GREGARINA LUTESCENS N. SP. INFECTING THE HARLEQUIN LADYBIRD HARMONIA AXYRIDIS (COLEOPTERA: COCCINELLIDAE).

Gregarina lutescens n. sp. is described from the alimentary canal of the harlequin ladybird or multicolored Asian lady beetle, Harmonia axyridis (Coleoptera: Coccinellidae) collected from prairie fleabane, Erigeron strigosus, at Peru State College, Peru, Nemaha Co., Nebraska. Our specimens differ from all 11 known species of Gregarina infecting coccinellid beetles worldwide by differences in size and relative shape, color, and association structure. Gregarina lutescens n. sp. is smaller than 7 known species infecting coccinellid beetles but larger than the other 4 known species based on confidence interval exclusion of means. Our specimens are unique among known species of interest in their quince-yellow cytoplasm and precocious but ephemeral serial associations of up to 5 satellites. Nucleotide sequence (18S) phylogenetic analyses place the new species basal to a member of an internal clade of Gregarina that comprises gregarines parasitizing chrysomelid beetles. Phylogenetically, the analysis recovered 3 major lineages within the gregarines, representing the superfamilies Gregarinoidea, Stenophoroidea, and Stylocephaloidea and indicating the propensity of gregarines to track host lineages and environments through evolutionary time. These findings confirm the polyphyletic nature of Gregarina, which currently comprises over 300 described species, only a handful of which have documented genetic sequences suitable for phylogenetic analysis. Recollection, redescription, and molecular clarification of gregarine species infecting coccinellids would likely result in identification of a unique clade that would be an excellent system for studying the effect of intraguild host competition on parasite diversification and community structure. Ecologically, patterns of prevalence in this study indicate that G. lutescens reproduces primarily in larval hosts but depends on infections in adult beetles to overwinter, reflecting the differential vagility and frost tolerance of larval and adult host life cycle stages.

REVISION OF CORONOEPIMERITUS WITH DESCRIPTION OF CORONOEPIMERITUS OTOENSIS N. SP. INFECTING THE ADMIRABLE GRASSHOPPER SYRBULA ADMIRABILIS (ORTHOPTERA: ACRIDIDAE).

Herein, we reaffirm the validity of Coronoepimeritus and its junior synonym, Quadruspinospora, provide new combinations for existing species of Quadruspinospora, recognize Monospinospora n. gen. to receive Coronoepimeritus monospinus, and describe Coronoepimeritus otoensis n. sp. from the alimentary canal of the admirable grasshopper, Syrbula admirabilis (Orthoptera: Acrididae), from Nemaha County (40°45'69.49″N, 95°71'60.39″W) near Peru, Nebraska. Our specimens differ from all known species by differences in total gamont length (TL), oocyst length (OL), and oocyst spine length (OSL). Coronoepimeritus atractomorphii n. comb. is most like the new species on the basis of OL and OSL (10 µm and 33 µm, respectively, for both species), but the new species possesses gamonts whose TL is less than 60% of that reported for C. atractomorphii (average TL, 703 µm vs. 1,020 µm, respectively). Nucleotide sequences (18S) and phylogenetic analyses confirm the placement of the new species in Coronoepimeritus, and the K/Θ test (the ratio between the average distance separating 2 species-level clades and the genetic diversity of these clades) is consistent with a species distinct from the only other known sequence sample in the group, Coronoepimeritus mexicana n. comb.

Environmental Persistence and Infectivity of Oocysts of Two Species of Gregarines, Blabericola migrator and Blabericola cubensis (Apicomplexa: Eugregarinida: Blabericolidae), Parasitizing Blaberid Cockroaches (Dictyoptera: Blaberidae).

For apicomplexan parasites using an oral-fecal transmission route with significant environmental exposure, the environmental persistence and infectivity of the oocyst has a direct impact on local infection dynamics, including the ability to withstand extended periods without readily available hosts. Herein we quantify the environmental persistence and infectivity of the oocysts of 2 septate gregarine species at controlled temperature and humidity and demonstrate that they can persist over multiple generational time spans. Species of Blabericola generally complete their endogenous life cycles from oocyst to oocyst within 10 days. The median residual environmental oocyst lifetime for Blabericola oocysts in this study is 21-28 days, but a significant number of oocysts of Blabericola migrator persisted and remained infective in the environment for up to 39 days while those of Blabericola cubensis persisted and remained infective for up to 92 days. Although long-lived relative to their own generational time, the oocysts of Blabericola species infecting cockroaches are short-lived relative to gregarines infecting tenebrionid beetles. For these gregarines, oocysts can persist in the environment and remain infective for up to 787 days. Mechanistically, environmental persistence and infectivity are probably energy-limited phenomena related to the amount of stored amylopectin and the basal metabolic rate of quiescent oocysts.

Effects of Developmental Temperature on Gametocysts and Oocysts of Two Species of Gregarines Blabericola migrator and Blabericola cubensis (Apicomplexa: Eugregarinida: Blabericolidae) Parasitizing Blaberid Cockroaches (Dictyoptera: Blaberidae).

Abiotic environmental conditions, especially temperature and humidity, have profound effects on the growth and development of gregarines, but these effects remain largely undocumented. Quantifying the effects of environmental conditions on the growth and development of exogenous gregarine ontogenetic stages is an important first step in understanding the transmission, population dynamics, and environmental persistence of gregarine infection. In this study, we examined the effect of 6 environmental temperatures (10, 18, 22, 27, 35, and 40 C) at constant humidity (0 mmHg vapor pressure deficit) on gametocyst development and oocyst viability in 2 gregarine species: Blabericola migrator and Blabericola cubensis parasitizing the Tiger-striped Hissing Cockroach, Princisia vanwaerebecki, and the Discoid Cockroach, Blaberus discoidalis, respectively. Temperature has a significant effect on gametocyst development and oocyst viability for both gregarine species. Gametocyst development for both gregarine species displays a similar threshold response to environmental temperature: 10 and 40 C represent extremes outside their developmental range, but within these extremes, the relationship between gametocyst development and temperature is weakly direct. Dehiscence increased with temperature from 68% at 18 C to 93% at 22 C and remained at that level through 35 C. Developmental temperature also has a meaningful but inverse effect on oocyst viability of both B. migrator and B. cubensis. For both species, oocyst viability is highest at 18 and 22 C and is significantly reduced at 27 and 35 C. Thus oocyst production and sporozoite viability are linked but environmentally independent phenomena. Overall, there is an acceptable developmental temperature zone for B. migrator and B. cubensis that ranges from 18 to 27 C, but production of viable sporozoites is greatest in a relatively narrow zone around 22 C. Prior studies have postulated that mechanisms that concentrate oocysts and hosts, such as host behavior or host microhabitat preference, increase the host-oocyst encounter rate and thus transmission. This study indicates that abiotic influences on gametocyst development may also lead to heterogeneous oocyst distributions in the environment and increase the likelihood of host-oocyst encounters.

Tomaculocystis corpulenta n. gen., n. sp. (Apicomplexa: Eugregarinorida) parasitizing the little yellow cockroach, Cariblatta lutea (Blattodea: Ectobiidae), in Alabama and Florida with recognition of Tomaculocystis cylindrosa n. comb. and Tomaculocystis mukundai n. comb. parasitizing ectobiid cockroaches in India.

Tomaculocystis corpulenta n. gen., n. sp. (Apicomplexa: Eugregarinorida: Septatorina: Gregarinidae) is described from populations of the little yellow cockroach, Cariblatta lutea (Blattodea: Ectobiidae), established in laboratory culture from samples collected in Alabama and Florida. Tomaculocystis n. gen. are differentiated from other members of Gregarina by a markedly elliptoid gametocyst inside a persistent, lomentiform hyaline epicyst; developmental organization and growth of the spore tubes from gametocyst surface tumidi; and dehiscence by extrusion of non-chain forming oocysts through spore tubes that barely extend beyond the epicyst wall. Gregarina cylindrosa, Gregarina discocephala, and Gregarina mukundai are recognized as members of Tomaculocystis, and G. cylindrosa is recognized as the senior synonym of G. discocephala. Thus, Tomaculocystis cylindrosa n. comb. and Tomaculocystis mukundai n. comb. are formed. Species of Tomaculocystis are distinguished based on gamont deutomerite and oocyst shape and size. The oocysts of T. corpulenta are broadly dolioform, lack 4 polar knobs, and possess distinct, unique polar plates. Oocysts of all other known species in the genus are more oblong in shape, possess 4 polar knobs, and lack the distinct polar plates observed in the oocysts of T. corpulenta. Host utilization and geographic distribution among gregarine genera parasitizing the cockroach family Ectobiidae reveal a pattern of host-parasite specificity linking gregarine genera with ectobiidid subfamilies. Overall patterns suggest a hypothesis of European endemicy for Gamocystis, but hypotheses for the origin and radiation of Tomaculocystis or species of Gregarina infecting cockroaches are confounded by the cosmopolitan spread of pest cockroach species among humans.

Blabericola rhyparobiae n. comb. (Apicomplexa: Eugregarinida: Blabericolidae), parasitizing the Madeira cockroach, Rhyparobia maderae (Dictyoptera: Blaberidae).

Blabericola rhyparobiae n. comb. (Apicomplexa: Eugregarinida: Blabericolidae) is redescribed from the Madeira cockroach, Rhyparobia maderae (Dictyoptera: Blaberidae: Oxyhaloinae: Nauphoetini). Oocysts within the genus are typically dolioform with nonprojecting polar plates. Those of B. rhyparobiae differ from all other species of Blabericola in being oblong rather than dolioform. Morphometrically, the oocysts of B. rhyparobiae are significantly narrower than those of any other known species in the genus; they are significantly longer than those of Blabericola haasi, Blabericola migrator, and Blabericola princisi, but they are significantly shorter than those of Blabericola cubensis (oocyst width 4.47 µm vs. 4.74 µm, 4.70 µm, 5.06 µm, 5.21 µm, respectively; oocyst length 8.98 µm vs. 7.94 µm, 7.93 µm, 8.85 µm, 9.26 µm, respectively). All 5 species are also distinguished by unique sporozoite-bearing cavity sizes and morphometric ratios. Gametocysts of Blabericola species are either orbicular (B. cubensis, B. princisi) or elliptoid (B. haasi , B. migrator, B. rhyparobiae). Among Blabericola species with elliptoid gametocysts, the gametocysts of B. rhyparobiae are intermediate in size relative to the much larger gametocysts of B. migrator and the much smaller gametocysts of B. haasi (gametocyst length 462.06 µm vs. 728.11 µm, 272.02 µm; gametocyst width 297.12 µm vs. 461.31 µm, 178.36 µm, respectively). No structurally unique feature of the gamont distinguishes among species of Blabericola, but gamonts of all 5 species differ morphometrically. Gamonts of B. rhyparobiae differ significantly from all other species in the genus in the primite's protomerite and deutomerite lengths, the satellite' deutomerite lengths, and the total length of both primite and satellite in association. The gamonts of B. rhyparobiae are significantly smaller than those of B. cubensis, B. migrator, and B. princisi but significantly larger than those of B. haasi and can be readily distinguished based on size alone (primite total length 460 µm vs. 563 µm, 800 µm, 547 µm, 316 µm, respectively; satellite total length 419 µm vs. 507 µm, 695 µm, 526 µm, 298 µm, respectively).

Protomagalhaensia richardsoni n. sp. (Apicomplexa: Eugregarinida: Blabericolidae): a new gregarine parasitizing the giant lobster cockroach, Henschoutedenia flexivitta (Dictyoptera: Blaberidae).

Protomagalhaensia richardsoni n. sp. (Apicomplexa: Eugregarinida: Blabericolidae) is described from the giant lobster cockroach, Henschoutedenia flexivitta (Dictyoptera: Blattaria: Blaberidae: Oxyhaloinae: Nauphoetini). Oocysts within the genus are dolioform with polar plates. Those of Protomagalhaensia granulosae, Protomagalhaensia wolfi, and Protomagalhaensia blaberae possess distinct apical spines and a sagittal depression that are absent or reduced in P. richardsoni and Protomagalhaensia cerastes. Oocysts of P. richardsoni are significantly longer with larger sporozoite-bearing cavities than those of P. blaberae, P. cerastes, P. granulosae, and P. wolfi (external oocyst length 8.07 µm vs. 7.42 µm, 7.50 µm, 6.87 µm, 7.56 µm, respectively; internal oocyst length 6.94 µm vs. 6.44 µm, 6.77 µm, 6.09 µm, 6.72 µm, respectively). All 5 species are also distinguished by unique oocyst length/width ratios. No unique morphological structure distinguishes among the gametocysts of Protomagalhaensia species, but gametocysts of P. richardsoni are significantly shorter than those of P. blaberae, P. cerastes, P. granulosae, and P. wolfi (gametocyst length 184.3 µm vs. 325.15 µm, 253.27 µm, 273.63 µm, 218.3 µm, respectively). No structurally unique morphological gamont feature distinguishes among species of Protomagalhaensia. Rather, species distinctions are morphometric in nature. In general, gamonts of P. richardsoni are readily distinguished from those of P. cerastes and P. wolfi based on size alone: the latter species being roughly half the size of P. richardsoni. Gamonts of P. richardsoni are most similar to those of P. granulosae and P. blaberae but with relatively smaller primites and more slender satellites.

Excystation signals do not isolate gregarine gene pools: experimental excystation of Blabericola migrator among 11 species of cockroaches.

An experimental excystation assay was used to test the potential species isolating effects of excystation signaling among gregarines. Oocysts of a single gregarine species, Blabericola migrator , were tested for activation, excystation, and sporozoite motility by using intestinal extracts from 11 species of cockroaches representing a cohesive phylogeny of 7 genera, 3 subfamilies, and 2 families of Blattodea. Sporozoite activation, excystation, and motility were observed for all excystation assay replications using intestinal fluid from blaberid hosts, but delayed activation or excystation was observed for all assay replications using intestinal fluid from hosts in the family Blattidae. The results illustrate a trend toward a generalized excystation signal among gregarines that is conserved across the host clade at a subfamily or family level but that is unlikely to play a significant role as a species-isolating mechanism among sibling gregarine species.

Metronidazole induces gametocytogenesis in gregarine associations maintained in vitro.

Gametocytogenesis was induced in mature associations of Protomagalhaensia wolfi and Protomagalhaensia blaberae maintained in vitro by inclusion of metronidazole in the culture medium. The response was neither strictly dosage dependent nor uniform across gregarine species. We hypothesize that metronidazole induces gregarine gametocytogenesis by disrupting PUF2 proteins responsible for the translational control of sexual development and gametocytogenesis in apicomplexans.

Synoptic revision of Blabericola (Apicomplexa: Eugregarinida: Blabericolidae) parasitizing blaberid cockroaches (Dictyoptera: Blaberidae), with comments on delineating gregarine species boundaries.

Complete synoptic redescriptions, including complete morphometric data for all life cycle stages, species recognition characters, and differential comparisons are presented for the 4 gregarine species comprising Blabericola . Blabericola cubensis ( Peregrine, 1970 ), Blabericola haasi (Geus, 1969), Blabericola migrator ( Clopton, 1995 ), and Blabericola princisi ( Peregrine, 1970 ) are redescribed from their type hosts, i.e., the discoid cockroach Blaberus discoidalis , the lobster cockroach Nauphoeta cinerea , the Madagascar hissing cockroach Gromphadorhina portentosa , and the Bolivian cockroach Blaberus boliviensis , respectively. These gregarine species descriptions are stabilized through deposition of extensive new voucher collections. Species of Blabericola are distinguished by differences in relative metric ratios, morphology of oocysts, and by relative metric ratios of mature gamonts in association. This work is discussed as a model for morphological species descriptions in the Eugregarinorida including the 6 principles for morphological gregarine species descriptions, i.e., a centroid and population variation approach, adequate sample size, partitioning developmental variation and sexual dimorphism, recognition and minimization of fixation and physiological artifacts to eliminate false morphotypes, and comparative data sets across multiple life cycle stages.

Revision of the genus Stictospora and description of Stictospora villani, n. sp. (Apicomplexa: Eugregarinida: Actinocephalidae) from larvae of the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae), in Michigan.

Stictospora villani n. sp. is described from larvae of the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) from southern Michigan. Stictospora villani is distinguished from known species of the genus by differences in gamont size and by structural details of the epimerite. In general, S. villani is smaller than other known species of the genus and possesses an epimerite with a margin of 20-24 sympetalous, pendulate, narrowly to very narrowly elliptoid lamina. Previously described species are characterized by epimerites with notably fewer marginal lamina. A heretofore unrecognized protistan is diagnosed from the coelomic fat bodies and tentatively placed within the Neogregarinorida. We conclude that previous workers have mistaken this neogregarine for the gametocyst and oocyst of Stictospora, which are described in this study for the first time. The generic diagnosis of Stictospora is revised to encompass the epimeritic variation of its constituent species and to correctly reflect the morphology of the oocyst.

Phylogeny of gregarines (Apicomplexa) as inferred from small-subunit rDNA and beta-tubulin.

Gregarines are thought to be deep-branching apicomplexans. Accordingly, a robust inference of gregarine phylogeny is crucial to any interpretation of apicomplexan evolution, but molecular sequences from gregarines are restricted to a small number of small-subunit (SSU) rDNA sequences from derived taxa. This work examines the usefulness of SSU rDNA and beta-tubulin sequences for inferring gregarine phylogeny. SSU rRNA genes from Lecudina (Mingazzini) sp., Monocystis agilis Stein, Leidyana migrator Clopton and Gregarina polymorpha Dufour, as well as the beta-tubulin gene from Leidyana migrator, were sequenced. The results of phylogenetic analyses of alveolate taxa using both genes were consistent with an early origin of gregarines and the putative 'sister' relationship between gregarines and Cryptosporidium, but neither phylogeny was strongly supported. In addition, two SSU rDNA sequences from unidentified marine eukaryotes were found to branch among the gregarines: one was a sequence derived from the haemolymph parasite of the giant clam, Tridacna crocea, and the other was a sequence misattributed to the foraminiferan Ammonium beccarii. In all of our analyses, the SSU rDNA sequence from Colpodella sp. clustered weakly with the apicomplexans, which is consistent with ultrastructural data. Altogether, the exact position of gregarines with respect to Cryptosporidium and other apicomplexans remains to be confirmed, but the congruence of SSU rDNA and beta-tubulin trees with one another and with morphological data does suggest that further sampling of molecular data will eventually put gregarine diversity into a phylogenetic context.