Perspectives in Phycology

Entomologia Generalis, Vol. 37 (2018), Issues 3–4, 197–230 Article Published in print July 2018

The Phenomenon of Metathetely, formerly known as Prothetely, in Raphidioptera (Insecta: Holometabola: Neuropterida)**

Horst Aspöck1, Viktoria Abbt2, Ulrike Aspöck3,4 and Axel Gruppe2*

1 Institute of Specific Prophylaxis and Tropical Medicine, Medical Parasitology, Medical University of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria 2 Chair of Zoology – Entomology, Technical University of Munich (TUM), Hans-Carl- von-Carlowitz-Platz 2, 85354 Freising, Germany 3 Natural History Museum Vienna, Department of Entomology, Burgring 7, 1010 Vienna, Austria 4 Department of Integrative Zoology, University of Vienna, Althanstraße 14, 1090 Vienna, Austria * Corresponding author: [email protected]

With 36 figures and 4 tables

Abstract: For completion of their life cycle, most snakefly species require two years, some only one, and others (at least single specimens) three years or more. In most species, the larvae of the final stage hibernate in a state of quiescence, pupate in spring and emerge asadults shortly thereafter. Hibernation starts when the temperature decreases, thus inducing quiescence in the larva. If the temperature decrease is withheld during the last hibernation, the larvae remain active and usually continue to molt, but will not pupate successfully in spring. Moreover, most of them will die prematurely and prior to that will often develop considerable pathomorphological alterations of the eyes, sometimes also the antennae, some develop wing pads and occasionally even pathomorphological modifications of the last abdominal segments. Until now, this phenomenon in Raphidioptera has been inaccurately referred to as “prothetely”; however, in reality, it represents “metathetely”. The degree and duration of lower temperatures in winter that are required for a normal pupation after hibernation have been presumed to be different among the species. So far, no standardized experiments have been carried out to clarify this. Here, we report on results of chilling the final larval stage of three species – Phaeostigma (Ph.) notata (Fabricius), Raphidia (R.) mediterranea (H. Aspöck, U. Aspöck & Rausch), and Mongoloraphidia (M.) sororcula (H. Aspöck & U. Aspöck) – from +20 °C to +4 °C for 4, 8, 12, 16, and 20 weeks. As expected considerable differences between the species were found: M. sororcula, which occurs in a region with markedly continental climate with a very cold winter, requires the

eschweizerbart_xxx 198 Horst Aspöck et al. longest period of chill for a normal metamorphosis. R. mediterranea, which occurs in Mediterranean areas with only short cold periods, needs only short periods of chilling for successful development and normal pupation. Ph. notata, which is distributed in large parts of extramediterranean Europe, necessitates a distinctly greater chilling than R. mediterranea, but less than M. sororcula. At any rate, a +4°C chilling for 20 weeks is sufficient to prevent metathetely in all species. Each metathetelously affected individual, even those from the same species, differs in some detail from all others. Presumably, these pathomorphological alterations are the result of an unsuccessful pupation, rather than an early appearance of imaginal characters. Thus, it is appropriate to term the phenomenon “metathetely” rather than “prothetely”. The physiological back- ground of pathomorphological alterations due to withholding the chill is still entirely unknown. Various forms of metathetely in the three species are shown in 28 figures. The decrease of temperature as a precondition of pupation or – generally spoken – of normal and successful metamorphosis of snakeflies is convincingly correlated with the distribution of extant Raphidioptera in the world.

Keywords: Agulla (A.) astuta (Banks, 1911), Agulla (A.) bicolor (Albarda, 1891), Agulla (A.) bractea (Carpenter, 1936), Mongoloraphidia (M.) sororcula (H. Aspöck & U. Aspöck, 1966), Phaeostigma (Ph.) notata (Fabricius, 1781), Phaeostigma (Magnoraphidia) major (Burmeister, 1839), Raphidia (R.) mediterranea H. Aspöck, U. Aspöck & Rausch, 1977, chilling duration, development, distribution, hibernation, larvae, pathomorphology, pupation, quiescence, rearing, temperature.

**Dedication

This publication is dedicated to the memory of Professor August Wilhelm Steffan (23/05/1933–03/11/2016), founder and former Editor-in-Chief of Entomologia Germanica, later Entomologia Generalis. He was famous and sometimes dreaded for his critical, meticulous and often painstaking scrutiny in editing manuscripts including the extensive usage of Latin terminology. Moreover, he was a convinced and also a convincing advocate of the use of German language in scientific publications. One of us (H.A.) had the honour to act as a guest editor of an issue of Entomologia Generalis devoted to German as a language in science. Over the years Prof. Steffan came to admit that English has become the lingua franca of our days, which enables scientists the world over to communicate more easily. Indeed, we have to be grateful for this international medium, nevertheless national languages must remain as valu- able options to publish scientific results. Two of us (H.A. and U.A.) had the pleasure of knowing Prof. Steffan personally; we had many substantive conversations with him and enjoyed his originality. Prof. Steffan was an outstanding figure in our branch of science, he will remain unforgettable.

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1 Introduction

1.1 Raphidioptera: Overview

Raphidioptera (snakeflies), the smallest order of Holometabola, comprises two families: Raphidiidae (with about 200 species) and Inocelliidae (with about 40 species) (H. Aspöck & U. Aspöck 2014b) (Figs. 1–6). The distributions of the two families in the world are similar: They comprise the arboreal parts of the Palaearctic region (including Northern parts of the Oriental realm at high altitudes), as well as arboreal parts of the west and southwest Nearctic region (including transition zones to the Neotropics at high elevations) (Fig. 7) (H. Aspöck & U. Aspöck 2012, 2013b, U. Aspöck et al. 1992, Haring et al. 2011). Snakeflies are lacking entirely in the Southern Hemisphere. Snakeflies are often apostrophized as “living fossils” since they had their greatest period in the Mesozoic, possibly with thousands of species and with a much larger distribution comprising tropical regions and the Southern Hemisphere. It was hypothesized that the K/T-impact (when an asteroid of a diameter of about 10 km slammed into our planet causing a dramatic climatic change, which led to the extinction of numerous organisms) had extinguished the majority of Raphidioptera, particularly those in the Southern Hemisphere. The extant snakeflies are restricted to regions in the Northern Hemisphere characterized by a distinct decrease of temperature in winter. This seems to be necessary for the development of all snakeflies to the imago stage in both families (H. Aspöck 1998, 2000, H. Aspöck & U. Aspöck 2009, 2013b, 2014, H. Aspöck et al. 1991, 2012, 2014, U. Aspöck & H. Aspöck 2007, 2009, U. Aspöck et al. 2014b). The development from egg to imago lasts in most species for two years, in a few species (possibly in all Agulla species and several Palaearctic species) only one year, in many species, however, longer, up to several years. Even within a species (possibly even within a population of a species) there is a surprising variation in the length of development, since some individuals need only one year, some two, some more (H. Aspöck 2002, U. Aspöck et al. 1994, Gruppe & Abbt in press). During hibernation, larvae exhibit dormancy at low temperatures in the form of quiescence in the sense of Müller (1970, 1992). At a sufficiently decreased temperature, the larva becomes inactive; however, if it is transferred to a higher (e.g. room) temperature – even from a temperature of −15 °C (or lower) – it will become active within seconds. In most cases the last overwintering stage is the final larval stage, and pupation occurs in spring. However, in a few genera (or clades) of Raphidiidae pupation takes place already in summer or autumn, yet in these cases a cold period is likewise necessary for the overwintering pupa to develop to the imago (H. Aspöck 2002, U. Aspöck et al. 1994). However, it is not known whether and how often larvae molt after the last hibernation and before pupation. If the final larval stage is not subjected to a decrease of temperature in winter, it will remain alive, be active, feed and molt, but after weeks or months it will molt to a peculiar larva with more or less markedly developed pupal characters. These larvae may molt again, however, sooner or later they die. This kind of disordered metamorphosis has usually, although incorrectly, been termed “prothetely”.

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Fig. 1. Phaostigma (Ph.) notata (Fabricius, 1781), male. Germany, Bavaria, Nürnberg, Stein, 9 May 2009. L. Weltner leg. (Photo: Leo Weltner, Nürnberg). Length of forewing: 11 mm.

Fig. 2. Raphidia (R.) mediterranea H. Aspöck, U. Aspöck & Rausch, 1977, female. Austria, Upper Austria, Mühlviertel, Pelmberg near Hellmonsödt, 24 June 2014. H. & U. Aspöck leg. (Photo: Harald Bruckner, Vienna). Length of forewing: 9 mm.

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Fig. 3. Phaostigma (Ph.) notata (Fabricius, 1781), larva. Austria, Styria, Gulsen, 5 May 2016. Ch. Komposch leg. (Photo: Harald Bruckner, Vienna). Total length: 13 mm.

Fig. 4. Raphidia (R.) mediterranea H. Aspöck, U. Aspöck & Rausch, 1977, pupa. Austria, Upper Austria, Mühlviertel, Pelmberg near Hellmonsödt, ex ovo from female 24 June 2014. H. & U. Aspöck leg. (Photo: Harald Bruckner, Vienna). Total length: 11 mm.

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Fig. 5. Inocellia crassicornis (Schummel, 1832), male. Austria, Lower Austria, Eichkogel, 18 May 2013. H. & U. Aspöck leg. (Photo: Harald Bruckner, Vienna). Length of forewing: 9.5 mm.

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Fig. 6. Fibla (Reisserella) pasiphae (H. Aspöck & U. Aspöck, 1971), female. Greece, Crete, Thripti mountains, 3 April 1989. H. Paulus leg. (Photo: Hannes Paulus, Vienna). Length of forewing: 15 mm.

Fig. 7. Distribution map of extant Raphidioptera of the world. From H. Aspöck & U. Aspöck 2013a.

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1.2 Prothetely and Metathetely: An Overview

The term prothetely1 was introduced by Kolbe (1903), who described the phenomenon in the final larval stage of Lepidoptera and Coleoptera that suffered from a disorder of metamorphosis which led to the development of individuals with larval, pupal and – according to the author – imaginal characters. Lengerken (1924a, 1924b, 1932) studied these disorders in Coleoptera (Tenebrionidae) and suggested that the appearance of pupal characters in larvae of beetles is rather an effect of a retardation of some characters of the pupa than a forestalling of some pupal characters. Thus he described these creatures as “metatelic” pupae, i.e. incompletely developed pupae. Until the middle of the 1930s this disorder of metamorphosis in holometabolous insects was known only in Coleoptera and Lepidoptera. Du Bois & Geigy (1935) were the first authors who reported this phenomenon in Neuropterida, namely in Megaloptera. In their rearing of Sialis lutaria L. they found three larvae with distinct pupal characters. They interpreted this as a convincing form of metathetely and referred to individuals with the disordered metamorphosis as “Larvenpuppe”, which is German for larval pupa. Piepho (1941) summarized what was known on these disorders in Coleoptera, Lepidoptera and Megaloptera and pointed out that they are the result of incomplete molts of a larva to a pupa, thus representing metately (sic!) and not prothetely. In caterpillars of Galleria mellonella, he induced metathetely by implantation of corpora allata, and he concluded that the secret of the corpora allata (i.e. the juvenile hormone) is responsible for development of metathetely. Eglin (1939) was the first to discover this phenomenon in a snakefly. He described and figured a “Larvenpuppe” (larval pupa) of Raphidia major (now: Phaeostigma (Magnoraphidia) major (Burm.)). Although he used neither the term prothetely nor metathetely, he clearly pointed out that the larval pupa was the result of an unsuccessful molt of the final larval stage. Moreover, Eglin (1939) did not expose his larvae to low temperatures in winter, and he complained that three larvae died during the winter, although they had shown perfect vitality into autumn. One of his larvae of Ph. major survived and passed the winter, yet developed into a “Larvenpuppe”. Nothing was published on this phenomenon in Raphidioptera for over 20 years, until Vannier & Condé (1962) dealt with the topic in an article on observations of the

1 Prothetely is derived from the Greek προθεω, προθειν /prothein = run ahead and τελος / telos = target, aim. It infers that characters of a later developmental stage appear in the post- embryonic development phase, e.g. pupal characters surface in a larva of a holometabolous insect. However, if the inappropriate characters are the result of retardation (e.g. pupal characters in the last larval stage), thus representing an unsuccessful or incomplete molt to the next stage, the term “metathetely” should be used, which is derived from the Greek μετάθεω, μετάθειν /metathein = to follow, to pursue in the sense of being behind schedule. Some authors (Kunike 1942) used “protely” and “metately” instead of “prothetely” and “metathetely”. These are clearly incorrect forms since the dynamic part of the term (thein = to run), which characterizes the development, is missing. The short, but inaccurate term, “metately” has unfortunately often been used (e.g. Weber 1933, 1954), while “metathetely” is the appropriate form.

eschweizerbart_xxx The Phenomenon of Metathetely 205 biology of snakeflies in France. The authors observed, described and figured a larva of Phaeostigma notata (Fbr.) which was very similar to that treated by Eglin (1939). The larva had been kept at room temperature until December and was then brought to an outdoor location with temperatures around 5 °C. Vannier & Condé (1962) labeled the condition of disordered metamorphosis with pathomorphological characters – “prothetely”. Other larvae of Ph. notata which they kept under the same conditions died, although some developed to adults. By 1970 the phenomenon of disordered pathomorphological individuals in Raphidioptera was under investigation, and it was suggested that it was caused by a disturbance of metamorphosis during captivity, but it was not known that withholding the decrease of temperature in winter was the key factor for the condition of disordered development then described under the term prothetely (H. Aspöck & U. Aspöck 1971). At the beginning of the 1970s, two of us (H.A. & U.A.) together with Hubert & Renate Rausch began to rear snakeflies of an increasing number of species, and as we kept the larvae at the beginning partly at room temperature even in winter we soon observed the disorders of metamorphosis in various forms. We initially identified these pathomorphological alterations as prothetely (H. Aspöck et al. 1974). We showed that this phenomenon could almost always be induced by preventing the final larval stage from overwintering in low temperatures. The disorders concerned particularly the eyes, meso- and metathorax having wing pads and last abdominal segments with peculiar appendages. However, the degree of “prothetely” differed markedly and no two individuals were identical, each specimen appeared differently. Usually the disordered specimens died – either soon after the molt which led to the disorder or sometimes after one or two further molts. In rare cases the disordered individuals pupated successfully, and the imago hatched with various morphological disorders, particularly in the wings and/or genital segments (H. Aspöck et al. 1974, 1991). We assumed there was a relationship between the decrease in temperature and the duration of the chill during hibernation with the occurrence of disordered characters, and that the degree of pathomorphological alterations was different in the various species. Metathetely is a phenomenon that occurs almost exclusively when rearing individuals. Although we and many colleagues (particularly H. & R. Rausch) have collected thousands of larvae of snakeflies in the field and continue to collect larvae, only once have we found a larva (of Raphidia (R.) ophiopsis L.) with disordered eyes and wing pads in nature. This individual was found under the bark of a pine-tree, which stood isolated and received intensive sunshine, so that the bark may have been often warmer; it is likely that this induced the metathetely. Summing up our knowledge of pathomorphological larvae of Raphidioptera, it is obvious that these individuals represent disordered pupae because the last larval instar is unable to pupate normally. Thus, the commonly used term prothetely must be replaced by metathetely. Although the phenomenon of metathetely is well known and often described in reared individuals, no standardized experiments with a definite number of specimens at different hibernation regimes (certain temperatures and certain time periods) had been carried out, and thus far, no experimental data were available.

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2 Experimental studies on the influence of a defined temperature and defined duration of exposure to coldness on the development of metathetely

The experiments were carried out in the framework of a master’s thesis on “Temperature model for the development of larvae of Raphidioptera” (Abbt 2016).

2.1 Materials and methods

2.1.1 Species examined Three Raphidioptera species, all belonging to the family Raphidiidae, were included:

1 Phaeostigma (Ph.) notata (Fabricius, 1781): This is an expansive polycentric extramediterranean-European faunal element (H. Aspöck et al. 1991), whose distribution comprises large parts of Europe as far east as the Ural. The species is euryoecious, the larvae are subcorticolous living on a great number of decidu- ous and particularly coniferous trees. The species occurs from sea level to the timber line. The development from egg to adult lasts usually two or three years. To establish the rearing of Ph. notata, females were collected by one of us (V.A.) in Freising, Bavaria, Germany (48.40506° N / 11.71474° E, 490 m) in May 2014. 2 Raphidia (R.) mediterranea H. Aspöck, U. Aspöck & Rausch, 1977: This species is a Mediterranean (probably a Balkanoponto-Mediterranean) faunal element, which has been introduced by unknown human activities to the open air museum of Pelmberg, Austria, where it develops in the thatched roof of the old farmhouse that has led to a mass occurrence of the species in the yard of the farmhouse every year (Rausch et al. 2016, Gruppe et al. 2017, H. Aspöck et al. 2017). In Mediterranean regions, the larvae develop in soil and detritus between roots of bushes, in higher elevations also under bark of trees. The exact locality is: Austria, Pelmberg near Hellmonsödt, 48.40889° N / 14.32028° E, 800 m. In July 2014, two of us (H.A. & U.A.) collected several females to establish a program of rearing this species. 3 Mongoloraphidia (M.) sororcula (H. Aspöck & U. Aspöck, 1966): This species is a stationary Mongolian faunal element, whose distribution is confined to a rela- tively small area in the northwest of Mongolia (H. Aspöck et al. 1991). Females of the species were collected by one of us (A.G.) in Mongolia in June 2012 and then continuously reared ab ovo in the laboratory at the Technical University of Munich (TUM) in Freising so that larvae of the first stage were available. In nature the development takes place between stones and gravels (Gruppe & Abbt in press) and probably also in the sandy soil between roots of Caragana (and other bushes). The development from egg to adult lasts mostly two years, sometimes one or three years (Gruppe & Abbt in press).

2.1.2 Rearing snakeflies under laboratory conditions All stages of development were kept in round plastic tubes of 25 mm diameter and 35 mm height with a non-airtight cap (H. Aspöck & U. Aspöck 2009). The tubes

eschweizerbart_xxx The Phenomenon of Metathetely 207 contained rolls of striped tissue paper of about 10 mm height as a shelter for the larvae as well as a substrate for females to lay eggs. Larvae as well as the females were fed with pieces of mealworms. To prevent cannibalism larvae were separated soon after they hatched from the eggs (usually in the second instar) and where then kept individually in a tube throughout the whole experiment. All larvae were reared ab ovo from females which had laid eggs in spring 2014. The larvae were kept in a climatic chamber (16 h light/8 h dark) at +20 °C until October. From October 2014 until March 2015 the larvae were kept at +4 °C (always dark), and then transferred again to +20 °C, at which temperature they were kept until 23 September 2015. For the hibernation experiments, full mature larvae were used, i.e. larvae before their second hibernation. (A development of two years was anticipated, although it can- not be excluded that some larvae might have had a three-year development). Larvae of all three species were divided into five equal groups and each group received a hibernation period of different lengths: 4, 8, 12, 16, and 20 weeks respectively at 4 °C (in darkness) (Table 1).

2.2 Results

Tables 2, 3 and 4 show the percentages of larvae which developed metathetely in correlation with different periods of exposure to chill at 4 °C. Fig. 8 summarizes the results of the experiments in graph form. The larvae of the three species reacted quite differently to the five different periods of hibernation at +4 °C. It is of interest that M. sororcula – a species occurring in a distinctly continental climate with very cold winters developed the highest percentage of metathetelous larvae (actually metathetelous pupae) compared to Ph. notata and R. mediterranea when exposed to +4 °C for short periods, while R. mediterranea largely tolerated even short periods of chilling. Moreover, it is clear that a period of 20 weeks of +4 °C is long enough to prevent larvae of all three species from developing metathetely. Figs 9–36 show various degrees of metathetely in Phaeostigma notata, Raphidia mediterranea and Mongoloraphidia sororcula that were observed in our experiments. The degree of pathomorphological alterations is in each case different, no individual is identical with another, and there is apparently no correlation with the length of the period of low temperature and the degree of metathetely. The pathomorphological alterations pertain to the head, thorax, and abdomen (H. Aspöck et al. 1974, 1991, Abbt 2016). The normal larva of Raphidiidae has 7 stemmata on each side, while pathomorphological larvae show various conspicu- ous modifications in the form of heavily pigmented swells (Figs 9, 11, 13, 32, 34, 36) in the region where the stemmata are situated in the normal larva. Also, the cuticular structure of the head differs in some of these individuals from that of normal larvae. In many cases the abnormal eyes are the only pathomorphological alterations. However, wing pads on meso- and metathorax are found quite often, they have varying lengths and sometimes even show traces of wing venation (Abbt 2016).

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Table 1. Hibernation of three species of Raphidiidae with 5 different periods of exposure to a chilling temperature of +4 °C. Duration of chilling 4 weeks 8 weeks 12 weeks 16 weeks 20 weeks Temperature + 4 °C + 4 °C + 4 °C + 4 °C + 4 °C End of hibernation 29/10/2015 27/11/2015 23/12/2015 20/01/2016 19/02/2016 period Number of larvae of 15 15 15 10 9 M. sorocula Number of larvae of 16 16 16 13 11 Ph. notata Number of larvae of 32 32 32 31 31 R. mediterranea Total number 63 63 63 54 51

Table 2. Development of metathetely in larvae of Phaeostigma (Ph.) notata after second hibernation. Duration of Number of larvae Total number of Percentage of exposure to 4 °C developing larvae larvae developing metathetely metathetely 4 weeks 7 16 43.7 % 8 weeks 6 16 37.5 % 12 weeks 0 16 0.0 % 16 weeks 0 13 0.0 % 20 weeks 0 11 0.0 % Total 13 72 18.0 %

Fig. 8. Development of metathetely in pupae of three species of Raphidiidae after chilling (+4°C) for 4, 8, 12, 16, and 20 weeks (see also tables).

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Fig. 9. Phaeostigma notata. Head of metathetelous pupa with pathomorphological development of eyes. Exposure to chill of +4°C: 4 weeks.

Fig. 10. Phaeostigma notata. Head of a metathetelous pupa with pathomorphological development of eyes and pathomorphologically sculptured integument of head. Exposure to chill of +4°C: 4 weeks.

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Fig. 11. Phaeostigma notata. Head of a metathetelous pupa with pathomorphological development of eyes. Exposure to chill of +4°C: 8 weeks.

Fig. 12. Phaeostigma notata. Head of a metathetelous pupa with pathomorphological development of eyes. Exposure to chill of +4°C: 8 weeks.

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Fig. 13. Phaeostigma notata. Head of a metathetelous pupa with pathomorphological development of eyes. Exposure to chill of +4°C: 8 weeks.

Fig. 14. Phaeostigma notata. Head of a metathetelous pupa with pathomorphological development of eyes. Exposure to chill of +4°C: 8 weeks.

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Fig. 15. Phaeostigma notata. Head of a metathetelous pupa with pathomorphological development of eyes. Exposure to chill of +4°C: 4 weeks.

Fig. 16. Phaeostigma notata. Pterothorax of a metathetelous pupa with long wing pads. Exposure to chill of +4°C: 4 weeks.

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Fig. 17. Phaeostigma notata. Pterothorax of a metathetelous pupa with short wing pads. Exposure to chill of +4°C: 8 weeks.

Fig. 18. Phaeostigma notata. Pterothorax of a metathetelous pupa with short wing pads. Exposure to chill of +4°C: 8 weeks.

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Fig. 19. Phaeostigma notata. Pterothorax of a metathetelous pupa with intermediate wing pads. Exposure to chill of +4°C: 8 weeks.

Fig. 20. Phaeostigma notata. Pterothorax of a metathetelous pupa with long wing pads. Exposure to chill of +4°C: 4 weeks.

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Fig. 21. Phaeostigma notata. Metathetelous pupa, head with pathomorphological development of eyes and pterothorax with four differently shaped wing pads. Exposure to chill of +4°C: 8 weeks.

Fig. 22. Phaeostigma notata. Metathetelous pupa, head with pathomorphological development of eyes, pterothorax with long wing pads. Exposure to chill of +4°C: 4 weeks.

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Fig. 23. Phaeostigma notata. Metathetelous pupa, male genital segments, lateral, with pathomorphological development of gonapophyses 8 and gonapophyses 9. Exposure to chill of +4°C: 8 weeks; gp = gonapophysis; gx = gonocoxite.

Table 3. Development of metathetely in larvae of Raphidia (R.) mediterranea after second hibernation. Duration of Number of larvae Total number of Percentage of exposure to 4 °C developing larvae larvae developing metathetely metathetely 4 weeks 1 32 3.1 % 8 weeks 0 32 0.0 % 12 weeks 1 32 3.1 % 16 weeks 0 31 0.0 % 20 weeks 0 31 0.0 % Total 2 158 1.3 %

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Fig. 24. Phaeostigma notata. Metathetelous pupa, male genital segments, ventral, with pathomorphological development of gonapophyses 8 and gonapophyses 9. Exposure to chill of +4°C: 8 weeks.

Table 4. Development of metathetely in larvae of Mongoloraphidia (M.) sororcula after second hibernation. Duration of Number of larvae Total number of Percentage of exposure to 4 °C developing larvae larvae developing metathetely metathetely 4 weeks 10 15 66.6 % 8 weeks 12 15 80.0 % 12 weeks 3 15 20.0 % 16 weeks 1 10 10.0 % 20 weeks 0 9 0.0 % Total 26 64 40.6 %

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Fig. 25. Phaeostigma notata. Metathetelous pupa, male genital segments, ventral, with pathomorphological development of gonapophyses 8 and gonapophyses 9. Exposure to chill of +4°C: 8 weeks; gp = gonapophysis; gx = gonocoxite; T = tergite.

In some individuals, pathomorphological alterations of the last abdominal segments were observed. They also differ amongst each other, but can usually be identified by alterations of certain structures of the genitalia, particularly gonocoxites and gonapophyses in males and the ovipositor in females (Figs 23, 25, 26) (Abbt 2016). Beutel & Ge (2007) published a comprehensive study on the morphology and anatomy of the head of a normal larva of Phaeostigma notata, which will be an impor-

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Fig. 26. Phaeostigma notata. Metathetelous pupa, female genital segments, lateral, with pathomorphological development of gonapophyses 8 and gonocoxites 9. Exposure to chill of +4°C: 4 weeks; gp = gonapophyses; gx = gonocoxite; T = tergite.

tant source of information in future studies on the head of metathetelous larvae of Ph. notata and of the evaluation of the various pathomorphological alterations.

3 Discussion

The phenomenon of “prothetely”, however, more accurately speaking of “metathetely”, has been known in Raphidioptera for several decades as singular events occurring sometimes in laboratory rearing of larvae of snakeflies. It was first observed in a larva of Phaeostigma major by Eglin (1939), then again by Vannier & Condé (1962). In both papers the authors traced back this disorder of metamorphosis with the

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Fig. 27. Phaeostigma notata. Metathetelous pupa, female genital segments, ventral, with pathomorphological development of gonapophyses 8 and gonocoxites 9. Exposure to chill of +4°C: 4 weeks.

Fig. 28. Phaeostigma notata. Metathetelous male prepupa, lateral. Exposure to chill of +4°C: 4 weeks.

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Fig. 29. Phaeostigma notata. Metathetelous male pupa, lateral. Exposure to chill of +4°C: 4 weeks.

Fig. 30. Raphidia mediterranea. Metathetelous pupa, head with pathomorphological development of eyes, pterothorax with long wing pads. Exposure to chill of +4°C: 12 weeks.

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Fig. 31. Raphidia mediterranea. Metathetelous pupa, head, with pathomorphological development of eyes. Eyes surrounded by circular brightening. Exposure to chill of +4°C: 4 weeks.

Fig. 32. Raphidia mediterranea. Metathetelous pupa, part of head with pathomorphological development of eyes. Eyes surrounded by circular brightening. Exposure to chill of +4°C: 4 weeks.

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Fig. 33. Raphidia mediterranea. Metathetelous pupa, last abdominal segments, ventral. Exposure to chill of +4°C: 12 weeks.

Fig. 34. Mongoloraphidia sororcula. Metathetelous pupa, head with pathomorphological development of eyes and with pathomorphological development of antennae. Exposure to chill of +4°C: 4 weeks.

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Fig. 35. Mongoloraphidia sororcula. Metathetelous pupa, head with pathomorphological development of eyes and with pathomorphological development of antennae. Exposure to chill of +4°C: 4 weeks.

Fig. 36. Mongoloraphidia sororcula. Metathetelous pupa, head with pathomorphological development of eyes. Exposure to chill of +4°C: 4 weeks.

eschweizerbart_xxx The Phenomenon of Metathetely 225 development of pathomorphological characters to disturbances during the rearing, but they failed to ascertain that withholding low temperature during the last hibernation of the full-grown larva induced this pathological development. Woglum & McGregor (1958, 1959) concluded from their rearing of snakeflies that chilling in winter is necessary for pupation in spring, but they did not observe disordered individuals. Only at the beginning of the 1970s, when we started to rear larvae of snakeflies in large numbers and when we kept them initially at room temperature permanently, it soon became obvious that withholding the chilling temperature during winter pre- vented fullgrown larvae from pupating and caused numerous pathomorphological individuals (H. Aspöck et al. 1974). In agreement with Du Bois and Geigy (1935) and Weber (1933, 1954) we are now convinced that it is correct to name this phenomenon “metathetely” (it had been previously and inaccurately termed “metately”, see footnote 1), since it is the unsuccessful trial of the mature larva to pupate with the development of pathomorphological pupal characters. Thus, it seems to be a retardation of achieving the next instar, i.e. the pupal stage in the form of development of only rudimentary characters of the pupa. Previously, we termed this phenomenon prothetely (H. Aspöck et al. 1974, H. Aspöck et al. 1991, H. Aspöck 2002), although it is actually metathetely, and the individuals should be considered metathetelous pupae2. An individual with wing pads and pathomorphological alterations of last abdominal segments can convincingly be addressed as a pupa after defective molt of the final larval stage. Admittedly, it is a dilemma to regard a larva which has only (slight) morphological alterations of the eyes (without any visible traces of wing pads or modifications of the last abdominal segments) – and which looks like a larva and behaves like a normal larva – as a pupa. Nevertheless, biologically speaking it is a metathetelous pupa. The phenomenon has been observed in many species of Raphidiidae (H. Aspöck et al. 1991), but most findings were made incidentally and no standardized experiments were carried out. We knew that the metathetely can probably easily be induced in apparently any snakefly which usually pupates after the last hibernation, and we suspected that in different species different chilling conditions in winter might be essential, however, no concrete data existed. Here we present conclusive data of the occurrence of metathetely in three species undergoing hibernation at +4 °C for five different periods (4, 8, 12, 16 and 20 weeks) beginning with the end of September (Abbt 2016). The three species are derived from separate genera: Mongoloraphidia, Phaeostigma, and Raphidia. Mongoloraphidia belongs to a clade which is the sister taxon of the rest of the Palaearctic Raphidiidae

2 An interesting and unusual case of metathetely in a larva of Mesosa curculionides (Cerambycidae: Lamiinae), which was found in nature, was described by Paulus (1971). Besides metathetelous characteristics (distinctly elongated antennae, wing pads) the larva had larval legs on the thoracic segments. Legs normally never occur in larvae of the subfam- ily Lamiinae, however, they do in other Cerambycidae. Paulus (1971) interpreted this as a kind of an “atavism”, since such phenomena were called at that time. Today we term this “re-expression” and the author suggested in agreement with modern views that the hormonal disorder which had led to metathetely might have also blocked the suppression of the development of extremities in this lamiine larva.

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(Haring et al. 2011), and within these the genera Phaeostigma and Raphidia are distantly apart from each other. Moreover, the three species have different distributions with dissimilar ecological and biological characteristics. Thus, it is not surprising that periods of chilling for a successful pupation showed marked differences. Mongoloraphidia sororcula, a species restricted to high altitudes in Mongolia required the longest period of chilling. Distinctly shorter periods are mandatory for Phaeostigma notata which occurs in large parts of extramediterranean Europe. Finally, Raphidia mediterranea, a Mediterranean faunal element occurring also in the coastal areas of Aegean islands rarely produces metathetelous pupae at chilling temperatures of +4 °C. In other words, this species tolerates even short periods of chilling. The experiments clearly show differences in the frequency of development of metathetely depending on the length of chilling temperature of +4 °C in winter in the three species. Thus, the question remains whether lower chilling temperature (eventually even below 0 °C), and perhaps for shorter periods of time would be sufficient to prevent larvae from developing metathetely. Based on our studies carried out throughout the past 50 years and now in a standardized form, it is unquestionable that a period of chilling during the final larval stage is a precondition for successful pupating. It is, however, also obvious that the necessary degree of chilling is quite different in the various species. Despite this Kovarik et al. (1991) arrived at the contrary conclusion that chilling is not necessary for successful pupation – at least in the Nearctic species Agulla bicolor (Albarda). Nonetheless, the authors reported on the occurrence of metathetelous larvae in their rearing – carried out at room temperature – and this is a strong hint that chilling of the final larval stage is likewise a precondition in Agulla bicolor for normal development of an entire population, i.e. the pupation of all individuals. It is conceivable that Agulla bicolor requires only a slight decrease of temperature during hibernation, and this may be fulfilled – at least partially – under conditions of varying room temperature. Woglum & McGregor (1958) studied the biology of the Nearctic snakeflyAgulla bractea Carpenter, found in orange orchards in California, and came to the conclusion that a period of chill is necessary for pupation. The authors compared specimens overwintering under natural conditions with individuals kept at room temperature. Larvae in nature (after a period of cold) pupated in spring and developed to adults. Larvae kept at room temperature in winter remained active and molted, and a few pupated despite lack of natural chill very late in May, but the majority died. In a further publication Woglum & McGregor (1959) reported on their extensive studies on another snakefly species, Agulla astuta (Banks), which was found to be common in citrus orchards in southern California. As with the other two above-mentioned Nearctic species – Agulla bicolor and Agulla bractea – Agulla astuta completes its life cycle in a single year. Eggs are laid in spring, larvae are active throughout the summer and enter hibernation by dormancy (quiescence) until March, when they continue their metamorphosis by entering the prepupal stage, followed by pupation and finally by molting to adults, which appear from April to June. However, when kept at room temperature in autumn and winter, larvae remained active (although somewhat reduced) and did not pupate in the following spring. On the contrary, they remained

eschweizerbart_xxx The Phenomenon of Metathetely 227 alive up to three seasons and when exposed to chill in winter, pupated afterwards, and at least some of them developed to adults. No cases of pathomorphological development were mentioned by Woglum & McGregor (1959). Although metathetely has so far been observed only in several Palaearctic species of Raphidiidae (however, in every species whose final larval stage was not exposed to cold) and at least in one species of Agulla (the dominant Nearctic genus), one may conclude that it may principally be induced in every species of the family. However, as was formerly supposed and as it could now be verified under standardized conditions, the degree of cold and the period of chilling in correlation to development of pathomorphological alterations are very different among the species and depend on the climatic conditions in the distribution area of a given species. Species with a distinctly Northern distribution in areas with continental climate (i.e. very cold winter temperatures below −30 °C or even below −40 °C) – e.g. Mongoloraphidia sororcula – require long periods of chilling to escape metathetely. Species occurring in the Southern regions of the distribution of the family with comparatively low chilling and short cold periods develop metathetely more rarely. At any rate, Raphidiidae and almost certainly Inocelliidae (H. Aspöck et al. 2012) – due to their largely identical distribution being restricted to regions with a distinct winter period – require a period of chill to induce pupation and complete the life cycle. So far, metathetely has not been observed in Inocelliidae, but this can be explained by the fact that species of the family have been reared after it had become obvious in Raphidiidae that decreased temperatures in winter were necessary. We and other researchers have been reluctant to sacrifice the few available larvae of Inocelliidae for such experiments. Nonetheless, we have no doubt that Inocelliidae can only pupate after a period of decreased temperature. Our explanation as to why the order Raphidioptera is restricted to certain regions of the Northern Hemisphere is the dependence of the metamorphosis on the winter temperature, i.e. chilling (H. Aspöck & U. Aspöck 2014a, b). In the Mesozoic, Raphidioptera occurred in tropical regions and in the Southern Hemisphere; these lines (families) most probably died out as a consequence of the dramatic events following the K/T-impact about 65 million years ago. Although we have acquired basic facts about the occurrence of metathetely in Raphidioptera, some fundamental questions remain open: What actually occurs when chilling is withheld? In other words, what are the physiological effects due to chilling? Does chilling activate or inactivate the release of juvenile hormone from the corpora allata at a particular time during hibernation (see above, Piepho 1941)? Depending on temperature and length of chilling, and possibly moreover, when the period of decreased temperature begins, a great variety and diver- sity in different species is to be expected. Additionally, at which phase in the life cycle for those species which overwinter in the pupal stage (H. Aspöck 2002: type 2) or which pupate later in the year (H. Aspöck 2002: type 3) is chilling necessary for normal development? Today we know how to collect large numbers of larvae and how to rear Raphidioptera successfully in the lab (H. Aspöck et al. 1991, H. Aspöck 2002,

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H. Aspöck & U. Aspöck 2009). Moreover, we have gained experience in the design of experiments on the development and chilling of Raphidioptera larvae (Abbt 2016). The next step would be to solve the puzzle of metathetely, i.e. the development of pathomorphological pupae, and the underlying biochemical pathways. A large research field is open for physiologists!

Acknowledgements: We want to express our warmest thanks to the colleagues who provided photographs: Mag. Harald Bruckner, Vienna (Figs 2–5), Univ.-Prof. Dr. Hannes Paulus, Vienna (Fig. 6), and Leo Weltner, Nürnberg (Fig. 1). Grateful thanks to cand. med. Alexandra R. Szewczyk, BA, Vienna, personal assistant of H. Aspöck, for her diligent and precise work with the manuscript. Grateful thanks to DDr John Plant (Guilford, Connecticut), for critically read- ing and considerably improving the manuscript and polishing the English.

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Manuscript received: 7 January 2018 Manuscript accepted: 18 January 2018

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