Global Gene Expression Analysis during Sporulation of the Aquatic Fungus Blastocladiella emersonii

The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. (C) 2011 Elsevier Inc. All rights reserved.

Distribution of an alien aquatic snail in relation to flow variability, human activities and water quality

1. Disturbance and anthropogenic land use changes are usually considered to be key factors facilitating biological invasions. However, specific comparisons of invasion success between sites affected to different degrees by these factors are rare.

2. In this study we related the large-scale distribution of the invading New Zealand mud snail (Potamopyrgus antipodarum) in southern Victorian streams, Australia, to anthropogenic land use, flow variability, water quality and distance from the site to the sea along the stream channel.

3. The presence of P. antipodarum was positively related to an index of flow-driven disturbance, the coefficient of variability of mean daily flows for the year prior to the study.

4. Furthermore, we found that the invader was more likely to occur at sites with multiple land uses in the catchment, in the forms of grazing, forestry and anthropogenic developments (e.g. towns and dams), compared with sites with low-impact activities in the catchment. However, this relationship was confounded by a higher likelihood of finding this snail in lowland sites close to the sea.

5. We conclude that P. antipodarum could potentially be found worldwide at sites with similar ecological characteristics. We hypothesise that its success as an invader may be related to an ability to quickly re-colonise denuded areas and that population abundances may respond to increased food resources. Disturbances could facilitate this invader by creating spaces for colonisation (e.g. a possible consequence of floods) or changing resource levels (e.g. increased nutrient levels in streams with intense human land use in their catchments).

Impacts of atrazine in aquatic ecosystems

A portion of all herbicides applied to forests, croplands, road sides, and gardens are inevitably lost to water bodies either directly through runoff or indirectly by leaching through groundwater into ephemeral streams and lakes. Once in the aquatic environment, herbicides may cause stress within aquatic communities and radically alter community structure. Atrazine is one of the most effective and inexpensive herbicides in the world and is consequently used more frequently than any other herbicide. Atrazine is frequently detected in aquatic waters, and has been known to affect reproduction of aquatic flora and fauna, which in turn impacts on the community structure as a whole. This paper presents a summary of the reported direct and indirect impacts of atrazine on aquatic organisms and community structure. The information can be used for developing improved management guidelines and legislation. It is concluded that a single universal maximum limit on the atrazine application in catchments, as suggested by many regulatory authorities, does not provide adequate protection of the aquatic environment. Rather, it is advocated that flexible limits on the application of atrazine be developed in line with the potential risk of contamination to surface and subsurface water and fragility of the aquatic environment.

Reconnaissance limnology of Tasmania VIII. Tasmanian coastal lagoons - epicentres of endemism in the Australian aquatic microbiota

Two clusters of coastal lagoons, one near Strahan on Tasmania's west coast, the other near St Helens on the north-east coast, are the prime known epicentres of novelty and endemism in the Australian freshwater algal flora. The algae inhabiting these acid, dystrophic lagoons have a very limited distribution. Other dystrophic lagoons may have one or two, but not all, of this suite of endemics. The Strahan dune lakes, especially Lake Garcia, also have the greatest microfaunal diversity yet recorded from any Tasmanian waterbody, including several endemic species. The St Helens sites are less rich in species of microfauna, perhaps because of climatic differences or perhaps because of less intensive sampling there, but they, too, contain endemic taxa. The lagoons in both areas lie outside the formal protection of national parks, but present land management does provide a measure of protection.

INVASIVE AQUATIC INVASION: THE SPREAD OF VARIABLE LEAF MILFOIL IN MAINE

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Purification and characterisation of endo-β-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor

Laminarinase and endo-β-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a V_max of 42.0 µmol reducing sugars produced min–¹ mg protein–¹, a K_m of 0.126% (w/v) and an optimum pH range of 5.5–7, and hydrolysed mainly β-1,3-glycosidic bonds. In addition to the hydrolysis of β-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of β-1,4-glycosidic bonds. It had a V_max of 19.6 µmol reducing sugars produced min^–1 mg protein^–1, a K_m of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-β-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4–7. It mainly hydrolysed β-1,4-glycosidic bonds, but was also capable of significant hydrolysis of β-1,3-glycosidic bonds. Two endo-β-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53±3 and 52 kDa, were purified from C. destructor. Endo-β-1,4-glucanase 1 was only capable of hydrolysing β-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-β-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

Terrestrial detritus supports the food webs in lowland intermittent streams of south-eastern Australia : a stable isotope study

1. Large amounts of terrestrial detritus enter many low-order forested streams, and this organic
material is often the major basal resource in the metazoan food webs of such systems. However,
despite their apparently low biomass, algae are the dominant food of organisms in a number of
aquatic communities which conventionally would have been presumed to be dependent on
allochthonous detritus, particularly those in the tropics and also in lowland intermittent streams
in arid Australia.
2. The dual stable isotope signatures (d13C and d15N) of potential primary food sources were
compared with the isotopic signatures of common aquatic animals in lowland intermittent
streams in south-eastern Australia, in both spring and summer, to determine whether
allochthonous detritus was an important nutritional resource in these systems. The isotopic
signatures of the major potential allochthonous plant food sources (Eucalyptus, Phalaris and
Juncus) overlapped, but were distinct from algae and the dominant macrophytes growing in the
study reaches. The isotopic signatures of biofilm were more spatially and temporally variable
than those of the other basal resources.
3. Despite allochthonous detritus having relatively high C : N ratios compared to other
potential basal resources, results from ISOSOURCE mixing model calculations demonstrated
that this detritus, and the associated biofilm, were the major energy sources assimilated by
macroinvertebrate primary consumers in both spring and summer. The importance of these
energy sources was also reflected in animals higher in the food web, including predatory
macroinvertebrates and fish. These resources were supplemented by autochthonous sources of
higher nutritional value (i.e. filamentous algae and macrophytes, which had relatively low
C : N ratios) when they became more prolific as the streams dried to disconnected pools in
summer.
4. The results highlight the importance of allochthonous detritus (particularly from Eucalyptus)
as a dependable energy source for benthic macroinvertebrates and fish in lowland intermittent
streams of south-eastern Australia. This contrasts with previous stable isotope studies
conducted in lowland intermittent streams in arid Australia, which have reported that the fauna
are primarily dependent on autochthonous algae.

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, Cherax destructor and Engaeus sericatus (Astacidae, Parastacidae)

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81–82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-β-1,4-glucanase, β-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-β-1,4-glucanase and β-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-β-d-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.

The development of biological objectives for streams in a single catchment: a case study on the catchment of western port bay, Victoria, Australia

Government environment protection policies for waterways have traditionally relied on water quality indicators and their objectives. In this paper we describe the development of biological objectives based on invertebrate indicators for inclusion in a government policy for the catchment of Western Port Bay, Victoria. The first step of defining segments (areas with streams in which the same objectives are applied) was problematic, requiring two different approaches, as follows. Site groups initially based on invertebrate community composition derived using multivariate techniques (ordination and classification) proved to be unsuitable for policy segments. Segment boundaries were subsequently defined using topographical (e.g. boundary of foothills and lowland plains), climate (e.g. rainfall) and land-use (e.g. urban) features. We used information and data from reference sites inside as well outside the catchment to derive specific biological objectives based on aquatic invertebrates for these segments. Objectives were specified for the following four indicators – number of invertebrate families, the SIGNAL index, the AUSRIVAS predictive model and the number of key families.

Effects of invertebrate predators and a pesticide on temporary pond microcosms used for aquatic toxicity testing

The effects of increased trophic complexity, through the addition of predatory notonectids (Anisops deanei), on temporary pond microcosms used for aquatic toxicity testing were studied. Replicate microcosms were established using sediment from a dried temporary pond, and treated with one of four concentrations of the organochlorine pesticide endosulfan (0, 1, 10 or 50 μg/L), in the presence or absence of six A. deanei. The tanks were sampled regularly for nine weeks following the addition of the predators and the entire contents of each tank counted after 12 weeks. Analysis using non-metric multidimensional scaling (MDS) and non-parametric MANOVA showed that both Anisops and endosulfan at concentrations >10 μg/L significantly altered community structure. However, an interaction between the effects of Anisops and the effects of endosulfan was not detected. The addition of Anisops did not increase the variability of response and thus did not reduce the sensitivity of the test method.

Spatial and temporal variation of infauna in an Australian estuary

Macroinvertebrates living within the sediments of the Hopkins River estuary were examined during 1997. Differences in the abundance and composition of faunal assemblages were identified between vegetated and unvegetated habitats and between the lower, middle and upper sections of the estuary. The numbers of taxa and individuals present were found to be similar to that of other periodically opened estuaries.