GREGARINES (APICOMPLEXA: EUGREGARINORIDA) PARASITIZING THE CAVE CRICKET TROGLOPHILUS (ORTHOPTERA: RAPHIDOPHORIDAE) IN THE SLOVENIAN KARST GREGARINE (APICOMPLEXA: EUGREGARINORIDA), KI PARAZITIRAJO V JAMSKIH KOBILICAH TROGLOPHILUS (ORTHOPTERA: RAPHIDOPHORIDAE) NA SLOVENSKEM KRASU Dušan DEVETAK 1* & Tone NOV AK 1 Abstract UDC 593.191:595.72(497.4) Dušan Devetak & Tone Novak: Gregarines (Apicomplexa: Eugregarinorida) parasitizing the cave cricket Troglophilus (Orthoptera: Raphidophoridae) in the Slovenian karst The cave crickets Troglophilus neglectus Krauss, 1879 and T. cavicola (Kollar, 1833) originating from caves in Slovenia were surveyed for gregarines. Two gregarine species were identified. Gregarina troglophili (Golemansky & Lipa, 1991) occurred in both cave cricket species, T. cavicola being reported a new host species. Besides, a new species, Gregarina bernardae sp. n., in- fecting T. neglectus, was identified and described. The two spe- cies often infest the same T. neglectus individuals, but can be well distinguished upon the shape and morphometric indexes of both solitary gregarines and associations. The deutomerite in solitary gamonts of G. troglophili is orbicular to broadly el- liptoid, while in G. bernardae sp. n. it is oblong to dolioform. In associations, the primite deutomerite in G. troglophili is orbicu- lar to broadly elliptoid, while in G. bernardae sp. n. it is shal- lowly oblong to oblong. In contrast to broadly obovoid to finely obdeltoid satellite deutomerite in G. troglophili, it is narrowly dolioform or dolioform to narrowly obdeltoid in G. bernardae sp. n. Both species are new to the faunal list of Slovenia, and to the list of cave-dwelling species in the country. We direct the attention to the parasite diversity of cave-dwelling animals – a promising, but understudied topic that might considerably contribute to the diversity of the subterranean fauna. Keywords: Conoidasida, Gregarinasina, Gregarina troglophili, Gregarina bernardae sp. n., parasite diversity, syntopy. Izvleček UDK 593.191:595.72(497.4) Dušan Devetak & Tone Novak: Gregarine (Apicomplexa: Eu- gregarinorida), ki parazitirajo v jamskih kobilicah Troglo- philus (Orthoptera: Raphidophoridae) na slovenskem krasu V jamskih kobilicah Troglophilus neglectus Krauss, 1879 in T. cavicola (Kollar, 1833) sva preverjala prisotnost gregarin. Prepoznala sva dve vrsti gregarin. Gregarina troglophili Gole- mansky & Lipa, 1991 je bila prisotna v obeh jamskih kobili- cah; T. cavicola je njen novo ugotovljeni gostitelj. Poleg te sva v T. neglectus našla novo vrsto, Gregarina bernardae sp. n., ki jo tu opisujeva. Obe vrsti sta pogosto v istih osebkih T. neglectus, vendar se dobro razlikujeta po obliki in morfometrijskih indek- sih posameznih gregarin kot asociacij. Devtomerit posameznih G. troglophili je kroglast do široko eliptičen, medtem ko je pri G. bernardae sp. n. podolgovat do sodčkast. V asociacijah je dev- tomerit primita pri G. troglophili kroglast do široko eliptičen, pri G. bernardae sp. n. pa plitvo podolgovat do podolgovat. Za razliko od široko do fino vzdolžno nesomerno jajčastega dev- tomerita satelita pri G. troglophili, je ta pri G. bernardae sp. n. ozko sodčkast do sodčkast ali ozko deltoidne oblike. V prispe- vku prikazujeva tudi sizigij pri obeh vrstah. Obenem želiva opozoriti na vrstno pestrost zajedavcev v podzemlju živečih organizmov – obetajoče, a slabo raziskano področje, ki bi lahko znatno prispevalo k povečanju diverzitete podzemeljske favne. Ključne besede: Conoidasida, Gregarinasina, Gregarina troglo- phili, Gregarina bernardae sp. n., raznovrstnost zajedavcev, so- bivanje v habitatu. ACTA CARSOLOGICA 50/2-3, 317-328, POSTOJNA 2021 1 Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroška cesta 160, SI-2000 Maribor, Slovenia, e-mails: dusan.devetak@guest.arnes.si; tone.novak@guest.um.si * Corresponding author Received/Prejeto: 18.11.2020 DOI: https://doi.org/10.3986/ac.v50i2-3.9305 COBISS: 1.01 INTRODUCTION For many reasons, karst attracts much attention among landscapes (Knez & Kranjc, 2009). Special concerns are dedicated to economy issues, such like bauxite deposits and economically interesting elements in these deposits (Mongelli et al., 2017), water supply (Blatnik et al., 2020) and an often outstanding biodiversity above and below ground with a high degree of endemism, and markedly different species assemblages (Williams, 2008; Dehar- veng & Bedos, 2018; Moldovan, 2018; Culver & Pipan, 2019). The endemism refers to free-living cave-dwelling biota (Bole et al., 1993; Moldovan, 2012; Sket, 2012), as well as to parasites of subterranean taxa. Scarce reports on parasites for subterranean species may refer to either their actual rarity or under-sampling (Humphreys, 2018). Unfortunately, the research in parasites of cave-dwelling species is rare; in this way, we are missing important data on diversity, and several interesting issues related to par- asitism in hosts specialized to living in the subterranean environment (Golemansky & Bonnet, 1994). Among parasites, prevalently ectoparasites, like Protozoa (Golemansky & Bonnet, 1994), Temnocephali- da (Matjašič, 1959), Acari (Dusbábek, 1998) and Diptera (Matile, 1994; Obame-Nkoghe et al., 2017), have been reported, mostly in cave-dwelling vertebrates. Among endoparasites, some Protozoa—mostly Apicomplexa and Ciliophora—(Golemansky & Bonnet, 1994; Muzzell et al., 1997), Trematoda and Nematoda (Muzzell et al., 1997) and Acanthocephala (Amin et al., 2019) were re- corded, both in vertebrates and invertebrates. In cave-dwelling Holarctic Orthoptera and Myr- iapoda, at the end of the past century, Golemansky and Bonnet (1994) counted 25 species of Gregarinida, which directed them to the conclusion that the number of en- doparasitic species is not lesser in subterranean hosts in comparison to the epigean hosts. However, in orthop- terans this is expected, since most of them are not tro- globiotic species and share both the hypogean and epi- gean ecophases in their life cycle (Novak & Kuštor, 1983; Dessuter-Grandcolas et al., 1998; Lipovšek et al., 2011). Thus, the Golemansky and Bonnet (1994) and Hum- phreys (2018) observations of under-sampling are still in power; a challenge for a systematic comparative study of endoparasites in cave-dwelling hosts. Gregarines parasitizing subterranean arthropods are poorly known; indeed, only sporadic findings from cave-dwelling centipedes, millipedes and insects have been hitherto reported (e.g., Watson, 1916; Semans, 1939, 1943; Théodoridès & Ormières, 1959; Golemansky & Lipa, 1991; Golemansky & Bonnet, 1994; Golemansky, 2015). Almost a dozen of gregarine species has been de- scribed in the cave crickets of the family Rhaphidophori- dae, most of them in Ellis'es and Watson's papers a hun- dred years ago in North America (Ellis, 1913a, 1913b; Watson, 1915, 1916; Semans, 1939, 1943; Desportes & Schrével, 2013). At the end of the 20 th century the only European rhaphidophorid gregarine, Gregarina troglo- phili Golemansky & Lipa, 1991, was described in Bul- garia, parasitizing the cave cricket Troglophilus neglectus Krauss (Golemansky & Lipa, 1991). While early papers on the gregarines parasitizing the Rhaphidophoridae provide only rough morphological description of soli- tary individuals and the description of their associations, Golemansky and Lipa (1991) report metric data suffi- cient to characterize both trophozoites and gamonts. During our ecological and biological research in subterranean habitats in Slovenia, we recorded, for the first time, one known, and one new gregarine species from two native cave cricket species, Troglophilus ne- glectus Krauss, 1879 and T. cavicola (Kollar, 1833). These two cave crickets are the most important non-troglobi- otic invertebrates in central Europe with respect to their mass and energy bulk in caves (Novak et al., 2013). Here we deal with the two gregarine species; we provide an emended description of Gregarina troglophili Goleman- sky & Lipa, 1991 and describe the newly-discovered spe- cies, Gregarina bernardae sp. n. MATERIAL AND METHODS Twenty-four adult and immature Troglophilus neglectus and three T. cavicola adults were taken alive manually from the following caves: Bečka jama (Cad. No. 6275, ac- cording to the Cave Register of the Karst Research Insti- tute ZRC SAZU and Speleological Association of Slove- nia; 45.66962° N, 15.34421° E, 185 m a.s.l.) near Metlika, Ferranova buža (Cad. No. 8085; 45.97627° N, 14.25206° E, 660 m a.s.l.) near Vrhnika, and Zguba jama (Cad. No. 6290; 45.79648° N, 14.21377° E, 561 m a.s.l.) near Posto- jna–all of them in the Slovenian Dinaric karst area–and transported to the Laboratory of Animal Physiology and Ethology at the University of Maribor. DUŠAN DEVETAK & TONE NOV AK ACTA CARSOLOGICA 50/2-3 – 2021 318 The crickets were eviscerated and the alimentary ca- nal (diagram in Novak & Kuštor, 1983, Fig. 5: digestive tube) dissected in insect Ringer’s solution (Laughton et al., 2011). Live gregarines were observed in Ringer’s so- lution. Permanent microscope slide preparations of gre- garines were made using wet smears of the cave crickets gut tissues with parasites, and fixed by flotation on hot fixative (ethanol, formaldehyde, and acetic acid) (Clop- ton & Hays, 2006). Then, the preparations were stained with Semichon’s acetocarmine (Semichon, 1924), de- hydrated in ethanol series, cleared in xylene series, and mounted in Canada balsam. Gregarines were photographed using a Nikon E 800 microscope with a mounted digital camera Nikon DN100. Thirty associations and 25 solitary gamonts of each species were measured. Measurements were taken from the digitized images of living specimens using Eclipse Net v. 1.16.3 image analysis software. Gregarine anatomical and ontogenetic nomenclature largely fol- lows that proposed by Levine (1971), deferring to Clop- ton (2009) on syzygy and association. Terminology for shapes of planes and solids follows Clopton (2004). Statistical analyses were performed in GraphPad Prism 6 software. Normality of data obtained was tested using the Shapiro-Wilk test. Given that the data tested were normally distributed, differences of means for two species were tested using the Student’s t-test. RESULTS In both Troglophilus neglectus and T. cavicola, Gregari- na troglophili was recorded; T. cavicola was for the first time found to be the host of this species. In addition to G. troglophili, a new Gregarina species was observed in T. neglectus only and is described here. Since the origi- nal description of the Gregarina troglophili Golemansky & Lipa, 1991 is based on a small number of individu- als (Golemansky & Lipa, 1991), we here provide a more complete description of the species, enabled by a larger sample. GREGARINES (APICOMPLEXA: EUGREGARINORIDA) PARASITIZING THE CAVE CRICKET TROGLOPHILUS (ORTHOPTERA: RAPHIDOPHORIDAE) IN THE SLOVENIAN KARST Figure 1. Live specimens of Grega- rina troglophili. A) solitary gam- ont; B) mature gamonts in asso- ciation; C) syzygy. Abbreviations: D – deutomerite; N – nucleus; P – protomerite; Pr – primite; Sa – satellite. ACTA CARSOLOGICA 50/2-3 – 2021 319 Gregarina troglophili Golemansky & Lipa, 1991 (Figures 1A–C) Material: Host: Troglophilus neglectus Krauss, 1879 Localities and prevalence: - Bečka jama, 15. 6. 2012, 3/4 individuals infected (G. troglophili is co-occurring with G. bernardae sp. n.). - Ferranova buža, 11. 6. 2012, 3/10 individuals infected; 28. 11. 2012, 3/5 individuals infected (G. troglophili is co-occurring with G. bernardae sp. n.). - Zguba jama, 6. 6. 2012, 1/5 individuals infected. Host: Troglophilus cavicola (Kollar, 1833) Localities and prevalence: - Ferranova buža, 11. 6. 2012, 1/3 individuals infected. New host report. DUŠAN DEVETAK & TONE NOV AK Table 1: Morphometric data for solitary gamonts of Gregarina troglophili and G. bernardae sp. n. N = 25 individuals. Statistically signifi- cant differences in bold. DL – deutomerite length; DWE – deutomerite width at equatorial axis; DWM – maximum deutomerite width; PL – protomerite length; PWE – protomerite width at equatorial axis; PWM – maximum protomerite width; SWT – p values of Shapiro-Wilk test; TL – total length. All measures in µm. G. troglophili G. bernardae sp. n. t-test Mean ± SD Range SWT Mean ± SD Range SWT df = 48 TL 220.6 ± 72.4 148 – 396 0.062 197.2 ± 73.5 110 – 311 0.062 t = 1.132 p = 0.263 PL 45.9 ± 7.4 35 – 58 0.120 37.2 ± 10.2 23 – 51 0.072 t = 3.433 p = 0.001 DL 174.6 ± 67.6 113 - 343 0.055 160.0 ± 63.8 86 – 261 0.051 t =0.790 p = 0.433 PWM 104.6 ± 38.6 72 – 190 0.062 61.0 ± 23.2 38 – 102 0.077 t = 4.835 p < 0.001 PWE 92.7 ± 33.5 66 – 165 0.051 58.9 ± 23 34 – 102 0.056 t = 4.157 p = 0.001 DWM 159.2 ± 71.7 102 – 320 0.071 79.7 ± 31 52 – 140 0.100 t = 5.087 p < 0.001 DWE 155.5 ± 69.8 100 – 305 0.110 77.5 ± 30.5 51 – 140 0.055 t = 5.120 p < 0.001 Table 2: Morphometric data for gamonts in associations of Gregarina troglophili and G. bernardae sp. n. N = 30 associations. Statistically significant differences in bold. PrDL – primite deutomerite length; PrDWE – primite deutomerite width at equatorial axis; PrDWM – maximum primite deutomerite width; PrPL – primite protomerite length; PrPWE – primite protomerite width at equatorial axis; PrPWM – maximum primite protomerite width; PrTL – primite total length; SaDL – satellite deutomerite length; SaDWE – satellite deutomerite width at equatorial axis; SaDWM – maximum satellite deutomerite width; SaPL – satellite protomerite length; SaPWE – satellite protom- erite width at equatorial axis; SaPWM – maximum satellite protomerite width; SaTL – satellite total length; SWT – P values of Shapiro- Wilk test. All measures in µm. G. troglophili G. bernardae sp. n. t-test Mean ± SD Range SWT Mean ± SD Range SWT df = 58 Primite PrTL 221.1 ± 77.3 92 – 365 0.440 310.3 ± 47.4 228 – 390 0.360 t = 5.393, p < 0.001 PrPL 45.9 ± 14.8 20 – 75 0.370 57.5 ± 11.1 38 – 76 0.300 t = 3.445, p = 0.001 PrDL 175.2 ± 66.4 72 – 295 0.180 252.7 ± 37.8 188 – 326 0.550 t = 5.561, p < 0.001 PrPWM 100.6 ± 37.8 50 – 193 0.051 106.0 ± 22.2 70 – 145 0.140 t = 0.675, p = 0.503 PrPWE 90.4 ± 32.9 47 – 173 0.062 103.7 ± 21.7 69 – 145 0.170 t = 1.854, p = 0.069 PrDWM 159.0 ± 63.9 68 – 315 0.075 126.9 ± 29.8 79 – 180 0.051 t = 2.487, p = 0.015 PrDWE 157.3 ± 63.3 66 – 312 0.077 123.3 ± 28.9 77 – 180 0.071 t = 2.673, p = 0.010 Satellite SaTL 203.8 ± 70.3 110 – 300 0.051 333.5 ± 37.3 277 – 408 0.071 t = 8.924, p < 0.001 SaPL 21.8 ± 5.7 10 – 31 0.260 30.4 ± 8.3 18 – 53 0.053 t = 4.673, p < 0.001 SaDL 182 ± 70.8 100 – 278 0.052 303.1 ± 39.4 242 – 375 0.062 t = 8.184, p < 0.001 SaPWM 88.5 ± 38.6 37 – 160 0.051 82.4 ± 18 58 – 120 0.051 t = 0.780, p = 0.438 SaPWE 79.5 ± 29.8 37 – 126 0.060 78.0 ± 16.9 53 – 110 0.100 t = 0.245, p = 0.807 SaDWM 138.8 ± 61.8 50 – 224 0.053 125.0 ± 30.1 87 – 195 0.051 t = 1.100, p = 0.276 SaDWE 132.2 ± 57.2 49 – 216 0.051 114.3 ± 26.3 80 – 183 0.051 t = 1.550, p = 0.125 ACTA CARSOLOGICA 50/2-3 – 2021 320 Description: - Solitary gamonts (Figure 1A): Total length 148–396 µm. Protomerite shallowly to broadly ovoid, mark- edly constricted at protomerite-deutomerite septum. Deutomerite orbicular to broadly elliptoid. Nucleus orbicular. Measurements and morphometric indexes of solitary gregarines are given in Tables 1 and 3. - Associations (Figure 1B): Gamonts anisomorphic; epimerite absent; associations caudo-frontal. - Primite: Total length 92–365 µm. Protomerite broadly or shallowly ovoid to shallowly elliptoid. Deutomerite orbicular to broadly elliptoid, constricted at protom- erite-deutomerite septum. Nucleus orbicular. Mea- surements and morphometric indexes of primites are presented in Tables 2 and 4. - Satellite: Total length 110–300 µm. Protomerite de- pressed or very depressed ovoid to very depressed oblong. Deutomerite broadly obovoid to finely ob- deltoid, not constricted at protomerite-deutomerite septum. Measurements and morphometric indexes of satellites are presented in Tables 2 and 4. Syzygy is presented in Figure 1C. Gregarina bernardae sp. n. (Figures 2-4) Material: Host: Troglophilus neglectus Krauss, 1879 Localities and prevalence: - Bečka jama, 15. 6. 2012, 3/4 individuals infected (the new species is co-occurring with G. troglophili). - Ferranova buža, 28. 11. 2012, 3/5 individuals infected (the new species is co-occurring with G. troglophili). Description: - Solitary individuals (Figures 2B, 4A): Total length 110–311 µm. Protomerite shallowly ovoid to shallow- ly oblong, markedly constricted at protomerite-deu- tomerite septum. Deutomerite oblong to dolioform. Nucleus orbicular. Measurements and morphometric indexes of solitary gregarines are presented in Tables 1 and 3. - Associations (Figures 2A, 3): Gamonts anisomorphic; epimerite absent; associations caudo-frontal. - Primite: Total length 228–390 µm. Protomerite shal- lowly oblong or shallowly dolioform to shallowly ovoid. Deutomerite shallowly oblong to oblong, con- stricted at protomerite-deutomerite septum. Nucleus orbicular. Measurements and morphometric indexes of primites are presented in Tables 2 and 4. - Satellite: Total length 277–408 µm. Protomerite shal- lowly or very shallowly oblong to depressed oblong. Deutomerite narrowly dolioform or dolioform to nar- rowly obdeltoid, constricted at protomerite-deutom- erite septum. Nucleus orbicular. Measurements and morphometric indexes of satellites are presented in Tables 2 and 4. Syzygy is presented in Figure 4B. Oocysts of Gregarina sp.: Unfortunately, we found oocysts exclusively in the Troglophilus individuals in- fested with both Gregarina species. Since oocysts of G. troglophili have not been described, we could not identify whether the oocysts belonged to G. troglophili or G. ber- nardae. GREGARINES (APICOMPLEXA: EUGREGARINORIDA) PARASITIZING THE CAVE CRICKET TROGLOPHILUS (ORTHOPTERA: RAPHIDOPHORIDAE) IN THE SLOVENIAN KARST Figure 2: Specimens of Gregarina bernardae sp. n. A) mature gam- onts in association; B) solitary gamont. For abbreviations see Fig- ure 1. ACTA CARSOLOGICA 50/2-3 – 2021 321 DUŠAN DEVETAK & TONE NOV AK Figure 3: Live specimens of Grega- rina bernardae sp. n. A, B) mature gamonts in association. For abbre- viations see Figure 1. Figure 4: Live specimens of Grega- rina bernardae sp. n. A) solitary gamont; B) syzygy. For abbrevia- tions see Figure 1. ACTA CARSOLOGICA 50/2-3 – 2021 322 GREGARINES (APICOMPLEXA: EUGREGARINORIDA) PARASITIZING THE CAVE CRICKET TROGLOPHILUS (ORTHOPTERA: RAPHIDOPHORIDAE) IN THE SLOVENIAN KARST Table 3: Morphometric indexes for solitary gamonts of Gregarina troglophili and G. bernardae sp. n. N=25 individuals. Statistically sig- nificant differences in bold. DL/DWM – deutomerite orbicularity; DL/PL – relative individual length; DWM/PWM – relative individual width; PL/PWM – protomerite orbicularity; TL/DL – relative deutomerite length; TL/DWM – individual orbicularity; TL/PL – relative protomerite length. G. troglophili G. bernardae sp. n. t-test Mean ± SD Range Mean ± SD Range df = 48 TL/PL 4.75 ± 1.06 3.65 – 7.58 5.18 ± 0.66 4.34 – 6.33 t = 1.717, p = 0.092 TL/DL 1.28 ± 0.18 1.15 – 1.38 1.24 ± 0.04 1.19 – 1.30 t = 2.717, p = 0.009 TL/DWM 1,44 ± 0.18 1.13 – 1.73 2.49 ± 0.39 1.98 – 3.02 t = 12.230, p < 0.001 PL/PWM 0.47 ± 0.09 0.28 – 0.59 0.64 ± 0.13 0.48 – 0.89 t = 5.236, p < 0.001 DL/DWM 1.12 ± 0.13 0.92 – 1.36 2.00 ± 0.30 1.57 – 2.36 t = 13.190, p < 0.001 DL/PL 3.75 ± 1.06 2.65 – 6.58 4.18 ± 0.66 3.34 – 5.33 t = 1.717, p = 0.092 DWM/PWM 1.49 ± 0.12 1.32 – 1.69 1.31 ± 0.09 1.13 – 1.41 t = 6.127, p < 0.001 Table 4: Morphometric indexes for gamonts in associations of Gregarina troglophili and G. bernardae sp. n. N = 30 associations. Statisti- cally significant differences in bold. PrDL/PrDWM – primite deutomerite orbicularity; PrDL/PrPL – relative primite length; PrDL/SaDL – relative gamont deutomerite length; PrDWM/PrPWM – relative primite width; PrDWM/SaDWM – relative gamont deutomerite width; PrPL/PrPWM – primite protomerite orbicularity; PrPL/SaPL – relative gamont protomerite length; PrPWM/SaPWM – relative gamont protomerite width; PrTL/PrDL – relative primite deutomerite length; PrTL/PrDWM – primite orbicularity; PrTL/PrPL – relative primite protomerite length; PrTL/SaTL – relative gamont length; SaDL/SaDWM – satellite deutomerite orbicularity; SaDL/SaPL – relative satel- lite length; SaDWM/SaPWM – relative satellite width; SaPL/SaPWM – satellite protomerite orbicularity; SaTL/SaDL – relative satellite deutomerite length; SaTL/SaDWM – satellite orbicularity; SaTL/SaPL – relative satellite protomerite length. G. troglophili G. bernardae sp. n. t-test Mean ± SD Range Mean ± SD Range df = 58 PrTL/PrPL 4.91 ± 1.34 3.40 – 9.19 5.45 ± 0.47 4.67 – 6.25 t = 2.091, p = 0.041 PrTL/PrDL 1.28 ± 0.07 1.12 – 1.42 1.23 ± 0.02 1.19 – 1.27 t = 3.655, p = 0.001 PrTL/PrDWM 1.42 ± 0.14 1.16 – 1.63 2.51 ± 0.30 2.06 – 3.16 t = 17.700, p < 0.001 PrPL/PrPWM 0.47 ± 0.1 0.32 – 0.63 0.54 ± 0.05 0.48 – 0.66 t = 3.587, p =0.001 PrDL/PrDWM 1.11 ± 0.12 0.91 – 1.37 2.04 ± 0.27 1.67 – 2.66 t = 17.100, p < 0.001 PrDL/PrPL 3.91 ± 1.34 2.40 – 8.19 4.45 ± 0.47 3.67 – 5.25 t = 2.089, p = 0.041 PrDWM/PrPWM 1.57 ± 0.17 1.34 – 1.90 1.19 ± 0.07 1.06 – 1.29 t = 10.990, p < 0.001 SaTL/SaPL 10.00 ± 4.2 4.58 – 17.40 11.80 ± 3.83 6.28 – 21.80 t = 1.746, p = 0.086 SaTL/SaDL 1.14 ± 0.07 1.06 – 1.28 1.10 ± 0.03 1.05 – 1.19 t = 2.664, p = 0.010 SaTL/SaDWM 1.58 ± 0.35 1.16 – 2.40 2.77 ± 0.49 1.69 – 3.47 t = 10.910, p < 0.001 SaPL/SaPWM 0.30 ± 0.15 0.13 – 0.55 0.39 ± 0.13 0.18 – 0.62 t = 2.389, p = 0.020 SaDL/SaDWM 1.37 ± 0.27 1.08 – 2.02 2.51 ± 0.42 1.58 – 3.12 t = 12.500, p < 0.001 SaDL/SaPL 8.95 ± 4.21 3.58 – 16.40 10.80 ± 3.83 5.28 – 20.80 t = 1.793, p = 0.078 SaDWM/SaPWM 1.55 ± 0.2 1.29 – 1.93 1.52 ± 0.12 1.19 – 1.65 t = 0.773, p = 0.442 PrTL/SaTL 1.09 ± 0.16 0.77 – 1.42 0.93 ± 0.08 0.80 – 1.07 t = 4.959, p < 0.001 PrPL/SaPL 2.22 ± 0.92 1.05 – 4.21 2.04 ± 0.75 1.09 – 3.41 t = 0.845, p = 0.402 PrDL/SaDL 0.97 ± 0.15 0.71 – 1.28 0.83 ± 0.07 0.75 – 0.97 t = 4.671, p < 0.001 PrPWM/SaPWM 1.21 ± 0.36 0.71 – 1.97 1.30 ± 0.21 0.95 – 1.63 t = 1.147, p = 0.256 PrDWM/SaDWM 1.23 ± 0.38 0.85 – 2.03 1.03 ± 0.17 0.69 – 1.30 t = 2.648, p = 0.010 ACTA CARSOLOGICA 50/2-3 – 2021 323 DUŠAN DEVETAK & TONE NOV AK Taxonomic summary: Superphylum Alveolata Cavalier-Smith, 1991 Phylum Apicomplexa Levine, 1980 Class Conoidasida Levine, 1988 Subclass Gregarinasina Dufour, 1828 Order Eugregarinorida Léger, 1900 Family Gregarinidae Labbé, 1899 Genus Gregarina Dufour, 1828 Gregarina bernardae sp. n. Devetak & Novak, 2021 - Species diagnosis: Solitary individuals with shallowly ovoid to shallowly oblong protomerite and oblong to dolioform deutomerite, on average 197.2 µm long and 79.7 µm wide. Associations caudo-frontal. Shallowly oblong or shallowly dolioform to shallowly ovoid pri- mite protomerite, on average 57.5 µm long and 106 µm wide (maximum width). Shallowly oblong to ob- long primite deutomerite, on average 252.7 µm long and 126.9 µm wide (maximum width). Shallowly or very shallowly oblong to depressed oblong satellite protomerite, on average 30.4 µm long and 82.4 µm wide (maximum width). Narrowly dolioform or doli- oform to narrowly obdeltoid satellite deutomerite, on average 303.1 µm long and 125 µm wide (maximum width). - Type host: Troglophilus neglectus Krauss, 1879 (Or- thoptera: Ensifera: Rhaphidophoridae). - Site of infection: Intestine. - Type locality: Bečka jama, Slovenia (Cad. No. 6275; 45.66962° N, 15.34421° E, 185 m a.s.l., Boldraž near Metlika). - Specimens deposited: The holotype is a hapantotype slide, deposited in the Laboratory of Animal Physiol- ogy and Ethology, FNM UM, University of Maribor, Maribor, Slovenia. The holotype is a solitary gregarine accompanied by an association on slide TROGNEG 2012-01 (Figure 2). The other solitary gregarines and associations in slides TROGNEG 2012-02 and TROG- NEG 2012-03 are paratypes. - Etymology: The specific epithet is given in honour of the first author’s wife, Bernarda Devetak, who has been supporting the first author during his field and lab studies. Remarks Gregarina bernardae sp. n. is clearly distinguished from G. troglophili Golemansky & Lipa, 1991 by both mor- phometric indexes and size in solitary gamonts (Tables 1, 3) and associations (Tables 2, 4). The t-test revealed the following significant differences in morphometric indexes of solitary individuals between the two spe- cies: relative deutomerite length, individual orbicular- ity (Figure 6C), protomerite orbicularity, deutomerite orbicularity (Figure 6D), and relative individual width (Table 3). Associations of Gregarina bernardae sp. n. are distinguished from those of G. troglophili by the follow- ing morphometric indexes: relative primite deutomer- ite length, primite orbicularity (Figure 5A), primite protomerite orbicularity, primite deutomerite orbicu- larity (Figure 5D), relative primite width, satellite or- bicularity (Figure 5B), satellite deutomerite orbicularity (Figure 6B), relative gamont length, and relative gamont deutomerite length (Table 4). Figure 5: Morphometric indexes of associations of Gregarina troglo- phili and G. bernardae sp. n. A) In- dexes of primite total length (PrTL) and maximum primite deutom- erite width (PrDWM). B) Indexes of satellite total length (SaTL) and maximum satellite deutomerite width (SaDWM). C) Indexes of satellite deutomerite length (SaDL) and satellite protomerite length (SaPL). D) Indexes of primite deu- tomerite length (PrDL) and maxi- mum primite deutomerite width (PrDWM). Circles – G. troglophili; triangles – G. bernardae sp. n. All measures in µm. ACTA CARSOLOGICA 50/2-3 – 2021 324 DISCUSSION Knowledge on gregarines in arthropods in Slovenia is scarce. Until now, gregarines have only been reported in some epigean insects and myriapods (Devetak et al., 2013, 2019; Devetak, 2014; Rueckert & Devetak, 2017), but not in cave-dwelling arthropods. This paper is the first report on gregarines in the cave crickets Troglophilus neglectus and T. cavicola of the family Rhaphidophori- dae from karstic caves in Slovenia. We found two spe- cies, Gregarina troglophili Golemansky & Lipa, 1991 and G. bernardae sp. n., sometimes in the same hosts – the syntopy posing a nice opportunity for their comparison. Our relatively rich collection of G. troglophili ena- bled us to provide a more comprehensive description in comparison to the original description (Golemansky & Lipa, 1991), and a comprehensive comparison of G. troglophili and G. bernardae sp. n. The two species sub- stantially differ in the shape of deutomerite. In solitary individuals, the shape of the deutomerite in G. troglophili is orbicular to broadly elliptoid, whilst in G. bernardae sp. n. it is oblong to dolioform. In associations, the primite deutomerite in G. troglophili is orbicular to broadly ellip- toid, whilst in G. bernardae sp. n. it is shallowly oblong to oblong. In contrast to a broadly obovoid to finely obdel- toid satellite deutomerite in G. troglophili, it is narrowly dolioform or dolioform to narrowly obdeltoid in G. ber- nardae sp. n. All the gregarines parasitizing in North American rhaphidophorids (Ellis, 1913a, 1913b; Watson, 1915, 1916; Semans, 1939) differ from G. troglophili by both the shape of solitary individuals and associations, but two of them, namely Gregarina ceutophili Semans, 1939 and G. hadenoeci Semans, 1939 resemble G. bernardae sp. n. In contrast to G. bernardae sp. n., the primite protomerite in G. ceutophili has sometimes blunt apex and satellite sep- tum is devoid of a marked constriction (Semans, 1939) which is constantly present in G. bernardae sp. n. asso- ciations. When taking into account morphometric data from the Semans’ original description (Semans, 1939), G. ha- denoeci significantly differs from G. bernardae sp. n. in morphometric indexes. Differences refer to the primite orbicularity (PrTL/PrDWM; G. hadenoeci: 2.43 vs G. bernardae sp. n.: 2.51), primite protomerite orbicularity (PrPL/PrPWM; 0.68 vs 0.54), primite deutomerite or- bicularity (PrDL/PrDWM; 1.87 vs 2.04), satellite orbicu- larity (SaTL/SaDWM; 1.91 vs 2.77), satellite protomerite orbicularity (SaPL/SaPWM; 0.5 vs 0.39), and satellite deutomerite orbicularity (SaDL/SaDWM; 1.57 vs 2.51). In addition to these differences, gregarines are regarded highly host-specific parasites, generally restricted to a single host species (Levine, 1988). We did not perform any molecular phylogenetic analyses. In conclusion, two gregarines reported here strict- ly differ from gregarines in other rhaphidophorid cave crickets. Although numerous papers have been pub- lished on gregarine species in Europe (see Desportes & Schrével, 2013) and there are several monographs (e.g., Lipa, 1967; Geus, 1969), our knowledge on the diversity GREGARINES (APICOMPLEXA: EUGREGARINORIDA) PARASITIZING THE CAVE CRICKET TROGLOPHILUS (ORTHOPTERA: RAPHIDOPHORIDAE) IN THE SLOVENIAN KARST Figure 6: Morphometric indexes of associations and solitary gam- onts of Gregarina troglophili and G. bernardae sp. n. A) Indexes of primite deutomerite length (PrDL) and primite protomerite length (PrPL). B) Indexes of satel- lite deutomerite length (SaDL) and maximum satellite deutomerite width (SaDWM). C) Indexes of total length (TL) and maximum deutomerite width (DWM). D) In- dexes of deutomerite length (DL) and maximum deutomerite width (DWM). Circles – G. troglophili; triangles – G. bernardae sp. n. All measures in µm. ACTA CARSOLOGICA 50/2-3 – 2021 325 and distribution of gregarines infesting the European subterranean taxa is still deficient. The finding of a new species suggests the existence of a variety of gregarines in cave-dwelling arthropods that have not been investigated with this regard. We would like to stress that the para- site diversity of cave-dwelling animals contributes to the whole diversity of the subterranean fauna; thus, it would be worth to devote our attention into this research. ACKNOWLEDGEMENT We would like to thank Andrea Bardůnek Valigurová (Brno, Czech Republic), Paweł Sienkiewicz (Poznań, Poland) and an anonymous reviewer for their insightful comments on an early version of the paper. This study was supported by the Slovenian Research Agency and the Research Programme Computationally Intensive Com- plex Systems (P1-0403). REFERENCES Amin, O.M., Heckmann, R.A., Fišer, Ž., Zakšek, V., Herlyn, H., Kostanjšek, R., 2019. 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