Invasion of synanthropic silverfish continues: first established populations of Ctenolepisma calvum (Ritter, 1910) revealed in the Czech Republic

Synanthropic silverfish species have spread over Europe as invaders in recent years. This paper reports the first occurrence of synanthropic silverfish Ctenolepisma calvum in the Czech Republic. Established populations of the species were revealed at two localities in Prague. At both sites, C . calvum occurred inside the buildings with room temperatures of 23.3–28.3 °C and moderate humidity 32.6–55.8%. From this point of view, our observations indicate that the climate restrictions suggested for Lepisma saccharinum and Thermobia domestica may not be effective against C . calvum . Thus, its suitable management is challenging for the future. This study also summarizes the available data on C. calvum distribution from the literature, social platforms and invasive species databases, which pointed out that only little is known about C . calvum and the species seems to be either under-recorded or rapidly spreading throughout Europe. In light of this, further monitoring, as well as risk assessment of this silverfish species, is highly needed


Introduction
Lepismatidae is an ancient family of primitive wingless insects containing more than 500 species, among which most live outdoors (Hage et al. 2020) or in association with ants (Molero-Baltanás et al. 2017).However, few lepismatid species, known also as silverfish, have successfully colonized the urban environment and become common nuisance pests.
Generally, the synanthropic silverfish species consume plant proteins, cellulose and starchy materials, and thus may cause damage to documents, books, books bindings, papier-mâché, photos, wallpapers and textiles (Mallis et al. 1958;Querner 2015).While the costs of silverfish mechanical damage in domestic settings are limited, valuable objects may become degraded or destroyed by silverfish in galleries, museums, libraries and collections (Aak et al. 2019).Moreover, the silverfish are known to be allergenic (Barletta et al. 2007) and carry opportunistic bacteria (Kulma et al. 2021), therefore the potential importance from the public health point of view should be taken into account.
Historically, only two representatives of Lepismatidae were known living indoors in the Czech Republic.Lepisma saccharinum Linnaeus, 1758, a synanthropic element of habitats with room temperatures of 22-27 °C and high humidity (75-92%) (DeVries and Appel 2013) such as bathrooms or basements, is a very common household pest occurring throughout the country (Rupeš et al. 1998).Thermophilic Thermobia domestica Packard, 1873 occurs in warm to hot (32-41 °C) and moist (76-85%) urban habitats (McIver and Harwood 1966;DeVries and Appel 2013), and so its presence in the Czech Republic is known particularly from the bakeries, boiler rooms, heating plants and steam tunnels.Interestingly, this species is also commercially available as feed for ornamental amphibians and lizards (Kulma et al. 2018).
At the turn of the century, Ctenolepisma lineatum Fabricius, 1775 was recorded in the outdoor environment of mild climate areas in the region of South Moravia (Štys and Rozkošný 1996;Král and Davidová-Vilímová 2001) and Rusek (2005) even listed the species in the Red List of Threatened Species in the Czech Republic.Nevertheless, the status of C. lineatum was re-evaluated and the species is now missing in the current Red List edition (Hejda et al. 2017).Recently, the facultative synanthropic life of this species was confirmed by its observation in indoor settings in South Moravia (Kulma unpublished observation).Finally, the invasive species Ctenolepisma longicaudatum Escherich, 1905 was detected causing damage to documents in offices of a private company on the edge of Prague (Kulma et al. 2018).In subsequent years this species has been recorded several times throughout the country, and it is therefore considered to be widespread (Kulma et al. 2021).Due to its low requirements of both temperature and humidity, C. longicaudatum is expected to even become one of the dominant Czech household pests, as happened for instance also in Faroe Islands (Thomsen et al. 2019), the Netherlands (Buijs 2019) and Norway (Aak et al. 2021).
While the populations of silverfish species affiliated to high humidity and temperature could be simply eradicated by a combination of climate regulation and cleaning (Querner 2015), the control of species with high bio-plasticity, such as C. longicaudatum, requires a different approach including usage of poisoned baits and dust insecticides (Mattsson and Kolbjørn Mohn 2019;Aak et al. 2020b;BPCA 2020).The correct identification of the silverfish species is thus a crucial tool to establish suitable management in the infested areas.In this paper, we aimed to update the list of synanthropic silverfish species in the Czech Republic and document the first reports of Ctenolepisma calvum (Ritter, 1910) in Prague.

Collection of specimens
Silverfish activity was monitored at two localities in Prague using either aspirators (EntoSphinx, Pardubice) or sticky traps (Samura, Prague).To reduce potential damage to the captured insects, the collected silverfish were transported alive in vials with strips of filter paper to the laboratory in the National Institute of Public Health in Prague (NIPH).Here, the specimens were preserved in plastic vials with 70% ethanol and morphologically identified using valid entomological keys (Wygodzinsky 1972 andAak et al. 2019).The voucher specimens are deposited at the Entomological collection of the NIPH, Czech Republic, and a few live individuals are maintained in the same institution for further investigation of the species.Some specimens are also preserved in the Zygentoma collection of the Department of Zoology of the University of Córdoba (Spain).The temperature and relative humidity were recorded at both sites using a thermohygrometer COMET-C3120 (Comet system, Rožnov pod Radhoštěm).

Identification
The body surface of the collected specimens is uniformly white, and the two lateral caudal appendages are approximately 2/3 of the body length, while the middle caudal appendage is approximately as long as the body.These characters suggest they may belong to C. calvum, but body length, color and length of antennae and caudal appendages can show intraspecific variability or not be correctly assessed because of loss of scales and breaking off of appendages.Moreover, although some characters used for identifying Lepismatidae can be observed using a stereomicroscope, most diagnostic characters necessary to distinguish species are difficult or impossible to detect with this method, because they refer to chaetotaxy and some of the setae can be detached and their insertions only visible with higher magnification.So, the identification was made combining characters observed with stereomicroscopy and those examined with light microscope.This latter method required the dissection of some specimens and mounting them on slides using Tendeiro medium (Molero-Baltanás et al. 2000).
The original descriptions of most species belonging to the genus Ctenolepisma are obsolete and require an update.This is the case of C. calvum, with the added problem that the diagnosis made by Ritter (1910) is partially incorrect.The available keys that include synanthropic species of Ctenolepisma were considered, but they are not completely appropriate since they do not include all the potential species that can enter into houses in Europe and/or do not include the appropriate microscopic characters to distinguish all these species (Wygodzinsky 1972;Aak et al. 2019).This led to a decision to base the identification on the comparison with all potential species with which it could be confused and using as a reference a topotype of C. calvum, i.e., a specimen of the species captured at the locality at which the original type was obtained.This specimen was collected in 1907 in Radanaputra (Ceylon), offered by S.L. Tuxen to L.F. Mendes, who mounted it on a slide (Mendes pers. comm.) and deposited it in the collection of CZ, IICT (Centro de Zoologia do Instituto de Investigacão Cientifica Tropical, Lisbon), now transferred to the collection of MUHNAC (Museu Nacional de História Natural, Entomology, Lisbon, Portugal); the second author had the opportunity to examine this specimen during a visit to the former institution.
The sex could be distinguished according to the presence or absence of an ovipositor (see Figure 1).

Photography
The few collected individuals were examined with a Leica DVM6 digital microscope equipped with Objektiv Plan APO FOV 43.75 and an additional high-resolution 10-megapixel camera.The object was illuminated by an integrated Coaxial LED ring light.Each field of view (FOV) was photographed as a series of images in various focal planes (Z-Stack) automatically calculated using Leica LAS X 3.0 software (Leica Microsystems CMS GmbH Wetzlar, Germany, 2020) into an all-in focus image, in which all elements are displayed in sharp focus.Basic adjustment of exposure and sharpness of the final images was done within the post-process in Adobe Lightroom Classic (Adobe Inc. 2021).Micrographs were taken using a Nikon DS-Fi1 digital camera on a Nikon Labophot light microscope, and plates were prepared using the GNU Image Manipulation Program (GIMP) ver.2.10.12.

Distribution maps
Distribution maps were downloaded from https://d-maps.com/and edited using Zoner Photo Studio software (Brno, Czech Republic).The distribution data were collected from the sources indexed at Google Scholar and Scopus databases, and from the on-line citizen science platforms iNaturalist (www.inaturalist.org),Observation (www.observation.org)and Global Biodiversity Information Facility (www.gbif.org).

Results
On 17 March 2021, the first author observed one silverfish crawling on the wall in a storage room (temperature 25.1 °C, humidity 36.0%) in the basement of the Faculty of Agrobiology, Food and Natural Resources at the Czech University of Life Sciences, Prague (CZU, 50°07′N; 14°22′E).On 22 March 2021, other two individuals were captured in a neighbouring room used for laboratory insect rearing .Here, the individuals were hidden behind towels.During the next few days, two other silverfish were collected using an aspirator by students maintaining the insectarium, kept alive and photographically documented.On 14 th and 18 th April 2021, two more individuals were found on sticky traps placed under and on top of the racks in the insectarium (see Figure 2A).By the date of manuscript submission, more than 20 silverfish were either trapped or captured alive by the students at this locality.From further laboratory investigation, the authors were able to confirm that adults of both sexes were present at both sites.Hence, the authors considered both populations as established especially due to observation of offspring at various developmental stages at the sites.Finally, the species was shown to occur at room temperatures 23.3-28.2°C with moderate to low humidity.
The maximum body length measured is 8 mm.Observed with a stereomicroscope, live silverfish are uniformly whitish with a slightly yellowishbrown or grey pigment in some appendages (Figures 1, 3, 4).Specimens preserved in ethanol turned a yellowish brown color.The shape of the last abdominal tergite is trapezoidal, shorter than wide, and the two lateral caudal appendages are approximately 2/3 of the body length, while the middle caudal appendage is approximately as long as the body.Tufts of feathered bristles are present in the frontal part of the head (Figure 4), a typical characteristic of Ctenolepisma and Thermobia species.The uniform color and the shape of the last abdominal tergite rule out the possibility that they are C. lineatum or T. domestica, synanthropic species that show When examined ventrally (Figure 1), the abdomen of the presumed C. calvum from Prague shows lateral bristle-combs in the abdominal sternites II-VIII, seems to lack abdominal bristle-combs and has only one pair of styli.This could eliminate C. targionii because this species has medial urosternal bristle-combs in segments II-VI and females bear two pairs of styli, but if the styli and the setae are detached, their sockets are not easily visible with stereomicroscope.Ctenolepisma rothschildi and C. longicaudatum also have two pairs of abdominal styli, and the ovipositor of C. longicaudatum is very long, surpassing the styli of segment IX by 2 or more times their length, but young specimens with about 7-8 mm have not developed the second pair and young females have a shorter ovipositor.
The dorsal chaetotaxy is not clearly visible (Figures 3, 4), but this does not imply that the species lacks dorsal bristle combs, which can be interpreted when following the key of Aak et al. (2019); in fact, infralateral bristle-combs of abdominal tergites can be observed ventrally (Figure 1).Probably, most macrochaetae are tumbled and their insertions are difficult to see, a usual problem with any species of silverfish.So, conclusive identification requires examining some specimens with a light microscope.
Comparing the characters of the Ctenolepisma specimens from Prague with the topotype of C. calvum from Ceylon, they match essentially in every diagnostic character.They bear 1+1 infralateral bristle-combs in the abdominal tergite I, 3+3 bristle-combs in the abdominal tergites II-V and 2+2 bristle-combs in the abdominal tergites VI-VIII.Most of the bristles (usually referred as macrochaetae by Lepismatidae taxonomists) are detached, so only their insertions are visible (Figures 5, 6).They also show 1+1 lateral bristle-combs in the abdominal sternites III-VIII and lack median combs in all abdominal sternites.The number of macrochaetae in each comb is low, which can explain the difficulty to detect them with other observation tools (urotergal combs have 1-4 and urosternal combs 4-7 macrochaetae).Only C. rothschildi has a similar abdominal chaetotaxy and the only significant difference is the number of pairs of styli; this is the species that can be considered closest to C. calvum and with which confusion is most likely, since it is considered by Irish (1995) as an anthropophilic and cosmopolitan species.
These details of the chaetotaxy of C. calvum were not indicated in the original description or in any other posterior descriptive note on the species, Figure 6.Micrograph of a lateral portion of the posterior margin of the pronotum of Ctenolepisma calvum where the insertion of a macrochaetae (C) is marked; it is accompanied by the insertion of another smaller seta.This matches with the description of this character by Wygodzinsky (1972) and with a specimen from Ceylon.The hind margin of the pronotum is marked with dashed blue lines.Photograph by R. Molero-Baltanás.so this is the first time they are reported.insertion of a second pair of styli has not been detected, so it is assumed that they bear only one pair of styli.These characters, and some others such as the length of the ovipositor relative to the styli, do not coincide with the original description of the species by Ritter (1910), but we assume that this description is partially incorrect, because the dorsal chaetotaxy was overlooked leading to probable confusion with some specimens of C. longicaudatum studied together with C. calvum and captured in the same locality.The dissected specimens show a character mentioned and designed by Wygodzinsky (1972), who considered this as essential to separate this species into a different genus, Peliolepisma: the reduction of the posterolateral bristlecombs of thoracic nota.In the remaining species of Ctenolepisma, these combs consist of two or more macrochaetae, while Wygodzinsky stated that in C. calvum they bear only one, which can be accompanied by few small setae.The topotype of C. calvum matches this description: all of its thoracic nota show in each lateral posterior margin only one insertion of a macrochaeta.In the specimens from Prague, some variability has been observed: several posterolateral bristle-combs consist of one (Figure 6) and some others of two or three macrochaetae.But this can be interpreted as an intraspecific variability that Wygodzinsky did not detect and that confirm the inconsistency of this character for erecting an independent genus.
All the dissected specimens were identified as C. calvum.The observed individuals are also believed to belong to this species.
Based on an overview of available data on C. calvum distribution, the Czech Republic has become the eighth European country where the occurrence of the species has been reported (see Figure 7).Nevertheless, as the identification on which most of these records are based is doubtful, this work provides the first confirmed report of the species in the continent using appropriate microscopic characters.The distribution maps including newly reported sites from Prague are displayed in Figure 8.

Discussion
The first reports of C. calvum at two localities within Prague city indicate that this species may follow C. longicaudatum and colonize the indoor environment throughout the Czech Republic and other Central European countries.Generally, little is known about C. calvum.In comparison with the other synanthropic silverfish species, only a little attention has been paid to this pest.Its origin and native range are confused.The first reports of C. calvum came from Ceylon (Ritter 1910;Crusz 1957) followed by Guyana and Cuba, where it was reported to be a common house lepismatid by Wygodzinsky (1972).The first reports from Europe came from Chemnitz, Germany (Landsberger and Querner 2018) and Norway (Hage et al. 2020), although no appropriate microscopic characters were used to identify conclusively this species.Online platforms recently provided some information about C. calvum presence in a few European countries such as Austria, Croatia, Finland, Kosovo, Italy, Luxembourg, Russia, Spain and Switzerland (GBIF 2021;iNaturalist 2021).However, the species identification is questionable in most of the cases.To the best of the authors' knowledge, no specific data on bionomy are available for this species.Considering this silverfish's tiny size (< 8 mm of total body length) and agility, the occurrence of the species might be likely under-recorded due to problems of capture.Also, misidentification with other silverfish species is likely.As C. calvum may co-occur with C. longicaudatum (Aak et al. 2021), the confusion of identification is probable.It is therefore possible that the species is overlooked and has already spread throughout Europe, and it is currently becoming more abundant.For instance, at the CZU, the species could not be present for a long time, at least in the infested rooms, because sticky traps serving for capture of escaped insects are placed under the racks and they are regularly checked.The first trapped individual was found a few days after the first observation of C. calvum presence at these rooms.At the second site, the silverfish was observed by the new owners of the flat during their first week there.By this time, the flat was empty for at least one month and former inhabitants claimed no previous experience with silverfish.The source of such infestations thus remains unknown.Generally, silverfish are transported by homeowners, employees or delivery service drivers in cardboard boxes and containers or clothes, which were previously stored in infested areas (Kulma et al. 2018).Special attention should therefore be paid to incoming deliveries at vulnerable areas such as archives, galleries, libraries and museums (Querner 2015).The rapid elimination of packing materials is thus also highly recommended to decrease the risk of infestation (Mattsson and Kolbjørn Mohn 2019).
Regarding pest management, climatic restrictions, which were typically recommended for control of synanthropic silverfish species in the past, are ineffective against silverfish with building-wide and more uniform spatial distribution, such as for instance the invasive C. longicaudatum (Aak et al. 2020a).Based on our observations, it is obvious that C. calvum thrives at room temperature with moderate humidity.Although damage caused by the species was not reported from the infested sites reported in this paper as well as by other authors (Landsberger and Querner 2018;Aak et al. 2019;Hage et al. 2020), even activity of the silverfish can cause discomfort to people inhabiting or working in infested buildings (Aak et al. 2019), especially in living rooms or kitchens.Moreover, C. longicaudatum was recently shown to transmit some opportunistic bacteria (F.Boiocchi 2021, pers.comm.), and because of similar bionomy, the public health impact of C. calvum is expected to be similar, although different species have distinctive requirements and could host different microbiomes.
In order to define pest management of the invasive silverfish species, further research on their spread is essential.Correct identification is essential to choose the appropriate pest control management measures.From this point of view, the major patterns specific to this species, which could be easily determined by insect control professionals, are the coloration and length of caudal appendages.Nevertheless, to avoid misidentifications, other morphological characters should be considered when a population of a species not previously recorded in a geographic area is detected for the first time, and expert assistance is recommended in these cases, since other similar synanthropic species could be present.
This work contributes to the knowledge by adding novel data on distribution, habitat and requirements of C. calvum, which was proved to be another indoor silverfish pest in Europe.Further research with emphasis on bionomy of the species is required.Also, the complicated identification of the species indicated that genus revision and updated descriptions of the most common and potential pests are desirable.Finally, we recommend raising awareness especially of pest controllers, staff of museums or galleries, and also of other stakeholders to take the occurrence of this species in the Czech Republic and all Europe into account.

Figure 1 .
Figure 1.Details of abdomen and ovipositor of Ctenolepisma calvum female.Only one pair of abdominal styli (orange arrow) is visible, as well as some lateral urosternal bristle-combs (yellow arrow) and several infralateral urotergal bristle-combs (green arrow).Note that the apex of the ovipositor slightly surpasses the apex of styli IX.Photograph by M. Petrtýl.

Figure 2 .
Figure 2. Individuals of Ctenolepisma calvum found in the sticky trap at the Czech University of Life Sciences Prague (A, C) and captured alive by citizens in a private flat (B).Photographs by M. Kulma.

Figure 3 .
Figure 3.The specimen of Ctenolepisma calvum captured at the Czech University of Life Sciences Prague.Photograph by M. Petrtýl.

Figure 4 .
Figure 4. Detail of head and thorax of Ctenolepisma calvum captured at the Czech University of Life Sciences Prague.Photograph by M. Petrtýl.

Figure 5 .
Figure 5. Micrograph of the right side of the abdominal tergite II of Ctenolepisma calvum, showing the insertions of the feathered bristles forming groups of two (combs of two macrochaetae).A: infralateral comb, not in focus because the bending of the tergite in its lateral part towards the ventral side of the insect.B: lateral comb.C: sublateral comb.All bristles are detached, but one is visible next to the comb C. Photograph by R. Molero-Baltanás.

Figure 7 .
Figure 7.The current (May 2021) distribution and years of first records of Ctenolepisma calvum in Europe.Countries with occurrences verified by local entomologists are shown black, unverified records from iNaturalist.organd gbif.org are in grey.No records were found on Observation.org.