1 2 3 1 Gregarines (Apicomplexa, Gregarinasina) in psocids (Insecta, Psocoptera) including a new 4 5 6 2 species description and their potential use as pest control agents. 7 8 3 9 a* b 10 4 Sonja Rueckert , Dušan Devetak 11 12 5 13 14 6 aSchool of Applied Sciences, Edinburgh Napier University, Sighthill Campus, Edinburgh, 15 16 7 EH11 4BN, Scotland, UK 17 18 8 bDepartment of Biology, Faculty of Natural Sciences and Mathematics, University of 19 20 9 Maribor, Koroška cesta 160, 2000 Maribor, Slovenia 21 22 23 10 24 * 25 11 Corresponding author. E-mail address: [email protected] 26 27 12 28 29 13 30 31 14 32 33 15 34 35 16 36 37 17 38 39 40 18 41 42 19 43 44 20 45 46 21 47 48 22 49 50 23 51 52 24 53 54 25 55 56 57 58 59 1 60 61 62 63 26 Abstract: 64 65 27 Gregarine apicomplexans are unicellular organisms that infect invertebrate hosts in marine, 66 67 28 freshwater and terrestrial habitats. The largest group of invertebrates infested on land is the 68 69 29 insects. The insect order Psocoptera (booklice) has recently gained wider interest due to 70 71 30 specimens occurring in stored food products and therefore being considered pest organisms. 72 73 31 Biological control agents are often used to eliminate pest organisms. In this study we 74 75 examined the psocid Dorypteryx domestica, an invasive psocid species that is spreading all 76 32 77 78 33 over the world. We were able to isolate and describe a new gregarine species (Enterocystis 79 80 34 dorypterygis sp. n.) infecting D. domestica. The trophozoites are panduri- or pyriform and 81 82 35 their association/syzygy is caudo-frontal. The surface is inscribed by longitudinal epicytic 83 84 36 folds covering the complete cell. Phylogenetic analyses of the SSU rDNA gene revealed an 85 86 37 only weakly supported relationship with two Gregarina species G. ormieri and G. 87 88 38 basiconstrictonea, both from tenebrionid beetles. Gregarines have been proposed to have 89 90 39 some potential as biological control agents for several insects. Identifying the gregarine 91 92 40 species infecting pest organisms like psocids is a first step and prerequisite for the probable 93 94 95 41 utilization of these parasites as biological control agents in the future. 96 97 42 98 99 43 Keywords 100 101 44 Apicomplexan parasites, Enterocystidae, phylogeny, biological control agent 102 103 45 104 105 106 46 107 108 47 109 110 48 111 112 49 113 114 50 115 116 117 118 2 119 120 121 122 51 Introduction 123 124 52 Psocoptera is an order of small soft-bodied hemimetabolous insects commonly called psocids, 125 126 53 barklice or booklice. There are around 2,000 species described in the world. The geographical 127 128 54 origin of many domestic species remains unknown, as psocids have been transported by 129 130 55 humans, via the holds of ships, in packing materials and trade goods (New, 1987). Dorypteryx 131 132 56 domestica (Smithers, 1958) is an interesting psocid species that was originally described from 133 134 human habitations in Zimbabwe, Africa and is an invasive species spreading all over the 135 57 136 137 58 world since 1973 (Lienhard, 1977). Since then it has been detected from at least 16 European 138 139 59 countries. 140 141 60 A considerable number of psocids occur in buildings, such as human dwellings, food stores, 142 143 61 warehouses and granaries (Baz and Monserrat, 1999; Mockford, 2003). In general, Psocoptera 144 145 62 feed on algae, fungi, lichens, particles of organic debris, small eggs and dead bodies of insects 146 147 63 (Mockford, 2003). Some species occur typically in domestic environments such as humid 148 149 64 rooms, basements, damp walls where they feed on fungal hyphae and spores of moulds (Baz 150 151 65 and Monserrat, 1999). A few psocid species occurring in buildings feed on the paste and 152 153 154 66 bindings of old books as well as on the fungal spores and hyphae, which invade the pages 155 156 67 (Mockford, 2003). In human dwellings, the main effect due to psocopteran presence in/on 157 158 68 buildings is lowering property values (New, 1987). In Spain for example, three psocid species 159 160 69 – one of them was Dorypteryx domestica – have been described as one cause for the 161 162 70 propagation of the alga Pleurococcus on recently constructed buildings (Baz and Monserrat, 163 164 71 1999), which is at least disfiguring, but might also have effects on the decay of the building 165 166 72 material. 167 168 73 This insect order has only recently become of greater interest, when a large number of 169 170 171 74 specimens have been found in stored products. Even though the infestation of food products 172 173 75 by psocids rarely causes health problems in humans such as asthmatic reactions and allergies 174 175 176 177 3 178 179 180 76 from house dust (Spieksma & Smits, 1975; Mockford, 2003) or skin diseases (Conci and 181 182 183 77 Franceschi, 1953; Agostini et al., 1982), it is still unhygienic and certain psocid species can 184 185 78 even serve as intermediate hosts of some ruminant infecting cestodes (Svadzhian, 1963; 186 187 79 Kuznetsov, 1966). 188 189 80 190 191 81 Some psocids, like Dorypteryx domestica, may occasionally become a nuisance in 192 193 82 habitations, or play a role as pest insects in stored food products. Parasites and predators are 194 195 83 known be important natural regulators of pest population densities in some pest insects. So 196 197 84 far, endoparasites (gregarines, cestodes, nematodes and fungi), ectoparasites (acari) and 198 199 parasitoids (Hymenoptera) have been reported for psocids. As parasites can shape the 200 85 201 202 86 community structure of their host organisms, it is important to gain knowledge on these 203 204 87 parasites. Records on the occurrence of for example gregarines in psocids are sparse (e. g. 205 206 88 Geus, 1969; Sarkar and Haldar, 1980; Devetak et al., 2013) and there is no literature about 207 208 89 protozoan pathogens found in natural populations of Dorypteryx domestica. In general 209 210 90 gregarine apicomplexans infect marine, freshwater and terrestrial invertebrates. Due to the 211 212 91 sheer number of insects in terrestrial habitats most eugregarines are described from terrestrial 213 214 92 hosts. However, gregarines have been reported from less than 1% of all invertebrate species, 215 216 leaving the gregarine fauna of 99% of potential invertebrate hosts to be discovered (Clopton, 217 93 218 219 94 2000). Even though most gregarine species are described from insects, there are only five 220 221 95 gregarine species described from around 12 psocopteran species (two only identified to genus 222 223 96 level) belonging to 10 genera in Germany, Switzerland and India. There are four species of 224 225 97 septate gregarines (Hyalospora psocorum, H. stenopoci, Liposcelius coronata and 226 227 98 Ancyrophora similis) belonging to two families (Hirmocystidae Grassé, 1953; 228 229 99 Actinocephalidae Léger, 1892) and three genera (Hyalospora Schneider, 1875; Liposcelius 230 231 100 Sarkar & Haldar, 1980; Ancyrophora Léger, 1892). There is also one aseptate gregarine 232 233 234 235 236 4 237 238 239 101 (Enterocystis bengalensis) of the family Enterocystidae Codreanu, 1940 and the genus 240 241 242 102 Enterocystis Zwetkow, 1926, known to infect psocids (Sarkar, 1983; Desportes and Schrével, 243 244 103 2013). All available descriptions of these species are based on line drawings only (von 245 246 104 Siebold 1839, Geus, 1969; Sarkar and Haldar, 1980; Sarkar, 1983). There are no 247 248 105 ultrastructural or molecular data available for the species infecting Psocoptera hosts 249 250 106 (Desportes and Schrével, 2013). 251 252 107 253 254 108 In the current study we set out to investigate Dorypteryx domestica from Slovenia for the 255 256 109 presence of gregarines. We studied the general morphology and phylogenetic position of the 257 258 gregarine found to parasitize D. domestica and we discuss the possibility of employing 259 110 260 261 111 gregarines as biological control agents. 262 263 112 264 265 113 Material and Methods 266 267 114 Collection and isolation of organisms 268 269 115 Specimens of Dorypteryx domestica were collected by the second author from a basement of 270 271 272 116 a house in Maribor, Slovenia (46°33'58.5"N 015°39'15.2"E). The gut content was released in 273 274 117 0.9% saline solution by teasing apart the intestines of the psocid with fine-tipped forceps 275 276 118 under a dissecting microscope (Zeiss Stemi 2000). The gut material was examined under an 277 278 119 inverted microscope (Zeiss Axiovert A1) and parasites were isolated with a handdrawn glass 279 280 120 pipette and washed three times in 0.9% saline solution, before being examined and 281 282 121 photographed under the inverted microscope or prepared for DNA extraction. 283 284 122 285 286 123 Light and scanning electron microscopy 287 288 289 124 Light micrographs of specimens were taken with a digital camera Nikon DN100 attached to a 290 291 125 microscope (Nikon E 800). Differential interference contrast (DIC) light micrographs were 292 293 294 295 5 296 297 298 126 taken with a 5 megapixel CMOS camera AxioCam Erc 5s, attached to an inverted microscope 299 300 301 127 (Zeiss Axiovert1). 302 303 128 Between 10 and 20 specimens of the isolated gregarine species were prepared for scanning 304 305 129 electron microscopy (SEM). Individuals were deposited directly into the threaded hole of a 306 307 130 Swinnex filter holder, containing a 10 µm polycarbonate membrane filter (Millipore Corp., 308 309 131 Billerica, MA), that was submerged in 10 ml of 0.9% saline solution within a small canister (2 310 311 132 cm diam. and 3.5 cm tall). A piece of Whatman No. 1 filter paper was mounted on the inside 312 313 133 base of a beaker (4 cm diam. and 5 cm tall) that was slightly larger than the canister. The 314 315 134 Whatman filter paper was saturated with 4% (w/v) OsO and the beaker was turned over the 316 4 317 canister. The parasites were fixed by OsO vapors for 30 min. Ten drops of 4% (w/v) OsO 318 135 4 4 319 320 136 were added directly to the saline solution and the parasites were fixed for an additional 30 321 322 137 min. A 10-ml syringe filled with distilled water was screwed to the Swinnex filter holder and 323 324 138 the entire apparatus was removed from the canister containing seawater and fixative. Filters 325 326 139 were washed with water and dehydrated with a graded series of ethyl alcohol. They were 327

328 140 critical point dried with CO2. Filters were mounted on stubs, sputter coated with 5 nm of gold, 329 330 141 and viewed under a scanning electron microscope (Hitachi). Some SEM data were presented 331 332 142 on a black background using Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, 333 334 CA). 335 143 336 337 144 338 339 145 DNA isolation, PCR, cloning, and sequencing 340 341 146 Two individual trophozoites were isolated from the dissected hosts, washed three times in 342 343 147 saline solution, and deposited into a 1.5-ml microcentrifuge tube. DNA was extracted using 344 345 148 the MasterPureTM Complete DNA and RNA Purification Kit (Epicentre Biotechnologies, 346 347 149 Madison, WI). Small subunit rDNA (SSU rDNA) sequences were PCR-amplified using a 348 349 150 total volume of 25µl containing 2 µl of primer, 2.5 µl of DNA template, 20.5 µl of dH2O and 350 351 352 353 354 6 355 356 357 151 one PuReTaq Ready-to-go PCR Bead (GE Healthcare, Quebec, Canada). The SSU rDNA 358 359 360 152 sequences from these species were amplified in one fragment (~1800 basepairs) using 361 362 153 universal eukaryotic PCR primers F1 5´-GCGCTACCTGGTTGATCCTGCC-3´ and R1 5´- 363 364 154 GATCCTTCTGCAGGTTCACCTAC-3´. PCR was performed using the following protocol: 365 366 155 After 4 cycles of initial denaturation at 94 °C for 4.30 min, 45 °C for 1 min and 72 °C for 1.45 367 368 156 min, 34 cycles of 94 °C for 30 sec (denaturation), 50 °C for 1 min (annealing), 72 °C for 1.45 369 370 157 min (extension), followed by a final extension period at 72 °C for 10 min. PCR products 371 372 158 corresponding to the expected size were gel isolated using the UltraCleanTM 15 DNA 373 374 159 Purification kit (MO Bio, Carlsbad, California) and cloned into the pSC-A-amp/kan vector 375 376 using the StrataClone PCR Cloning Kit (Stratagene, AgilentTechnologies, California). Eight 377 160 378 379 161 cloned plasmids were digested with EcoRI and screened for size. Two clones were sequenced 380 381 162 with ABI big dye reaction mix using vector primers and internal primers oriented in both 382 383 163 directions using the cycle sequencing technology on an ABI 3730XL sequencing machine 384 385 164 (eurofins Genomics, Germany). 386 387 165 . 388 389 166 The new SSU rDNA sequences were initially identified by BLAST analysis and subsequently 390 391 167 verified with molecular phylogenetic analyses (GenBank Accession number for Enterocystis 392 393 dorypterygis sp. n.: KY697695). 394 168 395 396 169 397 398 170 Molecular phylogenetic analysis 399 400 171 The new SSU rDNA sequence was aligned with 116 other SSU rDNA sequences, 401 402 172 representing the major lineages of gregarines (with an emphasis on terrestrial gregarines and 403 404 173 environmental sequences) and dinozoans as relevant outgroup. The 117-sequence alignment 405 406 174 was subsequently edited and fine-tuned using MacClade 4.08 (Maddison and Maddison, 407 408 175 2005). The program PhyML (Guindon and Gascuel 2003) was used to analyze the 117- 409 410 411 412 413 7 414 415 416 176 sequence alignment (774 unambiguously aligned sites; gaps excluded) with maximum- 417 418 419 177 likelihood (ML). Smart Model Selection selected a general-time reversible (GTR) model of 420 421 178 nucleotide substitutions (Posada and Crandall, 1998) that incorporated invariable sites and a 422 423 179 discrete gamma distribution (six categories) (GTR + G+ I + F model:  = 0.725 and fraction 424 425 180 of invariable sites = 0.187) under the Akaike Information Criterion (AIC) (Guindon et al., 426 427 181 2010). ML bootstrap analyses were performed on 500 pseudoreplicates, with one heuristic 428 429 182 search per pseudo-replicate (Zwickl, 2006), using the same program set to the GTR model +G 430 431 183 + I + F. Bayesian analysis of the 117-sequence dataset was performed using the program 432 433 434 184 MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001). The programme was set to operate using 435 436 185 the following parameters: nst=6, ngammacat=5, rates=invgamma. Parameters of Metropolis 437 438 186 Coupling Markov Chains Monte Carlo (mcmc) were set to: nchains=4, nruns=4, temp=0.2, 439 440 187 ngen=7000000, samplefreq=100, burninfrac= 0.5 (the first 50% of 70000 sampled trees, i.e. 441 442 188 the first 35000, were discarded in each run). The computation was performed on the CIPRES 443 444 189 Science Gateway V 3.3 (Miller et al., 2010). 445 446 190 447 448 191 449 450 192 Results 451 452 453 193 Of 31 investigated psocopteran specimens, 21 were infected with gregarines, giving an 454 455 194 infection prevalence of 68%. The mean intensity of infection was 13 gregarines per host with 456 457 195 a range of 6-24 gregarines per host. 458 459 196 460 461 197 Morphology of Enterocystis dorypterygis sp. n. 462 463 198 Enterocystis dorypterygis sp. n.: Trophozoites were isolated from the psocid Dorypteryx 464 465 199 domestica (Smithers, 1958) (Psocoptera; Psyllipsocidae). The cell morphology showed 466 467 200 general similarities with Enterocystis bengalensis from a psocid species (Psocathropos sp.) 468 469 470 471 472 8 473 474 475 201 described by Sarkar (1983). The cells were elongated and panduriform with a little 476 477 478 202 indentation at the anterior end of the cell, but no septum (Fig. 1). Trophozoites were 59.3 ± 479 480 203 6.9 µm (45.6– 69.0 µm, n = 35) long (mean ± SD; min–max; number) and 25.4±2.5 µm 481 482 204 (21.5–30.7 µm, n = 35) wide at their widest part. The anterior end was mostly a bit globular 483 484 205 and rounded, while the posterior end was more blunt (Fig. 1a-c). Some of the cells were more 485 486 206 pyriform with a long anterior neck-like region (Fig. 1a). The round nucleus [7.8 (5.6-10) µm x 487 488 207 8.7 (6.5-11), n = 21] was situated in the posterior half of the cell (Fig. 1a-b), but sometimes 489 490 208 shifted to the anterior half (Fig. 1c-d). Gametocysts were spherical and the diameter was 30 491 492 209 μm (25.5-39.2 μm; n=4). Associations between mature trophozoites (or gamonts) appear to be 493 494 caudo-frontal (Fig. 1c-d). The SEM micrographs demonstrated that the whole cell surface was 495 210 496 497 211 inscribed by longitudinal epicytic folds (~200) (Fig. 1e). Neither the anterior nor the posterior 498 499 212 end was free of folds (Fig. 1e-f). The epicytic folds appeared to be arranged in waves along 500 501 213 the longitudinal axis (Fig. 1e, g). In the middle of the cell, the density of folds was 6-8 502 503 214 folds/micron (Fig. 1g). Trophozoites were stiff and capable of gliding movements. 504 505 215 506 507 216 Molecular phylogeny of Enterocystis dorypterygis sp. n. 508 509 217 Molecular phylogenetic analyses of the 117-sequence dataset produced a tree topology with a 510 511 moderately supported clade of dinoflagellates (outgroup) and a moderately supported clade of 512 218 513 514 219 apicomplexan sequences (Fig. 2). The deeper branches within the tree were all poorly 515 516 220 resolved. Within the apicomplexans three clades were formed, consisting of (1) piroplasmids, 517 518 221 coccidians, cryptosporidians, rhytidocystids, monocystids, neogregarines and mainly 519 520 222 terrestrial eugregarines, (2) archi- and eugregarines from mainly polychaete hosts and (3) 521 522 223 mainly eugregarines from ascidian, polychaete, nemertean and crustacean hosts. The new 523 524 224 sequence of Enterocystis dorypterygis sp. n. clustered within the strongly supported clade of 525 526 225 mainly terrestrial eugregarines comprised of the genera Amoebogregarina, Gregarina, 527 528 529 530 531 9 532 533 534 226 Leidyana, Protomagalhaensia and the as archigregarine considered Caliculium. The new 535 536 537 227 sequence formed a low supported clade with two Gregarina species G. ormieri and G. 538 539 228 basiconstrictonea both from tenebrionid beetles. Those three sequences formed a sistergroup 540 541 229 to the strongly supported clade of Leidyana and Protomagalhaensia species. 542 543 230 544 545 231 Discussion 546 547 The majority of eugregarine species are reported from insects. Most of these belong to the 548 232 549 550 233 septate gregarines (trophozoite devided into protomerite and deutomerite by a septum) and 551 552 234 only a few belong to the aseptate gregarines (trophozoite composed of single compartment 553 554 235 lacking the septum). As they have been reported from less than 1% of the known insect 555 556 236 species the gregarine fauna of over 99% is still to be discovered (compare Clopton, 2000). A 557 558 237 good example here are the Psocoptera, of the ~2000 described psocid species, only 10 have 559 560 238 been reported to be infected with gregarine apicomplexans. In this study we were able to 561 562 239 describe a new gregarine species (Enterocystis dorypterygis sp. n.) from a new psocid host 563 564 240 species (Dorypteryx domestica). 565 566 567 241 568 569 242 Enterocystis dorypterygis sp. n. 570 571 243 The new gregarine species isolated from the psocopteran D. domestica is an aseptate 572 573 244 gregarine, and a first record of a gregarine infection in this psocid. The characteristic of a 574 575 245 missing septum distinguishes the new species from all described septate gregarines 576 577 246 (Hyalospora psocorum, H. stenopoci, Liposcelius coronata and Ancyrophora similis) 578 579 247 infecting Psocoptera (nine species including: Peripsocus alboguttatus, Amphigerontia 580 581 248 bifasciata, Psocus longicornis, P. quadripunctatus, Caecilius flavidus, Lachesilla quercus, 582 583 584 249 Mesopsocus unipunctatus, Graphopsocus cruciatus, Stenopsocus immaculatus). All nine 585 586 250 Psocoptera species are actually infected by H. psocorum (Geus, 1969), whereas all other 587 588 589 590 10 591 592 593 251 sepatate gregarine species are reported from just a single psocid host species (Desportes and 594 595 596 252 Schrével, 2013). There is only one known aseptate gregarine species (Enterocystis 597 598 253 bengalensis), which belongs to the family Enterocystidae Codreanu, 1940, and was described 599 600 254 by Sarkar (1983) from Psocathropos (syn. Psocatropos) sp. in India. Desportes (2013) 601 602 255 questions this identification and new species description as all other species belonging to the 603 604 256 family Enterocystidae infect the aquatic larvae of Ephemoptera and not any Psocopteridae. 605 606 257 The psocid genus infected with E. bengalensis is falsly named as Psocoptrips sp. in Desportes 607 608 258 (2013). The families/genera/species of aseptate gregarines known to infect terrestrial and 609 610 259 freshwater invertebrates, are all recorded from other invertebrates than Psocoptera. The newly 611 612 described gregarine species is most similar to Enterocystis bengalensis than any other 613 260 614 615 261 Enterocystis species, based on the morphology of the trophozoites and associations. Due to its 616 617 262 smaller size and the different host species the described gregarine is considered a new 618 619 263 Enterocystis species. 620 621 264 622 623 265 Molecular phylogeny of Enterocystis 624 625 266 Up to this date there is no reference sequence of any Enterocystis species available in any of 626 627 267 the public databases such as GenBank. The sequence of our newly described species E. 628 629 dorypterygis sp. n. clustered within the highly supported clade of mainly terrestrial gregarines 630 268 631 632 269 (Fig. 2). The closest relatives were species of the genus Gregarina infecting Tenebrionidae, 633 634 270 Leidyana and Protomagalhaensia both infecting Blattaria, all of which are septate 635 636 271 eugregarines. This is an example of another aseptate eugregarine clustering within a clade of 637 638 272 septate eugregarines, all infecting arthropods and in this case insects. It has been questioned 639 640 273 before, if the separation of septate and aseptate gregarines, estblished by Chakravarty (1959) 641 642 274 reflects the actual phylogenetic relationships of eugregarines (e.g. Rueckert et al., 2011). The 643 644 275 current study and the study by Rueckert et al. (2011) clearly show that the septate 645 646 647 648 649 11 650 651 652 276 eugregarines do not form a monophyletic clade. Therefore, the taxonomic separation of 653 654 655 277 sepatate and aseptate eugregarines based on a morphological feature (the septum) should be 656 657 278 deemed invalid. What still remains uncertain is the evolutionary history of certain septate and 658 659 279 aspetate gregarines infecting insects. At the moment there are two possible scenarios: i) 660 661 280 according to Leander (2006, 2008) a lecudinid stem group gave rise to all other eugregarine 662 663 281 lineages and the eugregarines of insects became comparmentalized by forming a transverse 664 665 282 septum between cell regions, so there might be some lineages that remained without a septum 666 667 283 or ii) it could be a loss of the septum giving rise to secondary aseptate gregarines as was 668 669 284 suggested by Grassé (1953). The latter one also indicating that the septum is not a reliable 670 671 character in gregarine taxonomy. One example of such an aseptate gregarine in an insect is 672 285 673 674 286 the genus Gamocystis, which only presents a septum in a very early trophozoite stage 675 676 287 (Clopton, 2000; Desportes and Schrével, 2013). No septum was detected in any of the 677 678 288 observed trophozoit stages of E. dorypterygis sp. n. in this study. 679 680 289 681 682 290 Based on our current knowledge we have decided to assign the gregarine species infecting the 683 684 291 psocopteran Dorypteryx domestica to the genus Enterocystis. The two species Enterocystis 685 686 292 dorypterygis sp. n. and E. bengalensis will be validated, as soon as molecular sequence data 687 688 of the type species of Enterocystis or any other species belonging to this genus become 689 293 690 691 294 available. 692 693 295 694 695 296 Gregarines as possible biological control agents 696 697 297 Insects encompass a great number of pest species and so far several have been recorded as 698 699 298 hosts of gregarine apicomplexans. The present knowledge on the interactions between pest 700 701 299 insects and their pathogens is still insufficient. Gregarines infecting mosquitos have been 702 703 300 mentioned in the literature as potential biological control agents with opposing views for a 704 705 706 707 708 12 709 710 711 301 few decades (Lantova and Volf, 2014). Whereas, the possibility of utilizing gregarines as pest 712 713 714 302 control agents in cockroaches, grasshoppers, fleas, beetles and flies has only recently entered 715 716 303 any form of discussion. 717 718 304 Lopes and Alves (2005) for example tested the effect of gregarines on the susceptibility of 719 720 305 cockroaches towards control measures. Blatella germanica adults infected with gregarines 721 722 306 were more susceptible to the treatment with the fungus Metarhizium anisopliae and 723 724 307 triflumuron than healthy cockroaches. Studies have shown that gregarines have an impact on 725 726 308 fecundity, feeding and mortality rates in economically important grasshopper species, 727 728 309 justifying the utilization of gregarine apicomplexans in biological control monitoring of these 729 730 pest species (Pushkala and Muralirangan, 1997; Johny et al., 2000). Cat fleas, 731 310 732 733 311 Ctenocephalides felis, are infected with the gregarine Steinina ctenocephali and Alarcon et al. 734 735 312 (2011) were able to confirm its potential as biological control agent for this cat parasite. A 736 737 313 few studies have also been carried out on beetles. Due to their high infection rate in the grey 738 739 314 corn weevil, Tanymecus dilaticollis, gregarines play a role as natural regulators of the beetle’s 740 741 315 population density (Takov et al., 2013). A few gregarines species have been reported in bark 742 743 316 beetles (Curculionidae: Scolytinae) to date (Takov et al., 2011; Pernek et al., 2009; Yaman 744 745 317 and Baki, 2010). Consequently, they could play a role as pest control agents against bark 746 747 beetles in the future. 748 318 749 750 319 Among Diptera or flies, phlebotomine sand flies (Psychodidae) and mosquitos (Culicidae) are 751 752 320 important vectors of human pathogens. Gregarines infecting phlebotomine flies of the genera 753 754 321 Lutzomyia and Phlebotomus were studied (Lantova et al., 2011; McCarthy et al., 2011) and 755 756 322 the results suggested that they could possibly be an efficient control method of phlebotomine 757 758 323 populations. Despite the fact that gregarines increased the mortality of immature stages in 759 760 324 Phlebotomus sergenti and negatively affected the survival of adult flies, their potential for use 761 762 325 in pest control is questionable as a result of several factors, including this pathogen’s strict 763 764 765 766 767 13 768 769 770 326 host specificity (Lantova et al., 2011). While the study of gregarines in Lutzomyia longipalpis, 771 772 773 327 the vector of visceral leishmaniasis, suggested that they are a possible efficient control agents 774 775 328 under natural conditions (McCarthy et al., 2011). 776 777 329 In mosquitoes, the susceptibility of Culex bitaeniorhynchus to two species of Ascogregarina 778 779 330 parasites naturally infecting Aedes mosquitoes was determined (Mourya and Soman, 2000). 780 781 331 The gregarines caused high mortality of Culex mosquitoes, but were not able to complete their 782 783 332 life cycle in the unnatural hosts. The survival of infected mosquitoes was significantly 784 785 333 reduced. 786 787 334 788 789 Gregarines do infect numerous invertebrates that are classified as pest organisms due to their 790 335 791 792 336 negative impact on for example crops and human health. Understanding the gregarine-host 793 794 337 interactions is crucial to make any progress in the possible utilization of gregarines to control 795 796 338 aforementioned pests. The identification of gregarine species infecting these pest organisms 797 798 339 examplified in this study by E. dorypterygis sp. n. infesting Dorypterix domestica is a first 799 800 340 and essential step in that direction. 801 802 341 803 804 805 342 Taxonomic Summary 806 807 343 Superphylum Alveolata Cavalier-Smith, 1991 808 809 344 Phylum Apicomplexa Levine, 1980, emend. Adl et al., 2005 810 811 345 Class Conoidasida Levine, 1988 812 813 346 Subclass Gregarinasina Dufour, 1828 814 815 347 Order Eugregarinorida Léger, 1900 816 817 348 Family Enterocystidae Codreanu, 1940 818 819 820 349 Genus Enterocystis Zwetkow, 1926 821 822 350 Enterocystis dorypterygis sp. n. Devetak and Rueckert, 2017 823 824 825 826 14 827 828 829 351 Species diagnosis: Trophozoites elongated and panduriform, little indentation at anterior end, 830 831 832 352 but no septum. Trophozoites on average 59.3 µm long and 25.4 µm wide. Anterior end 833 834 353 globular and rounded, posterior end more blunt. Some cells pyriform with a long anterior 835 836 354 neck-like region. Round nucleus situated in the posterior half of the cell, sometimes shifted to 837 838 355 the anterior half. Gametocysts spherical, diameter 30 μm. Associations caudo-frontal. Cell 839 840 356 surface inscribed by longitudinal epicytic folds (~200), including anterior and posterior end, 841 842 357 arranged in waves along longitudinal axis. Density of folds 6-8 folds/micron. Trophozoites 843 844 358 stiff, capable of gliding movements. 845 846 359 Type host: Dorypteryx domestica (Smithers, 1958) (Psocodea: Psyllipsocidae). 847 848 Site: Intestine. 849 360 850 851 361 Type locality: Maribor, Slovenia (46°33'58.5"N 015°39'15.2"E). 852 853 362 Type micrographs: Figs. 1a, e. 854 855 363 DNA sequence: Small subunit rDNA (Genbank Accession number: xxx). 856 857 364 Etymology: Species-group name dorypterygis is is a noun in the genitive case (Article 858 859 365 11.9.1.3 of the ICZN 1999) derived from the genus-group name Dorypteryx, referring to the 860 861 366 host of the new Enterocystis species. 862 863 367 864 865 866 368 Acknowledgements 867 868 369 We are grateful Dr. Arturo Baz (University of Alcalá, Madrid, Spain) for the identification of 869 870 370 the psocopteran host. 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PhD thesis, University 1220 517 1221 1222 518 of Texas at Austin. 1223 1224 519 1225 1226 520 Figure legends 1227 1228 521 Fig. 1: Differential interference contrast (DIC) light micrographs and scanning electron 1229 1230 522 micrographs (SEM) of Enterocystis dorypterygis sp. n. from psocopteran Dorypteryx 1231 1232 1233 523 domestica. A-B) Differently shaped trophozoite cells with a flattened or rounded anterior end 1234 1235 524 (mucron, arrowhead). The nucelus (n) is visible in the posterior half of the cell. C-D) Two 1236 1237 1238 1239 21 1240 1241 1242 525 gamonts in caudo-frontal syzygy. The nucleus (n) here lies in the anterior part of the cell. The 1243 1244 1245 526 attachment zone is marked with a double arrowhead. E) SEM of a trophozoite cell showing 1246 1247 527 epicytic folds (arrow) running longitudinallyand undulating (asterisk) along the whole cell 1248 1249 528 including the mucron area (arrowhead). F) Higher magnification SEM of the anterior end with 1250 1251 529 epicytic folds covering the mucron area. G) Higher magnification SEM of the longitudinal 1252 1253 530 epicytic folds (arrows). Scale bars: A – 25µm; B – 15µm; C – 25µm; D – 35µm; E – 10µm; F 1254 1255 531 – 2.5µm; G – 2.5µm. 1256 1257 532 1258 1259 533 Fig. 2: Phylogenetic tree of gregarine apicomplexans using dinoflagellate species as outgroup. 1260 1261 1262 534 This gamma-corrected maximum likelihood tree (-ln L = 17107.63238,  = 0.725, fraction of 1263 1264 535 invariable sites = 0.187, 6 rate categories) inferred using the GTR model of substitution on an 1265 1266 536 alignment of 117 small subunit (SSU) rDNA sequences and 774 unambiguously aligned sites. 1267 1268 537 Numbers at the branches denote bootstrap percentage (top) and Bayesian posterior 1269 1270 538 probabilities (bottom). When both values were below 50% or 0.50 numbers were not reported. 1271 1272 539 Black dots on branches denote Bayesian posterior probabilities and bootstrap percentages of 1273 1274 540 0.95/95% or higher. 1275 1276 1277 541 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 22