First, hemocytes aggregatearound the target, then lyse and release substan-ces into the hemolymph to attract other hemo-cytes, until a tight capsule forms. This capsulethen melanizes and kills the parasite. The processstarts within a few minutes after invasion or im-plantation (only 1 min is required for the attach-ment of hemocytes to foreign objects) and within2–24 h the typical three-layered cellular envelopehas formed around the parasite (Gotz and Boman,1985). The immune response of Polistes dominulusafter the invasion of Xenos vesparum differs fromthis general plan: first, the encapsulation isdelayed (incomplete capsules were seen only 48 hafter infection); second, the capsule surrounds apseudo-target, the triungulin’s exuvium, while thereal target—the living parasite—is not affected.Control experiments of nylon implants (a wide-spread technique, due to its ‘‘spectacular’’ effects,Siva-Jothy et al., 2005) have shown that theimmune system of P. dominulus immatures is ableto encapsulate and melanize foreign objects after24 h. ‘‘The use of nonliving immune stimuli allowsfocusing on the unfolding of the immune responseas such, excluding effects due to the dynamicbehavior of a real parasite’’; moreover, this ap-proach is useful to understand ‘‘the complex tem-poral dynamics’’ of host–parasite interaction, ‘‘aparticularly relevant but often neglected aspect’’(Korner and Schmid-Hempel, 2004).

As far as timing, the first 48 h are critical: dur-ing this time, triungulins generally undergo 1stmolting without any interference. After that,encapsulation does take place but it involves apseudo-target, hence it is ineffective; the livingparasite is not recognized as nonself, therefore it isnot attacked by hemocytes during its subsequentendoparasitic development. Moreover, the proteo-lytic cascade that leads to the production and thedeposit of melanin must be locally compromised,because we didn’t observe any sign of melanizationeither around the living parasite or within thecapsule.

We hypothesize that Xenos vesparum may adopta complex strategy of immunity elusion, involvingboth active suppression and passive avoidance ofthe host defense reaction. The former is critical atthe beginning of infection: the parasite is able todelay and transiently inhibit encapsulation, and tolocally suppress melanization. The specificity ofthis host–parasite interaction allows the wasp todefend itself from other injuries during its larval

development and through adulthood: xenobiotics,but also bacteria, fungi, viruses and other meta-zoan parasites. In a similar way, the hemocytes ofthe host Ephestia kuhniella (Lepidoptera, Pyrali-dae) may be transiently altered in proximity to theeggs of Venturia canescens (Hymenoptera, Ichneu-monidae) but otherwise they activate the immunedefense during the development of this parasitoid(Kinuthia et al., 1999; for a specific active suppres-sion by hymenopteran parasitoids, see also Schmidtet al., 2001; Pennacchio et al., 2003; Giron andStrand, 2004).

In regard to passive avoidance, there may besurface chemical properties that allow Xenos ves-parum to safely inhabit the Polistes dominulushemocoel from 2nd instar to the extrusion of thefemale cephalothorax or the male puparium.Mechanisms of passive elusion of insect immunityare well-documented in several host–parasitoidsystems: from the development of parasitoids inlocations protected from circulating hemocytes andencapsulation (like the nerve ganglion), to ‘‘surfacefeatures that prevent the host from recognizingthe parasitoid as nonself ’’ (Schmidt et al., 2001).In our case, laboratory dissections did not revealany fixed site where X. vesparum settles. Conse-quently, the elusion of cellular encapsulationmight depend on peculiar features present on theouter surface of the parasite cuticle. Suitableexamples of these mechanisms are the proteinsthat coat the eggs of parasitoids in the genus Cote-sia (Hymenoptera, Braconidae) (Asgari et al.,1998) or the ‘‘fibrous layer’’ protecting the eggs ofthe parasite Macrocentrus cingulum (Hymenop-tera, Braconidae) when deposited into the larvalhost Ostrinia furnacalis (Lepidoptera, Piralidae;Hu et al., 2003).

From this perspective, the ‘‘ecdysless molting’’ inStrepsiptera could be read as a ‘‘preadaptation foran endoparasitic lifestyle’’ (Kinuthia et al., 1999).The cuticle of the Xenos vesparum 2nd instar larvadoes not elicit the immune reaction of the hostand, without ecdysis, this outer layer remains theunique host–parasite interface during the wholeendoparasitic development. Moreover, the multi-layered envelope of the parasite (three or more,depending on sex) makes it unlikely that immuno-suppressive substances are released through thisthick cuticle; it is more likely that the parasite’spermanent cuticle adsorbs self-substances from thewasp hemolymph. In our experiments, specimensthat have already undergone 1st molting are ableto avoid the host defense reaction if implanted in anaive larval wasp, suggesting that they could havean innate camouflage mechanism, a cuticularchemical insignificance. Preliminary studies on theepicuticular hydrocarbon profiles of X. vesparum(Beani et al., 2005b) agree with a chemical mim-icry hypothesis: the chemical profiles of cephalo-thorax and cephalotheca were more similar to host



hydrocarbons than the profiles of the 1st instarlarvae and the adult males of X. vesparum, theonly free living stages of this parasite.

Failure of Parasite Development

The three encapsulated Xenos vesparum speci-mens (see Results) can be seen as an imbalance ofthe ‘‘labile equilibrium’’ mentioned above: in thesecases, the first molting is anomalous and the para-site’s 2nd instar larva fails to move away quicklyenough from the triungulin’s exuvium, and is thusentrapped in the forming capsule. Dissections ofnaturally infected larvae of Polistes dominulus(Hughes et al., 2003) have shown a partial encap-sulation of ‘‘molting 1st instars’’ in few specimens(2 of 18), and the authors were doubtful as towhether it was an example of successful defense;certainly, in the case of fully encapsulated 2ndinstar larvae, the immune response of the host iseffective since, within a capsule, an organismalmost always dies (Schmidt et al., 2001), becauseit cannot take up nutrients and oxygen.

In these samples of unsuccessful parasitism,TEM observation of one specimen (probably a 2ndinstar, being next to the triungulin’s exuvium)revealed two additional exuvia overlapping thelarval cuticle. We hypothesize that the additionalcuticle layers are the result of two unexpected‘‘ecdysless’’ moults, actuated by the parasite inorder to strengthen the defense barrier againsthost hemocytes: an ultimate attempt to confrontthe encapsulation process. It becomes clear thattoo precise expectations and comparisons withother parasites are unfeasible, because ‘‘strepsip-terans are the only parasitic insects (includingparasitoids) to sequentially parasitize disparatestages of the same holometabolous host’’ (Hughesand Kathirithamby, 2005) and so large a spectrumof hosts: it follows that the strategies adopted bythese organisms have to be flexible and often lieoutside the schemes of the other host–parasite sys-tems.

ACKNOWLEDGMENTS

We are grateful to Drs. David Mercati and Euge-nio Paccagnini and to Prof. Pietro Lupetti for theirhelp in the preparation of the experiments.

LITERATURE CITED

Asgari S, Theopold U, Wellby C, Schmidt O. 1998. A proteinwith protective properties against the cellular defense reac-tions in insects. Proc Natl Acad Sci USA 95:3690–3695.

Beani L. 2006. Crazy wasps: When parasites manipulate thePolistes phenotype. Ann Zool Fennici 43:564–574.

Beani L, Giusti F, Mercati D, Lupetti P, Paccagnini E, TurillazziS, Dallai R. 2005a. Mating of Xenos vesparum (Rossi)(Insecta: Strepsiptera) revisited. J Morphol 265:291–303.

Beani L, Theodora P, Dallai R, Turillazzi S. 2005b. Epicuticularhydrocarbons of Xenos vesparum (Strepsiptera Stylopidae),parasite of Polistes dominulus (Hymenoptera Vespidae): Pre-liminary data. Redia 87:167–169.

Brodeur J, Boivin G. 2004. Functional ecology of immature par-asitoids. Annu Rev Entomol 49:27–49.

Feener DH, Brown BV. 1997. Diptera as parasitoids. Annu RevEntomol 42:73–97.

Giron D, Strand MR. 2004. Host resistance and the evolution ofkin recognition in polyembryonic wasps. Proc Biol Sci 6:S395–S398.

Gotz P, Boman HG. 1985. Insect immunity. In: Kerkut GA, Gil-bert LI, editors. Comprehensive Insect Physiology, Biochemis-try and Pharmacology. Oxford: Pergamon. pp 453–485.

Grimaldi D, Kathirithamby J, Schawaroch V. 2005. Strepsip-tera and triungula in Cretaceous amber. Insect Syst Evol 36:1–20.

Gupta AP. 1985. Cellular elements in the hemolymph. In: Ker-kut GA, Gilbert LI, editors. Comprehensive Insect Physiology,Biochemistry and Pharmacology. Oxford: Pergamon. pp 400–451.

Hu J, Zhu XX, Fu WJ. 2003. Passive evasion of encapsulationin Macrocentrus cingulum Brischke (Hymenoptera: Braconi-dae), a polyembryonic parasitoid of Ostrinia furnacalisGuenee (Lepidoptera: Pyralidae). J Insect Physiol 49:367–375.

Hughes DP, Kathirithamby J. 2005. Cost of strepsipteran mac-roparasitism for immature wasps: Does sociality modulatevirulence? Oikos 110:428–434.

Hughes DP, Beani L, Turillazzi S, Kathirithamby J. 2003. Prev-alence of the parasite Strepsiptera in Polistes as detected bydissection of immatures. Insect Soc 50:62–69.

Hughes DP, Kathirithamby J, Beani L. 2004a. Prevalence ofthe parasite Strepsiptera in adult Polistes wasps: Fieldcollection and literature overview. Ethol Ecol Evol 16:363–375.

Hughes DP, Kathirithamby J, Turillazzi S, Beani L. 2004b.Social wasps desert the colony and aggregate outside ifparasitized: Parasite manipulation? Behav Ecol 15:1037–1043.

Kathirithamby J. 1989. Review of the order Strepsiptera. SystEntomol 14:41–92.

Kathirithamby J. 2000. Morphology of the female Myrmecolaci-dae (Strepsiptera) including the apron and an associatedstructure. Zool J Linn Soc 128:269–287.

Kathirithamby J, Spencer Smith D, Lomas MB, Luke BM.1984. Apolysis without ecdysis in larval development of astrepsipteran. Elenchus tenuicornis (Kirby). Zool J Linn Soc82:335–343.

Kathirithamby J, Ross LD, Johnston JS. 2003. Masqueradingas self? Endoparasitic Strepsiptera (Insecta) enclose them-selves in host-derived epidermal bag. Proc Natl Acad Sci USA100:7655–7659.

Kinuthia W, Li D, Schmidt O, Theopold U. 1999. Is the surfaceof endoparasitic wasp eggs and larvae covered by a limitedcoagulation reaction? J Insect Physiol 45:501–506.

Korner P, Schmid-Hempel P. 2004. In vivo dynamics of animmune response in the bumble bee Bombus terrestris.J Invertebr Pathol 87:59–66.

Lavine MD, Strand MR. 2002. Insect hemocytes and their rolein immunity. Insect Biochem Mol Biol 32:1295–1309.

Maeta Y, Goukon K, Kitamura K, Miyanaga R. 2001. Factorsthat determine the positions where Pseudoxenos iwatai Esaki(Strepsiptera: Stylopidae) extrudes from the host abdomen.Symposium papers on Strepsiptera presented at the XXIInternational Congress of Entomology, Foz do Iguacu, August20–26, 2001.

Pennacchio F, Strand R. 2006. Evolution of developmental strat-egies in parasitic Hymenoptera. Annu Rev Entomol 51:233–258.

Pennacchio F, Tranfaglia A, Malva C. 2003. Host-parasitoid an-tagonism in insects: New opportunities for pest control? AgroFood Industry Hi Tech 14:53–56.



Pohl H. 1998. Die Primarlarven der Facherflugler—Evolutio-nare Trends (Insecta, Strepsiptera), PhD Dissertation, I–V,Technische Universitat Darmstadt, pp 1–252.

Rolff J, Siva-Jothy MT. 2003. Invertebrate ecological immunol-ogy. Science 301:472–475.

Rowley AF, Ratcliffe NA. 1981. Insects. In: Ratcliffe NA, RowleyAF, editors. Invertebrate Blood Cells, Vol 2. London: Aca-demic Press. pp 421–488.

Schmid-Hempel P. 2005. Evolutionary ecology of insect immunedefenses. Annu Rev Entomol 50:529–551.

Schmid-Hempel P, Ebert D. 2003. On the evolutionary ecologyof specific immune defense. Trends Ecol Evol 18:27–32.

Schmidt O, Theopold U, Strand M. 2001. Innate immunity andits evasion and suppression by hymenopteran endoparasi-toids. BioEssays 23:344–351.

Siva-Jothy M, Moret Y, Rolff J. 2005. Insect immunity: An evo-lutionary ecology perspective. Adv Insect Physiol 32:1–48.

Strambi C, Strambi A. 1973. Influence du developpement duparasite Xenos vesparum Rossi (Insecta, Strepsiptere) sur lesysteme neuroendocrinien des femelles de Polistes (Hymenop-tere, Vespide) au debut de leur vie imaginale. Arch AnatMicrosc Morphol Exp 62:39–54.

Vinson SB, Iwantsch GF. 1980. Host regulation by insect para-sitoids. Q Rev Biol 55:143–165.



Documents

Developmental strategy of the endoparasite Xenos vesparum (strepsiptera, Insecta): Host invasion and elusion of its defense reactions