Diurnal variation in relative photosynthetic performance in giant kelp Macrocystis pyrifera (Phaeophyceae, Laminariales) at different depths as estimated using PAM fluorometry

Pulse-amplitude modulated (PAM) fluorometry allows instantaneous estimates of photosynthetic rates, but may well produce variable measurements of photosynthetic activity depending on time of day, recent light history, internal fluctuations, and environmental variability. To investigate this, we compare estimates of diurnal variability in relative photosynthetic performance for the giant kelp, Macrocystis pyrifera (L.) C. Agardh, obtained from PAM fluorometry at three depths during 3 days characterized by different light conditions, and for two different blade ages. Sampling in the mid morning, late morning, early afternoon and late afternoon, we examined diurnal changes in relative photosynthetic performance in meristematic tissue and older blades occurring near the bottom, in the mid water, and at the water surface. Measures of maximum relative electron transport rates (rETR_max), minimum saturating irradiance (E_k), photosynthetic efficiency (α) and maximum quantum yield (F_v/F_m) show that giant kelp blades in the mid water and near the bottom exhibit little to no photosynthetic changes during the day. Near the surface, however, blades exhibit photosynthetic characteristics similar to light-adapted species in that they begin the day acclimated to low light, acclimate to increasing irradiance during the day, and end the day acclimated to low light. Consequently, while estimates of rETR_max were highest during the midday for all sample depths and days, they were also always highest near the surface for both old blades (112.16 ± 8.7, 98.6 ± 14.7, 70.16 ± 5.7) and meristematic tissue (109.0 ± 9.0, 86.9 ± 1.9, 59.2 ± 11.6, surface, mid water and bottom, respectively). Similar patterns were observed for E_k for both old blades (169.2 ± 5.4, 88.0 ± 11.2, 83.8 ± 5.2) and meristematic tissue (138.4 ± 11.5, 96.6 ± 4.69, 68.4 ± 10.6). In contrast, estimates of F_v/F_m were lowest near the surface during the midday for both old blades (0.6 ± 0.02, 0.73 ± 0.69, 0.75 ± 0.01) and meristematic tissue (0.58 ± 0.02, 0.69 ± 0.05, 0.74 ± 0.01, surface, mid water and bottom, respectively). These patterns coincided with similar patterns in ambient light, which was most variable and reached its greatest values near the surface during the midday.     

In situ detection of antibiotic-resistance elements in single Bacillus cereus spores

Rapid detection of Bacillus spores is a challenging task in food and defense industries. In situ labeling of spores would be advantageous for detection by automated systems based on single-cell analysis. Determination of antibiotic-resistance genes in bacterial spores using in situ labeling has never been developed. Most of the in situ detection schemes employ techniques such as fluorescence in situ hybridization (FISH) that target the naturally amplified ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a plasmidic or chromosomal origin and is present in low copy numbers in the cell. The main challenge in the development of low-target in situ detection in spores is the permeabilization procedure and the signal amplification required for detection. This study presents permeabilization and in situ signal amplification protocols, using Bacillus cereus spores as a model, in order to detect antibiotic-resistance genes. The permeabilization protocol was designed based on the different layers of the Bacillus spore. Catalyzed reporter deposition (CARD)–FISH and in situ polymerase chain reaction (PCR) were used as signal amplification techniques. B. cereus was transformed with the high copy number pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to chloramphenicol and erythromycin, respectively. In addition, a rifampicin-resistant B. cereus strain, conferred by a single-nucleotide polymorphism (SNP) in the chromosome, was used. Using CARD–FISH, only the high copy number plasmid pC194 was detected. On the other hand, in situ PCR gave positive results in all tested instances. This study demonstrated that it was feasible to detect antibiotic-resistance genes in Bacillus spores using in situ techniques. In addition, in situ PCR has been shown to be more sensitive and more applicable than CARD–FISH in detecting low copy targets.     

Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.     

Septal pore complex morphology in the Agaricomycotina (Basidiomycota) with emphasis on the Cantharellales and Hymenochaetales

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales.It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.     

Predator-prey interactions from in situ time-lapse observations of a sublittoral mussel bed in the Gulf of Trieste (Northern Adriatic)

Hexaplex trunculus (Linnaeus, 1758) is one of the most abundant and widespread muricid gastropods in the Northern Adriatic Sea, but relatively little is known about the feeding ecology of this predator. We examined the activity of H. trunculus on a sublittoral mussel bed at 24 m depth through in situ time-lapse observations and bulk samples. The camera photographed a 0.25 m² section of the mussel bed at 6-min intervals for ~ 23 h. Photos were examined frame-by-frame for gastropod movement and activities, especially interactions between H. trunculus and Mytilus galloprovincialis (Lamarck, 1819). Our survey indicates high activity-levels of H. trunculus on the sea floor: all gastropods made minor movements, most made major movements, and most left the field of view during the study-interval. On average, individuals remained stationary for only 7.3 h. Two predation attempts on Mytilus involving conspecific competition were documented, and one Hexaplex was consuming a mussel at the onset of the deployment. Additionally, 487 M. galloprovincialis from four diver-taken 0.25 m² quadrates were measured and examined for traces of marginal chipping and drilling predation. Mytilus from surface samples ranged from 11.1 mm to 95.5 mm in length, and one of the four samples had a significantly different average shell length from the others. 114 H. trunculus were collected and measured. Hexaplex ranged from 22.1 mm to 86.1 mm and the mean shell length did not differ among samples, though they were overwhelmingly medium and large. Predation frequency (the ratio of successfully preyed upon bivalves to the total number of bivalves sampled) is high at the studied site (> 55%), and large gastropods preferred a chipping mode of predation to drilling, supporting earlier laboratory studies showing a preference for M. galloprovincialis and this predation strategy. Prey effectiveness (the ratio of failed predatory attacks to total predatory attacks) is also high (63.8%), and no evidence of a size refuge was found. Feeding in H. trunculus is highly facultative, calling for caution when using drill holes to estimate predation intensities; whenever possible, traces of multiple predation modes should be considered.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Germination rate and sporeling development of Chondracanthus chamissoi (Rhodophyta, Gigartinales) varies along a latitudinal gradient on the coast of Chile

Chondracanthus chamissoi (C. Agardh) Kützing is an economically important red seaweed with an extended latitudinal distribution along the south-east Pacific. Here we report on the seasonal in vitro germination of carpospores and tetraspores from four populations distributed from 27 to 41° S on the Chilean coast. Our results show that both types of spores exhibited a different physiological behavior related to the geographic origin of the specimens. Germination occurred throughout the year for both spore types in the four populations. However, for the northern locations (Calderilla, La Herradura and Puerto Aldea) germination was higher in spring, while for the southern location (Lechagua), germination was higher in summer. The growth rate of carposporelings and tetrasporelings varied seasonally in all locations studied, with higher growth in spring. Among all, carposporelings from Lechagua specimens reached the highest growth rates (9.3 ± 0.2% d⁻¹). However, spores from Herradura and P. Aldea had a good germination and SGR in all seasons and would be good candidates to start spores-based cultivation of this valuable resource in Chile.     

Molecular and morphological relationships between two closely related species, Turbinaria ornata and T. conoides (Sargassaceae,Phaeophyceae)

We employed four markers (two chloroplastic, one mitochondrial and one nuclear) to investigate whether Turbinaria ornata and Turbinaria conoides, two morphologically similar brown algae, are distinct species. The molecular data revealed reciprocal monophyly, thus confirming the two algae as discrete species. Nevertheless, taxonomical ambiguities remain. T. conoides is more morphologically variable than T. ornata. We identified an atypical T. conoides morphotype whose blade possesses intramarginal teeth, a morphological characteristic of the T. ornata group that is normally absent within T. conoides. The molecular data indicate, nonetheless, that this morphotype belongs genetically to the T. conoides group. Thus, the results cast doubt upon the validity of using intramarginal teeth for species diagnosis.     

Entamoeba histolytica, heterologous expression and in situ immunolocalization of ornithine decarboxylase (EhODC)

Previous studies from this laboratory have dealt with the purification and biochemical characterization of ornithine decarboxylase (ODC) from Entamoeba histolytica. Enzyme compartmentalization has been described as a major mechanism in the regulation of polyamine metabolism. However, the subcellular location of ODC in the human parasite has remained unresolved. To examine this issue, we cloned the full-length gene (Ehodc) encoding for the parasite enzyme, whose open reading frame encodes for a peptide of 412 amino acids with an estimated molecular mass of 46 kDa that exhibits similarity to other ODCs. Heterologous overexpression of the gene allowed us to purify the recombinant protein (rEhODC) by metal affinity chromatography. The purified polypeptide was used to raise heteroclonal antibodies that were utilized to localize the enzyme in situ by immunofluorescence and confocal microscopy. EhODC was observed to be associated with the plasma membrane, in vesicles close to the plasma membrane and in the EhkOs organelle.     

Cytochrome oxidase I sequences reveal possible cryptic diversity in the cosmopolitan symbiotic copepod Nesippus orientalis Heller, 1868 (Pandaridae: Siphonostomatoida) on elasmobranch hosts from the KwaZulu-Natal coast of South Africa

Over the past decade, numerous molecular phylogenetic studies uncovered cryptic diversity within the Copepoda, yet very few investigations focused on symbiotic copepods. Here we report mitochondrial DNA cytochrome oxidase I diversity in the cosmopolitan elasmobranch symbiont Nesippus orientalis off the KwaZulu-Natal coast of South Africa. Analysis of partial COI sequences of copepods sampled from a diversity of shark hosts, revealed the presence of two divergent clades. Diversity within the clades does not appear to be structured based on host species, host individual, geographic locality or time of sampling. However, divergence between the two clades seems to be related to host species. Phylogenetic analyses of representatives from the two clades, along with Nesippus spp., Caligus spp. and Lepeophtheirus spp. outgroups, further supports the distinction between the two clades. Future molecular phylogenetic investigations of widespread copepod symbionts most likely will reveal far greater levels of biodiversity than currently recognized.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

A male cone of Pseudofrenelopsis dalatzensis with in situ pollen grains from the Lower Cretaceous of Northeast China

A Classopollis-containing male cone found in close association with Pseudofrenelopsis dalatzensis from the Lower Cretaceous Dalazi Formation of Yanji Basin, eastern Jilin, China was studied using scanning electron microscopy and described as Classostrobus dalatzensis sp. nov. The small oval cone, borne on the top of a stalk (fertile short shoot), consists of helically arranged microsporophylls, each with a rhomboid and expanded distal head. The short shoot is divided into indistinct nodes and internodes with fine longitudinal striations. Cuticles of the microsporophyll and the internode of the short shoot are generally similar to Pseudofrenelopsis dalatzensis internode cuticles, all of them having papillate outer surfaces. Stomata are similar in structure and arrangement, with papillae of subsidiary cells overhanging the stomatal pit. Epidermal cells are rectangular or isodiametrical, each with a robust papilla on the periclinal wall. Hypodermal cells are present between the stomatal files. The internal structure of the in situ Classopollis pollen grains was studied in detail. Pollen grain is small, with thin sexine, poorly developed nexine and may be immature. This is the first record of in situ pollen grains Classopollis in China of which the internal structure is known in detail.     

Irradiance and nutrient limitation of Dicytota spp. populations on Conch Reef, Florida Keys, USA

The photosynthetic performance, pigmentation and response to nutrient enrichment of a Dictyota community were studied over a 32 m depth gradient on Conch Reef, Florida Keys, USA. Dictyota spp. was the dominant space occupier on Conch Reef. During summer months from 1994 to 2001, mean percent cover was 43% at 7 and 21 m depths. Percent cover of Dictyota spp. was markedly lower at 32 m relative to shallower sites. The metabolism of Dictyota menstrualis and Dictyota pulchella were negatively impacted by attenuation of irradiance with increased depth such that ETR_max and P/R ratio decreased by 85% and 47%, respectively in samples from 7 to 32 m. Decreased cover of Dicytota spp. at 32 m relative to shallower sites may be the result of the inability of this species complex to acclimate to low irradiance levels as indicated by a lack of change in α and photosynthetic pigment content with increased depth.The response of D. menstrualis populations on Conch Reef to nutrient enrichment was variable. During August 2000, a natural enrichment experiment was conducted as D. menstrualis photosynthetic performance was surveyed both during and for days after a period of cool, nutrient-rich internal tidal bores bathed the 21 m site. No changes in in situ photosynthetic performance were observed either during or for 2 to 4 days after this natural event suggesting no nutrient limitation. In August 2001, a manipulative nutrient enrichment experiment was conducted with D. menstrualis from 7, 21 and 32 m. Increases in Φ_II were found in individuals exposed to nutrient enrichment from all depths, indicating at that time D. menstrualis was nutrient-limited on Conch Reef. Variation in the physiological response to nutrient enrichment may be the result of the frequency of internal tidal bores on Conch Reef in the months prior to our experiments. Variation in the responses by Dictyota spp. to irradiance and nutrient enrichment suggests that long-term monitoring over relevant temporal and spatial scales is necessary to accurately characterize limits on productivity and spread of this weedy species assemblage.     

Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota)

Torrubiella is a genus of arthropod-pathogenic fungi that primarily attacks spiders and scale insects. Based on the morphology of the perithecia, asci, and ascospores, it is classified in Clavicipitaceae s. lat. (Hypocreales), and is considered a close relative of Cordyceps s. 1., which was recently reclassified into three families (Clavicipitaceae s. str., Cordycipitaceae, Ophiocordycipitaceae) and four genera (Cordyceps s. str, Elaphocordyceps, Metacordyceps, and Ophiocordyceps). Torrubiella is distinguished morphologically from Cordyceps s. lat. mainly by the production of superficial perithecia and the absence of a well-developed stipitate stroma. To test and refine evolutionary hypotheses regarding the placement of Torrubiella and its relationship to Cordyceps s. lat., a multi-gene phylogeny was constructed by conducting ML and Bayesian analyses. The monophyly of Torrubiella was rejected by these analyses with species of the genus present in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, and often intermixed among species of Cordyceps s. lat. The morphological characters traditionally used to define the genus are, therefore, not phylogenetically informative, with the stipitate stromata being gained and/or lost several times among clavicipitaceous fungi. Two new genera (Conoideocrella, Orbiocrella) are proposed to accommodate two separate lineages of torrubielloid fungi in the Clavicipitaceae s. str. In addition, one species is reclassified in Cordyceps s. str. and three are reclassified in Ophiocordyceps. The phylogenetic importance of anamorphic genera, host affiliation, and stipitate stromata is discussed.     

The anamorphic genus Monotosporella (Ascomycota) from Eocene amber and from modern Agathis resin

The anamorphic fungal genus Monotosporella (Ascomycota, Sordariomycetes) has been reco-vered from a piece of Early Eocene Indian amber, as well as from the surface of extant resin flows in New Caledonia. The fossil fungus was obtained from the Tarkeshwar Lignite Mine of Gujarat State, western India, and was part of the biota of an early tropical angiosperm rainforest. The amber inclusion represents the second fossil record of Sordariomycetes, as well as the first fossil of its particular order (either Savoryellales or Chaetosphaeriales). The fossil fungus is distinguished from extant representatives by possessing both short conidiophores and small two-septate pyriform conidia, and is described as Monotosporella doerfeltii sp. nov. Inside the amber, the anamorph is attached to its substrate, which is likely the degraded thallus of a cladoniform lichen. The extant New Caledonian species is assigned to Monotosporella setosa. It was found growing on semi-solidified resin flows of Agathis ovata (Araucariaceae), and is the first record of Monotosporella from modern resin substrates.     

Isolation of anti-Candida albicans compounds from Markhamia obtusifolia (Baker) Sprague (Bignoniaceae)

An increase in clinical cases of Candidiosis globally as well as fungal resistance to drugs prompted the search for novel anti-Candida albicans agents from plant sources. Leaf extracts of Markhamia obtusifolia were screened for activity against C. albicans in vitro. An acetone extract obtained following serial exhaustive extraction contained mainly the active components with at least four active zones on the bioautogram. Bioassay guided fractionation of this extract led to the isolation of three compounds which inhibited the growth of three C. albicans strains. Based on spectroscopy studies (NMR and MS), the compounds were identified as 3β-hydroxyurs-12-en-28-oic acid, ursolic acid (1) 3β, 19α-dihydroxyurs-12-en-28-oic acid, pomolic acid (2) and 2β, 3β, 19α -trihydroxy-urs-12-en-28-oic acid, 2-epi-tormentic acid (3). The most active compound was 3β, 19α-dihydroxy-12-ursen-28-oic acid (2) with a minimum inhibitory concentration (MIC) value of 12.5 µg/mL for C. albicans isolated from dog and 25.0 µg/mL for C. albicans from cat and ATCC 90028 at 24 h following incubation. However, at 48 h of incubation MICs were > 400 µg/mL for all the three compounds isolated. This study indicated that M. obtusifolia could be a potential source of active principles against C. albicans.