ELECTRON MICROSCOPY OF MITOSIS IN A RADIOSENSITIVE GIANT AMOEBA

Various aspects of the ultrastructure of the dividing nuclei in the large radiosensitive amoeba Pelomyxa illinoisensis are demonstrated. Evidence of nuclear envelope breakdown is presented, and membrane fragments are traced throughout metaphase to envelope reconstruction in anaphase and telophase. Annuli in the nuclear envelope and its fragments are shown throughout mitosis. During metaphase and anaphase some 15 to 20 mitochondria are aligned at each end of the spindle, and are called polar mitochondria. The radioresistant amoebae Pelomyxa carolinensis and Amoeba proteus do not have polar mitochondria, and Pelomyxa illinoisensis is unique in this regard. The shape of the P. illinoisensis interphase nucleoli differs from that in the two radioresistant species, and certain aspects of nucleolar dissolution in the prophase vary. Helical coils in the interphase nucleoplasm are similar to those in the radioresistant amoebae. A "blister" phase in the flatly shaped telophase nuclei of P. illinoisensis is described which is interpreted to be the result of a rapid nuclear expansion leading to the formation of the normal spherical interphase nuclei.     

Starvation effects on the ultrastructure of amoeba mitochondria

Summary Mitochondria in non-starved giant amoebae, Pelomyxa carolinensis, contain tubules lying at random in the matrix. Many mitochondria in starved amoebae have enlarged tubules aligned in a zigzag pattern. Tubules within the zigzag region are separated by very little matrix material. Some of these altered mitochondria are found in 70% of amoebae starved for only 24 hours, and in nearly all P. carolinensis starved for 8 days or longer. The percentage of such altered mitochondria increases from zero in most well-fed amoebae, to about 60% after two weeks of continuous starvation. Most P. carolinensis starved at 25° C survive less than three weeks. Microfilament bundles are observed in the matrix of some mitochondria in amoebae starved for more than two days.Work supported by the U. S. Atomic Energy Commission.The authors acknowledge the assistance of Miss Doris Jean Buer and Miss Patricia Ann Sustarsic.     

Stratification within centrifuged amoeba nuclei

Summary Two species of large, fresh water amoebae were ultracentrifuged and studied with the electron microscope. Emphasis was placed on the stratification of the nucleoplasm, including nucleoli, within the confines of the nuclear envelope during interphase. Three major strata were found in the nuclei of both amoeba species, namely the centripetal nucleoplasm, the middle chromatin stratum, and the centrifugal nucleolar mass. In the highly radioresistant A. proteus, the nucleolar mass separated into a centripetal electron-opaque layer and a centrifugal electron-lucent layer. The latter layer appears to be missing from the radiosensitive P. illinoisensis. The nature of these nucleolar layers and their possible relationship to differences in radiosensitivity between the two species of amoebae is discussed. The contents of the heavier of the two nucleolar layers in A. proteus might be resistant to radiation damage and may possess radiorestorative capacity.Work supported by U.S. Atomic Energy Commission. A part of the work was reported at the 17th Meeting of the Society of Protozoologists, Boulder, Colorado in 1964.     

Inoculation of Paenibacillus illinoisensis alleviates root mortality, activates of lignification-related enzymes, and induction of the isozymes in pepper plants infected by Phytophthora capsici

To investigate the variations of the enzymes responsible for lignification, after inoculation with Phytophthora capsici and/or Paenibacillus illinoisensis KJA-424, in relation to biocontrol of Phytophthora blight in pepper, roots of two-month-old plants were inoculated with P. capsici inoculation (P), and co-inoculation of P. capsici and P. illinoisensis cell cultures (P + A). Root mortality of pepper plants induced by inoculation of P. capsici was completely recovered by co-inoculation with antagonistic KJA-424. At day 7, peroxidase (POD) activity increased by 36.7% in P-treated roots but by 7.1% only in P + A-treated, compared with control. Polyphenol oxidase (PPO) activity increased for 3 days and then drastically decreased in P-treated roots but maintained a constant level in control and P + A-treated. At day 7, PPO activity in P-treated leaves decreased but recovered to the level of control in the P + A-treated. Three major POD isozymes (45, 53, and 114 kDa) were shown in P-treated roots, while two major (53 and 114 kDa) in control and P + A-treated, suggesting that the 45 kDa of POD was actively induced in P-treated roots but not induced in P + A-treated roots. A PPO isozyme of 80 kDa was induced in P-treated roots but not induced by co-treated with KJA-424. In leaves, the POD isozyme of 45 kDa appears to be systemically induced in P-treated only. The PPO isozyme of 80 kDa in leaves was not induced by pathogen challenge but recovered by co-inoculated with P. illinoisensis. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, activates of lignification-related enzymes and induction of the isozymes in pepper plants infected by P. capsici.     

A study on histones in amoeba nuclei by indirect immunofluorescence method

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

Variation in the SSUrDNA of the Genus Protostelium Leads to a New Phylogenetic Understanding of the Genus and of the Species Concept for Protostelium mycophaga (Protosteliida,Amoebozoa)

Members of the genus Protostelium (including P. mycophaga, P. nocturnum, and P. okumukumu) are protosteloid amoebae commonly found in terrestrial habitats on dead plant matter. They, along with the closely allied nominal genus Planoprotostelium, containing the single species Pl. aurantium, all have an amoeboid trophic stage with acutely pointed subpseudopodia and orange lipid droplets in the granuloplasm. These amoebae form stalked fruiting bodies topped with a single, usually deciduous spore. The species are identified based on their fruiting body morphologies except for Pl. aurantium which looks similar to P. mycophaga in fruiting morphology, but has amoebae that can make flagella in liquid medium. We built phylogenetic trees using nuclear small subunit ribosomal DNA sequences of 35 isolates from the genera Protostelium and Planoprotostelium and found that (1) the nonflagellated P. nocturnum and P. okumukumu branch basally in the genus Protostelium, (2) the flagellate, Pl. aurantium falls within the genus Protostelium in a monophyletic clade with the nominal variety, P. mycophaga var. crassipes, (3) the cultures initially identified as Protostelium mycophaga can be divided into at least three morphologically recognizable taxa, P. aurantium n. comb., P. apiculatum n. sp., and P. m. rodmani n. subsp., as well as a paraphyletic assemblage that includes the remainder of the P. mycophaga morphotype. These findings have implications for understanding the ecology, evolution, and diversity of these amoeboid organisms and for using these amoebae as models for other amoeboid groups.     

Treatment of Paenibacillus illinoisensis suppresses the activities of antioxidative enzymes in pepper roots caused by Phytophthora capsici infection

Summary The activity of antioxidative enzymes after inoculation of pepper (Capsicum annuum L. Chungok) with a pathogen, Phytophthora capsici (P), the causal agent of Phytophtora blight and dual inoculation of pathogen and an antagonist, Paenibacillus illinoisensis KJA-424 (P+A), were measured and compared with that of non-inoculated (C) roots. Root mortality was significantly reduced by about 84% in P+A treatment compared with P treatment alone. When compared to the non-inoculated (C) roots, malondialdehyde (MDA) concentration gradually decreased by 52.4% in 7 days only in P-treated roots and hydrogen peroxide (H₂O₂) was not significantly affected by the treatment for 5 days but significantly decreased in the P+A-treated roots at day 7. P-treatment continuously induced peroxidase (POD) and superoxide dismutase (SOD), resulting in significant increases of 36.7% and 27.7% at day 7, respectively, compared to the control. In P+A-treated roots, the activities of POD and SOD also increased for 5 days but returned to the control level at day 7. Catalase activity fluctuated but again increased over the 7-day period following P+A inoculation. These results indicate that an antagonist P. illinoisensis KJA-424 alleviated root mortality and suppressed the elevated activities of POD and SOD in the root of pepper plant root caused by P.␣capsici infection.     

An Ixodes minor and Borrelia carolinensis enzootic cycle involving a critically endangered Mojave Desert rodent

Microtus californicus scirpensis is an endangered, isolated subspecies of California vole. It requires water pools and riparian bulrush (Schoenoplectus americanus) and occupies some of the rarest habitat of any North American mammal. The minimally vegetated, extremely arid desert surrounding the pools is essentially uninhabitable for Ixodes species ticks. We describe an enzootic cycle of Borrelia carolinensis in Ixodes minor ticks at a site 3500 km distant from the region in which I. minor is known to occur in Tecopa Host Springs, Inyo County, eastern Mojave Desert, California. Voles were live‐trapped, and ticks and blood samples queried by PCR and DNA sequencing for identification and determination of the presence of Borrelia spp. Between 2011–2013, we found 21 Ixodes minor ticks (prevalence 4–8%) on Amargosa voles and Reithrodontomys megalotis. DNA sequencing of 16S rRNA from ticks yielded 99% identity to I. minor. There was 92% identity with I. minor in the calreticulin gene fragment. Three ticks (23.1%), 15 (24%) voles, three (27%) house mice, and one (7%) harvest mice were PCR positive for Borrelia spp. Sequencing of the 5S‐23S intergenic spacer region and flagellin gene assigned Amargosa vole Borrelia strains to B. carolinensis. Ixodes minor, first described in 1902 from a single Guatemalan record, reportedly occurs only in the southeast American on small mammals and birds. The source of this tick in the Mojave Desert and time scale for introduction is not known but likely via migratory birds. Borrelia strains in the Amargosa ecosystem most closely resemble B. carolinensis. B. carolinensis occurs in a rodent‐I. minor enzootic cycle in the southeast U.S. although its epidemiological significance for people or rodents is unknown. The presence of a tick and Borrelia spp. only known from southeast U.S. in this extremely isolated habitat on the other side of the continent is of serious concern because it suggests that the animals in the ecosystem could be vulnerable to further incursions of pathogens and parasites.     

Hybridization and rapid differentiation after secondary contact between the native green anole (Anolis carolinensis) and the introduced green anole (Anolis porcatus)

In allopatric species, reproductive isolation evolves through the accumulation of genetic incompatibilities. The degree of divergence required for complete reproductive isolation is highly variable across taxa, which makes the outcome of secondary contact between allopatric species unpredictable. Since before the Pliocene, two species of Anolis lizards, Anolis carolinensis and Anolis porcatus, have been allopatric, yet this period of independent evolution has not led to substantial species‐specific morphological differentiation, and therefore, they might not be reproductively isolated. In this study, we determined the genetic consequences of localized, secondary contact between the native green anole, A. carolinensis, and the introduced Cuban green anole, A. porcatus, in South Miami. Using 18 microsatellite markers, we found that the South Miami population formed a genetic cluster distinct from both parental species. Mitochondrial DNA revealed maternal A. porcatus ancestry for 35% of the individuals sampled from this population, indicating a high degree of cytonuclear discordance. Thus, hybridization with A. porcatus, not just population structure within A. carolinensis, may be responsible for the genetic distinctiveness of this population. Using tree‐based maximum‐likelihood analysis, we found support for a more recent, secondary introduction of A. porcatus to Florida. Evidence that ~33% of the nuclear DNA resulted from a secondary introduction supports the hybrid origin of the green anole population in South Miami. We used multiple lines of evidence and multiple genetic markers to reconstruct otherwise cryptic patterns of species introduction and hybridization. Genetic evidence for a lack of reproductive isolation, as well as morphological similarities between the two species, supports revising the taxonomy of A. carolinensis to include A. porcatus from western Cuba. Future studies should target the current geographic extent of introgression originating from the past injection of genetic material from Cuban green anoles and determine the consequences for the evolutionary trajectory of green anole populations in southern Florida.     

The Free Amino Acid Levels of Pelomyxa carolinensis,Amoeba dubia and Proteus*

SYNOPSIS. The free amino acid extracts of 3 species of the free-living amoebae, Pelomyxa carolinensis, Amoeba dubia and A. proteus, were studied. Both the free amino acid patterns and levels in each species were different. Altho it appears that the free amino acid patterns were not altered by culture routines or starvation, the free amino acid levels were altered by these factors. The possibility of using free amino acid levels as a criterion for recognizing genetic differences is discussed.     

The Interaction of Naegleria fowleri Amoebae with Murine Macrophage Cell Lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D₁, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowler bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

Importance of fragmentation-tolerant species as seed dispersers in disturbed landscapes

Forest fragmentation can negatively affect plants if animal seed-dispersers become locally extinct in fragments. We conducted a 2-year experiment to evaluate the importance of tree squirrels (Sciurus) as seed dispersers for Quercus, Carya, and Juglans, and to assess dispersal consequences in patches where fragmentation-sensitive eastern gray squirrels (Sciurus carolinensis) are absent. We accounted for fates of ∼15,700 seeds from five tree species in four exclosure treatments at 18 fragments during a high (2003–2004) and low seed (2004–2005) year. Two treatments excluded Sciurus to mimic disperser loss. We sampled nut-tree seedling density at 259 sites across eight watersheds, half of which were too fragmented to support S. carolinensis, but supported fragmentation-tolerant fox squirrels (Sciurus niger). Autumn-to-spring seed survival was low (∼1%) for all species during low seed production. During high seed production, survival was higher for Juglans nigra (20%) and Carya ovata (16%) than for three Quercus species (∼4% for Quercus palustris and Quercus rubra in two exclosure types; ∼1% for Quercus alba in all treatments). Survival of J. nigra, C. ovata, and Q. rubra was ≥2.1–7.7 times higher for seeds in exclosures that Sciurus could access. Seed displacement distance was higher in the low seed than the seed-rich year, but the proportion of seeds surviving to greater distances was higher in seed-rich years for all seed types except Q. rubra. This affirms the importance of masting to seed survival and dispersal, but also suggests an advantage to trees of producing seed in non-mast years. Seedling densities were comparable in watersheds with and without S. carolinensis. These results demonstrate the importance of tree squirrels as dispersers of nut-bearing trees, but suggest that fragmentation may not disrupt dispersal of certain species if losing S. carolinensis from disturbed landscapes is compensated for by fragmentation-tolerant fox squirrels (S. niger).     

Interactions Between Aphaenogaster carolinensis (Hymenoptera: Formicidae) and Four Sympatric Ant Species

Individual ants of Aphaenogaster carolinensis Wheeler were placed in two way combinations with four sympatric ant species (Aphaenogaster lamellidens Mayr, Crematogaster lineolata Say, Pachycondyla chinensis Emery and Prenolepis imparis Say) to determine whether their interspecific behavior was dependent upon the ant species they were confronted with. The tendency of A. carolinensis to initiate and terminate interactions, and the durations of those interactions, were measured for all combinations. There was no significant effect of species on initiation or termination. There was a significant effect of species on the duration of interactions that were both initiated and terminated by A. carolinensis. Durations of interactions with A. lamellidens were significantly longer than with other species. The conclusion is that the interspecific behavior of A. carolinensis is dependent in part on the species it is interacting with.     

Feeding Characteristics of an Amoeba (Lobosea: Naegleria) Grazing Upon Cyanobacteria: Food Selection, Ingestion and Digestion Progress

Bacterivory by heterotrophic nanoflagellates and ciliates has been widely studied in aquatic environments, but data on the grazing of amoebae, are still scarce. From the water samples of Dianchi Lake (Kunming, Yunnan Province, China), we isolated an amoeba, designated as Naegleria sp. strain W2, which had potent grazing effects on some kind of cyanobacteria. The food selection mechanism and the digestion process of the amoeba were investigated in batch experiments. Predation experiments showed that filamentous cyanobacteria (e.g., Anabaena, Cylindrospermum, Gloeotrichia, and Phormidium) were readily consumed, with clearance rates ranging from 0.332 to 0.513 nL amoeba⁻¹ h⁻¹. The tight threads (Oscilltoria) and aggregates (Aphanizomenon) could not be ingested; however, their sonicated fragments were observed inside food vacuoles, suggesting that their morphologies prevent them from being ingested. Live video microscopy noted that unicellular Chroococcaceae (e.g., Synechococcus, Aphanocapsa, and Microcystis) were excreted after ingestion, indicating that food selection takes place inside food vacuoles. To determine whether the tastes or the toxins prevented them from being digested, heat-killed cells were retested for predation. Digestion rates and ingestion rates of the amoebae for filamentous cyanobacteria were estimated from food vacuole content volume. Through a “cold-chase” method, we found that the food vacuole contents declined exponentially in diluted amoebae cells, and digestion rates were relatively constant, averaging about 1.5% food vacuole content min⁻¹ at 28°C. Ingestion strongly depended on the satiation status of the amoebae, starved amoebae fed at higher rates compared with satiated amoebae. Our results suggest that the food selection and food processing mechanisms of the amoeba are similar to those of interception feeding flagellates; however, filamentous cyanobacteria cannot obtain a refuge under the grazing pressure of phagotrophic amoebae, which may widen our knowledge on the grazing of protists.     

Display Behavior and Habitat Use in Single and Mixed Populations of Anolis carolinensis and Anolis sagrei Lizards

Introduced species can have a variety of effects on the behavior and ecology of native species. We compared display behavior and habitat use of introduced Anolis sagrei and native Anolis carolinensis lizards across three sites in Southern Louisiana. The chosen sites were similar in that they were all located in urban settings with clumped vegetation. The first site contained only A. sagrei, the second supported sympatric A. sagrei and A. carolinensis populations, and the third site harbored only A. carolinensis. We found that (1) A. carolinensis perched significantly higher when A. sagrei was present, consistent with previous studies, whereas perch height of A. sagrei was not altered by the presence of A. carolinensis; (2) A. carolinensis in single and mixed sites exhibited different proportions of display types, with individuals at the mixed Tulane site performing significantly more C displays than those at the single site; and (3) Anolis sagrei at the Tulane mixed site exhibited less push‐ups than those in the site with A. sagrei alone. These data suggest that the arrival of congeners can affect display behavior of anoles, although such effects are different for the natives and the invaders.     

Fish humps in two Colorado River fishes: a morphological response to cyprinid predation?

Extant fishes endemic to the upper Colorado River of the American southwest include only cyprinids and catostomids. A curious attribute in species of both groups is the presence of a large nuchal hump. Largest cyprinid humps occur in humpback chub, Gila cypha, and largest catostomid humps occur in razorback sucker, Xyrauchen texanus. Several authors have suggested the humps confer a hydrodynamic advantage to life in fast flow, but this premise has not been confirmed with experimental work. To test the role of humps in Colorado River fishes, we subjected whole-body casts of preserved specimens with humps and with humps removed to controlled flows in an experimental tank. These tests confirmed that humps increased drag coefficients for X. texanus and G. cypha with no additional lift component. High energetic costs of locomotion and position-holding with a large hump, and the additional metabolic expense of forming large humps, suggest that the humps are not relict structures. Instead, we argue that these large humps represent convergent evolution prompted by predation from a cyprinid piscivore. Colorado pikeminnow, Ptychocheilus lucius, top piscivore in the Colorado River system, is the only native fish capable of consuming large X. texanus and G. cypha, and it also is sympatric with them. However, lack of jaw teeth and a relatively small jaw gape limit the maximum prey size that P. lucius can consume. Based on gape size, about 55% of X. texanus and 71% of G. cypha could be consumed by even the largest P. lucius. However, vulnerability would increase to 73 and 83% respectively if these species did not have humps. Coevolution tends to favor predator defense mechanisms in prey most vulnerable to such a voracious predator. Development of a large nuchal hump provides a deep body that is difficult or impossible for P. lucius to ingest.     

What’s on your boots: an investigation into the role we play in protist dispersal

D. M. Wilkinson (2010, Journal of Biogeography, 37 , 393–397) suggested that anthropogenic dispersal is an understudied and potentially important factor in terrestrial protist biogeography. We investigated human footwear as a potential vector of dictyostelids, a diverse group of amoebae that includes both geographically restricted and cosmopolitan species. Eighteen pairs of boots were examined and dictyostelids were isolated from nearly all samples larger than 5.0 g. In total, six dictyostelid isolates were recovered, corresponding to four species –Dictyostelium minutum, D. sphaerocephalum, D. leptosomopsis and a new species, Polysphondylium sp. 1. Myxogastrid amoebae and acrasid‐like aggregations were also observed. Thus anthropogenic dispersal of naked amoebae appears to occur. The possible role of variations in dictyostelid fruiting body morphologies in dispersal potential is also discussed. These results support Wilkinson’s proposal and suggest that dictyostelids may be a useful group with which to study anthropogenic dispersal of terrestrial protists.     

Distribution and ecology of freshwater sponges in Connecticut

A survey of Connecticut lakes and rivers revealed the presence of 7 species of freshwater sponge: Spongilla lacustris, Ephydatia muelleri, Eunapius fragilis, Anheteromeyenia ryderi, A. argyrosperma, Corvomeyenia carolinensis, and Corvospongilla novaeterrae in order of decreasing frequency of occurrence. Corvomeyenia carolinensis has not been reported previously beyond its type locality in South Carolina. In addition, microscleres of Spongilla lacustris, Anheteromeyenia-like megascleres, Ephydatia muelleri-like megascleres, and smooth megascleres (amphioxeas), which could not be assigned to a particular species, were found in surface sediments from lake cores. Spongilla lacustris inhabiting small rivers produced brown, thick-capsuled gemmules during the summer and yellow, thin-capsuled gemmules during the fall. The thick-capsuled gemmules, but not the thin-capsuled gemmules, are tolerant of desiccation; and populations of Spongilla lacustris and Ephydatia muelleri survived severe drying of their habitats during the summer.