Description of the new type of polaroplast of Microsporidia from Mesocyclops leuckartii (Copepoda: Cyclopidae)

The polaroplast of microsporidium from copepod Mesocyclops leuckartii was studied by transmission electron microscopy. The polaroplast differs from all previously described types by complex structure.     

Epizootiological studies of Amblyospora camposi (Microsporidia: Amblyosporidae) in Culex renatoi (Diptera: Culicidae) and Paracyclops fimbriatus fimbriatus (Copepoda: Cyclopidae) in a bromeliad habitat

The epizootiology of Amblyospora camposi was studied in a natural population of Culex renatoi, a bromeliad-inhabiting mosquito, and its intermediate host, Paracyclops fimbriatus fimbriatus, over a 2-year period. Twenty Eryngium cabrerae plants were sampled monthly from January 2003 to January 2005 and the prevalence of A. camposi in P.f. fimbriatus and Cx. renatoi populations was determined. The monthly prevalence rates of meiospore infections in Cx. renatoi larvae never exceeded 5.5% and was detected in 50% of the monthly samples. Meiospores were available in plants over the course of the study at a mean concentration of 2 x 10(4) meiospores/ml. Within each plant the parasite was maintained by horizontal transmission. P.f. fimbriatus with vegetative stages and mature spores were found regularly in bromeliads suggesting efficient meiospore infectivity to field copepod populations. The mean concentration of spores from copepods found in plants was 8 x 10(2) spores/ml. Infections in copepods were detected in 54% of the monthly samples with a prevalence rate ranging from 0.55 to 17.4% and an overall average of 5.1%. Vegetative stages in fourth instar mosquito larvae (probably derived from the horizontal pathway via spores formed in copepods) were detected in 12.5% of the monthly samples with an overall prevalence rate of 1.1%. Infections in female and male adults were detected in 20.8% of the monthly samples with an overall average of 4.1% and 6.8%, respectively.     

The phylogenetic position of Ovavesicula popilliae (Microsporidia) and its relationship to Antonospora and Paranosema based on small subunit rDNA analysis

Comparative small subunit rDNA sequence analyses, indicate that Ovavesicula popilliae, a microsporidian parasite of the Japanese beetle, Popillia japonica, represents a distant sister group to Paranosema and Antonospora. These three genera represent a second major group (the Nosema/Vairimorpha clade representing the first) of Microsopridia which infect terrestrial insects, suggesting independent origins for both groups. Phylogenetic analyses of Ovavesicula and other Microsporidia having a multi-sporous sporogony reveal that this condition is found in several unrelated taxa implying either that multi-sporous sporogony is the ancestral condition for Microsporidia or that it has multiple origins.     

Life cycle and description of Amblyospora camposi n. sp. (Microsporidia: Amblyosporidae) in the mosquito Culex renatoi (Diptera, Culicidae) and the copepod Paracyclops fimbriatus fimbriatus (Copepoda, Cyclopidae)

The life cycle of Amblyospora camposi n. sp. is described from the mosquito Culex renatoi and the copepod Paracyclops fimbriatus fimbriatus collected in the leaf axils of the plant Eryngium cabrerae in Argentina. Meiospores of A. camposi (5.8 x 4.1 microm) were infectious per os to female adults of the copepod P. f. fimbriatus. All developmental stages in the copepod had unpaired nuclei, with sporulation involving the formation of a sub-persistent, sporontogenic, interfacial envelope and the production of a second type of uninucleate spore. These spores, formed in the ovaries of P. f. fimbriatus, were large, pyriform, and measured 10.70 x 3.85 microm. When ingested they infected C. renatoi larvae to initiate a sequence that involves schizogony and gametogony and ends with plasmogamy and nuclear association to form diplokaryotic meronts. Oblong ovate binucleate spores (7.86 x 2.96 microm) are formed in the adult mosquito and are responsible for vertical transmission to the filial generation. This is the first report of an Amblyospora species from a mosquito that inhabits the small-water bodies held in parts of terresterial plants (phytotelmata).     

The phylogenetic position of Ovavesicula popilliae (Microsporidia) and its relationship to Antonospora and Paranosema based on small subunit rDNA analysis

Comparative small subunit rDNA sequence analyses, indicate that Ovavesicula popilliae, a microsporidian parasite of the Japanese beetle, Popillia japonica, represents a distant sister group to Paranosema and Antonospora. These three genera represent a second major group (the Nosema/Vairimorpha clade representing the first) of Microsopridia which infect terrestrial insects, suggesting independent origins for both groups. Phylogenetic analyses of Ovavesicula and other Microsporidia having a multi-sporous sporogony reveal that this condition is found in several unrelated taxa implying either that multi-sporous sporogony is the ancestral condition for Microsporidia or that it has multiple origins.     

Phylogenetic position of Octosporea muscaedomesticae (Microsporidia) and its relationship to Octosporea bayeri based on small subunit rDNA analysis

Comparative phylogenetic analysis of the small subunit rDNA sequence of Octosporea muscaedomesticae (Flu, 1911) (type species) (Microsporidia) isolated from the blowfly Phormia regina (Diptera:Calliphoridae) is presented. Neighbor Joining bootstrap, Maximum Parsimony and Maximum Likelihood analyses with 38 microsporidian taxa representing five major clades of Microsporidia placed O. muscaedomesticae on a separate branch within a clade containing parasites of freshwater hosts. O. muscaedomesticae differed from Octosporea bayeri, a parasite of the microcrustacean, Daphnia magna (Cladocera:Daphniidae) by 29% demonstrating that the latter microsporidium is not closely related to the type species at the generic level, and should not be placed within the genus Octosporea, a conclusion that is further supported by morphological and developmental differences. Considering the number of disparately related hosts from which Octosporea species have been previously described based mostly on developmental and morphological characters it is likely that many will not fit the current definition of the genus, and it is possible that molecular analysis of these species will show that this genus as defined represents a polyphyletic grouping of unrelated taxa.     

Life cycle,ultrastructure and molecular phylogeny of Hyalinocysta chapmani (Microsporidia: Thelohaniidae), a parasite of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae)

The complete life cycle of the microsporidium Hyalinocysta chapmani is described from the primary mosquito host Culiseta melanura and the intermediate copepod host Orthocyclops modestus. Infections are initiated in larval C. melanura following the oral ingestion of uninucleate spores from infected copepods. Spores germinate within the lumen of the midgut and directly invade fat body tissue where all development occurs. Uninucleated schizonts undergo binary division (schizogony) followed by karyokinesis (nuclear division) to form diplokaryotic meronts. Merogony is by synchronous binary division. The onset of sporogony is characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of the diplokaryon resulting in the formation of eight ovoid meiospores enclosed within a sporophorous vesicle. Most infected larvae die during the fourth stadium and there is no evidence of a developmental sequence leading to vertical transmission. Hyalinocysta chapmani is horizontally transmitted to O. modestus via oral ingestion of meiospores. Infections become established within ovarian tissue of females and all parasite development is haplophasic. Uninucleate schizonts divide by binary division during an initial schizogonic cycle. Newly formed uninucleate cells produce a thin sporophorous vesicle and undergo repeated nuclear division during sporogony to produce a rosette-shaped, multinucleated sporogonial plasmodium with up to 18 nuclei. This is followed by cytoplasmic cleavage, sporogenesis, and disintegration of the sporophorous vesicle to form membrane-free uninucleate spores. Infected females eventually die and there is no egg development. The small subunit rDNA sequence of H. chapmani isolated from meiospores from C. melanura was identical to the small subunit rDNA sequence obtained from spores from O. modestus, corroborating the laboratory transmission studies and confirming the intermediary role of O. modestus in the life cycle. Phylogenetic analysis was conducted with closely related microsporidia from mosquitoes. Hyalinocysta chapmani did not cluster within described Amblyospora species and can be considered a sister group, warranting separate genus status.     

Two new species of Paracyclops (Copepoda: Cyclopoida,Cyclopidae) from Africa

Paracyclops longispina n. sp. and Paracyclops altissimus n. sp. are described. New characters derived from detailed examination of body and limb ornamentation are used to differentiate them from other Paracyclops species. Paracyclops longispina n. sp. resembles Paracyclops fimbriatus (Fischer, 1853) and Paracyclops imminutus Kiefer, 1929. It differs from the former by the presence of a well-developed spinular row near the base of the inner setae on the antennal coxobasis in both sexes, and from the latter in the structure of the seminal receptacle and the position of the mid-distal spinular row on the posterior surface of the coxa of leg 1. Paracyclops altissimus n. sp. can be distinguished from other members of genus mainly by the structure of the seminal receptacle and leg 5. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogeny of Mesocyclops (Copepoda: Cyclopidae) inferred from morphological characters

Evolutionary relationships were investigated in the genus Mesocyclops, a pantropical freshwater cyclopid group. In the phylogenetic analyses that involved all 71 known species, and used 81 morphological characters (265 character states) mainly of the adult females, two different approaches were applied: global parsimony, and a new distance method based upon the recognition of sister‐groups on the basis of minimal distances iteratively corrected for unique character states (MICSEQ). In coding of the characters, half of which showed intraspecific variation, the ‘scaled’ method was employed, which assumes that any trait between its absence and fixed presence passes through a polymorphic stage. Impact of the reference points on topology of the trees generated by the parsimony method was tested in three ways where the outgroups comprised: (1) nine species representing six genera of two subfamilies; (2) three species from two genera supposedly not distant from Mesocyclops; and (3) one presumably close and one distant relative of Mesocyclops. The trees generated by the parsimony‐based and corrected distance methods agreed as to the monophyly of the following groups: reidae‐clade (M. reidae, M. chaci, M. yutsil); rarus‐clade (Mesocyclops annae, M. pseudoannae, M. splendidus, M. rarus, M. paludosus, M. darwini, M. dayakorum); annulatus‐clade (Mesocyclops intermedius, M. ellipticus, M. paranaensis, M. annulatus, M. tenuisaccus); meridianus‐clade (Mesocyclops meridionalis, M. varius, M. venezolanus, M. brasilianus, M. pseudomeridianus, M. meridianus); major‐clade (Mesocyclops major, M. pilosus, M. insulensis); dussarti‐clade (M. dussarti, M. dadayi, M. isabellae, M. thermocyclopoides); pubiventris‐clade (M. pubiventris, M. medialis, M. brooksi, M. notius). A majority of the analyses support a clade of the ‘true’Mesocyclops including all ingroup species except the reidae‐group, and point to monophyly of the Old World species lacking medial spine on P1 basipodite. There were, however, some components for which the two procedures, regardless of the outgroup choice and/or character set, suggested different relationships. Basal relationships of Mesocyclops[between M. edax (North and Central America), the Neotropical species (M. longisetus, M. araucanus, M. evadomingoi, meridianus‐ and annulatus‐clade), Old World group (P1 basipodite without medial spine) and the rarus‐clade (Old World; P1 basipodite with medial spine)] remained unresolved. © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society, 2006, 147 , 1–70.     

3 Phylogenetic affinity of the palmelloid green algae, verdigellas and palmophyllum (chlorophyta), based on analyses of nuclear-encoded small subunit rDNA sequences

Palmophyllum, Verdigellas and Palmoclathrus are marine palmelloid green algae with morphologies ranging from closely adherent crusts, peltate discs, to upright branched thalli. Thalli of these taxa are comprised of small spherical cells embedded within a dense mucilaginous matrix. Taxonomic affinities of these palmelloid genera, however, has remained uncertain. Previous studies of Palmophyllum and Verdigellas classified these algae within the Palmellaceae, but the complete absence of data regarding reproduction have blurred ordinal designations. Generally, these algae have been classified as members of the Tetrasporales within the class Chlorophyceae, but the Chlorococcales has also been proposed. Global analyses of eukaryotic nuclear-encoded small subunit rDNA sequences based on parsimony (MP), neighbor joining (NJ) and likelihood (ML) methods confirm the placement of Palmophyllum and Verdigellas as a monophyletic group within the Chlorophyta, but class and ordinal affinities were not clearly resolved. ML suggested that Verdigellas and Palmophyllum are members of a clade with coccoid prasinophyte algae at the base of the Chlorophyta, while NJ and ML suggested that the palmelloid genera formed a basal lineage of the Viridiplantae. A consistent feature of all analyses, however, is that Verdigellas and Palmophyllum did not group with the chlorophycean orders, Tetrasporales or Chlorococcales. Results will be discussed in the context of taxonomy, character evolution, and implications for green plant evolution. (Supported in part by NSF grants DEB-0128952 to MWF, DEB-0129030 to MAB, and DEB-0128977 to FWZ)     

Evolution of ruminant Sarcocystis (Sporozoa) parasites based on small subunit rDNA sequences

We present an evolutionary analysis of 13 species of Sarcocystis, including 4 newly sequenced species with ruminants as their intermediate host, based on complete small subunit rDNA sequences. Those species with ruminants as their intermediate host form a well-supported clade, and there are at least two major clades within this group, one containing those species forming microcysts and with dogs as their definitive host and the other containing those species forming macrocysts and with cats as their definitive host. Those species with nonruminants as their intermediate host form the paraphyletic sister group to these clades. Most of the species have considerable genotypic differences (differing in more than 100 nucleotide positions), except for S. buffalonis and S. hirsuta. There is a large suite of genotypic differences indicating that those species infecting ruminant and nonruminant hosts have had very different evolutionary histories, and similarly for the felid- and canid-infecting species. Furthermore, the rDNA sequences that represent the different structural regions of the rRNA molecule have very different genotypic behavior within Sarcocystis. The evolution of these regions should be functionally constrained, and their differences can be explained in terms of the importance of the nucleotide sequences to their functions.     

Application of method of OTE staining of ultrathin sections based on example of microsporidia (Protozoa: Microsporidia)

A comparative analysis of ultrastructure of some organelles stained by different methods of staining of ultrathin sections has been performed using the example of intracellular parasites, i.e., microsporidia. The distinctive peculiarities are revealed and the advantages and disadvantages are substantiated of the traditional method of contrasting with uranyl acetate and the nontraditional method of contrasting using black Chinese tea extract, i.e., oolong tea extract (OTE). The OTE-staining method dies the basic intracellular structures of microsporidia, which is a matter of taxonomic significance; it also reveals additional layers of the polar filament with more distinct boundaries between them. However, traditional UA-staining better reveals some structures (membranes, layers of envelope of mature spores, structure of the rough endoplasmic reticulum, Golgi complex, nuclear chromatin) and provides for higher general contrast. The OTE solution is safe to use and can be longer kept in light at room temperature without losing its activity. However, the OTE-method is time-consuming. Hence, this staining method has both advantages and disadvantages. On the whole, it can be used as an alternative to the traditional staining with uranyl acetate.     

Epizootiology of Hyalinocysta chapmani (Microsporidia: Thelohaniidae) infections in field populations of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae): a three-year investigation

The epizootiology, transmission dynamics and survival strategies employed by the microsporidium Hyalinocysta chapmani were examined in field populations of its primary mosquito host, Culiseta melanura and its intermediate copepod host, Orthocyclops modestus over a three-year period in an aquatic subterranean habitat. H. chapmani was enzootic and was maintained in a continuous cycle of horizontal transmission between each host. There were three distinct periods during the summer and fall when developing mosquito larvae acquired infections; each was preceded by or coincident with the detection of infected copepods. Results were corroborated in laboratory bioassays, wherein transmission was achieved in mosquito larvae that were reared in water and sediment samples taken from the site during the same time periods. The highest infection rates, ranging from 60% to 48%, were repeatedly observed during the first six weeks of larval development. These were coincident with the most sustained collections of infected copepods obtained during the year and highest levels of infection achieved in the laboratory transmission studies. The high prevalence rates of lethal infection observed in larval populations of C. melanura at this site are among the highest recorded for any mosquito-parasitic microsporidium and clearly suggest that H. chapmani is an important natural enemy of C. melanura. H. chapmani appears to overwinter in diapausing mosquito larvae but may also persist in copepods. The absence of vertical transmission in the life cycle of H. chapmani and the sole reliance on horizontal transmission via an intermediate host are unique survival strategies not seen among other mosquito-parasitic microsporidia. The epizootiological data suggest that this transmission strategy is a function of the biological attributes of the hosts and the comparatively stable environment in which they inhabit. The subterranean habitat is inundated with water throughout the year; copepods are omnipresent and C. melanura has overlapping broods. The spatial and temporal overlap of both hosts affords abundant opportunity for continuous horizontal transmission and increases the likelihood that H. chapmani will find a target host. It is hypothesized that natural selection has favored the production of meiospores in female host mosquitoes rather than congenital transfer of infection to progeny via ovarian infection as a strategy for achieving greater transmission success.     

Characteristics of the reproductive cycles and development times of Copidodiaptomus numidicus (Copepoda: Calanoida) and Acanthocyclops robustus (Copepoda: Cyclopoida)

The female reproductive cycles of the calanoid copepod Copidodiaptomus numidicus and the cyclopoid Acanthocyclops robustus were characterized by periodic changes in the oviducts. Females oscillated between a gravid and a non-gravid condition, and the whole cycle was strongly dependent on temperature. In both species, the maturation of new oocytes took place when the old egg sacs were still being carried, thus ensuring a rapid clutch succession. The embryonic and post-embryonic development duration of C.numidicus and A.robustus was investigated at five and four different temperatures, respectively. Embryonic development time was related to oviductal cycle duration at each temperature to estimate the minimum amount of non-ovigerous females expected under non-limiting mate and food conditions. Copidodiaptomus numidicus females spent 35-19% of the cycle without carrying eggs at 7-20C. Acanthocyclops robustus females spent 31-33% of the cycle without carrying eggs at the same temperature interval. Both naupliar and copepodid durations were inverse functions of temperature with nauplii developing at a faster rate than copepodids. In general, A.robustus showed reproductive advantage over C.numidicus due to a faster oviductal cycle and shorter embryonic and post-embryonic development times. However, the influence of food quality may be crucial. Acanthocyclops robustus copepodid development may lengthen well beyond the corresponding development stages of C.numidicus when growing only on algae. This retardation of development affects especially the later development stages of the cyclopoid.      

A new species of Metacyclops from a hyporheic habitat in North Vietnam (Crustacea,Copepoda, Cyclopidae)

A new species of Metacyclops is described from hyporheic waters and small rock depression with leaf litter in North Vietnam, the Tam Đao Mountains). Metacyclops amicitiae sp. n. can be distinguished from its congeners by the unique combination of the following characters: 12-segmented antennule, distal segment of P4 endopodite bearing a single apical spine, and the surface ornamentation of the intercoxal sclerites in P1–P4 (pilose on the distal margin of P1-P4 and spinulose on the caudal surface of P4). The latter character separates the new Metacyclops from its closest relative, Metacyclops ryukyuensis, known only from the Ryukyu Islands (Ishigaki). The genus Metacyclops with the new species described herein is also for the first time recorded from Vietnam. An identification key is provided to the south and east Asian species of the genus.     

Revision of the African species of the genus Mesocyclops Sars, 1914 (Copepoda: Cyclopidae)

A revision is made of the African Mesocyclops species, based on type material (when extant) and on collections from various parts in Africa.Diagnostic characteristics are: presence or absence of spinule patterns on antennular segments, structure of the hyaline membrane of the 17th antennular segment, spine pattern on the basipodite of the antenna, armature of the maxillulary palp, presence or absence of a spine on the basipodite of P₁, armature of the connecting lamella, coxopodite and basipodite of P₄, armature of the apical spines of Enp₃P₄, armature of the last thoracic segment, genital segment and the other abdominal segments, armature of the furcal rami and structure of the receptaculum seminis.First, the taxonomical status of M. leuckarti (Claus) is redefined; this species does not occur in Africa and its geographical range is restricted to Europe and the western part of Northern Asia. On the African continent, twelve other taxa are found. Four are described as new to science: M. kieferi sp.n., M. dussarti sp.n., M. spinosus sp.n. and M. aequatorialis similis subsp.n. The remainder are: M. major Sars, M. tenuisaccus (Sars), M. paludosus Lindberg, M. salinus Onabamiro, M. ogunnus Onabamiro, M. aspericornis (Daday), M. rarus Kiefer and M. aequatorialis aequatorialis (Kiefer).Their geographical distribution is discussed. Eleven taxa are restricted to the African continent (including the Arabian Peninsula and the Canary Islands). One taxon is also found in the Oriental Region. Madagascan representatives are briefly mentioned.The importance of breeding and cross-breeding experiments should be stressed in copepod taxonomy. Experiments were performed on African species and M. leuckarti to evaluate the morphological characters used in the present study, and intraspecific morphological variability was examined up to and including the third generation (Van de Velde, in press).In future one should critically reconsider the so-called cosmopolitan species; they may indeed represent a complex of related taxa, each with a geographical range more restricted than that of the species-complex itself.Abstracted from the author's Ph.D. thesis, State University of Ghent, 1982.Abstracted from the author's Ph.D. thesis, State University of Ghent, 1982.     

Chamaebotrys prolifera sp. nov. (Rhodymeniaceae, Rhodophyta) from Puerto Rico, Caribbean Sea, based on morphology and small subunit rDNA sequences

A new rhodymeniacean species, Chamaebotrys prolifera , is described from a shallow water habitat in Puerto Rico, representing the first occurrence of the genus in the Atlantic Ocean. Plants, to 5 cm across, are decumbent and comprised of compressed vesicles that are originally proliferously branched at the perimeter. Older vesicles become branched from their dorsal surfaces as well. Branches are septate at their origin and become irregularly shaped with age. Anastomoses between adjacent vesicles is common. Individual vesicles measure to 15 mm in broadest dimension. Vesicle walls consist of two layers of medullary cells and two layers of cortical cells. Tetrasporangia, which occur in diffuse nemathecia, are spherical, to 30 µm in diameter and are cut off terminally from an inner cortical cell. Cystocarps are hemispherical measuring to 800 µm in diameter and 350 µm in height. Spermatangia are apparently cut off randomly from outer cortical cells across the thallus surface. Molecular evidence confirms placement of Chamaebotrys within the Rhodymeniaceae.