Further records of two spider-parasitoids of the genus Polysphincta ( Hymenoptera , Ichneumonidae , Ephialtini ) from Central Europe , with notes on their host interactions

A polysphinctine wasp Polysphincta longa, associated with the orb web building spider Araneus angulatus, was recorded for the first time in Slovakia. Its congener Polysphincta tuberosa was recorded in association with a new spider host Araneus sturmi. New records of Polysphincta species from the Czech and Slovak Republics are presented. The final stage larvae of both, P. longa and P. tuberosa, induce a specific alteration in web architecture: the spider constructed a unique 3D tangle of silk – so called “cocoon web” – to protect the parasitoid during the pupal stage. The host range of wasps belonging to the genus Polysphincta in Europe is discussed.

Polysphincta is a moderately large genus within the Polysphincta group of genera (Ephialtini, Ichneumonidae, Hymenopte ra), which are koinobiont parasitoids (a parasitoid, whose host continues to feed and grow after parasitization) exclusively associated with spider hosts.The genus is presently represen ted by five valid species in Europe: Polysphincta boops Tschek, 1869, P. longa Kasparyan, 1976, P. rufipes Gravenhorst 1829, P. tuberosa Gravenhorst, 1829 and P. vexator Fitton, Shaw & Gauld, 1988(Yu et al. 2012).All of them are known to be strictly associated with species of the family Araneidae (e.g.Fitton et al. 1988, Yu et al. 2012), but their host spectrum involves multiple species; with the exception of P. longa (see below) which uses a single host species (Fitton et al. 1988, Schmitt et al. 2012, Yu et al. 2012, Fritzén & Shaw 2014, Ko renko et al. 2014).Polysphincta longa is probably widely distri buted across Europe, but presumably is often misidentified as the morphologically similar species P. boops (Fritzén & Shaw 2014).Araneus angulatus Clerck, 1757 recently turned out to be an exclusive host of this species (Fritzén & Shaw 2014).
Interestingly, a host behavioural manipulation has evolved in the final stage larvae of these wasps.Shortly before killing the spider host, the final stage larvae of several polysphincti nes manipulate the webbing behaviour of their hosts, which spin a special web structure called a 'cocoon web' in order to establish a safe place for pupation.The cocoon web, a term coined by Eberhard (2000), is a web construction which is built by the spider host under the influence of the parasitoid's final stage larva.The cocoon web is stronger than the nor mal web and presumably provides a more durable support for the wasp's cocoon (e.g.Eberhard 2000, Korenko et al. 2014).Some polysphinctine parasitoids make use of original struc tures of spider's normal web for protection during the pupal stage, as was documented for P. rufipes (Schmitt et al. 2012).
No modification of spider web building behaviour of P. longa was observed by Fritzén & Shaw (2014).
Here we present new records for P. longa and P. tuberosa from Slovakia and the Czech Republic, the host records, and descriptions of web architecture modification induced by the parasitoid's final stage larva.The host range of wasps of the genus Polysphincta in Europe is also discussed.
Spiders were collected by beating bushes and tree bran ches up to a height of two meters above ground.A square shaped net (1 m 2 area) was used and each collected spider was inspected for the presence of parasitoid larva.A parasitized Araneus angulatus was reared in a glass arena with a 400 × 400 mm base, 550 mm height and with a Yshaped twig in stalled across the arena to provide space for building a web.A parasitized Araneus sturmi (Hahn, 1831) was reared in glass arena with a 200 × 50 mm base and 200 mm height.Other parasitized spider hosts (Araniella spp.and Araneus quadratus Clerck, 1757) were reared in tubes with a 15 mm diameter and 100 mm height.Laboratory reared Drosophila flies or houseflies (Musca domestica Linnaeus, 1758) were provided every three days until the spiders were killed by the parasitoid final stage larvae.The web building behaviour of manipulated A. angulatus and A. sturmi was observed until the larvae killed and consumed the spiders and then pupa ted.Photographs were taken using an EOS 500D singlelens reflex digital camera (Canon Inc.).Microscopic observations of spider silk were performed in various parts of the cocoon web of A. angulatus (the wall of the silk chamber, threads in the chamber surroundings and the dragline thread).Speci mens are deposited in the collection of S.K. (Department of Agroecology and Biometeorology, CULS, Czech Republic) and Kamil Holý (Department of Entomology, Crop Research Institute, Czech Republic).

Field observation
The orb web weaving spider A. angulatus is a relatively rare arboreal species.During our excursions, we found only one specimen, and it was parasitized by a polysphinctine larva.After rearing of the parasitoid larva to adulthood in the la boratory, the parasitoid was identified as P. longa.The wasp is recorded for the first time in Slovakia.Other araneid hosts, Araniella spp., A. quadratus, A. sturmi were observed in higher numbers at several localities beyond that of A. angulatus, but the presence of parasitoid larvae was sporadic (N = 8).All eight larvae on these araneid spiders were P. tuberosa.

Laboratory observation
The parasitized Araneus angulatus (body length 8 mm) was placed in an experimental arena containing a Yshaped twig (20th September 2016).The larva sat transversely at the an terior apex of the spider's opisthosoma just above the pedicel (Fig. 1).The spider built only one strong silk line between the ends of the arms of the Yshaped twig at an early stage.Most of the time, the spider sat on the bark at the end of a twig.Only one orb web was built (11th October 2016), when the spider captured prey and fed.The spider's capture web was 42 cm wide with 16 radial lines and 25/24 spi ral lines in the upper/lower part of the orb.The web was kept by the spider for two days, then the fly was provided as prey and the web was damaged by the spider.After feeding, the spider did not build any other orb web.After two weeks (24th October 2016), the larva doubled its body length to  2).The cocoon web of P. longa was decorated by various forms of silk tufts (Fig. 3), which were produced after modification of the spider's behaviour presumably induced by the larval effect.The tufts were produced in various parts of the cocoon web.The highest number of tufts was on a wall of the silk chamber, which surrounded the parasitoid pupa (Fig. 3a), on threads in the surroundings of the chamber (Fig. 3b) and on a frame thread (Fig. 3c).
The parasitoid larva paralysed and killed the spider, and built its cocoon at the centre of this 3D tangle.The cocoon was fusiform, white, sparsely spun with an opening at a distal end and oriented horizontally at an angle of 40°.On the next day (25th October 2016), the larva pupated and three days la ter meconium (the first excrement after pupation) appeared in the lower part of the cocoon (27th October 2016).One adult female emerged after 8 days (5th November 2016).
Araneus sturmi (body length 4.5 mm) had a parasito id larva sitting transversely at the dorsal and posterior side of the spider's opisthosoma (Fig. 4).Under the influence of the final stage larva of P. tuberosa the spider built a unique threedimensional (3D) structure in the upper corner of the experimental arena with a high density of threads (Fig. 5).No tuftlike structure was observed.The cocoon was fusiform, yellowish white, sparsely spun with an opening at a distal end and oriented horizontally as for P. longa.

Host utilisation and specificity of European Polysphincta
Our study supports the view of Fritzén & Shaw (2014) that P. longa is exclusively associated with A. angulatus.This arbo real spider is a relatively rare species and prefers natural forest habitats, where it builds a large orb web mostly in the higher strata of canopies.The host's rareness seems to be one of the reasons why P. longa has been seldom reared from hosts.In addition, P. longa is potentially more abundant in Europe than previously thought because Fritzén & Shaw (2014) reexami ned material from several parts of Europe and revealed that P. longa was misidentified as P. boops, which is also associated with arboreal araneid spiders but attacks only the genus Araniella (e.g.Fitton et al. 1988, Fritzén & Shaw 2014, Korenko et al. 2014).
The hosts of both parasitoids occur in tree crowns but their microhabitat preferences, the sizes and orientations of their orb webs, and their body sizes differ considerably.Large vertical orb webs of A. angulatus were located in high strata of the tree crown and were often constructed across two trees.In contrast, the much smaller Araniella species build a rela tively small, mostly horizontally oriented orb web between tree twigs, sometimes covering only one tree leaf (Kůrka et al. 2015).Both parasitoids, P. longa and P. boops, share forest canopy habitats sympatrically, possibly causing the confusion.These two related parasitoid species presumable evolved their own hostsearching behaviour towards closelyrelated but slightly different spider lineages.
The Holarctic P. tuberosa, morphologically similar to the Palaearctic P. boops, also prefers small arboreal araneid spiders, but its host range is much wider than that of P. boops (e.g.Fit ton et al. 1988, Korenko et al. 2014).Although it attacks vari ous taxa, their ecology (web architecture, habitat preference) is similar (Kůrka et al. 2015).Another Polysphincta occurring in Europe, P. rufipes, is widely distributed across the Palaearctic (Yu et al. 2012).The species attacks araneid spiders such as Larinioides or Zygiella, which build a protection chamber at the side of the orb web (e.g.Fitton et al. 1988, Schmitt et al. 2012).Another congener is P. vexator distributed in the Bri tish Isles and Scandinavia (Yu et al. 2012), which seems to be associated with grassy peat bogs and mosses, where its major host spider A. quadratus is common (Fitton et al. 1988).A single record reared from Larinioides cornutus (Clerck, 1757) is also present (Fitton et al. 1988).
In total, five wasp species of the genus Polysphincta occur so far in Europe.Their host range is restricted to the spider family Araneidae, but each host preference is varied (Tab.1).The widest host range is documented in P. tuberosa, which attack three araneid genera (Fitton et al. 1988, Korenko et al. 2014).In contrast, P. boops seems to be strictly associated only with spiders of the genus Araniella (Korenko et al. 2014).Polysphincta longa seems to attack only A. angulatus (Fritzén & Shaw 2014, this study).

Manipulation of web-building behaviour
We observed, although only once, that the webbuilding be haviour of A. angulatus was modified by the final stage larva of P. longa.The spider built a unique structure corresponding to a cocoon web (a 3D tangle produced by the manipulated spider), which seems to serve to protect the parasitoid during the pupal stage.Our observation did not agree with Fritzén & Shaw (2014), who saw no modification of spider web building   behaviour.The explanation for this difference seems to lie in the size of the experimental arena.Fritzén & Shaw (2014) used a rearing arena of small size, whereas our observation was conducted in a large arena where the spider had enough space to build both a normal and a cocoon web.The utilisation of spider web structures by a parasitoid was also documented in P. rufipes (the parasitoid uses the normal web structure -spider shelter built at the side of normal web) (Schmitt et al. 2012) and in P. boops and P. tuberosa (parasi toid induces building of unique cocoon web) (Korenko et al. 2014).Similar cocoon web architecture could also be expected in the other European species of this genus, P. vexator.
Considering all available data, the utilisation of a 3D web structure (for protection during pupal stage) seems to be ty pical for wasps of the genus Polysphincta in Europe.These protecting constructions can make use of the spider's normal structures (the spider retreat of P. rufipes) or can be achie ved via a set of unique spider behaviours newly induced by the parasitoids (the 3D tangle of P. boops, P. tuberosa and P. longa).The cocoon web of P. longa uniquely contained many silk tufts of various forms which were produced by the spider after the parasitoid larva reached its final stage and modified the spider's behaviour.These structures were never observed in P. boops and P. tuberosa (Korenko et al. 2014, unpubl. data).Takasuka et al. (2015) found similar silk tufts on the cocoon webs of Cyclosa argenteoalba Bösenberg & Strand, 1906 un der the influence of the parasitoid ichneumonid Reclinervellus nielseni (Roman, 1923).Takasuka et al. (2015) showed that tuft decoration reflects UV light, possibly to prevent dama ge caused by collision of large insects and birds.The same function is expected in the tufts present on the cocoon web induced by P. longa.

Fig. 3 :
Fig. 3: Silk tufts produced by the spider under the influence of P. longa larva a. on the wall of the silk retreat; b. on threads in the surroundings of the retreat; c. on the dragline thread; Scales: 2 mm