From yeast to hypha: defining transcriptomic signatures of the morphological switch in the dimorphic fungal pathogen Ophiostoma novo-ulmi.

BACKGROUND: Yeast-to-hypha transition is a major morphological change in fungi. Molecular regulators and pathways that are involved in this process have been extensively studied in model species, including Saccharomyces cerevisiae. The Mitogen-Actived Protein Kinase (MAPK) cascade, for example, is known to be involved in the yeast-to-pseudohypha switch. Yet the conservation of mechanisms regulating such morphological changes in non-model fungi is still poorly understood. Here, we investigate cell remodeling and transcriptomic modifications that occur during this morphological switch in the highly aggressive ascomycete fungus Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. RESULTS: Using a combination of light microscopy, scanning electron microscopy and flow cytometry, we demonstrate that the morphological switch occurs in less than 27 h, with phenotypic cell modifications being detected within the first 4 h. Using RNAseq, we found that over 22% of the genome of O. novo-ulmi is differentially expressed during the transition. By performing clustering analyses of time series gene expression data, we identified several sets of genes that are differentially expressed according to distinct and representative temporal profiles. Further, we found that several genes that are homologous to S. cerevisiae MAPK genes are regulated during the yeast-to-hypha transition in O. novo-ulmi and mostly over-expressed, suggesting convergence in gene expression regulation. CONCLUSIONS: Our results are the first report of a time-course experiment monitoring the morphological transition in a non-model Sordariomycota species and reveal many genes of interest for further functional investigations of fungal dimorphism.

Patterns of colonization and spread in the fungal spruce pathogen Onnia tomentosa.

The basidiomycetous fungus Onnia tomentosa is one of the most widespread root rot pathogens in North America. Although the disease is more severe on spruce and pine trees, this pathogen can infect several coniferous species. To study the population structure of O. tomentosa, we harvested 180 basidiocarps in a 45-year-old white spruce plantation in western Quebec in autumn 1997 and extracted DNA directly from individual basidiocarps. Using a combination of spatial coordinates and molecular data based on the analysis of two mitochondrial and three nuclear loci, we measured the average genet size and molecular diversity and assessed the relative contribution of basidiospores and vegetative growth to the stand colonization. Most of the sampled basidiocarps that clustered spatially belonged to the same genet. A total of 37 discrete multilocus genets of an average size of 3.42 m were obtained. The genet size distribution was skewed towards smaller genets (<3 m) that displayed higher diversity than the larger genets (>3 m). The nuclear loci were in Hardy-Weinberg equilibrium in the larger genets, but not in the smaller genets, which displayed a deficiency of heterozygotes. This suggests a Wahlund effect, whereby different colonization events resulted in expected heterozygosity higher than observed heterozygosity. Using an estimate of the growth rate of the fungus, only a few of the largest genets were approximately the age of the plantation. These observations are consistent with the colonization by basidiospores subsequent to site preparation and tree planting followed by secondary colonization events and vegetative spread.

A PCSK9 variant and familial combined hyperlipidaemia.

BACKGROUND: Our discovery in 2003 of the first mutations of PCSK9 gene causing autosomal dominant hypercholesterolaemia (ADH) shed light on an unknown factor that strongly influences the level of circulating low density lipoprotein cholesterol (LDL-C). PCSK9 gain of function mutations cause hypercholesterolaemia by a reduction of LDL receptor levels, while PCSK9 loss of function variants are associated with a reduction of LDL-C values and a decreased risk of coronary heart disease. METHODS AND RESULTS: We report an insertion of two leucines (p.L21tri also designated p.L15_L16ins2L) in the leucine stretch of the signal peptide of PCSK9 that is found in two of 25 families with familial combined hyperlipidaemia (FCHL). This mutant is associated with high total cholesterol and LDL-C values in these families and is found also in a patient with familial hypercholesterolaemia and her father. CONCLUSION: PCSK9 variants might contribute to FCHL phenotype and are to be taken into consideration in the study of this complex and multigenic disease with other genes implicated in dyslipidaemia.

First Report of Erysiphe palczewskii Powdery Mildew of Siberian Pea Tree (Caragana arborescens) in Wisconsin and Quebec.

Shoots affected by powdery mildew were collected from Siberian pea trees in July 2009 on the University of Wisconsin-Madison campus and on the campus of Université Laval, Quebec City, Quebec. This exotic shrub or small tree is infrequently planted in Wisconsin and three shrubs in a group that were affected are the only examples known on the UW-Madison campus. In Quebec City, Siberian pea tree is more commonly used as an ornamental, often in hedges (as is the case of the affected plants on the Université Laval campus). In both locations, <10% of foliage was visibly affected, but incidence was greater on shoots closer to the ground than on higher shoots. White-to-grayish mycelium was present on leaves and young stems and sometimes completely covered both upper and lower leaf surfaces. Dark brown-to-black chasmothecia were numerous on leaf blades, petioles, and young stems, but were most abundant on lower surfaces of leaves. Morphology of chasmothecia, including appendages with distinctive terminal dichotomous branching, (1) was consistent with descriptions and illustrations of the fungus Erysiphe palczewskii Jacz. (synonym Microsphaera palczewskii) (1-4) thought to be native to Asia, but also known as an invader of Europe where it occurs on the same host. For a sample from Université Laval, mean diameter of chasmothecia was 113 µm, mean appendage length was 185 µm, and barrel-shaped conidia that lacked fibrosin bodies averaged 30 × 14 µm. Asci contained oval, yellow ascospores with mean dimensions of 20 × 12 µm. DNA was extracted from chasmothecia, and nuclear rDNA sequences (633 nucleotides) of the Wisconsin (GenBank Accession No. GQ497277) and Quebec (GenBank Accession No. GQ497276) specimens differed by only one nucleotide. The sequences that were obtained most closely matched GenBank sequences for Oidium spp. (98%) and Erysiphe spp. (97%). Further observations indicated that the same pathogen affected Siberian pea trees planted as ornamentals at several locations separated by ≥15 km in the metropolitan Quebec area. This report extends the eastern known limit of E. palczewskii in the United States, previously known from collections in Alaska (2), Washington (4), Idaho (4), North Dakota (3), and Minnesota (3). To our knowledge, this is the first report of this disease in Canada, and it indicates that the distribution of E. palczewskii is transcontinental. Specimens from Madison, WI and Quebec, QC have been deposited in the U.S. National Fungus Collections (BPI 879152) and the Rene Pomerleau Herbarium of the Canadian Forest Service Laurentian Forestry Centre (QFB-22601). References: (1) U. Braun. Beih. Nova Hedwigia 89:1, 1987. (2) D. A. Glawe and G. A. Laursen. Online publication. doi:10:1094/PHP-2005-1017-01-BR. Plant Health Progress, 2005. (3) D. A. Glawe et al. Online publication. doi:10.1094/PHP-2006-0117-01-BR. Plant Health Progress, 2006. (4) C. Nischwitz and G. Newcombe. Plant Dis. 87:451, 2003.

Genetic Diversity in Poplar Leaf Rust (Melampsora medusae f. sp. deltoidae) in the Zones of Host Sympatry and Allopatry.

ABSTRACT Poplar leaf rust caused by Melampsora medusae f. sp. deltoidae is a widespread disease in North America, where epidemics occur within zones of sympatry and allopatry of telial hosts (Populus spp.) and aecial hosts (Larix spp.). To test the hypothesis that epidemics originate in the zone of sympatry where the rust can complete its life cycle, populations in sympatry and allopatry were analyzed with single-strand conformational polymorphism for codominant detection of alleles directly from uredinia. More alleles were detected in rust populations in the zone of host sympatry than in allopatry. Almost all alleles found in the zone of allopatry were a subset of the allelic diversity present in the zone of host sympatry. Distance analyses clustered populations according to geographic origin, but not sampling year or type of stand (plantation or natural stands). Large differences in allelic and genotypic frequency were observed between years in allopatry but not in sympatry, suggesting new colonizations in allopatric populations. Our results point to a dynamic and complex pattern of inoculum dissemination in polar leaf rust. The hypothesis most consistent with our results is that populations in sympatry represent a source of inoculum for epidemics, with some annual recolonization in allopatry, possibly via intermediate population jumps.

Simultaneous induction of jasmonic acid and disease-responsive genes signifies tolerance of American elm to Dutch elm disease.

Dutch elm disease (DED), caused by three fungal species in the genus Ophiostoma, is the most devastating disease of both native European and North American elm trees. Although many tolerant cultivars have been identified and released, the tolerance mechanisms are not well understood and true resistance has not yet been achieved. Here we show that the expression of disease-responsive genes in reactions leading to tolerance or susceptibility is significantly differentiated within the first 144 hours post-inoculation (hpi). Analysis of the levels of endogenous plant defense molecules such as jasmonic acid (JA) and salicylic acid (SA) in tolerant and susceptible American elm saplings suggested SA and methyl-jasmonate as potential defense response elicitors, which was further confirmed by field observations. However, the tolerant phenotype can be best characterized by a concurrent induction of JA and disease-responsive genes at 96 hpi. Molecular investigations indicated that the expression of fungal genes (i.e. cerato ulmin) was also modulated by endogenous SA and JA and this response was unique among aggressive and non-aggressive fungal strains. The present study not only provides better understanding of tolerance mechanisms to DED, but also represents a first, verified template for examining simultaneous transcriptomic changes during American elm-fungus interactions.

A glycosyl hydrolase activity of mammalian reovirus sigma1 protein can contribute to viral infection through a mucus layer.

The mammalian reovirus sigma1 protein is responsible for viral attachment to host cells and hemagglutination properties of the virus. In the present study, sequence similarity between sigma1 and chicken-type lysozymes prompted us to investigate additional functions of the sigma1 protein. Expression in Pichia pastoris yeast cells showed that sigma1 can actually cleave lysozyme substrates, including complex sugars found in bacterial cell walls. Replacement by site-directed mutagenesis of acidic amino acid residues in sigma1 by their respective isosteric, uncharged, amino acid residues has allowed us to identify Glu36 and Asp54 as the catalytic pair involved in sigma1-mediated glycosidase activity. The enzyme appears inactive in virions but its activity is unmasked upon generation of infectious subviral particles (ISVPs) by partial proteolytic removal of the outer capsid proteins. Purified sigma1 protein and ISVPs can also hydrolyze mucins, heavily glycosylated glycoproteins that are a major component of the mucus layer overlaying the intestinal epithelium. Furthermore, reovirus infection of epithelial Madin Darby canine kidney cells was inhibited tenfold in cells expressing mucin at their apical surface, while this inhibition was overcome by ISVPs. Unmasking of sigma1 mucinolytic activity in the intestine, consecutive to proteolytic cleavage of virions to ISVPs, thus likely contributes to the known increase in infectivity of reovirus ISVPs compared to complete virions. This work presents the first evidence that some mammalian viruses have evolved mechanisms to facilitate their penetration through the protective barrier of the mucus layer in the intestinal tract.

Identification of a gamma-interferon-responsive element in the promoter of the human macrophage scavenger receptor A gene.

In the present study, we demonstrate gamma-interferon (gamma-IFN)-inducible scavenger receptor A (SR-A) mRNA expression during the early stages of THP-1 and blood monocyte differentiation. Predominant induction of SR-A type II mRNA parallels the increased accumulation of cholesteryl esters under these conditions. A potential signal transducer and activator of transcription (STAT1) binding site (gamma-interferon activation site) in the SR-A promoter demonstrates gamma-IFN-inducible DNA binding activity and is most likely responsible for the gamma-IFN-dependent expression of an SR-A promoter-luciferase fusion construct. In contrast, gamma-IFN inhibits SR-A expression in mature macrophages as well as after prolonged gamma-IFN incubation of THP-1 monocytes. Taken together, these results demonstrate opposite effects of gamma-IFN on SR-A expression and activity during the early versus late stages of monocyte maturation. gamma-IFN-induced STAT1 activation, leading to increased SR-A expression, could therefore play an important role in the initial steps of foam cell formation and xanthomatosis.

Involvement of oxysterols and lysophosphatidylcholine in the oxidized LDL-induced impairment of serum albumin synthesis by HEPG2 cells.

Oxidized low density lipoproteins (Ox-LDLs) are increasingly thought to be a key element in atherogenesis. We have previously reported that serum albumin has important antioxidant properties and that a reduced synthesis of albumin may represent a crucial point in the overall antioxidant defense. In the present work, we aimed at determining whether Ox-LDL could modulate albumin synthesis in cultured human hepatocytes (HepG2 cells). With the use of enzyme immunoassay and radiolabeled leucine incorporation followed by specific immunoprecipitation, Ox-LDL was found to lead to a dose-dependent decrease in albumin secretion. Moreover, the protein synthesis and mRNA levels were decreased in the presence of Ox-LDL, as assessed by Northern blot analysis. Because oxysterols and lysophospholipids are key components of Ox-LDL, we tested the effects of oxysterols (7-ketocholesterol and 25-hydroxycholesterol) and lysophosphatidylcholine on albumin secretion and expression. In our experimental conditions, we found that incubations with oxysterols or lysophosphatidylcholine at pathophysiological concentrations similar to those measured in Ox-LDLs reproduced the above-mentioned inhibitory effects on albumin synthesis. On the basis of our in vitro data, we propose that this newly described biological effect of Ox-LDL might partly explain the findings of epidemiological studies indicating that reduced levels of serum albumin are associated with increased mortality.

Magnesium, zinc, and copper status of US Navy SEAL trainees.

Magnesium, zinc, and copper status of 270 US Navy Sea, Air, and Land (SEAL) trainees was determined from dietary intakes and biochemical profiles. Although mean intakes exceeded the Recommended Dietary Allowances or the Estimated Safe and Adequate Intake range, intakes of 34%, 44%, and 37% of the trainees were below the recommendations for Mg, Zn, and Cu, respectively. Mean plasma concentrations were 0.85 +/- 0.004 mmol/L, 13.4 +/- 0.2 mumol/L, and 16.5 +/- 0.2 mumol/L for Mg, Zn, and Cu respectively. Mean 24-h urinary excretions were 5.7 +/- 0.2 mmol, 11.1 +/- 0.3 mumol, and 0.05 +/- 0.003 mumol, for Mg, Zn, and Cu, respectively. The data provided by the present study should be useful for comparing other physically active male populations.

A prospective view on phosphatases and replicative senescence.

Addition of phosphate to proteins by kinases, or its removal by phosphatases, is probably the control mechanism most often used by cells to maintain homeostasis. This mechanism presents the advantage of being fast, versatile, and easily reversible. It is used by all organisms from bacteria to man. Although more is known about the kinases, recent studies are beginning to shed light on the role of phosphatases, the enzymes that are responsible for terminating the effects of phosphorylation. These enzymes are perfect candidates for controlling all the crucial check points during cell cycle traverse, and as such, will be found to be responsible for many important decision in the life of a cell, including entry into replicative senescence.

Developmental expression of myelin proteolipid, basic protein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase transcripts in different rat brain regions.

RNA was extracted from five different rat brain regions during development, starting from embryonic day 15 (E15) until postnatal day 60 (P60). These RNA preparations were analyzed by both Northern and dot blot for their content of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), myelin proteolipid protein (PLP), and myelin basic protein (MBP) -specific transcripts. CNPase mRNA was readily detectable at E15 and PLP mRNA at P1 in all brain regions examined. In contrast, expression of MBP mRNA followed a caudorostral gradient. It was first observed at P1 in the mesencephalon and at P9-P11 in the olfactory bulb. Expression of these three transcripts displayed two types of developmental profiles. One was termed biphasic because the specific mRNA level increased regularly and then reached a plateau level. The other developmental profile was termed triphasic, because there was a gradual increase in the level of specific transcripts with a sudden appearance of a sharp peak followed by a decline to a plateau level. When the triphasic pattern was observed, the date of the peak appearance was probe-, but not region-, dependent. It was P15 for CNPase, P18 for MBP, and P21 for PLP. As these peaks occurred at a time during development when myelination was the most active, we postulate the existence of a transient external signal, perhaps neuronal, which would be responsible for this increased amount of myelin-related transcripts.

Juxtaglomerular cell tumor of the kidney: report of two cases with a papillary pattern.

We report the clinicopathologic, immunohistochemical, and electron microscopic study of two cases of juxtaglomerular cell tumor of the kidney with a hitherto unreported dominant papillary pattern. Both tumors were associated with high blood pressure that did not respond to medical therapy, but that returned to normal after removal of the kidney. They were well delineated, tan, and had no necrosis. The cores of the papillary structures consisted of polygonal cells found to express renin by immunohistochemistry and to contain renin protogranules by electron microscopy. The papillary fronds were covered by one layer of cuboidal epithelial cells that did not stain for renin and had ultrastructural features reminiscent of the collecting duct epithelium. These tumors must be differentiated from malignant papillary tumors of the kidney, such as papillary clear cell carcinoma, transitional cell carcinoma, and collecting duct carcinoma.

Physiologic properties of myelin proteins revealed by their expression in nonglial cells.

The transfection paradigm described herein can be used to investigate the functional properties of individual nervous system proteins in ways that have not been explored before. In particular, observations on the "structural" proteins of myelin are being made that have already yielded certain unique insights into the physiologic properties of these polypeptides. The ease with which site-directed mutagenesis procedures can be applied to these systems should eventually enable us to define with great precision the "functional domains" within each myelin protein.

Hormonal and metabolic responses of untrained, moderately trained, and highly trained men to three exercise intensities.

Untrained, moderately trained (runners, 15 to 25 mi/wk), and highly trained (runners, greater than 45 mi/wk) men participated in graded treadmill exercise at 50%, 70%, and 90% of their maximal oxygen consumption to quantify the relation between intensity of exercise and sympathetic nervous system and metabolic responses. Sympathetic system activation was noted at all intensities tested and was proportional to the relative exercise intensity. The magnitudes of the norepinephrine (NE) and epinephrine (E) responses were similar in all three groups of men at each relative exercise intensity and correlated with the magnitudes of change in levels of circulating plasma adrenocorticotropin hormone, cortisol, lactate (La), phosphate (Pi), and glucose (GI). The magnitudes of change in concentrations of La, Pi, and GI were also similar for the three groups at each relative exercise intensity. In contrast, a lower degree of sympathetic system activation in response to a given absolute workload was noted in the moderately and highly trained men as compared to that of the untrained men. Sympathetic and metabolic responses to exercise are similar under conditions of comparable relative exercise intensities, regardless of conditioning level. The sympathetic-adrenal medullary system is more sensitive to exercise than the hypothalamic-pituitary-adrenal axis. For a given absolute workload, the degree of activation significantly lower in trained individuals.

Effects of exercise and conditioning on clotting and fibrinolytic activity in men.

Sixty healthy men in three physical fitness categories (sedentary, on no organized fitness program; joggers, running 5-15 miles/wk; and marathoners, running greater than 50 miles/wk) were evaluated for changes in blood clotting and fibrinolytic activity before and after maximum exercise on a treadmill according to the Bruce protocol. The rate of blood clotting, as measured by prothrombin times, partial thromboplastin times and thrombin times, was accelerated by exercise (all P less than 0.005). The ability of euglobulin clots and plasma clots to lyse incorporated 125I-fibrin, termed 125I-euglobulin clot lysis (IEL) and 125I-plasma clot lysis (IPCL), were used as indexes of fibrinolytic activity. Marathoners had greater increases in fibrinolytic activity with exercise (76% compared with 63% for joggers and 55% for sedentary subjects by IEL; 427% compared with 418% for joggers and 309% for sedentary subjects by IPCL; all P less than 0.05). Fibrin degradation products increased with exercise (P less than 0.005 for the total group of 60 subjects). The absolute concentrations of alpha 2-plasmin inhibitor, alpha 2-macroglobulin, and antithrombin III increased with exercise (all P less than 0.005), but when concentrations were corrected for acute shifts of plasma water during exercise, the quantity of these inhibitors actually decreased (all P less than 0.005). The changes in clotting assays with exercise were not significantly correlated with changes in whole blood lactate, blood pyruvate, or rectal temperatures. Fibrinolytic assays before and after exercise correlated poorly to moderately with blood lactates (IEL: r = 0.441 and r = 0.425, respectively; IPCL: r = 0.294 and r = 0.544, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of a novel nucleoside analogue (Troxatyl, troxacitabine, BCH-4556) and AraC against leukemic human tumor xenografts expressing high or low cytidine deaminase activity.

PURPOSE: Troxacitabine (beta-L-dioxolane cytidine, BCH-4556; Troxatyl, BioChem Pharma Inc.) is a novel nucleoside analogue, which in experiments demonstrated potent antitumor activity against both leukemias and solid tumors. Since troxacitabine is a cytidine nucleoside analogue like AraC (1-beta-D-arabinofuranosylcytosine), which is currently used in the treatment of acute myelogenous leukemia, we compared the in vivo antileukemic activity of troxacitabine with that of AraC in human leukemia xenograft models. METHODS: The antiproliferative activity of troxacitabine and AraC was analyzed on hemapoietic cell lines by use of a thymidine incorporation assay. For in vivo studies, we compared troxacitabine with AraC by using equitotoxic schedules of the two nucleosides optimized for therapeutic activity. The antileukemic activity of both drugs was evaluated by measurement of their effect on the percent increased lifespan. RESULTS: AraC had good in vitro antiproliferative activity (IC50 = 14 nM) but was ineffective in vivo against the HL60 promyelocyte leukemia cell line (treated vs control, T/C = 105%). Troxacitabine, which in contrast to AraC is not a substrate for cytidine deaminase, showed potent in vitro and in vivo activity in the same model (IC50 = 53 nM and T/C = 272% to 422%). The poor in vivo activity of AraC against HL60 leukemia cells could be due to the high cytidine deaminase (CDA; EC 3.5.4.5) activity in this cell line. This hypothesis was tested with CCRF-CEM T-lymphoblastoid leukemia cells which have undetectable levels of CDA activity. Short-term exposure of these leukemia cell lines to both drugs indicated that AraC was indeed significantly more effective in the CCRF-CEM cell line than in HL60. In contrast, the antiproliferative activity of troxacitabine was similar for both cell lines. These observations were extended to in vivo studies. Mice bearing CCRF-CEM tumor xenografts were treated with AraC and troxacitabine. In this model, T/C values were comparable for both drugs and ranged from 138% to 157%. CONCLUSIONS: Our findings indicate that troxacitabine is likely to be effective not only against solid tumors with high CDA activity but also in leukemias which have developed resistance to AraC due to increased CDA levels; this suggests that troxacitabine is a promising agent for the treatment of cancer. Indeed, significant antileukemic activity has been observed with troxacitabine in a phase I clinical trial in patients with primary refractory or relapsed acute myeloid leukemias (AML).

Apolipoprotein E and atherosclerosis: insight from animal and human studies.

Major advances have been made in our understanding of the role of apolipoprotein E (apoE) in the onset and development of atherosclerosis. Increasing evidence from both animal and human studies suggests that apoE is able to protect against atherosclerosis by: a) promoting efficient uptake of triglyceride-rich lipoproteins from the circulation; b) maintaining normal macrophage lipid homeostasis; c) playing a role in cellular cholesterol efflux and reverse cholesterol transport; d) acting as an antioxidant; e) inhibiting platelet aggregation; and f) modulating immune function. In humans, apoE is polymorphic, and this genetic variation has a strong effect on its antiatherogenic characteristics. Thus, compared to the epsilon3 allele, the epsilon4 allele promotes atherosclerosis, whereas the epsilon2 allele is either pro- or anti-atherogenic, depending on the influence of both environmental and genetic factors. ApoE and its gene are prime targets for therapeutic intervention aimed at preventing or treating atherosclerotic vascular disease.

Fluorescence determination of microconcentrations of chlorambucil after photoactivation.

A fluorescence assay is described which measures the alkylating activity of chlorambucil or its isocyanate derivative after photoactivation in the presence of dimethyl sulfoxide. This assay has a lower limit of sensitivity of 100 ng/mL and RSD of less than 10% for chlorambucil. The method requires less than 5 micrograms of alkylating agent and the fluorophore produced is stable for at least 24 h.

Rubidium marking of Anopheles stephensi (Diptera: Culicidae).

Immature Anopheles stephensi Liston were reared in untreated water and water containing eight 2-fold dilutions of rubidium (Rb) from 1,000 to 7.8 ppm to determine the concentration that allowed reliable detection and produced the least toxic effects as measured by adult emergence, weight, and survival. The amount of Rb detected in mosquitoes increased positively with increasing concentrations in the rearing water. Concentrations > or = 31.2 ppm Rb in the rearing water provided high and consistent detection levels of > or = 3,500 ppm Rb/mg of adult mosquito. There were no adverse effects of Rb on the weights of mosquitoes. However, increased Rb concentrations in the rearing water were associated with decreased emergence and survival. After 8 d, percentage emergence from Rb concentrations of 0-31.2 ppm was > or = 50%. At day 21, Rb concentrations of 0-31.2 ppm showed < or = 29% reduction of female survival compared with controls. The recommended concentration for reliable Rb detection with minimal toxic effects in An. stephensi was 32 ppm.