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The oxidative addition of Pd to Si-H bonds is a crucial step in a variety of catalytic applications, and many aspects of this reaction are poorly understood. One important yet underexplored aspect is the electronic effect of silane substituents on reactivity. Herein we describe a systematic investigation of the formation of silyl palladium hydride complexes as a function of silane identity, focusing on electronic influence of the silanes. Using [(μ-dcpe)Pd] 2 (dcpe = dicyclohexyl(phosphino)ethane) and tertiary silanes, data show that equilibrium strongly favours products formed from electron-deficient silanes, and is fully dynamic with respect to both temperature and product distribution. A notable kinetic isotope effect (KIE) of 1.21 is observed with H/DSiPhMe 2 at 233 K, and the reaction is shown to be 0.5 th order in [(μ-dcpe)Pd] 2 and 1 st order in silane. Formed complexes exhibit temperature-dependent intramolecular H/Si ligand exchange on the NMR timescale, allowing determination of the energetic barrier to reversible oxidative addition. Taken together, these results give unique insight into the individual steps of oxidative addition and suggest the initial formation of a σ-complex intermediate to be rate-limiting. The insight gained from these mechanistic studies was applied to hydrosilylation of alkynes, which shows parallel trends in the effect of the silanes' substituents. Importantly, this work highlights the relevance of in-depth mechanistic studies of fundamental steps to catalysis., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: Yersinia entomophaga is an entomopathogenic bacterium that is active against scarab beetles, among other insects. In New Zealand, the African black beetle, Heteronychus arator (Coleoptera: Scarabaeidae), is a major pest of pastures and arable crops but very few control options exist and no insecticides are registered for use in established pastures., Results: In laboratory bioassays, H. arator adults were susceptible to a bait containing Y. entomophaga at low doses. This bait was more effective against H. arator adults during spring than autumn in small-scale field plots (320 mm diameter). A large-scale field trial (40 × 40 m plots) reduced adult numbers substantially: approximately twice as many beetles were captured in pitfall traps from untreated plots compared with plots treated with the Y. entomophaga bait at 70 kg ha -1 . This single bait application in spring also reduced subsequent larval populations in summer., Conclusions: Heteronychus arator is a difficult pest to manage using chemical insecticides. This biopesticide with Y. entomophaga as the active ingredient offers a new solution for New Zealand pastures, with potential for application to other crops affected by H. arator and for control of other pests. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Background: Porina is the common name for moths and larvae of the genus Wiseana (Lepidoptera: Hepialidae), some of which are significant pasture pests in New Zealand. Because of environmental concerns and the non-target effects of insecticide control measures, biological alternatives for the control of insect pests such as porina are required., Results: Using a food preference assay and time-lapse photography, a range of low-cost food ingredients were assessed for their palatability to porina larvae. Lead candidates were combined into extruded bait variants, allowing assessment of their palatability to porina larvae. A composite bait consisting of palatable ingredients was developed, into which the porina-active entomopathogen Yersinia entomophaga was incorporated. A 7 day minimum median lethal dose of approximately 6.0 × ±1 × 10 6 Y. entomophaga cells per 0.02 g of bait was defined. Field trials showed that the mean change in larval density over time differed between treatments, with Y. entomophaga bait applied at 87 kg ha -1 resulting in a mean 65% reduction in larval density relative to the control plots, and diflubenzuron treatment resulting in a mean 77% reduction relative to the control plots. The mean dry matter yields over the course of the trial were highest for diflubenzuron (5029 kg ha -1 ), followed by the Y. entomophaga (4783 kg ha -1 ) and control (4673 kg ha -1 ) treatments., Conclusions: The bacterium Y. entomophaga applied as a composite bait offers an environmentally sustainable approach for porina pest control. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Metal (oxy)hydroxides (MO x H y , M = Fe, Co, Ni, and mixtures thereof) are important materials in electrochemistry. In particular, MO x H y are the fastest known catalysts for the oxygen evolution reaction (OER) in alkaline media. While key descriptors such as overpotentials and activity have been thoroughly characterized, the nanostructure and its dynamics under electrochemical conditions are not yet fully understood. Here, we report on the structural evolution of Ni 1-δ Co δ O x H y nanosheets with varying ratios of Ni to Co, in operando using atomic force microscopy during electrochemical cycling. We found that the addition of Co to NiO x H y nanosheets results in a higher porosity of the as-synthesized nanosheets, apparently reducing mechanical stress associated with redox cycling and hence enhancing stability under electrochemical conditions. As opposed to nanosheets composed of pure NiO x H y , which dramatically reorganize under electrochemical conditions to form nanoparticle assemblies, restructuring is not found for Ni 1-δ Co δ O x H y with a high Co content. Ni 0.8 Fe 0.2 O x H y nanosheets show high roughness as-synthesized which increases during electrochemical cycling while the integrity of the nanosheet shape is maintained. These findings enhance the fundamental understanding of MO x H y materials and provide insight into how nanostructure and composition affect structural dynamics at the nanoscale.

African black beetle, Heteronychus arator (Scarabaeidae), is an exotic pest of pastures in northern New Zealand. Both adults and larvae feed on pasture grasses. Adults disperse by walking (short range) or flying (long range). Dispersal flights are triggered by warm night temperatures in spring and autumn. Short range adult dispersal in search of mates, food or oviposition sites is poorly understood. This study investigated walking activity of H. arator adults over three seasons in New Zealand pastures. Adult walking activity was monitored using pitfall traps along fence lines and in pasture plots on a dairy farm in Waikato, New Zealand, in spring 2013, spring 2014, and autumn 2015. Beetle populations were reduced by application of a biopesticide bait to compare walking activity between treated and control plots for up to 26 days post-treatment. Marked beetles were released into the pasture plots to measure the distance traveled by recaptured individuals. Trap catches along the fence lines were correlated with air temperatures in 2013. Trap catches were male biased in spring 2014 compared with autumn 2015. Trap numbers in the control plots were nearly double that of treated plots in both seasons. More beetles were caught in the pitfall traps at the edges of the treated plots than in the center. Trap catches were consistent throughout the control plot in spring 2014, but in autumn 2015 more beetles were caught in the center of the control plot than at the edges. Few marked beetles were recaptured with dispersal rates estimated as <0.5 m per day. Warmer temperatures encouraged short range dispersal in H. arator. Males were more active than females during the spring mating season. Edge effects were strong and should be considered in the design of field experiments.

Here we report the draft genome of Yersinia entomophaga type strain MH96T. The genome shows 93.8% nucleotide sequence identity to that of Yersinia nurmii type strain APN3a-cT, and comprises a single chromosome of approximately 4,275,531 bp. In silico analysis identified that, in addition to the previously documented Y. entomophaga Yen-TC gene cluster, the genome encodes a diverse array of toxins, including two type III secretion systems, and five rhs-associated gene clusters. As well as these multicomponent systems, several orthologs of known insect toxins, such as VIP2 toxin and the binary toxin PirAB, and distant orthologs of some mammalian toxins, including repeats-in-toxin, a cytolethal distending toxin, hemolysin-like genes and an adenylate cyclase were identified. The genome also contains a large number of hypothetical proteins and orthologs of known effector proteins, such as LopT, as well as genes encoding a wide range of proteolytic determinants, including metalloproteases and pathogen fitness determinants, such as genes involved in iron metabolism. The bioinformatic data derived from the current in silico analysis, along with previous information on the pathobiology of Y. entomophaga against its insect hosts, suggests that a number of these virulence systems are required for survival in the hemocoel and incapacitation of the insect host.

The Yersinia entomophaga toxin complex (Yen-TC) is the bacterium's main virulence determinant. Because of its high insect activity, methods were developed to allow the routine isolation and purification of Yen-TC from an overnight bacterial culture using size exclusion chromatography. Here we outline an overnight purification procedure using a 100-ml culture volume, where approximately 2 mg of Yen-TC, with an approximate purity of 95-98 %, can be routinely obtained.

The bacterium Burkholderia sp. Ha185 readily solubilizes inorganic phosphate by releasing the low molecular weight organic anion, 2-ketogluconate. Using random transposon mutagenesis and in silico analysis, a mutation that caused almost complete abolition of phosphate solubilization was located within hemX, which is part of the hem operon. Burkholderia sp. Ha185 HemX is a multidomain protein, predicted to encode a bifunctional uroporphyrinogen-III synthetase/uroporphyrin-III C-methyltransferase, which has not previously been implicated in phosphate solubilization. Complementation of hemX restored the ability of the mutant to solubilize phosphate in both plate and liquid cultures. Based on a combination of organic-anion profiling, quantitative polymerase chain reaction and in silico analyses, hemX was confirmed to be solely responsible for hydroxyapatite solubilization in Burkholderia sp. Ha185. It is proposed that the biosynthesis of a yet to be determined redox cofactor by HemX is the main pathway for generating 2-ketogluconate via a haem-dependent gluconate 2-dehydrogenase in Burkholderia sp. Ha185., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Organic phosphorus (P) is abundant in most soils but is largely unavailable to plants. Pseudomonas spp. can improve the availability of P to plants through the production of phytases and organic anions. Gluconate is a major component of Pseudomonas organic anion production and may therefore play an important role in the mineralization of insoluble organic P forms such as calcium-phytate (CaIHP). Organic anion and phytase production was characterized in 2 Pseudomonas spp. soil isolates (CCAR59, Ha200) and an isogenic mutant of strain Ha200, which lacked a functional glucose dehydrogenase (Gcd) gene (strain Ha200 gcd::Tn5B8). Wild-type and mutant strains of Pseudomonas spp. were evaluated for their ability to solubilize and hydrolyze CaIHP and to promote the growth and assimilation of P by tobacco plants. Gluconate, 2-keto-gluconate, pyruvate, ascorbate, acetate, and formate were detected in Pseudomonas spp. supernatants. Wild-type pseudomonads containing a functional gcd could produce gluconate and mineralize CaIHP, whereas the isogenic mutant could not. Inoculation with Pseudomonas improved the bioavailability of CaIHP to tobacco plants, but there was no difference in plant growth response due to Gcd function. Gcd function is required for the mineralization of CaIHP in vitro; however, further studies will be needed to quantify the relative contribution of specific organic anions such as gluconate to plant growth promotion by soil pseudomonads.

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ~4 × 10(7) CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ΔTC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ~3 Y. entomophaga or Y. entomophaga ΔTC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 × 10(7) CFU ml(-1) of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ~3 × 10(9) CFU ml(-1) of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The anti-feeding prophage (Afp), a phage-tail-like particle that causes cessation of feeding in the New Zealand grass grub, Costelytra zealandica, is encoded by 18 open reading frames (afp1-18). C-terminal truncations of afp14 resulted in shortened Afp particles, suggesting that Afp14 is involved in Afp length determination. We constructed an Afp assembly system (afp1-18), wherein Afp14 was truncated after the N-terminal 88 residues. This construct, when expressed in trans in Escherichia coli expressing a N-terminal 98-amino acid Afp14 construct, yielded fully assembled Afp but no assembled Afp was detected in the case of a N-terminal 96-amino acid Afp14 construct. These results suggested that the 98 N-terminal, amino acid residues of Afp14 is crucial for the initiation of Afp assembly via baseplate formation. Trans-based expression of wild-type afp14 resulted in Afp particles of varying lengths, all of which were shorter than the wild-type Afp particle. On the other hand, similar expression of Afp14 harboring a C-terminal extension (KLLEH(6)) resulted in elongated Afp particles. This information, combined with bioinformatics data, allowed us to propose a model delineating the mechanism and role of Afp14 in the maturation of the Afp particle., (© 2015 John Wiley & Sons Ltd.)

The bacterium Yersinia entomophaga was isolated from larvae of the New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), found in soil. Following ingestion of a lethal dose of bacteria, larvae of C. zealandica reduced feeding activity and movement. After approximately 4h infected larvae convulsed and regurgitated dark digestive fluid and expelled frass pellets leaving the midgut empty and the larva amber in appearance. In the initial stages of infection, ingested bacteria were mostly contained within the peritrophic membrane and expelled with the gut fluid or transferred into the hind gut. While few Y. entomophaga were associated with the midgut epithelial cells, by 24h cells were swelling and bursting with vesicles being expelled into the midgut lumen. By 48h, bacteria had entered the haemocoel and the midgut cells had further deteriorated. After 72h, the cellular remnants were totally detached from the basal membrane the infected insects were filled with bacteria and moribund or dead with septicaemia. Mortality was directly proportional to dose and time after infection. By applying a range of doses, the LD50 was determined as 2.9×10(4)Y. entomophaga per C. zealandica larva, with an LT50 of 2.94days for doses of>1×10(5) per larva. Ingestion of low doses of bacteria did not inhibit feeding activity but led more slowly to death. By time of death, Y. entomophaga had multiplied, approximately 500 fold, in the cadavers of the infected larvae., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The ABC toxin complexes produced by certain bacteria are of interest owing to their potent insecticidal activity and potential role in human disease. These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic. The carboxy-terminal region of the C protein is the main cytotoxic component, and is poorly conserved between different toxin complexes. A general model of action has been proposed, in which the toxin complex binds to the cell surface via the A protein, is endocytosed, and subsequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells. Toxin complexes have been visualized using single-particle electron microscopy, but no high-resolution structures of the components are available, and the role of the B protein in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex formed between the B and C proteins, determined to 2.5 Å by X-ray crystallography. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, C-terminal region of the C protein like the shell of an egg. The shell is decorated on one end by a β-propeller domain, which mediates attachment of the B-C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example, to our knowledge, of a structure that contains rearrangement hotspot (RHS) repeats, and illustrates a marked structural architecture that is probably conserved across both this widely distributed bacterial protein family and the related eukaryotic tyrosine-aspartate (YD)-repeat-containing protein family, which includes the teneurins. The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein encapsulation and delivery.

The Serratia entomophila antifeeding prophage Afp, forms a phage-tail-like particle that acts on the New Zealand grass grub, Costelytra zealandica with a 3-day LD50 of approximately 500 Afp particles per larva. Genes (afp1-18) encoding components of Afp were expressed and their products purified allowing morphological assessment of the products by transmission electron microscopy (TEM). Expression of afp1-15 resulted in the formation of a non-sheathed structure termed the tube-baseplate complex or TBC, composed of an irregular-length tube attached to a baseplate with associated tail fibres. Expression of afp1-16 produced mature, normal-length Afp particles, whereas coexpression of afp16 with afp1-15 in trans resulted in the formation of aberrant Afp particles of variable lengths. A C-terminally truncated Afp16 mutant resulted in a phenotype intermediate between mature Afp and TBC. The addition of purified Afp16 to Afp unravelled by acidic treatment resulted in the formation of shorter tubes when specimen pH was adjusted to 7 than those formed in the absence of Afp16. Analysis of TEM images of purified Afp16 revealed a hexameric ring-like structure similar to that formed by gp3 of phage T4 and gpU of phage λ. Our results suggest that Afp16 terminates tube elongation and is involved in sheath formation., (© 2013 John Wiley & Sons Ltd.)

Aims: The genes involved in choline transport and oxidation to glycine betaine in the biopesticidal bacterium Serratia entomophila were characterized, and the potential of osmoprotectants, coupled with increased NaCl concentrations, to improve the desiccation tolerance of this species was investigated., Methods and Results: Serratia entomophila carries sequences similar to the Escherichia coli betTIBA genes encoding a choline transporter and dehydrogenase, a betaine aldehyde dehydrogenase and a regulatory protein. Disruption of betA abolished the ability of Ser. entomophila to utilize choline as a carbon source. Quantitative reverse-transcriptase PCR analysis revealed that betA transcription was reduced compared to that of the upstream genes in the operon, and that NaCl and choline induced bet gene expression. Glycine betaine and choline increased the NaCl tolerance of Ser. entomophila, and osmotically preconditioned cultures survived better than control cultures following desiccation and immediately after application to agricultural soil., Conclusions: Addition of glycine betaine and NaCl to growth medium can greatly enhance the desiccation survival of Ser. entomophila, and its initial survival in soil., Significance and Impact of the Study: Serratia entomophila is sensitive to desiccation and does not persist under low soil moisture conditions. Techniques described here for enhancing the desiccation survival of Ser. entomophila can be used to improve formulations of this bacterium, and allow its application under a wider range of environmental conditions., (© 2012 AgResearch.)

Quantum dots (QDs) are an increasingly important class of nanoparticle, but little ecotoxicological data for QDs has been published to date. The effects of mercaptosuccinic acid (MSA)-capped QDs (QDs-MSA) and equivalent concentrations of cadmium (Cd) from cadmium chloride on growth and reproduction of the nematode Caenorhabditis elegans (Rhabditidae) were assessed in laboratory experiments. Growth from larvae to adults of C. elegans was unaffected by exposure to 1 µM fluorescent QDs-MSA, but adults produced more embryos and laid them prematurely. Furthermore, C. elegans exposed to QDs-MSA (1 µM) showed a high percentage of embryo mortality (19.2 ± 0.5, p < 0.001, percentage ± standard deviation) compared with unexposed nematodes (11.6 ± 0.4). An egg-laying defect phenotype was also observed at high frequency in response to 1 µM QDs-MSA exposure (38.3 ± 3.6%, p < 0.01; control 10.0 ± 2.2%). This resulted in a reduced mean life span (20.5 ± 1.1 d, p < 0.05) compared with the control (24.6 ± 1.0 d). Cadmium also caused reduced life span in C. elegans, but a low incidence of egg-laying defects was observed, suggesting that Cd and QDs-MSA affected C. elegans by different mechanisms. Furthermore, egg-laying defects caused by QDs-MSA responded to the addition of the anticonvulsant ethosuximide and to a lesser extent to the neurotransmitter serotonin, suggesting that QDs-MSA might have disrupted motor neurons during the reproduction process., (Copyright © 2012 SETAC.)

Yersinia entomophaga MH96, which was originally isolated from the New Zealand grass grub, Costelytra zealandica, produces an orally active proteinaceous toxin complex (Yen-Tc), and this toxin is responsible for mortality in a range of insect species, mainly within the Coleoptera and Lepidoptera. The genes encoding Yen-Tc are members of the toxin complex (Tc) family, with orthologs identified in several other bacterial species. As the mechanism of Yen-Tc activity remains unknown, a histopathological examination of C. zealandica larvae was undertaken in conjunction with cultured cells to identify the effects of Yen-Tc and to distinguish the contributions that its individual subunit components make upon intoxication. A progressive series of events that led to the deterioration of the midgut epithelium was observed. Additionally, experiments using a cell culture assay system were carried out to determine the cellular effects of intoxication on cells after topical application and the transient expression of Yen-Tc and its individual components. While observations were broadly consistent with those previously reported for other Tc family members, some differences were noted. In particular, the distinct stepwise disintegration of the midgut shared features associated with both apoptosis and necrotic programmed cell death pathways. Second, we observed, for the first time, a contribution of toxicity from two chitinases associated with the Yen-Tc complex. Our findings were suggestive of the activities encoded within the subunit components of Yen-Tc targeting different sites along putative programmed cell death pathways. Given the observed broad host range for Yen-Tc, these targeted loci are likely to be widely shared among insects.

Yersinia entomophaga MH96 is a native New Zealand soil bacterium that secretes a large ABC-type protein toxin complex, Yen-Tc, similar to those produced by nematode-associated bacteria such as Photorhabdus luminescens. Y. entomophaga displays an exceptionally virulent pathogenic phenotype in sensitive insect species, causing death within 72 h of infection. Because of this phenotype, there is intrinsic interest in the mechanism of action of Yen-Tc, and it also has the potential to function as a novel class of biopesticide. We have identified genes that encode chitinases as part of the toxin complex loci in Y. entomophaga MH96, P. luminescens, Photorhabdus asymbiotica and Xenorhabdus nematophila. Furthermore, we have shown that the secreted toxin complex from Y. entomophaga MH96 includes two chitinases as an integral part of the complex, a feature not described previously in other ABC toxins and possibly related to the severe disease caused by this bacterium. We present here the structure of the Y. entomophaga MH96 Chi1 chitinase, determined by X-ray crystallography to 1.74 Å resolution, and show that a ring of five symmetrically arranged lobes on the surface of the Yen-Tc toxin complex structure, as determined by single-particle electron microscopy, provides a good fit to the Chi1 monomer. We also confirm that the isolated chitinases display endochitinase activity, as does the complete toxin complex., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Toxin complex (Tc) proteins are a class of bacterial protein toxins that form large, multisubunit complexes. Comprising TcA, B, and C components, they are of great interest because many exhibit potent insecticidal activity. Here we report the structure of a novel Tc, Yen-Tc, isolated from the bacterium Yersinia entomophaga MH96, which differs from the majority of bacterially derived Tcs in that it exhibits oral activity toward a broad range of insect pests, including the diamondback moth (Plutella xylostella). We have determined the structure of the Yen-Tc using single particle electron microscopy and studied its mechanism of toxicity by comparative analyses of two variants of the complex exhibiting different toxicity profiles. We show that the A subunits form the basis of a fivefold symmetric assembly that differs substantially in structure and subunit arrangement from its most well characterized homologue, the Xenorhabdus nematophila toxin XptA1. Histopathological and quantitative dose response analyses identify the B and C subunits, which map to a single, surface-accessible region of the structure, as the sole determinants of toxicity. Finally, we show that the assembled Yen-Tc has endochitinase activity and attribute this to putative chitinase subunits that decorate the surface of the TcA scaffold, an observation that may explain the oral toxicity associated with the complex.

Through transposon mutagenesis and DNA sequence analysis, the main disease determinant of the entomopathogenic bacterium Yersinia entomophaga MH96 was localized to an ~32-kb pathogenicity island (PAI) designated PAI(Ye₉₆). Residing within PAI(Ye₉₆) are seven open reading frames that encode an insecticidal toxin complex (TC), comprising not only the readily recognized toxin complex A (TCA), TCB, and TCC components but also two chitinase proteins that form a composite TC molecule. The central TC gene-associated region (~19 kb) of PAI(Ye₉₆) was deleted from the Y. entomophaga MH96 genome, and a subsequent bioassay of the ΔTC derivative toward Costelytra zealandica larvae showed it to be innocuous. Virulence of the ΔTC mutant strain could be restored by the introduction of a clone containing the entire PAI(Ye₉₆) TC gene region. As much as 0.5 mg of the TC is released per 100 ml of Luria-Bertani broth at 25°C, while at 30 or 37°C, no TC could be detected in the culture supernatant. Filter-sterilized culture supernatants derived from Y. entomophaga MH96, but not from the ΔTC strain grown at temperatures of 25°C or less, were able to cause mortality. The 50% lethal doses (LD₅₀s) of the TC toward diamondback moth Plutella xylostella and C. zealandica larvae were defined as 30 ng and 50 ng, respectively, at 5 days after ingestion. Histological analysis of the effect of the TC toward P. xylostella larva showed that within 48 h after ingestion of the TC, there was a general dissolution of the larval midgut.

Some strains of Serratia entomophila and S. proteamaculans cause amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The disease determinants of S. entomophila, are encoded on a 153,404-bp plasmid, termed pADAP for amber disease associated plasmid. The S. proteamaculans strain 143 (Sp143) exhibits an unusual pathotype, where only 60-70% of C. zealandica larvae infected with the bacterium succumb to disease. DNA sequence analysis of the Sp143 pU143 virulence associated region identified high DNA similarity to the pADAP sep virulence associated region, with DNA sequence variation in the sepA gene and the variable region of the sepC component. No pADAP anti-feeding prophage orthologue was detected in the Sp143 genome. The region of pADAP replication was cloned and found to replicate in S. entomophila but not in Escherichia coli. DNA sequence analysis of the plasmid pSG348 repA gene from the French isolate of Serratia grimesii, identified 93% DNA identity to the pADAP repA gene. A comparison of the pU143 virulence associated region with the completed pADAP nucleotide sequence is given., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The sheath of the Serratia entomophila antifeeding prophage, which is pathogenic to the New Zealand grass grub Costelytra zealandica, is a 3-fold helix formed by a 4-fold symmetric repeating motif disposed around a helical inner tube. This structure, determined by electron microscopy and image processing, is distinct from that of the other known morphologically similar bacteriophage sheaths.

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and alpha-amylase were still detected in the midgut of diseased larvae.

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.

Serratia entomophila and Serratia proteamaculans cause amber disease of the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae). Three genes required for virulence, sepABC, are located on a large plasmid, pADAP. The translated products of the sep genes are members of the toxin complex (Tc) family of insecticidal toxins that reside in the genomes of some Enterobacteriaceae. Each of the sep genes was placed either singly or as various combinations under the control of an inducible arabinose promoter, allowing their inductive expression. Western Immunoblot confirmed that each of the Sep proteins migrated at their predicted size on sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. Bioassays of sonicated filtrates derived from the various arabinose-induced para-SEP constructs showed that only when sepA, sepB and sepC were coexpressed were amber disease symptoms observed in grass grub larvae. Fourteen days after ingestion of the Sep protein filtrate, approximately 64% of the larvae reverted from a diseased to a healthy phenotype. Redosing the revertents with a fresh Sep protein filtrate reinitiated the amber pathotype, indicating that the Sep proteins are needed to be continuously present to exert an effect.

The Serratia entomophila antifeeding prophage (Afp) is thought to form a virus-like structure that has activity towards the New Zealand grass grub, Costelytra zealandica. Through the trans based expression of AnfA1, an RfaH - like transcriptional antiterminator, the Afp, was able to be induced. The expressed Afp was purified and visualized by electron microscopy. The Afp resembled a phage tail-like bacteriocin, exhibiting two distinct morphologies: an extended and a contracted form. The purified Afp conferred rapid activity towards C. zealandica larvae, causing cessation of feeding and a change to an amber colouration within 48 h postinoculation, with increased dose rates causing larval mortality.

Some strains of Serratia entomophila and S. proteamaculans cause amber disease of the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae). Three genes required for virulence, sepABC, are located on a large plasmid, pADAP. Sequence analysis suggests that the sepABC gene cluster may be part of a horizontally mobile region. This study presents evidence for the putative mobility of the sep genes of pADAP. Southern blot analysis showed that orthologues of the sep genes reside on plasmids within S. entomophila, S. liquefaciens, S. proteamaculans, and a plasmid from Yersinia frederiksenii. Three plasmids hybridized to the pADAP sep virulence-associated region but not the pADAP replication and conjugation regions. Subsequent DNA sequence analysis of the Y. frederiksenii sep-like genes, designated tcYF1 and tcYF2, showed that they had 88% and 87% DNA identity to sepA and sepB, respectively. These results indicate that the sep genes are part of a discrete horizontally mobile region.

Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.

We have previously shown that exposure to exogenous androgens causes female sticklebacks (Gasterosteus aculeatus) to produce the glue protein, spiggin, in their kidneys. This protein can be quantified by an enzyme-linked immunosorbent assay developed and validated at the Centre for Environment, Fisheries and Aquaculture Science. Here we report the development of an in vivo test for the detection of environmental antiandrogens. The system involves the simultaneous exposure of female sticklebacks to 17alpha-methyltestosterone (a model androgen) at 500 ng/L and suspected environmental antiandrogens over a period of 21 days. The spiggin content of the kidneys is then measured, and any antiandrogenic activity is evaluated by comparing the spiggin levels of female fish exposed to antiandrogens to those of female fish exposed solely to the model androgen. The assay detects the antiandrogenic activity of flutamide, vinclozolin (both used at 250 microg/L), linuron (at 150 microg/L), and fenitrothion (at 15 and 150 microg/L). These results provide the first evidence of in vivo antiandrogenic activity of both linuron and fenitrothion in teleosts. Although there are other suggested fish species that could be used for this purpose, the stickleback is the only widely available species in which it is now possible to study both estrogenic and antiandrogenic end points in the same individual. Furthermore, the species is endemic and ubiquitous in Europe, and it possesses many ecological traits that make it better suited than other potential species for field research into endocrine disruption.

The in vitro aryl hydrocarbon receptor (AhR) agonist potency of offshore produced water effluents, collected from the United Kingdom Continental Shelf, was determined using the dioxin responsive (DR)-chemically activated luciferase expression (CALUX) assay. Octadecylsilane (C18) solid phase extraction (SPE) extracts of produced water were exposed to DR-CALUX cells for 24h in order to investigate the contribution in potency from compounds that are stable to metabolism by the CALUX cells during exposure. The stable AhR agonist potency determined over 24h was highly variable and ranged from 1 to 430 ng TCDD TEQ(CALUX)l(-1). These data reflect the highly variable composition of produced water discharges from different production fields. It is recommended that further work be performed to characterise the full range of stable dioxin like AhR agonists present in offshore produced water discharges using techniques such as bioassay-directed analysis.

The DR-CALUX assay has been utilised for the bio-analytical screening of a number of estuarine sediments for dioxin-like activity. Total sediment extracts (samples containing all extracted compounds) and cleaned-up extracts (samples with the most stable compounds isolated from the total extracts) were screened. The concentration of the stable dioxin-like compounds in the cleaned-up sediment extracts was between 1.0 and 106 pgTEQCALUX g(-1) dry weight. The majority of sediments contained levels of dioxin-like compounds that were above concentrations that are considered to be a low risk to aquatic organisms. The CALUX bio-analytical approach showed some disparity with the traditional analytical approach. The reasons for these differences have been identified tentatively: firstly, the DR-CALUX assay responds to all dioxin-like compounds, and secondly, it measures non-additive effects. The dioxin-like activity of compounds in sediment total extracts, which contain both labile and stable compounds, were also assessed and were six orders of magnitude higher than the cleaned-up samples. This suggests the vast majority of the total dioxin-like activity is attributable to labile compounds. Overall, the DR-CALUX assay is shown to be a useful tool in the assessment of dioxin-like activity in estuarine sediments.

Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae) cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 155-kb plasmid, pADAP, carries the genes sepA, sepB, and sepC, which are essential for production of amber disease symptoms. Transposon insertions in any of the sep genes in pADAP abolish gut clearance but not cessation of feeding, indicating the presence of an antifeeding gene(s) elsewhere on pADAP. Based on deletion analysis of pADAP and subsequent sequence data, a 47-kb clone was constructed, which when placed in either an Escherichia coli or a Serratia background exerted strong antifeeding activity and often led to rapid death of the infected grass grub larvae. Sequence data show that the antifeeding component is part of a large gene cluster that may form a defective prophage and that six potential members of this prophage are present in Photorhabdus luminescens subsp. laumondii TTO1, a species which also has sep gene homologues.

The in vitro estrogen receptor (ER) agonist potency and C1 to C9 alkyl substituted phenol content of offshore produced water effluents collected from the UK sector of the North Sea were determined using a combination of bio-analytical and chemical analysis techniques. An in vitro reporter gene assay was used to determine ER agonist potency, whilst gas chromatography coupled to mass spectrometry (GC-MS) was used to quantify the concentration of alkylphenols. The in vitro ER agonist potency was highly variable and ranged from less than the limit of detection (theoretically 0.03 ng 17beta-estradiol (E2) l(-1)) to 91 ng E2 l(-1). C1 to C5 alkylphenol concentrations were also highly variable ranging from 5 to 1600 microg l(-1) with a median concentration of 206 microg l(-1). These data reflect the highly variable composition of produced water discharges from different fields. The observed poor correlation of the alkylphenol isomer content and ER agonist activity suggests that other compounds present in the produced water discharges may be responsible for the ER agonist activity observed. It is recommended that further work be performed to characterise the full range of ER agonists present in offshore produced water discharges.

The estrogen receptor (ER) agonist potency of offshore produced water discharges was examined via bioassay-directed chemical analysis. The in vitro estrogen receptor (ER) and androgen receptor (AR) agonist potency of five produced water samples collected from oil-production platforms in the British and Norwegian sectors of the North Sea was determined by using the yeast estrogen and androgen screens. Produced water samples were extracted in situ on the production platforms by using large-volume solid-phase extraction. All five extracts tested positive for the presence of ER agonists, whereas no AR agonist activity could be detected. By using the yeast estrogen screen assay in association with bioassay-directed fractionation, attempts were made to identify the ER agonist compounds present in the produced water extracts. The fractionation procedure used cyano-amino-bonded silica normal-phase high-performance liquid chromatography to isolate estrogenic compounds from produced water extract followed by full-scan gas chromatography-electron-impact mass spectrometry (GC-(EI)MS) to identify them. Isomeric mixtures of C1 to C5 and C9 alkylphenols contributed to the majority of the ER agonist potency measured in the samples.

Some strains of the Enterobacteriaceae Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The genes responsible for this disease reside on a large, 155-kb plasmid designated amber disease-associated plasmid (pADAP). Herein, we report the DNA sequencing of approximately 50 kb upstream and 10 kb downstream of the virulence-encoding region. Based on similarity with proteins in the current databases, and potential ribosome-binding sites, 63 potential ORFs were determined. Eleven of these ORFs belong to a type IV pilus cluster (pilL-V) and a further eight have similarities to the translated products of the plasmid transfer traH-N genes of the plasmid R64. In addition, a degenerate 785-nt direct repeat flanks a 44.7-kb region with the potential to encode three Bacillus subtilis Yee-type proteins, a fimbrial gene cluster, the sep virulence-associated genes and several remnant IS elements.

Twenty-six pesticidal compounds and samples of stormwater from two different agricultural catchments were screened for oestrogenic activity using an in vitro recombinant yeast-based assay. Only six fungicides showed an oestrogenic response with low comparative biological activity of 5000 to 2.5 million times less potent than 17beta-estradiol (E2), a natural steroidal oestrogen. Concentrations of biological activity expressed as E(2) equivalents for the headwater stream stormwater samples ranged from <0.01 to 0.11 ng E2/l. These values are at least one order of magnitude below levels that have been documented to produce oestrogenic effects in fish and are therefore considered to represent a low risk to associated headwater stream communities. The potential sources of the oestrogenic activity measured in the headwater streams are discussed., (Copyright 2002 Elsevier Science B.V.)

We report the development and validation of a novel in vivo biomarker test for waterborne androgens. During breeding, male sticklebacks (Gasterosteus aculeatus) manufacture a glue protein, spiggin, in their kidneys that they use to build their nests. Spiggin production is under the control of androgens. Until now, however, it has only been possible to quantify its production by measurement of the height of kidney epithelial cells. In the present study, we report the development of an enzyme-linked immunosorbent assay (ELISA) for spiggin and demonstrate its application to the measurement of spiggin in the kidneys of female sticklebacks that have been exposed to androgens in water. Results from the ELISA procedure revealed a strong correlation with measurement of kidney epithelial cell height (r2 = 0.93). However, the ELISA was much quicker and had a considerably higher response range (100,000-fold vs fourfold). Clear, graded responses in spiggin production were obtained by exposing intact females to increasing concentrations of 17a-methyltestosterone and 5alpha-dihydrotestosterone over three-week test periods. The lowest effective concentrations for these two steroids were 100 ng/L and 3 micorg/L, respectively. Female sticklebacks that were exposed to pulp mill effluent also produced spiggin in their kidneys. Possession of an androgen-regulated protein by the female stickleback makes it a unique bioassay organism for detecting androgenic contamination in the aquatic environment.

Environmental androgens are a group of compounds that to date have received very little attention. In this study, a yeast-based androgen screen (YAS) was used to determine the level of in vitro androgenic activity in seven United Kingdom estuaries. Surface water, sediment pore water, and sediment particulate material solvent extracts collected from Southampton Water, the Thames, Mersey, Tees, Tyne, Clyde, and Forth were tested for in vitro androgenic activity. Eleven of the 41 surface water samples collected displayed androgenic activity >2 ng dihydrotestosterone (DHT) equivalents/L (3-9 ng DHT/L), while eight of the 39 sediment pore waters collected showed activity >45 ng DHT/L (51-187 ng DHT/L). High levels of androgenic activity were determined in the solvent extracts of sediments, with 10 of 39 samples exhibiting a level of androgenic activity >454 ng DHT/kg (1,020-15,300 ng DHT/kg). In vitro YAS testing of five selected sewage treatment works (STW) effluents entering these estuaries showed that measurable levels (34-635 ng DHT/L) of androgenic activity were observed in those receiving only primary treatment (Howdon STW and Irvine Valley Sewer) at the time of the survey. A toxicity identification evaluation (TIE) study of Irvine Valley Sewer effluent using the YAS assay was used to identify the natural steroids/steroid metabolites dehydrotestosterone, androstenedione, androstanedione, 5beta-androstane-3alpha,11beta-diol-17-one, androsterone, and epi-androsterone as responsible for 99% of the in vitro activity determined in the effluent.

A series of constitutive green fluorescent protein (pGFPuv) derivatives of the bacterium Serratia entomophila (Enterobacteriaceae) were constructed, allowing the fate of cells causing amber disease ingested by the New Zealand grass grub (Costelytra zealandica, Coleoptera: Scarabaeidae) to be monitored. Examination of tissue and contents of the alimentary tract over time from ingestion, under fluorescence microscopy, revealed that the major site of S. entomophila colonisation in the grass grub is intestinal particulate matter. Visual examinations showed that wild type pathogenic strain persisted in high numbers in the grass grub intestinal tract, notably in the area of the hindgut, but the S. entomophila pADAP-free strain 5.6RC and the pADK mutant derivatives (pADK-4, -10, -13) that gave a non-feeding without gut clearance phenotype, were unable to colonise the gut. The indiscriminate colonisation of the intestinal tract particulate matter by pathogenic bacteria, rather than the colonisation of a specific site of activity, suggests that the bacterial toxins are induced and released from the bacteria while they live freely in the grass grub intestinal tract.

As a result of the introduction of tighter discharge limits and effluent treatment processes at source, the concentration of alkylphenol ethoxylates and nonylphenol present in the final effluent discharge from a sewage treatment works that treats trade effluent from the textiles industry was reduced. The estrogenic effects of the final effluent discharge to the Aire River were compared over a four-year period during which various treatment measures were introduced. Male rainbow trout exposed to the effluent on four occasions in consecutive years (1994-1997) showed a reduction in the level of induced vitellogenesis between 1994 and 1997. A marked decrease in gonadosomatic index (GSI) and increase in heptaosomatic index (HSI) was measured in fish exposed to the effluent in 1994. In successive years, these differences diminished, and in the case of the GSI no measurable difference was observed between fish exposed to the final effluent or those in the control group in 1997. However, an increase in HSI was still measurable in 1997 in fish exposed to the final effluent and at sites farther downstream. The reduction in the effects of the effluent paralleled the reduction in the concentration of nonylphenol as well as its mono- and diethoxylates, which have been demonstrated to produce estrogenic effects in trout exposed to these compounds in the laboratory. This study demonstrates that the setting of more restricted discharge limits for known estrogenic chemicals of industrial origin can lead to significant reductions in the estrogenic activity of the watercourses into which the effluents are discharged.

Chemical analyses were combined with a biological assay to investigate the main estrogenic chemicals as they passed through a sewage treatment works (STW) and entered a river. The STW studied was unusual in that it received wastewater from the textile trade. This wastewater was shown to contain high concentrations of alkylphenol polyethoxylates and their degradation products, such as nonylphenol. High-performance liquid chromatography fractionation, combined with biological assay, showed that the majority of the estrogenic activity was contributed by the alkylphenolic chemicals and the natural estrogens 17beta-estradiol and estrone. Despite removal of a high proportion of the alkylphenolic chemicals by the various treatment processes within the STW, concentrations in the final effluent were still high compared to most other STW effluents in the United Kingdom. The effluent was very estrogenic to caged fish, as was the river water 2 and 5 km downstream of the STW, even though less so. Using various approaches, attempts were made to determine which group of chemicals contributed most to the estrogenic activity of the effluent. The analysis suggested that, in this unusual situation, the alkylphenolic chemicals may contribute the majority of the estrogenic activity of the effluent. However, this conclusion was based on a number of uncertainties that are presently unresolved and hence can be considered only tentative.

Some strains of the Enterobacteriaceae Serratia entomophila and S. proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The virulence determinants of the disease reside on a large plasmid designated pADAP (amber disease-associated plasmid). A BamHI, EcoRI, and HindIII restriction cleavage map of pADAP was constructed by means of cloning restriction fragments. Each fragment was mapped, and neighboring fragments of mapped clones were systematically isolated from libraries using DNA probes constructed from previously cloned fragments. Through the use of sniff sequencing from the distal ends of a number of pADAP subclones the location of putative IS elements and genes involved in replication and conjugation were identified and assigned on the map. The location of the amber disease virulence-associated region was also mapped. The final map of pADAP spans 155 kb, 40 kb larger than the previous estimate., (Copyright 2002 Elsevier Science.)

This report describes the identification of important estrogenic compounds in surface and sediment pore-water samples collected from the Tyne and Tees estuaries (United Kingdom) through the application of toxicity identification evaluation (TIE) procedures. The Tyne and Tees estuaries represent estuaries that have been historically impacted by industrial activities and continue to receive treated domestic sewage and industrial effluent. In 1998, Dabholm Gut on the Tees received a mixture of treated and untreated effluent, while Howdon sewage treatment works (STW) discharged primary treated effluents. An estrogenically active water sample collected from Howdon STW on the Tyne was shown to contain 17beta-estradiol, androsterone, and an unknown estrogenic compound(s). Most of the activity contained in a sample collected from the Dabholm Gut combined discharges on the Tees was also due to 17beta-estradiol with additional activity from nonylphenol and (tentatively) bis(2-ethylhexyl)phthalate. The only sediment pore-water sample to demonstrate estrogenic activity was collected from Dabholm Gut.

The transient movement of pesticides at biologically active concentrations during storm events is considered to be a cause of biological impoverishment in some headwater streams. The programme of work described sought to identify compounds that are the cause of toxic effects during such events. Along with targeted pesticide analysis, toxicity identification evaluation (TIE) procedures were used to identify compounds with a demonstrated toxic effect. These procedures were specifically directed towards isolating and attributing toxicity to classes of organic contaminants in samples collected from an English headwater stream during a storm event. The organic load was isolated by means of solid-phase extraction (SPE). Bioassay of the SPE extract at x 100 whole water concentrations confirmed that the samples contained substances toxic to Daphnia magna, although the raw samples were not toxic. Targeted pesticide analysis identified simazine and diuron as the major pesticides present and, using a toxicity unit (TU) approach. were shown to be responsible for a significant amount of the observed concentrate toxicity during a runoff event. However, they were not present in sufficient quantities to be totally responsible for a more toxic later event. By simplification of the SPE isolate using reverse-phase HPLC, fractions from which were tested for toxicity, the cause of concentrate toxicity in the later event was isolated to two discrete fractions. GC-MS analysis of these fractions identified nonylphenol. endosulfan sulphate and pendimethalin as present, with the majority of toxicity attributed to nonylphenol (NP). The main advantage of the TIE approach is that it allows biological active compounds with a demonstrated effect to be identified that may not be selected by more traditional techniques.

Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, or sepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.

The purpose of this study was to determine whether different topographies of disfluent behavior form a response class. A within-subject, repeated reversals (ABAB) design was used to analyze the stuttering behavior of three adult stuttering speakers. A single type of stuttering behavior was punished for each subject while frequencies of occurrence of other types were concurrently measured. The results showed that: (1) stuttering behaviors displayed direct behavioral covariation for all subjects, illustrating the existence of a response class; and (2) the response classes observed included both kernel and accessory features of stuttering. The results are discussed in terms of the literature on response classes and two-factor learning theory of stuttering with special emphasis on the implications of these results for our understanding of the development of stuttering.

Airborne levels of isopropyl alcohol when used as a disinfectant in a horizontal laminar-airflow hood were measured during a 10-day period to determine whether levels conformed to the Occupational Safety and Health Administration (OSHA) guidelines. An infrared vapor analyzer was positioned in the airflow of the six-foot horizontal laminar-airflow hood for 10 days to continuously monitor the levels of isopropyl alcohol under usual procedural conditions. A continuous printout of the levels of isopropyl alcohol allowed determination of the eight-hour time-weighted average level. The mean (+/- S.D.) eight-hour time-weighted average level of isopropyl alcohol in the laminar-airflow hood was 99.6 +/- 30.6 parts per million (ppm). By OSHA standards, the eight-hour time-weighted average airborne level of isopropyl alcohol in the work place cannot exceed 400 ppm. Usual conditions in the laminar-airflow hood at this institution complied with the OSHA standard for airborne isopropyl alcohol.