Description of the larval stage of Drusus mixtus (Pictet, 1834) (Trichoptera: Limnephilidae: Drusinae) with notes on its ecology and zoogeography

Abstract

In the Swiss Jura adults of Drusus mixtus and unknown Drusinae larvae which could not be identified with existing keys were sampled. Based on ripe pupae, the unknown larvae were identified as D. mixtus. The association was confirmed by specimen rearing in aquaria.

Based on morphology, larvae of Drusus mixtus key out together with D. croaticus in existing keys. D. mixtus is separated from the latter species by the shape of the anteromedian metanotal sclerites which are broadly triangular, whereas in D. croaticus the sclerites are almost parallel-sided, resembling a stretched rectangle. In addition, the two species are geographically well separated: D. croaticus is restricted to the confines of Croatia and Slovenia, whereas D. mixtus is only present in Switzerland and eastern France. With this present paper, all Central European Drusinae species except Drusus chapmani McL, 1901 (France, Switzerland) and D. noricus Malicky, 1981, an endemic from the Saualpe (Carinthia, Austria), are known in the larval stage.

Introduction

Caddisflies are considered primary indicator taxa in monitoring water quality (Barbour et al., 1999; Barbour & Yoder, 2000; Wright et al., 2000; AQEM consortium, 2002; Graf et al., 2002; Hering et al., 2006). This also fully applies to the subfamily Drusinae, where all members are restricted to water quality classes I or I-II and are used as bioindicators (sensitive species) (Moog et al., 2002; Graf et al., 2002).

Unfortunately, no comprehensive and integrated effort has been made to complete the available keys to larval Drusinae. Thirty species are reported from the Alps, including 24 species reported from Austria, Germany and Switzerland (Lubini-Ferlin & Vicentini 2005; Malicky 1999, 2004, 2009; Robert 2001, 2004). From the Balkan Peninsula, 24 additional species are known, many of them endemics (Malicky 2004, 2005). For caddisfly larvae in Austria and its surroundings, Waringer & Graf (1997, 2004) summarised the present knowledge on larval taxonomy. Since then, several new descriptions of Central European Drusinae larvae were published (Waringer et al., 2000, 2007, 2008a, b, Graf et al., 2005; Graf et al., 2009, 2010) which provided additional information. From the Dinaric western Balkan, seven species have been described so far (Kučinić et al., 2008, 2010a, b, c; Previšić, personal communiciation).

In the present paper we take a step at completing the larval taxonomy of subfamily Drusinae in Central Europe by providing a description of the larva of Drusus mixtus. This species has been included in existing keys using selected morphological characters that were documented by SEM photography (e.g. Pitsch, 1993: Figs. 304, 305, 315, 317) or macrophotography (Waringer & Graf, 1997); an in-depth description, however, is lacking to date. This description is based on larval material from the Swiss Jura which was collected in the Lionne, a tributary of the Lac de Joux in the west of the canton Waadt (Vaud; Switzerland) at 1004 m a.s.l. The species affiliation was confirmed by last instar larvae and ripe pupae from the same location which have been reared to the adult stage (three males) by Prof. H.-W. Bohle. This material enabled us to describe the larva in detail, permitting a better integration of D. mixtus in existing keys.

Material and methods

Larvae (13 last (fifth) instars, 3 fourth instars) of Drusus mixtus were collected by T.P on 18 April 1986 at the Lionne, a tributary of the Lac de Joux in the west of the canton Waadt (Switzerland), at three sampling stations: (1) La Lionne near l’Abbaya (6°20’ W, 46°39’N; 1000 m a.s.l); (2) Venoge near the great spring at l’Isle (6°24’ W, 46°37’N; 661 m a.s.l), spring distance: 0 m; (3) spring of the Orb (6°21’ W, 46°42’N; 770 m a.s.l.), spring distance: 0-300 m. Association between larvae and adult male specimens was based on rearings of last instar larvae and ripe pupae collected at the same location. Larval instar determination was based on head capsule widths of larval sclerites enclosed in pupal cases (fifth instars) and by extrapolating head width data series of known Drusinae species of similar final head width (fourth instars). In addition, 62 fifth instar larvae of Drusus croaticus were collected by A.P and M.K. in 2006 at several sites in Croatia (Gacka springs, Kupica spring, Čabranka spring, Zeleni Vir and Bijela Rijeka spring in the Plitvice Lakes National Park). Our material of Drusus trifidus consisted of 5 last instar larvae collected by W.G. in April, 2008 at several spring sites in the National Park Kalkalpen, Upper Austria.

Larvae were investigated and photographed using a Nikon SMZ 1500 binocular microscope with DS-Fi1 camera and NIS-elements F 2.30 and D 3.1 software.

Results

Description of the fifth instar larva of Drusus mixtus

Biometry

The body length of final instar larvae ranges from 8.7 to 12.5 mm, the head width from 1.43 to 1.60 mm (n= 13).

Head

Head capsule granulated, ellipsoid in shape and hypognathous (Figs. 1, 2). Coloration chestnut to dark or reddish brown, dorsally darker and laterally lighter; muscle attachment spots black brown (Fig. 2). Around the eyes, a yellowish ring is present (Fig. 4). The head capsule lacks the additional setae, spines or spinule areas known from other Drusinae larvae (e.g. Ecclisopteryx spp., Drusus trifidus, D. bosnicus group). Frontoclypeus bell-shaped, with narrow central constriction (Fig. 2). Antennae short, with short cylindrical base and with one prominent lateral seta; antennae situated halfway between eye and anterior head margin (Fig. 4). At each parietale, only the full set of ten primary setae (Wiggins, 1998) is present; setae 9 and 14 long and conspicuous (Fig. 4). Each side of the frontoclypeus with six primary setae, three of them along the anterior border. Labrum reddish-brown to brown, with setal brush at anterolateral margins and sparse setation on dorsal area. Ventral apotome bell-shaped, brownish. The black brown mandibles lack terminal teeth along edges as well as ridges in the central concavity.

Figs.1 - 6.

Drusus mixtus, fifth instar larva. 1: Head, thorax and first abdominal segment, dorsal view; 2: Head, frontal view; 3: First to third abdominal segment, right lateral view; arrow: setal area 3; 4: Head and pronotum, right lateral view; 5: First abdominal sternum; 6: Right midleg femur, posterior view. Scale bars: 1mm.

Thorax

Pronotum reddish-brown to blackbrown, with granular surface (Fig. 14). Posterior and lateral margins thickened and darkly sclerotized (Figs. 1, 4). In profile, the dorsal line of the pronotum is evenly rounded, thereby creating a small dorsal hump in its posterior third (Fig. 4, 14). Along the anterior border, setae are lacking at the pronotal midline (Fig. 2). Setae present in large numbers only on lateral pronotal sides (Fig. 4); spines that are present in other Drusinae (e.g. D. trifidus: Fig. 13) are lacking. The prosternite is inconspicuous and a prosternal horn is present. The mesonotum is completely covered by two brown sclerites lighter in colour than the pronotum; lateral and posterior margins darkly sclerotized (Fig. 1). The metanotum is partially covered by three pairs of medium-brown sclerites with the anterior metanotal sclerites being large and ovoidal; their median separation is distinctly smaller than their maximum extension along the body axis (Figs. 1, 16). A row of setae is present between the posteromedian sclerites; in addition groups of setae are situated between each lateral and posteromedian sclerite (Fig. 1). Legs brownish with numerous setae on coxa, trochanter and femur; tibia and tarsus with only a small number of setae (Figs. 7-12). Coxa and femur of fore leg (Figs. 7, 8) much wider than in mid and hind leg (Figs. 9-12). Additional setae are present at anterior and posterior faces of all femora (Figs. 6-12); ventral trochanteral brush present at fore legs only. Number of ventral edge setae on fore to hind leg femora is 3-5 each (Figs. 7- 12). Row of dorsal setae at mid and hind tibiae restricted to the distal third of the segment (Figs. 9-12).

Figs.13 - 18.

Figs.13 - 14: Dorsal section of pronotum of fifth instar larvae, right lateral view. 13: Drusus trifidus; 14. D. mixtus. Fig. 15: Drusus mixtus, fifth instar larva, tip of abdomen, dorsal view. Figs. 16 - 17: Metathorax and first abdominal segment of fifth instar larvae, dorsal view. 16: Drusus mixtus; arrow: fused setal areas 1; 17: D. croaticus; Fig. 18: Drusus mixtus, fifth instar larva, case. Scale bars: 1mm.

Figs.7 - 12.

Drusus mixtus, fifth instar larva. 7, 9, 11: Right fore, mid and hind leg, anterior view; 8, 10, 12: Right fore, mid and hind leg, posterior view. Scale bars: 1mm.

Abdomen

First abdominal segment with one dorsal and two lateral fleshy protuberances (Fig. 3). Setal areas sa 1 (sensu Wiggins, 1998) fused, thereby creating a continuous transverse row of setae anterior of the dorsal protuberance; however, setae are lacking posterior of the dorsal protuberance (Fig. 16, arrow). Setal area sa 3 covering the anterodorsal section of each lateral protuberance (Fig. 3, arrow). At the first abdominal sternum, setal areas sa 1, 2 and 3 are fused creating a continuous field of setae (Fig. 5). The setal bases at the central section of the first abdominal sternum are mostly small and inconspicuous except two larger bases near the midline which occasionally fuse with neighbouring smaller setal bases (Fig. 5). At the eighth abdominal dorsum, the number of posterodorsal setae (pds) is 4-6, consisting of 2 long and 2-4 short setae.

All gills consisting of single filaments (Fig. 3). At maximum, dorsal gills are present from the second (presegmental position) to the seventh (presegmental position). Ventral gills range from second (presegmental) to seventh segment (postsegmental). Lateral gills are present from the second (presegmental) to the fourth segment (postsegmental position). The lateral fringe extends from the posterior third of the second to the anterior half of the eighth abdominal segment. Light brown sclerite on ninth segment ovoid, with darker muscle attachment spots and irregular outline (Fig. 15). Along the posterior border, eight long and several shorter setae are present. Anal prolegs of the limnephilid type, brownish, with light muscle attachment spots. Anal claw dark brown, with one small accessory hook.

Case

The larval case is 8.4 – 13.5 mm in length, distinctly curved, tapering posteriorly (the width at anterior opening is 2.8 – 3.5 mm and at the posterior opening 1.7 – 2.7 mm; n= 13), and consists completely of mineral particles (Fig 18).

Morphological separation of fifth instar larvae of Drusus mixtus from other European Trichoptera

A summary of morphological features for the identification of limnephilid and Drusinae larvae is given in Waringer (1985). Within the framework of the limnephilid key by Waringer & Graf (1997, 2004) and Waringer et al. (2010), Drusus mixtus is separated from other species by the following features:

• -metanotum covered by three pairs of small sclerites (Fig. 1);
• -head and pronotum without a thick layer of woolly hairs (Figs. 2, 4);
• -head capsule without groups of additional spines, without central concavity and rims surrounding the frontoclypeus (Figs. 2, 4);
• -first abdominal sternum without a large median sclerotized patch (Fig. 5);
• -pronotum without ridge; in profile, dorsal outline evenly rounded in its posterior third, thereby creating a small dorsal hump (Figs. 4, 14);
• -Mandibles lacking terminal teeth along edges as well as ridges in the central concavity (Fig. 4);
• -Mid and hind leg femora faces with additional setae (Fig. 6);
• -Anteromedian metanotal sclerites large, ovoidal, their median separation distinctly smaller than their maximum extension along the body axis (Fig. 1, 16);
• -Long setae are lacking from the centre of the anterior pronotal margin (Fig. 2);
• -Pronotum without numerous light spines (Fig. 14).

At this position Drusus mixtus keys out together with Drusus croaticus Marinković (Kučinić et al., 2008). Morphologically, the two species are very similar; however, there are minute differences in the shape of the anterior metanotal sclerites: in D. mixtus, these sclerites are slightly triangular, with a wider median edge (length: width ratio < 2.0; Fig. 16). In D. croaticus, the anterior metanotal sclerites are more rectangular (length: width ratio ≥ 2.0; Fig. 17). In addition, the two species are geographically well separated: D. croaticus is restricted to the confines of Croatia and Slovenia within ecoregion 5 sensu Illies (1978) (Dinaric western Balkan) whereas D. mixtus is present in Switzerland and France (ecoregions 4 and 8: Alps and Western highlands) (Illies, 1978; Malicky & Barnard, 2009; Graf et al., 2008).

Phenology, habitat, and distribution

Our last and penultimate instar larval samples of D. mixtus on 18 April phenologically fit the reported flight period of this species which is on the wing from June to August. The presence of last and penultimate instar larvae in summer, combined with the flight period till August suggests a life cycle similar to that of D. biguttatus Pictet (Waringer, unpublished data) with first and second instars present in summer and autumn and third to fifth instars overwintering. With respect to longitudinal zonation patterns, Pitsch (1993) observed very low spring distances in D. mixtus (≤ 0.2 km), indicating that the species is restricted to springs and the hypocrenal and epirhithral region of small streams (Graf et al., 2008). At our sampling locations, D. mixtus was sympatric with Rhyacophila sp.sensu stricto, Synagapetus dubitans McL., Plectrocnemia geniculata McL., Plectrocnemia conspersa Curtis, Philopotamus ludificatus McL., Hydropsyche dinarica Marinković-Gospodnetić, Silo nigricornis Pictet, Drusus annulatus Stephens, Melampophylax mucoreus Hagen, Micropterna/Stenophylax sp., Potamophylax cingulatus and Sericostoma sp.

D. mixtus is a western species; records exist from the Swiss and French (Loue River) Jura and adjacent foothills, the Doubs river system and Savoy where the Jura meets the Alpine chain (Schmid, 1956; Verneaux et al., 2003; Lubini-Ferlin & Vicentini 2005). At the Mouthier site of the Upper Loue River (French Jura; 374 m a.s.l.), D. mixtus was recorded together with Rhyacophila tristis Pictet., Glossosoma conformis Neboiss, Agapetus fuscipes Curtis, Ecclisopteryx guttulata (Pictet), Chaetopteryx villosa (Fabricius), Halesus radiatus (Curtis), Potamophylax cingulatus (Stephens), Silo nigricornis (Pictet), Odontocerum albicorne (Scopoli) and another 25 Trichoptera species (Verneaux et al., 2004). The distance to the spring of this karstic outlet was 5.4 km, its width 14 m, the mean low water discharge 4.6 m³ s⁻¹, the slope 4‰ and the conductivity was 345 μS cm⁻¹ (Verneaux et al., 2004).

Discussion

According to Schmid (1956) D. mixtus belongs to the mixtus group, the largest and most heteogenous subgroup of the genus Drusus. In the adult stage, this subgroup is characterised by the presence of prominent lobes of the spinule field at the eighth abdominal tergites, and a lateral concavity of the ninth segment can be frequently observed. The upper appendices are of medium size and concave at their upper sides. In profile, the intermediate appendices, which show a tendency for reduction, are fitted with two teeth (one apical and one basal). Finally, the tenth abdominal segment is frequently open at its ventral side. Besides D. mixtus, D. biguttatus (Pictet), D. spelaeus Ulmer, D. improvisus McL., D. brunneus Klapalek, D. trifidus McL. and D. bolivari McL. belong to the subgroup (Schmid, 1956).

Recent results of phylogenetic studies based on molecular genetic data (Pauls et al., 2008) supports monophyly of the subfamily when compared with outgroups from subfamily Limnephilinae. However, at the level of species groups, there are differences with respect to species grouping concepts based on adult morphology (e.g. Schmid, 1956): whereas genus Metanoea is monophyletic, Drusus is polyphyletic with Anomalopterygella, Ecclisopteryx and Metanoea nested within. In addition, Ecclisopteryx is not monophyletic. The same analysis included four species of the mixtus group (D. biguttatus, D. mixtus, D. brunneus, D. trifidus), that fall into two well-supported subclades (5b, 5d in Fig. 3 of Pauls et al., 2008): D. brunneus and D. trifidus form a well-supported subclade (5b) whereas D. biguttatus and D. mixtus are grouped as sister taxa in another well-supported subclade also comprising D. adustus (McL.), D. melanchaetes McL., Metanoea flavipennis Pictet, M. rhaetica Schmid, Drusus nigrescens Meyer-Dür and D. monticola McL.

In addition to epilithic grazers, such as Drusus mixtus, carnivorous filterers (e.g. Drusus muelleri (McL) with serrated mandible edges and filtering setae and bristles, and omnivorous generalists with teeth on mandible edges (e.g. D. alpinus (Meyer-Dür) were identified in the phylogeny of Pauls et al. (2008).

With respect to feeding type evolution, either a progression from ancestral omnivorous shredders (e.g. Drusus alpinus) to both filtering carnivores (e.g. D. chrysotus) and epilithic grazers (e.g. D. mixtus) or a progression from filtering carnivores to omnivorous shredders and epilithic grazers is possible. Based on the fact that most Limnephilids are known to be shredders, the first scenario seems to be more likely. In addition, based on the ancestral character state reconstructions by Pauls et al. (2008), the mandible with teeth appears to be the ancestral state, which is maintained in the carnivorous filterers and omnivore generalist shredders. The spoon-shaped grazer mandible as it is present in D. mixtus appears to be derived, having lost the teeth on the mandible edge. As pointed out by Weaver and Morse (1986), feeding specialisation in Trichoptera may have opened opportunities to colonise new ecological niches and could have strongly promoted diversification. Drusinae mainly inhabit mountain streams where exposed gravel and boulders overgrown by epilithic algae represent an abundant microhabitat. Some Drusinae species evolved spoon-shaped mandibles fully suitable for scraping such algae and for colonizing such habitats. However, these new ecological niches are not without challenges and riks: epilithic algae are most abundant at lotic stream sections and grow at the upper surface of the substrate. This forces grazers and other scraper species to expose themselves more to higher current velocities during feeding than omnivorous generalists that feed near the banks (König & Waringer, 2008). This results in a significant over-representation of scraper Drusinae species in the drift when compared with their relative abundance on the stream bed (Bacher & Waringer 1996). In addition, predation risks at the upper surface of boulders are high, because foraging larvae are an easy prey for trout and water birds.

With respect to the other functional feeding types found in Drusinae, the acquisition of filtering bristles seems to be a derived character, too. With few exceptions, all Limnephilidae are shredders (Graf et al., 2002). Other feeding types are only found in the Drusinae and sporadically among other genera (Melampophylax and Micropterna). Melampophylax mucoreus, M. nepos and Micropterna testacea, for example, are Limnephilinae grazers with spoon-shaped mandibles which resemble the mandible morphology of D. mixtus (Waringer et al., 2009). Considering this number of derived grazers, changes in feeding ecology may be responsible for much of the diversification within the group.

Summary

Based on larval morphology, D. mixtus is morphologically very similar to D. croaticus. However, the two species differ slightly in the shape of the anterior metanotal sclerites: in D. mixtus, these sclerites have a wider median edge (length: width ratio < 2.0). In D. croaticus, the anterior metanotal sclerites are much more elongate and more or less rectangular (length: width ratio ≥ 2.0; Fig. 17). In addition, the two species are geographically well separated: D. croaticus is restricted to Croatia and Slovenia whereas D. mixtus is exclusively present in Switzerland and France.