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.     

Postingestive sorting of living and heat-killed Chlorella within the sea scallop, Placopecten magellanicus (Gmelin)

Suspension-feeding bivalves may enhance the energy value of their food supply by sorting particles both before and after ingestion. Previous research has indicated that the sea scallop (Placopecten magellanicus (Gmelin) (Mollusca: Bivalvia)) is capable of sorting particles within the gut both on the basis of physical properties (particle size and density) as well as chemical properties. In this study, the ability of the sea scallop to sort living from dead material solely on the basis of chemical properties was tested. The microalga Chlorella (Chlorophyta: Chlorophyceae) was chosen as the test particle because its thick cell wall remains physically intact following heat treatment, while its carbon, nitrogen, and chlorophyll a content declines. Scallops were fed a mixture of radiolabelled live and heat-killed Chlorella. We demonstrate that P. magellanicus can distinguish between living and dead algae, retaining live Chlorella cells longer than heat-killed cells. This ability to detect the subtle chemical differences between living algal material and detrital material would enhance the digestive efficiency of this species by reducing the amount of energy expended, digesting poor-quality materials. This paper presents the first study of the ability of a bivalve to distinguish between two physically identical but nutritionally different forms of the same species of microalgae.     

Thermal biology of the southernmost lizards in the world: Liolaemus sarmientoi and Liolaemus magellanicus from Patagonia,Argentina

We determined the efficiency of thermoregulation by the southernmost liolaemids Liolaemus sarmientoi and L. magellanicus from Patagonia, Argentina (51°S), by measuring body (T_b), microenvironmental, and operative temperatures in the field, and preferred body temperatures in the laboratory (T_pref). L. sarmientoi was found to be a poor thermoregulator, whereas L. magellanicus was deemed to be a constrained thermoconformer. Among all known species of Liolaemus, L. sarmientoi and L. magellanicus had the lowest T_b's when tested in the field; however, their T_pref's were similar to other liolaemids. These data suggest that these southernmost liolaemid species have not evolved appropriate thermoregulatory behaviors or made adequate physiological adaptations to face the extreme thermal challenges of their environment.     

A putative serine protease,SpSsp1, from Saprolegnia parasitica is recognised by sera of rainbow trout, Oncorhynchus mykiss

Saprolegniosis, the disease caused by Saprolegnia sp., results in considerable economic losses in aquaculture. Current control methods are inadequate, as they are either largely ineffective or present environmental and fish health concerns. Vaccination of fish presents an attractive alternative to these control methods. Therefore we set out to identify suitable antigens that could help generate a fish vaccine against Saprolegnia parasitica. Unexpectedly, antibodies against S. parasitica were found in serum from healthy rainbow trout, Oncorhynchus mykiss. The antibodies detected a single band in secreted proteins that were run on a one-dimensional SDS-polyacrylamide gel, which corresponded to two protein spots on a two-dimensional gel. The proteins were analysed by liquid chromatography tandem mass spectrometry. Mascot and bioinformatic analysis resulted in the identification of a single secreted protein, SpSsp1, of 481 amino acid residues, containing a subtilisin domain. Expression analysis demonstrated that SpSsp1 is highly expressed in all tested mycelial stages of S. parasitica. Investigation of other non-infected trout from several fish farms in the United Kingdom showed similar activity in their sera towards SpSsp1. Several fish that had no visible saprolegniosis showed an antibody response towards SpSsp1 suggesting that SpSsp1 might be a useful candidate for future vaccination trial experiments.     

Inhibition of virulent and hypovirulent Cryphonectria parasitica growth in dual culture by fungi commonly isolated from chestnut blight cankers

Chestnut blight cankers, caused by the fungus Cryphonectria parasitica, are prone to invasion by other microorganisms as the canker ages. This microbial community has the potential to alter canker expansion, which may influence the probability that the canker girdles the infected stem. Hypoviruses infect the pathogen mycelium directly and are known to decrease pathogen virulence (i.e. hypovirulent). These viral infections can slow pathogen growth, decreasing the rate of canker expansion and lowering the probability of girdling. Saprophytic fungi also invade the expanding canker and may antagonize C. parasitica leading to reduced pathogen growth. The combined effects of fungal antagonism and a hypovirulent pathogen could work in combination to reduce the probability of girdling the infected stem. We assessed the ability of different fungal taxa, isolated from low severity cankers, to inhibit the growth of virulent and hypovirulent forms of C. parasitica in dual culture tests on two cultural media. Percent growth inhibition of virulent C. parasitica by potentially antagonistic fungi ranged from 2 % to 34 %, while inhibition of hypovirulent C. parasitica ranged from 18 % to 54 %. Only one isolate, identified as Umbelopsis isabellina (UmbelopsisWS) inhibited the virulent form of the pathogen more than the hypovirulent form. All three Trichoderma isolates caused the greatest growth inhibition of virulent C. parasitica, but they, like all other fungal isolates tested, inhibited the hypovirulent form of the pathogen more than the virulent form. These results suggest that commonly occurring fungi in chestnut blight cankers, including Trichoderma, may inhibit the hypovirulent C. parasitica more than virulent C. parasitica. Thus, the presence of other fungi in cankers may not enhance the effect of hypovirulent C. parasitica to delay cankers from girdling a stem but instead intensify canker development.     

Storage glucan biosynthesis in Cyanidium, Chlorella and Prototheca: evidence for endosymbiosis?

The storage glucans of Chlorella pyrenoidosa and Prototheca zopfii are identical and consist of a linear polyglucan akin to amylose and a branched amylopectin component. The branched glucans of these algae differ markedly from that formed by the hot-springs alga, Cyanidium caldarium. The more highly branched Cyanidium glucan appears to be formed by branching glucosyltransferases which are different from those of the other two algae. The relevance of the data to the possibility of Cyanidium being a Prototheca-like Chlorella that has acquired symbiotic Cyanobacteria as chloroplasts is discussed.     

浒苔干粉末提取物对东海原甲藻和中肋骨条藻的克生作用

研究不同溶剂的浒苔干粉末组织提取液对两种赤潮藻—东海原甲藻和中肋骨条藻生长的克生效应。结果表明, 浒苔提取物中确实含有可以影响赤潮藻类生长的克生物质,克生作用具有较明显的浓度效应,添加浓度低时可能会表现为一定的促进生长的作用,添加浓度较高时表现为抑制作用,添加浓度越大,抑制作用越强,即“低促高抑”的特点,这与浒苔提取物对其他微藻的作用相似。其中,蒸馏水提取物对这两种赤潮藻的克生作用小于有机溶剂提取物,有机溶剂中,甲醇和乙酸乙酯提取物对这两种赤潮藻的克生效果最好,正己烷相对较差。根据相似相溶原理,可以初步推断最有效的克生物质应为具有相对较高的极性的有机物。两种赤潮藻对克生物质的敏感程度不同。东海原甲藻对克生物质的敏感性高于中肋骨条藻。蒸馏水、甲醇、乙酸乙酯、正己烷的浒苔干粉末提取物影响东海原甲藻生长的致死作用阈值浓度分别为5.00 g/L、0.50 g/L、0.50 g/L、0.63 g/L（相当于浒苔新鲜藻体浓度为22.00 g-wet/L、2.20 g-wet/L、2.20 g-wet/L、2.75 g-wet/L）;影响中肋骨条藻生长的致死作用阈值浓度则分别为20.00 g/L、1.25 g/L、1.25 g/L、2.50 g/L（相当于新鲜藻体浓度为88.00 g-wet/L、5.50 g-wet/L、5.50 g-wet/L、11.00 g-wet/L）。     

Investigations on cellular defense reactions with Galleria mellonella against Bacillus thuringiensis

In contrast to a great number of foreign particles (bacteria and inorganic materials), cells of some strains of Bacillus thuringiensis are not phagocytosed in the first hours after injection into the hemocoel of Galleria mellonella. This phenomenon is not caused by the production of β-exotoxin or exoenzymes, because heat-killed cells are not phagocytosed and the phagocytosis of latex particles is not prevented by the presence of living B. thuringiensis. The phagocytosis of heat-killed B. thuringiensis subtoxicus can be encouraged by treatment of the cells and by simultaneous injection of latex particles. A factor stimulating the phagocytosis is discussed. It is induced by the injection of phagocytosable latex particles into the hemocoel but not by injection of living or killed B. thuringiensis subtoxicus.     

Galleria melonella as an experimental in vivo host model for the fish-pathogenic oomycete Saprolegnia parasitica

Oomycetes are eukaryotic pathogens infecting animals and plants. Amongst them Saprolegnia parasitica is a fish pathogenic oomycete causing devastating losses in the aquaculture industry. To secure fish supply, new drugs are in high demand and since fish experiments are time consuming, expensive and involve animal welfare issues the search for adequate model systems is essential. Galleria mellonella serves as a heterologous host model for bacterial and fungal infections. This study extends the use of G. mellonella for studying infections with oomycetes. Saprolegniales are highly pathogenic to the insects while in contrast, the plant pathogen Phytophthora infestans showed no pathogenicity. Melanisation of hyphae below the cuticle allowed direct macroscopic monitoring of disease progression. However, the melanin response is not systemic as for other pathogens but instead is very local. The mortality of the larvae is dose-dependent and can be induced by cysts or regenerating protoplasts as an alternative source of inoculation.     

Divergence of the mitochondrial electron transport chains from the green alga Chlamydomonas reinhardtii and its colorless close relative Polytomella sp.

Compelling evidence exists that the colorless algae of the genus Polytomella arose from a green Chlamydomonas-like ancestor by losing its functional photosynthetic apparatus. Due to the close relationship between the colorless and the green chlorophyte, Polytomella sp. appeared as a useful indicative framework for structural studies of Chlamydomonas reinhardtii mitochondria. However, comparative studies reported here unexpectedly revealed significant differences between the mitochondrial respiratory systems of the two algae. Two-dimensional blue native/SDS-PAGE of isolated mitochondria indicated that cytochrome-containing respiratory complexes III and IV in the two chlorophytes contrast in size, subunit composition and relative abundance. Complex IV in Polytomella is smaller than its counterpart in C. reinhardtii and occurs in two forms that differ presumably in the presence of subunit COXIII. The cytochrome c and the iron-sulfur Rieske protein of both chlorophytes revealed structural differences on the amino acid sequence level. Under comparable culture conditions, the colorless alga exhibits lower levels of cytochrome c and complex IV but a higher respiratory activity than the green alga. Cytochrome c levels were also found to be differently regulated by the growth conditions in both algae. The divergence between the respiratory systems in the two related chlorophytes can be viewed as a consequence of the loss of photosynthetic activity and/or of the adaptation to the environment via the acquisition of a more flexible, heterotrophic metabolism. Our understanding of mitochondrial function and evolution is expected to be greatly enhanced via further parallel studies of photosynthetic/non-photosynthetic algae, for which this study forms an incentive.     

Genetic diversification of the chestnut blight fungus Cryphonectria parasitica and its associated hypovirus in Germany

Chestnut blight in south-western Germany was first reported in 1992 and is since expanding in distribution. Here we investigated the invasion history of Cryphonectria parasitica and its associated hypovirus. For this, we characterized 284 isolates collected between 1992 and 2012 for hypovirulence, vegetative compatibility (vc), mating type, and microsatellite haplotype. A total of 27 haplotypes and 15 vc types were observed, although the C. parasitica population analyzed is currently dominated to 50 % by one haplotype and to 64 % by the vc type EU-2. Structure analysis indicated two divergent genetic pools. Over 66 % of the haplotypes belonged to a pool probably originating from northern Italy. Further diversification is expected due to ongoing sexual recombination, but also to new migration and additional introductions. Cryphonectria hypovirus 1 (CHV-1) was found in four of five C. parasitica populations from Baden-Württemberg. Genetic analysis of the 35 CHV-1 isolates obtained revealed that they all belong to the German subtype, although they have clearly diverged from the first German hypovirus isolated in 1992. Our study suggests that C. parasitica has been introduced into Germany several times from two different gene pools, whereas the hypovirus most probably has a single origin.     

Experimental studies on the marine algae Iridaea and Gigartina

Field and laboratory studies of the economically important marine algae Iridaea cordata (Turner) Bory and Gigartina exasperata Harvey & Bailey (Rhodophyta: Gigartinales) have been made in order to establish information essential to the aquaculture of these species. The development of a simple and successful method for experimental transplantation of these species which permits periodic removal, measurement, and photography of the transplanted algae is described and its feasibility for successfully transplanting these algae to habitats in which they do not occur naturally, demonstrated. The growth rates of such transplanted algae and the maximum size which they attain are comparable to those of plants in the population from which the transplanted algae were taken.In field experiments, the optimum depth for maximum growth rates was 3–5 m below M.L.L.W. Laboratory experiments using a crossed-gradients (light and temperature) incubator indicated that optimum growth rates are obtained at temperatures of 10–14 °C and light intensities of 8.9–16.0 × 10³ erg/cm² sec (cool-white fluorescent); such light intensities are substantially higher than the optimum for many of the red algae that have been grown in laboratory culture. These results support the biological feasibility of cultivating these algae in the inland waters of the Pacific Northwest.     

Performance and thermal sensitivity of the southernmost lizards in the world, Liolaemus sarmientoi and Liolaemus magellanicus

Locomotor activity performance of reptiles is largely temperature dependent and, in harsh environments with short activity periods during the day and throughout the year, plays a vital role in the fitness of the species. This particular study focuses on the performance and the thermal sensitivity for running, at different body temperatures, of the two southernmost species of lizards in the world, Liolaemus sarmientoi and Liolaemus magellanicus, studied at two locations in the south of Santa Cruz province, Argentina (51°S, 70°W and 50°S, 72°W; 133 m asl). The speed of sprint and long runs was measured in the field to determine the physiological performance of lizards at different air temperatures. In both species speed increases with the temperature, and they reach the highest performance at high temperatures. The difference between activity and thermal optima suggests that L. magellanicus has colonized its actual environment recently, and that it has not had enough time for its physiological mechanisms to evolve and achieve a maximum performance at the cold temperatures they have to tolerate at present. In contrast, L. sarmientoi achieved a high performance over a wider range of temperatures that included temperatures lower than the preferred temperatures in the lab, which they can generally find in their environment.     

Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing

Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica.     

Recruitment and metamorphosis of Haliotis larvae induced by molecules uniquely available at the surfaces of crustose red algae

Crustose red algae induce substratum-specific settlement, attachment and metamorphosis of the planktonic larvae of Haliotis rufescens Swainson (gastropod mollusc), upon direct contact by the larvae with any of a number of algal species tested. Larvae are not induced by contact with intact foliose red, brown or green macroalgae. Geniculate red algae are only slightly active. Larval settlement and metamorphosis are shown to be triggered by a class of chemical inducers associated with macromolecules and found in extracts of all species of crustose, geniculate, and foliose red algae tested; these inducers are not found in extracts of brown or green macroalgae. The substratum specificity of larval settlement and metamorphosis is shown to result from the unique availability of these inducers at the surfaces of the crustose red algae. Using a newly-developed improved method of purification based upon size-separation by gel-filtration, followed by ion-exchange chromatography over a diethylaminoethyl (DEAE)-acrylamide matrix, the principal inducer of Haliotis larval settlement and metamorphosis has been resolved from the red algal phycobiliproteins. Sensitivity of this inducer to reduction in molecular weight by digestion with trypsin demonstrates that this inducer is associated with protein.     

Allelopathic activity of Elodea canadensis and Elodea nuttallii against epiphytes and phytoplankton

Elodea nuttallii and Elodea canadensis have both been introduced from North America to Europe. They are now common in many water bodies where they often form dominating stands. It was suggested that negative relationships between Elodea and phytoplankton or epiphytic covers exist, probably due to the release of growth inhibiting allelochemicals. This would be an effective strategy to avoid light limitation caused by algae and cyanobacteria. We investigated the allelopathic potential of both E. nuttallii and E. canadensis against different photoautotrophs, focussing on epiphytic algae and cyanobacteria isolated from different submersed macrophytes and culture strains. Methanolic extracts of both species inhibited the growth of most of these organisms. Only a culture strain of Scenedesmus brevispina was stimulated. Further separation of extracts yielded several active fractions, indicating that hydrophilic and slightly lipophilic compounds were responsible for growth reduction. At least some of the activity seems to be related to phenolic substances, but flavonoids in these species are inactive. Since growth declined also in a moderately lipophilic fraction of culture filtrate of E. nuttallii, we assume that active compounds were exuded in the water. Allelopathy might thus be relevant in situ and suppress cyanobacteria and algae. We furthermore found differences in the susceptibility of target organisms, which could (1) at least partly be a result of adaptation to the respective host plants and (2) indicate that allelopathic interference might reduce the abundance of some species, especially cyanobacteria, in epiphytic biofilms.     

In situ tests on inducible defenses in Dictyota kunthii and Macrocystis integrifolia (Phaeophyceae) from the Chilean coast

Numerous experimental studies have reported inducible defenses in macroalgae, but most of them have been conducted in laboratory environments where algae were maintained detached from the substratum and in artificial flow regimes. The results of those experiments might not reflect the natural situation, which can only be studied in situ. We examined whether the brown macroalgae Dictyota kunthii (C. Agardh) Greville and Macrocystis integrifolia (Bory) show inducible defenses following exposure to different grazing levels (direct, water-borne cues from nearby grazed conspecifics, presence of a non-grazing herbivore and natural grazing) in field experiments, striving to maintain natural conditions as much as possible. We measured palatability of algae after exposure to different grazing levels by using live algae and agar-based food containing non-polar extracts. M. integrifolia showed no induction of defenses (at least not of non-polar compounds), suggesting constitutive defenses, absence of defenses (tolerance) or use of another strategy to avoid herbivory. These results are similar to those from previous laboratory experiments. In D. kunthii, defense was induced after two weeks of direct grazing by amphipods under field conditions. Water-borne cues from nearby grazed conspecifics, presence of a non-grazing herbivore and natural grazing did not induce defenses. Induction of defense in response to direct grazing agrees with results from a previous laboratory study, but while indirect cues induced defenses in the laboratory, there was no measurable induced defense in the field. Probably chemical cues from grazers are diluted quickly in the field, not reaching concentrations that cause induction of defenses. This might be the reason why in some algae induction by direct grazing is a more important defensive strategy than induction by water-borne cues. The results from our study also suggest that laboratory experiments showing induced defenses in response to grazed neighbours or mere grazer presence need to be interpreted with caution.     

The algal flora of a subtidal Zostera bed in ventry bay,Southwest Ireland

Ventry Bay in County Kerry, southwest Ireland, was surveyed in June 1977 to a depth of 17 m using SCUBA and a grid of stations which were 400 m apart. The principal substrate was sand. Zostera marina L. was dominant and widespread from 0.5 to 13 m below Chart Datum and is the deepest-growing Zostera in Northwest Europe. Sixty species of algae were found and their occurrence and that of Zostera marina was subjected to cluster analysis resulting in one distinct cluster of Zostera marina, Ulva lactuca L., Gracilaria verrucosa (Huds.) Papenf., Polysiphonia elongata (Huds.) Spreng., Ectocarpus sp., Ceramium rubrum (Huds.) C. Ag. and Brongniartella byssoides (Good. et Woodw.) Schmitz. These algae are so common and widespread in the subtidal zone that their co-occurrence with Zostera is not considered to be significant. Zostera occurs virtually as monospecific stands in sand and the co-occurrence of any macroalgae is largely fortuitous and dependent on the heterogeneity of the substrate.     

Emerging new crown symptoms on Castanea sativa (Mill.): Attempting to model interactions among pests and fungal pathogens

In the 2015–2016 growing seasons, two novel symptoms were assessed on the crown of trees in orchards and coppices of chestnut groves in Central Italy. The first symptom was flagging of annual shoots with green leaves undergoing sudden wilt and turning brown later in the season. The second symptom consisted of leaves on annual shoots turning yellow before wilting in absence of flagging represented the second symptom. Samples were collected along transects in early summer, late summer and winter, and processed in the laboratory. The flagging symptom was associated in early summer with the presence of C. parasitica in cryptic dried buds on stems from the previous year's growth. The pathogen was also found in dormant buds in winter, suggesting that the infection could take place in summer during the Chinese gall wasp oviposition period. Cryphonectria parasitica was also isolated from abandoned galls in winter supporting the hypothesis that galls are a potential source of inoculum for crown infections. Aetiology of yellowing was not clarified and no fungal taxa were specifically associated with this symptom. Gnomoniopsis castanea, C. parasitica and, in early summer, Colletotrichum acutatum were the most abundant fungal taxa isolated from chestnut shoots and buds.