First record of the insect pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) infection in larvae and pupae of Rhizophagusgrandis Gyll. (Coleoptera, Rhizophaginae) from Turkey

The predator beetle Rhizophagus grandis Gyll. (Coleoptera, Rhizophaginae) is one of the most important biological control agents, mass-bred and used to suppress populations of an important pest: the great spruce bark beetle, Dendroctonus micans. The achlorophyllous alga Helicosporidium sp. was first discovered in the pest. Later it was also found in the predator, but only in the adults. In this study, the pathogenic alga Helicosporidium sp. was discovered in larvae and early pupae of R. grandis for the first time. The morphological characteristics of the pathogenic alga were revealed by light and electron microscopy. Infection rates of Helicosporidium sp. in the larvae and pupae of R. grandis were 23.5% and 6.25%, respectively.     

Pathogenesis of Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) in susceptible noctuid larvae

Helicosporidium sp. is a unique, achlorophyllous green alga that has been reported to infect various insect orders, including Lepidoptera, Diptera, and Coleoptera. The infectious cyst stage is ingested by the host, ruptures in the midgut lumen, and releases a filamentous cell. Histopathological examinations using larvae of a susceptible noctuid host, Spodoptera exigua, showed both cysts and filamentous cells affiliated with the microvillar lining of the midgut epithelium. A considerable proportion of the ingested cysts (22-39%) were recovered in feces collected 24 h after ingestion. A small number of filamentous cells passed the midgut epithelium and entered the hemocoel within 4-24 h after cyst ingestion. After 48 h, vegetative cell stages were detected in the hemolymph, followed by a 4- to 5-day period of increasing multiplication. Cyst differentiation in the colonized hemolymph began 6-7 days after the treatment.     

Coccomyxa sp. (Chlorophyta: Chlorococcales), a new pathogen in mussels (Mytilus galloprovincialis) of Vigo estuary (Galicia, NW Spain)

In this work, we describe the occurrence of irregular shaped green aggregations in the mantle, gill filaments, adductor muscle, visceral mass and haemolymph of wild mussels (Mytilus galloprovincialis) collected from the Vigo estuary (Galicia, NW Spain). Microscopic examination of these masses revealed that they consist of intracellular green algae which are spherical to oval in shape, 5 μm in length and 3 μm in width, without flagella and with a smooth surface. The algal cells present a small single nucleus, a mitochondrion, 1-2 parietal chloroplasts and lack pyrenoids. Reproduction is by formation of 2-4 autospores or daughter cells. Pigment analysis reveals the presence of photopigments typical of green algae in addition to alloxanthin, diadinoxanthin and diatoxanthin. These carotenoids are noted for the first time in a parasitic chlorophyte. The signs of infection, together with the morphological observations, suggest that this parasitic algae may be Coccomyxa parasitica. However, further molecular studies are required for confirmation. This is the first report of Coccomyxa algae parasitizing the species M. galloprovincialis.     

Host-parasite relationship of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica in the Argentinean Patagonian coast

The association of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica is described. The identity of the green alga was confirmed by molecular studies; the alga was found within the hemocytes that infiltrate the connective tissue of the geoduck siphons. Cytological characteristics of hemocytes were not altered by algal infection; very often the algae were seen enveloped by a digestive vacuole within the hemocyte cytoplasm, evidencing diverse degrees of resorption. Connective cells of siphons were rarely infected by C. parasitica. The mean prevalence of C. parasitica was higher (82%) in San Matías Gulf (42°00′S, 65°05′W) than in San José Gulf (45%) (40°32′S, 64°02′W); except for spring, when the two locations showed no differences in prevalences (80%). Independently of location, season and host size, infected geoducks showed lower condition index values than uninfected ones. Regarding other bivalve species, only one specimen of the razor clam Ensis macha was found infected, and none of the oysters Ostrea puelchana and Pododesmus rudis and scallop Aequipecten tehuelchus was parasitized by the green alga.     

The life cycle and host specificity of Psychodiella sergenti n. sp. and Ps. tobbi n. sp. (Protozoa: Apicomplexa) in sand flies Phlebotomus sergenti and Ph. tobbi (Diptera: Psychodidae)

Two new gregarines in the recently erected genus Psychodiella (formerly Ascogregarina), Psychodiella sergenti n. sp. and Psychodiella tobbi n. sp., are described based on morphology and life cycle observations conducted on larvae and adults of their natural hosts, the sand flies Phlebotomus sergenti and Phlebotomus tobbi, respectively. The phylogenetic analyses inferred from small subunit ribosomal DNA (SSU rDNA) sequences indicate the monophyly of newly described species with Psychodiella chagasi. Ps. sergenti n. sp. and Ps. tobbi n. sp. significantly differ from each other in the life cycle and in the size of life stages. The sexual development of Ps. sergenti n. sp. (syzygy, formation of gametocysts and oocysts) takes place exclusively in blood-fed Ph. sergenti females, while the sexual development of Ps. tobbi n. sp. takes place also in males and unfed females of Ph. tobbi. The susceptibility of Phlebotomus perniciosus, Phlebotomus papatasi, Ph. sergenti, Ph. tobbi, and Phlebotomus arabicus to both gregarines was examined by exposing 1st instar larvae to parasite oocysts. High host specificity was observed, as both gregarines were able to fully develop and complete regularly the life cycle only in their natural hosts. Both gregarines are considered as serious pathogens in laboratory-reared colonies of Old World sand flies.     

An investigation on the antioxidant status of the invasive alga Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman, et Boudouresque (Caulerpales, Chlorophyta)

Caulerpa racemosa var. cylindracea is a potential invader of the Mediterranean Sea and 11 Mediterranean countries are under threat from this alga. In the present study, in order to investigate seasonal changes in the antioxidant status of C. racemosa var. cylindracea, antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and lipid peroxidation (LPO) levels were determined in C. racemosa var. cylindracea samples, which were collected in May 2003, September 2003, December 2003, and March 2004 from Turkish coastlines. SOD and CAT activities showed similar trends. These enzymes increased from May to December, then sharp decreases were observed. On the other hand, GSH-Px enzyme activities showed a fluctuation between May 2003 and March 2004. In contrast to increases in SOD and CAT activities up to December 2003, LPO level decreased in this period. No significant correlation was observed between antioxidant status and solar radiation. In conclusion, the antioxidant status of C. racemosa var. cylindracea is strictly not affected by both solar radiation and seawater temperature; however, the growth of epiphytes on fronds may change antioxidant status. Further investigations are strongly warranted to understand the contributions of non-enzyme-based antioxidants such as glutathione, vitamin E, and vitamin C.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

Pseudomonas asturiensis sp. nov., isolated from soybean and weeds

Five strains of gram negative bacteria, isolated from soybean (LPPA 221^T, 222 and 223) and weeds (LPPA 816 and 1442), were analyzed by a polyphasic approach. The isolates showed variation in their phenotypic traits and were placed in the Pseudomonas fluorescens lineage, based on 16S rRNA gene sequence phylogeny, as a single but well separated cluster. MLSA analysis based on gyrB and rpoD sequences clustered the strains in a single branch in the Pseudomonas syringae group, and revealed P. viridiflava as closest relative. DNA–DNA hybridizations showed medium levels of DNA–DNA relatedness with the type strain of P. viridiflava (50%) and lower levels (<32%) with other type strains of the P. syringae group, supporting classification within a novel species of the genus Pseudomonas. The strains can be distinguished from species of the P. syringae group by the fatty acid C_17:0 cyclo that is present in a low amount (2.5%) and from P. viridiflava by their inability to assimilate d-tartrate and d-sorbitol, and by the formation of red colonies on TTC medium. For this new species, the name Pseudomonas asturiensis sp. nov. is proposed. The type strain is LPPA 221^T (=LMG 26898^T = CECT 8095^T).     

The attached form of the endangered freshwater alga Aegagropila linnaei Kützing (Chlorophyta) is found in the Zuideindigerwiede, The Netherlands

The freshwater green macroalga Aegagropila linnaei used to occur in several locations in The Netherlands in unattached growths forms (including the enigmatic ‘lake balls’) more than 60 years ago. A recent survey of most of the historical collection sites of the species reported the attached growth form for the first time for The Netherlands, but only in miniscule quantities and from one location only. Here we report on the finding of the attached form from the Zuideindigerwiede, one of the two historical collection sites that had not been surveyed previously. Unattached growth forms do not seem to occur anymore in The Netherlands, and the species as a whole must be regarded as rare and endangered.     

Evidence for a facultative mutualist nutritional relationship between the green coccoid alga Bracteacoccus sp. (Chlorophyceae) and the zoosporic fungus Rhizidium phycophilum (Chytridiomycota)

Symbiotic interactions between fungi and photosynthetic partners are common among derived fungal lineages. The only fungal-phototroph interactions thus far reported from the early diverging zoosporic fungi are parasitic in nature. Rhizidium phycophilum is a terrestrial, saprotrophic chytrid, which appears to be able to enter a facultative mutualism with a coccoid green alga in the absence of refractory organic material, such as pollen and chitin. Liquid and solid culturing methods were used in a series of differential fitness experiments in conjunction with microscopic analyses to characterize the interaction between R. phycophilum and the alga. The alga in this partnership is identified as a member of the genus Bracteacoccus. Under certain culturing conditions, algal cells grown in coculture with R. phycophilum were shown to grow larger and more prolifically than when cultured axenically under the same conditions. Additionally, dialysis experiments demonstrate that R. phycophilum does not parasitize Bracteacoccus sp., and can be cultured in media infused with unknown algal exudates. Rhizidium phycophilum and Bracteacoccus sp. represent the first facultative positive interaction between a zoosporic fungus and a photoautotroph and may prove a tractable system for modelling interactions between early fungi and plants.     

Grazing of the invasive alga Codium fragile ssp. tomentosoides by the common periwinkle Littorina littorea: Effects of thallus size, age and condition

We examined the potential of herbivory by the common periwinkle Littorina littorea to limit recruitment and vegetative re-growth of the invasive green alga Codium fragile ssp. tomentosoides in a series of manipulative field experiments in tidepools on a wave-exposed rocky shore in Nova Scotia, Canada. Snails were excluded or included from circular plots (14 to 20 cm diameter) with cages to compare growth and survival of C. fragile against procedural (partial cages) or natural (uncaged) control plots. Our results show that L. littorea may restrict growth and survival of C. fragile by grazing new recruits (< 2 cm thallus length), fronds of adult thalli that are bleached and necrotic, and residual holdfasts (< 2 mm thickness) of detached thalli (artificially severed to mimic wave dislodgement). Once recruits grow beyond a critical size (~ 3 cm), or damaged tissues or holdfasts regenerate, grazing of C. fragile by L. littorea appears to be limited and ineffectual. Our experimental results corroborate correlative evidence from previous studies that herbivory by L. littorea limits the abundance of C. fragile in tidepools on the Atlantic coast of Nova Scotia, particularly pools in the high intertidal zone where these snails are abundant. Lower on the shore, littorinid grazing and physical stressors that render algae more vulnerable to grazers (e.g. UV radiation and freezing) are less intensive, and probably have less of a regulatory effect on populations of C. fragile.     

Definition of a novel symbiovar (sv. retamae) within Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma

In this paper we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Spain and Morocco from root nodules of Retama sphaerocarpa and Retama monosperma. All the strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium lablabi CCBAU 23086^T, with 99.41% identity with respect to the strain Ro19^T. Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII were divergent in Ro19^T and B. lablabi CCBAU 23086^T, with identity values of 95.71%, 93.75% and 93.11%, respectively. These differences were congruent with DNA–DNA hybridization analysis that revealed an average of 35% relatedness between the novel species and B. lablabi CCBAU 23086^T. Also, differential phenotypic characteristics of the new species were found with respect to the already described species of Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose to classify the group of strains isolated from R. sphaerocarpa and R. monosperma as a novel species named Bradyrhizobium retamae sp. nov. (type strain Ro19^T = LMG 27393^T = CECT 8261^T). The analysis of symbiotic genes revealed that some of these strains constitute a new symbiovar within genus Bradyrhizobium for which we propose the name “retamae”, that mainly contains nodulating strains isolated from Retama species in different continents.     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.     

Giardia duodenalis: Kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG₁, IgG_2a, IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10¹, 10², 10³, 10⁴, 10⁵) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.     

Helicosporidium infection of the great European spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

An infection of the great European spruce bark beetle Dendroctonus micans (from Turkey) by the parasitic green alga Helicosporidium is described. This is the first time that Helicosporidium has been found to infect a bark beetle (Scolytidae) and the first time that a naturally infected beetle has been reported for the Eurasian continent. The typical cysts of Helicosporidium contain three ovoid cells and one helical, filamentous cell. The morphological characteristics are revealed by light and electron microscopy. Distinct electron-dense inclusions with a peculiar ultrastructure may represent pyrenoids. Since D. micans is an important pest, the discovery of a natural pathogen may offer a chance for biological control.