The peritrophic membrane of the gastropod Megathura crenulata: structure,composition, and site of formation

Peritrophic membranes (PMs) are acellular layered structures secreted around ingested materials by the gut epithelium. Most studies on PMs have focused on those of insects and crustaceans due to their potential ability to block the movement of pathogens from ingested materials into the body, and their possible use as unique targets relevant to pest management. While PMs are known to occur in other taxa, their distribution is spotty and little is known about their role in these other species. The gastropod Megathura crenulata produces a true PM, which has a chitinous matrix that makes up nearly half its wet weight. Unlike arthropod PMs, which are released by delamination from the microvilli of their gut cells, the chitinous matrix of the M. crenulata PM is secreted from epithelial cells lining most regions of its gut. Although its mode of synthesis is unique, it may serve the same functions as proposed for other PMs, including regulating diffusion, binding metabolites, restricting protease activity, blocking pathogens, and providing lubrication. In arthropods, numerous proteins with chitin‐binding specificities have been identified, consistent with the proposed functions. Analysis of PMs in M. crenulata showed several integral proteins associated with the membrane, suggesting that the PM in this mollusc may be involved in complex functions like those seen in the arthropods.     

The antitumor activity of the hydrolysates of chitinous materials hydrolyzed by crude enzyme from Bacillus amyloliquefaciens V656

Chitin, colloidal chitin and water-soluble chitosan were hydrolyzed by crude enzyme solution produce by Bacillus amyloliquefaciens V656. The hydrolysates with 12 h hydrolysis contained optimal (GlcNAc)₆ and showed higher antitumor activity. Among those chitinous materials, the most effective one was the hydrolysates of water-soluble chitosan, which inhibited the growth of CT26 cells and reduced the survival rate to 34% in 1 day. Since the hydrolysate of water-soluble chitosan contained the optimal hexamer/(GlcNAc)₆ at 12 h, it is conjectured that the antitumor activity should be related to (GlcNAc)₆. This conjecture was further affirmed by experiment with pure (GlcNAc)₆. However, This phenomenon might be due to the synergistic effect of the oligomers (GlcNAc)_n, n = 1–6 in the hydrolysates. The antitumor effect of the chitinous hydrolysates is worth further investigation.The aim of this study was to investigate the induced apoptosis in CT26 cells by the hydrolysates of chitinous materials. It was found that the hydrolysates (A, B and C) inhibited the survival of CT26 cells in a concentration- and time-dependent manner. The hydrolysates induced characteristic DNA fragmentation of the CT26 cells. These results suggested that the hydrolysates from chitinous materials are potent apoptosis-inducing agents for CT26 cells.     

Evaluation of the environmental specificity of Fluorescence In Situ Hybridization (FISH) using Fluorescence-Activated Cell Sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil

We explicitly tested for the first time the ‘environmental specificity’ of traditional 16S rRNA-targeted Fluorescence In Situ Hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridized population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51 ± 1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted-recovered fluorescent populations (n = 3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz^® method for the extraction of bacterial cells from soil.     

The structure of the egg-shell of Porrocaecum ensicaudatum (Nematoda: Ascaridida)

Wharton D. A. 1979. The structure of the egg-shell of Porrocaecum enslcaudatum (Nematoda: Ascaridida). International Journal for Parasltology9: 127–131. The egg-shell of Porrocaecum ensicaudatum is oval with an opercular plug at either end. The shell consists of three layers: an inner lipid layer, a middle chitinous layer and an outer vitelline layer. The vitelline layer has strands of particulate material attached to its outer surface. The chitinous layer consists of 8.5 nrn fibrils which are made up of a chitin microfibril core surrounded by a protein coat. The fibrils are oriented randomly or in parallel, there being no indication of helicoidal architecture.The chitinous layer varies in thickness to form a pattern of interconnecting ridges on the surface of the egg. This pattern presumably increases the shell's structural strength.     

Association of a luminous Vibrio sp., taxonomically related to Vibrio harveyi, with Clytia linearis (Thornely, 1900) (Hydrozoa, Cnidaria)

A previously unknown association between a luminous Vibrio sp., taxonomically related to the species Vibrio harveyi and a common member of the shallow/mid water communities of the Mediterranean Sea, the hydrozoan Clytia linearis is described. All the specimens of C. linearis observed under blue light excitation showed both a natural luminescence appearing as a series of fine dots due to clytin, and a clear fluorescence on the external side of the perisarc around the colonies due to the presence of luminous bacteria. Luminous bacteria were isolated from the surface of C. linearis, their phenotypic characterization as isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a Vibrio sp. taxonomically related to V. harveyi. The association of the V. harveyi-related species with C. linearis, as already suggested for another hydroid, Aglaophenia octodonta, could be explained with the activity of these bacteria of feeding on the chitinous structures present in these hydroids. Moreover, the adhesion of the luminous bacterium (here referred to as Vibrio sp. CL1) on C. linearis may contribute to the survival of this Vibrio species in the marine environment providing a suitable growth habitat.     

Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA)

We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation.     

Effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in European corn borer (Ostrinia nubilalis) larvae

European corn borer (ECB; Ostrinia nubilalis (Hubner)) larvae (third instar) fed 0.05% w/w wheat germ agglutinin (WGA) in their diet for 72 h showed very little increase in body weight, whereas weight of control larvae increased nearly fourfold. Light and transmission electron microscopy studies showed that the morphology of the peritrophic membrane (PM) changed within 24 h after ECB larvae fed on the WGA diet. Whereas the PM in the anterior region of the midgut was a thin membranous structure in control larvae, the WGA-fed larvae secreted a multiple-layered and unorganized PM that contained embedded food particles, bacteria, and pieces of disintegrated microvilli. Gold-labeled WGA was localized specifically in the PM and microvilli. The PM of WGA-fed larvae was inundated with dark-staining amorphous structures that, when incubated with anti-WGA, showed heavy WGA localization. The antibody label indicated that most of the ingested WGA was found in the PM, with lesser amounts on the microvillar surface and the least amount within the epithelium. After 72 h, the middle portion of the mesenteron revealed a thin, compact PM in the control larvae, whereas the PM of the WGA-fed larvae was multilayered and discontinuous, which allowed plant cell-wall fragments to penetrate into the microvilli of the epithelium. Scanning electron microscopy of PMs from fifth instar ECB larvae fed the WGA diet revealed a breakdown in the chitinous meshwork by 48 h after initiation of feeding. The endo-PM surface from control larvae was smooth and intact, whereas the PM of WGA-fed larvae showed disintegration of the meshwork and a reduced proteinaceous matrix. This allowed bacteria and food particles to penetrate through the PM into the ectoperitrophic space and directly contact the microvilli. Therefore, WGA, a protein inhibitor of larval growth, interferes with the formation and integrity of the PM, which exposes the brush border to ingested material. This, in turn, appears to stimulate secretion of additional PM layers, the concomitant disintegration of the microvilli, and cessation of feeding.     

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism

The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography‐tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α‐amylase) or other reactions (β‐1,3‐glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C‐Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase‐2 (McCHS‐2), chitinase (McCHI), and β‐N‐acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS‐2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS‐2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.     

Nucleotide Activation of the Ca-ATPase

We have used fluorescence spectroscopy, molecular modeling, and limited proteolysis to examine structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA). The Ca-ATPase in sarcoplasmic reticulum vesicles from fast twitch muscle (SERCA1a isoform) was selectively labeled with fluorescein isothiocyanate (FITC), a probe that specifically reacts with Lys-515 in the nucleotide-binding site. Conformation-specific proteolysis demonstrated that FITC labeling does not induce closure of the cytoplasmic headpiece, thereby assigning FITC-SERCA as a nucleotide-free enzyme. We used enzyme reverse mode to synthesize FITC monophosphate (FMP) on SERCA, producing a phosphorylated pseudosubstrate tethered to the nucleotide-binding site of a Ca²⁺-free enzyme (E2 state to prevent FMP hydrolysis). Conformation-specific proteolysis demonstrated that FMP formation induces SERCA headpiece closure similar to ATP binding, presumably due to the high energy phosphoryl group on the fluorescent probe (ATP·E2 analog). Subnanosecond-resolved detection of fluorescence lifetime, anisotropy, and quenching was used to characterize FMP-SERCA (ATP·E2 state) versus FITC-SERCA in Ca²⁺-free, Ca²⁺-bound, and actively cycling phosphoenzyme states (E2, E1, and EP). Time-resolved spectroscopy revealed that FMP-SERCA exhibits increased probe dynamics but decreased probe accessibility compared with FITC-SERCA, indicating that ATP exhibits enhanced dynamics within a closed cytoplasmic headpiece. Molecular modeling was used to calculate the solvent-accessible surface area of FITC and FMP bound to SERCA crystal structures, revealing a positive correlation of solvent-accessible surface area with quenching but not anisotropy. Thus, headpiece closure is coupled to substrate binding but not active site dynamics. We propose that dynamics in the nucleotide-binding site of SERCA is important for Ca²⁺ binding (distal allostery) and phosphoenzyme formation (direct activation).     

The structure of the egg-shell of gLobodera rostochiensis (Nematoda: Tylenchida)

Perry R. N., Wharton D. A. and Clarke A. J. 1982. The structure of the egg-shell of Globodera rostochiensis (Nematoda: Tyienchida). International Journal for Parasitology12: 481–485. The ultrastructure and histochemistry of the egg-shell of Globodera rostochiensis are described. The eggshell consists of an outer vitelline layer, a chitinous layer and an inner lipid layer. The vitelline layer is not unit membrane-like and has strands of particulate material attached to its outer surface. The chitinous layer is made up of fibres consisting of a chitin microfibril core, surrounded by a protein coat. The lipid layer contains lipoprotein membranes. These vary in number, the most commonly observed pattern being two or three membranes loosely associated with the inner surface of the egg-shell.     

Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model

Introduction

In this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting the C57BL/6 mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary to a sequence of the H. pylori 16S rRNA gene, was used. First, the potential cytotoxicity and genotoxicity of the probe was assessed by commercial assays. Further, the performance of the probe for detecting H. pylori at different pH conditions was tested in vitro, using fluorescence in situ hybridization (FISH). Finally, the efficiency of FIVH to detect H. pylori SS1 strain in C57BL/6 infected mice was evaluated ex vivo in mucus samples, in cryosections and paraffin-embedded sections by epifluorescence and confocal microscopy.

Results

H. pylori SS1 strain infecting C57BL/6 mice was successfully detected by the Cy3_HP_LNA/2OMe_PS probe in the mucus, attached to gastric epithelial cells and colonizing the gastric pits. The specificity of the probe for H. pylori was confirmed by microscopy.

Conclusions

In the future this methodology can be used in combination with a confocal laser endomicroscope for in vivo diagnosis of H. pylori infection using fluorescent LNA probes, which would be helpful to obtain an immediate diagnosis. Our results proved for the first time that FIVH method is applicable inside the body of a higher-order animal.     

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores.Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.     

Three new species of Hatschekia Poche, 1902 (Copepoda: Siphonostomatoida: Hatschekiidae) parasitic on boxfishes (Pisces: Tetraodontiformes: Aracanidae and Ostraciidae) in Japanese waters

Three new species of Hatschekia Poche, 1902 are described from the gill filaments of three species of boxfishes captured off southern Japan: H. pseudostracii n. sp. on Kentrocapros aculeatus (Houttuyn) (Aracanidae); H. bibullae n. sp. on Lactoria diaphana (Bloch & Schneider) (Ostraciidae); and H. kuroshioensis n. sp. on Tetrosomus concatenates (Bloch) (Ostraciidae). Of the 93 currently valid species in the genus, these new species differ from the 87 species which lack four stout processes on the posterior margin of the intercoxal sclerites of legs 1 and 2. Those processes are present on the remaining six species and the three new species. Of these nine species, H. pseudostracii n. sp. is distinguished by having a T-shaped chitinous frame on the cephalothorax, the leg 1 exopod twice as long as the endopod and a small parabasal papilla. H. bibullae n. sp. can be differentiated by a combination of morphological features as follows: a well-developed, thumb-shaped parabasal papilla, the leg 1 exopod twice as long as the endopod and a trunk lacking posterior lobes. H. kuroshioensis n. sp. can be recognised by bearing a T-shaped chitinous frame on the cephalothorax, the leg 1 exopod is three times as long as the endopod and the trunk lacks posterior lobes.     

Unusual swimming mechanism in Calycopsis borchgrevinki (Hydrozoa)

Calycopsis borchgrevinki (Browne, 1910) from Antarctic waterswas studied histologically. The entire bell is enclosed in achitin-like skin, supported by chitin-like rods in the radialmesogleal fibres. In the tentacles a thick mesogloea is presentwithout chitinous structures. The gonads are embedded in thestomach folds. It is postulated that these characters are adaptationsto ecological conditions in Antarctic waters.     

Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥10² cells ml⁻¹ was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.     

Dynamic role of microvilli in peritrophic membrane formation

Although the peritrophic membrane (PM) is a common extracellular construction in many invertebrate groups, evidence of the location of its secretion has never been reported. In this study a specific marker for chitin has been developed, enabling a separate examination of secretion of the chitinous and proteinaceous components of the PM in the millipede, Glomeris marginata. Chitin appears first at the base of the microvilli (MV), synchronized in adjacent cells along the entire length of the midgut. Evidence showing that it originates at the plasma membrane is discussed. Proteinaceous components appear to be added from the MV to the chitinous sheet as it moves along the MV toward the lumen. Precedence for such a dynamic role for MV in formation of extracellular structures is reviewed. The completed PM extends around individual items in the gut contents as well as forming a multilayered envelope; this may enhance both its digestive and protective functions.     

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3)

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC₆(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.     

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo.     

Using an online phycocyanin fluorescence probe for rapid monitoring of cyanobacteria in Macau freshwater reservoir

Monitoring of cyanobacteria and their toxins are traditionally conducted by cell counting, chlorophyll-a (chl-a) determination and cyanotoxin measurements, respectively. These methods are tedious, costly, time consuming, and insensitive to rapid changes in water quality and cyanobacterial abundance. We have applied and tested an online phycocyanin (PC) fluorescence probe for rapid monitoring of cyanobacteria in the Macau Storage Reservoir (MSR) that is experiencing cyanobacterial blooms. The relationships among cyanobacterial abundance, biovolume, cylindrospermopsin concentration, and PC fluorescence were analyzed using both laboratory and in-the-field studies. The performance of the probe was compared with traditional methods, and its advantages and limitations were assessed in pure and mixed cyanobacterial cultures in the laboratory. The proposed techniques successfully estimated the species including Microcystis and Cylindrospermopsis, two toxic species recently observed in the MSR. During February–November, 2010, the PC probe detected high correlations between PC and cell numbers (R ² = 0.71). Unlike the chl-a content, which indicates only the total algal biomass, the PC pigment specifically indicates cyanobacteria. These results support the PC parameter as a reliable estimate of cyanobacterial cell number, especially in freshwater bodies where the phytoplankton community and structure are stable. Thus, the PC probe is potentially applicable to online monitoring of cyanobacteria.     

Feeding Activity, Salivary Amylase Activity, and Superficial Damage to Soybean Seed by Adult Edessa meditabunda (F.) and Euschistus heros (F.) (Hemiptera: Pentatomidae)

Greenhouse and laboratory studies were conducted to evaluate feeding activity and superficial damage to soybean seed by the brown-winged stink bug, Edessa meditabunda (F.), and the Neotropical brown stink bug, Euschistus heros (F.). Soybean plants (cv. BRS 282), at R6 stage of development were used. Thirty pairs of each species were used individually for 48?h. Two daily observations (9:00?AM and 3:00?PM) were taken to record the number of bugs (feeding/resting) on plant parts. Harvested seeds imbibed in tetrazolium solution were photographed for measurement of the damaged surface. Adult E. meditabunda significantly preferred soybean stems (19.7 bugs) to pods (2.7). Feeding/resting was similar at 9:00?AM (mean number of 28.0 bugs) and 3:00?PM (24.3). Euschistus heros equally fed/stayed on stems (7.3 bugs) and pods (6.9), although most bugs (12.3) remained on the cage net; feeding/resting on all plant structures amounted to 13.7 bugs at 9:00?AM and 17.7 bugs at 3:00?PM. Amylase activity was greater for E. heros (41.61?±?0.89?U/mg) and almost none for E. meditabunda (2.35?±?0.14?U/mg). The superficial damage to seeds was significantly greater for E. meditabunda (22. 9?mm²) compared to E. heros (12.5?mm²). However, E. meditabunda caused less shrinkage of the seed tegument, while E. heros damage was deeper and seeds showed reduction in size.