Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae.

A novel Bacillus thuringiensis crystal protein with a silent activity against the Colorado potato beetle is described. The crystal proteins are produced as bipyramidal crystals. These crystals contain a protein of 129 kDa with a trypsin-resistant core fragment of 72 kDa. Neither a spore-crystal mixture nor in vitro-solubilized crystals are toxic to any of several Lepidoptera and Coleoptera species tested. In contrast, a trypsin-treated solution containing the 72-kDa tryptic core fragment of the protoxin is highly toxic to Colorado potato beetle larvae. The crystal protein-encoding gene was cloned and sequenced. The inferred amino acid sequence of the putative toxic fragment has 37, 32, and 33% homology to the CryIIIA, CryIIIB, and CryIIID toxins, respectively. Interestingly, the 501 C-terminal amino acids show 41 to 48% amino acid identity with corresponding C-terminal amino acid sequences of other crystal proteins. Because of the toxicity of the fragment to the Colorado potato beetle and because of the distinct similarities of the toxic fragment with the other CryIII proteins, this gene was given a new subclass name (cryIIIC) within the CryIII class of coleopteran-active crystal proteins. CryIIIC represents the first example of a crystal protein with a silent activity towards coleopteran insect larvae. Natural CryIIIC crystals are not toxic. Toxicity is revealed only after an in vitro solubilization and activation step.     

Cranial morphological scaling and relative prey size limitations for a native predator in an invaded system

Over evolutionary time, predator-prey interactions have shaped and constrained functional and behavioral traits of piscivorous fishes. The endangered Colorado Pikeminnow Ptychocheilus lucius, a large endemic piscivore of the Colorado River Basin, encounters a substantially altered prey base that differs in behaviors and morphologies compared to the historical suite of native prey. To assess physical limitations of Colorado Pikeminnow predation, we conducted a feeding experiment with two species of nonnative prey (spined and despined Channel Catfish Ictalurus punctatus and Red Shiner Cyprinella lutrensis) and quantified scaling of cranial morphology in this predator. In our predation experiments, Colorado Pikeminnow (215–312 mm total length) consumed both spined and despined Channel Catfish as well as Red Shiner but only consumed prey less than 20% of the predator’s total length. Previous feeding trials using smaller Colorado Pikeminnow, with native and nonnative prey species, indicated they consumed prey up to 35% of their total length, suggesting relative prey size limits may decrease as this predator grows. Morphological measurements also suggested relative prey size suitability may decrease as Colorado Pikeminnow become larger, with head depth and width demonstrating isometric scaling at small sizes and shifting to negative allometry as fish get larger. Together, these data suggest an ontogenetic shift in the head morphology of Colorado Pikeminnow may decrease the relative size of prey available to these predators. In severely altered systems, understanding trophic characteristics that limit overall predator resource availability will be critical for conservation of piscivorous fishes.     

Two new bacterial pathogens of Colorado potato beetle Coleoptera: Chrysomelidae)

Other than Bacillus thuringiensis Berliner, few bacteria are lethal to the Colorado potato beetle (Leptinotarsa decemlineata [Say]), a major pest of potatoes and eggplant. Expanded use of biologicals for the control of Colorado potato beetle will improve resistance management, reduce pesticide use, and produce novel compounds for potential use in transgenic plants. Using freeze-dried, rehydrated artificial diet in pellet form to screen bacteria lethal to other insects, we determined that strains of Photorhabdus luminescens killed Colorado potato beetle larvae. The LC50 for second instar larvae of strain HM5-1 was 6.4 +/- 1.87 x 10(7) cells per diet pellet. In an attempt to find additional naturally occurring P. luminescens strains toxic to Colorado potato beetle larvae, we recovered, from soil, bacteria that produced a purple pigment. This bacterial strain, identified as Chromobacterium sp. by 16S ribosomal DNA sequencing, was also toxic to Colorado potato beetle larvae within 3 d. The LC50 for second instar larvae for these bacteria was 2.0 +/- 0.79 x 10(8) cells per diet pellet, while the LC50 was approximately 1 log lower for third instar larvae. P. luminescens appeared to kill by means of a protein toxin that may be similar to the described lepidopteran protein toxins. Based on the heat and acid stability, the toxin or toxins that Chromobacterium sp. produces, while not fully characterized, do not appear to be typical proteins. In both bacteria, the toxins are made after exponential growth ceases.     

Sequence determination and analysis of the full-length genome of colorado tick fever virus, the type species of genus Coltivirus (Family Reoviridae)

The Colorado tick fever virus (CTFV) is the type species of genus Coltivirus, family Reoviridae. Its genome consisting of 12 segments of dsRNA was completely sequenced. It was found to be 29,174 nucleotides long (the longest of all Reoviridae genomes characterized to date). Conserved sequences at the 5' end (SACUUUUGY) and at the 3' end (WUGCAGUS) of the 12 segments were identified. The analysis of the putative proteins deduced from the nucleotide sequences permitted to identify functional motifs. In particular, the VP1 was identified unambiguously as the viral RNA dependent RNA pylmerase (RDRP) (VP1pol), with a GDD located at a similar position to Reoviridae RDRPs. In other genes, RGD cell-binding, NTPAse, single strand binding protein and kinase motifs were identified. Comparison with Reoviridae proteins showed significant similarities to RDRPs (CTFV-VP1) and sigma C protein of orthoreovirus (CTFV-VP6). Similarities to nonviral enzymatic proteins, such as methyltransferases, NTPAses, RNA replication factors, were also identified.     

Telomeres and double-strand breaks - all's well that "ends" well. ..

Bailey, S. Telomeres and Double-Strand Breaks - All's Well that "Ends" Well. ... Radiat. Res. 169, 1-7 (2008). Sometimes one's life (including one's science) makes a lot more sense when viewed from the perspective of time, reflected back on over a number of years. That has indeed been the case for me. Strangely enough, the story begins with chromosomes and "ends" with telomeres, both at Colorado State University. And, just as with chromosomes, a lot happened in between. Telomeres were first identified based on their function-they protected the physical ends of chromosomes from interaction with broken DNA ends created by ionizing radiation. While I was at Los Alamos National Laboratory, the sequence of human telomeres was discovered, making probes available that allowed us to re-examine and provide direct support of these early observations; thus began my fascination with telomeres. Chromosome orientation in situ hybridization (CO-FISH) also came onto the scene while I was in Los Alamos. This strand-specific modification of standard FISH, especially when combined with telomeric sequence probes, has proven to be a powerful approach that provides information not available by any other means. Applications have included pericentric inversion detection, distinction between leading- and lagging-strand telomeres, and identification of telomere-double-strand break (DSB) fusions. We also provided the first direct evidence that DSB repair proteins (DNA-PK in particular) are required for mammalian telomeric end capping, and we have been characterizing telomere dysfunction in NHEJ and HR repair-deficient backgrounds ever since. Cells must correctly distinguish between DNA ends represented by telomeres and DNA ends produced by DSBs if all is to end well. Just as these studies have provided new insight into the complex, often surprising, interactions at DNA ends, they also provoke new questions. Whereas it is now well established that DSB repair proteins associate with telomeres, most recently we've been asking whether the reverse scenario holds: Do telomere proteins interact with DSBs? We find that DSBs induced by ionizing radiations are not sufficient to recruit the essential telomere protein TRF2 as an early damage response, so perhaps this interplay is a one-way street. The rest of the story waits to unfold.     

New tricks for ubiquitin and friends

The foothills of the Rocky Mountains provided a spectacular setting for the American Society for Cell Biology (ASCB) meeting entitled ‘Non-traditional functions of ubiquitin and ubiquitin-like proteins’, Colorado College, Colorado Springs, CO, USA, on 11–14 August, 2002. Organizers Linda Hicke and Cecile Pickart put together an excellent programme of talks covering functions of ubiquitin other than its well known role in proteasomal targeting. The increasingly diverse biological processes in which the ubiquitin-like proteins (UBLs) are involved, also featured. One of the aims of the meeting was to bring together researchers working directly with ubiquitin and UBLs, and also those who have found that their favourite molecule or process is somehow influenced by these small versatile tags. As a result, delegates were treated to a diverse and highly stimulating meeting.     

Nitrogen cycling in desert biological soil crusts across biogeographic regions in the Southwestern United States

Biological soil crusts (BSCs) are thought to be important in the fertility of arid lands as gateways for carbon (C) and nitrogen (N). Studies on the Colorado Plateau have shown that an incomplete internal N cycle operates in BSCs that results in significant exports of dissolved organic N, ammonia and nitrate into the bulk soil through percolating water, thus mechanistically explaining their role as a N gateway. It is not known if this pattern is found in other arid regions. To examine this, we measured rates of major biogeochemical N-transformations in a variety of BSCs collected from the Colorado Plateau and the Mojave, Sonoran and Chihuahuan Deserts. Dinitrogen fixation and aerobic ammonia oxidation were prominent transformations at all sites. We found anaerobic ammonia oxidation (anammox) rates to be below the detection limit in all cases, and at least 50-fold smaller than rates of N₂-fixation, making it an irrelevant process for these BSCs. Heterotrophic denitrification was also of little consequence for the flow of N, with rates at least an order of magnitude smaller than those of N₂-fixation. Thus we could confirm that despite the demonstrable differences in microbial community composition and soil material, BSCs from major biogeographic regions in arid North America displayed a remarkably consistent pattern of internal N cycling. The implications for arid land fertility drawn from previous studies in the Colorado Plateau appear applicable to BSCs across other arid regions of the Southwestern United States.     

Analysis of regional species distribution models based on radio-telemetry datasets from multiple small-scale studies

The identification of core habitat areas and resulting prediction maps are vital tools for land managers. Often, agencies have large datasets from multiple studies over time that could be combined for a more informed and complete picture of a species. Colorado Parks and Wildlife has a large database for greater sage-grouse (Centrocercus urophasianus) including 11 radio-telemetry studies completed over 12 years (1997–2008) across northwestern Colorado. We divided the 49,470-km² study area into 1-km² grids with the number of sage-grouse locations in each grid cell that contained at least 1 location counted as the response variable. We used a generalized linear mixed model (GLMM) using land cover variables as fixed effects and individual birds and populations as random effects to predict greater sage-grouse location counts during breeding, summer, and winter seasons. The mixed effects model enabled us to model correlations that may exist in grouped data (e.g., correlations among individuals and populations). We found only individual groupings accounted for variation in the summer and breeding seasons, but not the winter season. The breeding and summer seasonal models predicted sage-grouse presence in the currently delineated populations for Colorado, but we found little evidence supporting a winter season model. According to our models, about 50% of the study area in Colorado is considered highly or moderately suitable habitat in both the breeding and summer seasons. As oil and gas development and other landscape changes occur in this portion of Colorado, knowledge of where management actions can be accomplished or possible restoration can occur becomes more critical. These seasonal models provide data-driven, distribution maps that managers and biologists can use for identification and exploration when investigating greater sage-grouse issues across the Colorado range. Using historic data for future decisions on species management while accounting for issues found from combining datasets allows land managers the flexibility to use all information available. © 2013 The Wildlife Society.     

Comparative antigenicity and immunogenicity of hepadnavirus core proteins

The hepatitis B virus core protein (HBcAg) is a uniquely immunogenic particulate antigen and as such has been used as a vaccine carrier platform. The use of other hepadnavirus core proteins as vaccine carriers has not been explored. To determine whether the rodent hepadnavirus core proteins derived from the woodchuck (WHcAg), ground squirrel (GScAg), and arctic squirrel (AScAg) viruses possess immunogen characteristics similar to those of HBcAg, comparative antigenicity and immunogenicity studies were performed. The results indicate that (i) the rodent core proteins are equal in immunogenicity to or more immunogenic than HBcAg at the B-cell and T-cell levels; (ii) major histocompatibility complex (MHC) genes influence the immune response to the rodent core proteins (however, nonresponder haplotypes were not identified); (iii) WHcAg can behave as a T-cell-independent antigen in athymic mice; (iv) the rodent core proteins are not significantly cross-reactive with the HBcAg at the antibody level (however, the nonparticulate "eAgs" do appear to be cross-reactive); (v) the rodent core proteins are only partially cross-reactive with HBcAg at the CD4+ T-cell level, depending on MHC haplotype; and (vi) the rodent core proteins are competent to function as vaccine carrier platforms for heterologous, B-cell epitopes. These results have implications for the selection of an optimal hepadnavirus core protein for vaccine design, especially in view of the "preexisting" immunity problem that is inherent in the use of HBcAg for human vaccine development.     

Synthesis and deposition of oocyte envelopes in the Colorado beetle,Leptinotarsa decemlineata say

Summary Electron microscopical and autoradiographic methods demonstrate that the secretion vesicles (SV), which are condensed by the Golgi-complexes of the follicle cells of the Colorado beetle, contain proteins which can be labelled with ³H-leucine. The labelled proteins are transported to the oocyte during vitellogenesis. At the end of yolk deposition, a few SV, situated just above the microvilli, disintegrate and give rise to the two layers of the vitelline membrane (VM). During the laying down of the VM or perhaps at a slightly earlier stage a layer is deposited beneath the basement membrane of the follicle cells. This layer may be important in inducing the formation of the egg membranes. Once the VM has formed, the follicle cells degenerate completely. The chorionic inner layer arises from the breakdown of SV, while the chorionic outer layer is formed from the degenerated follicle cells.Dr. A. de Loof gratefully acknowledges a mandate as Aangesteld Navorser of the National Foundation of Scientific Research in Belgium. He also thanks Prof. Dr. A. Gillard for his very helpfull criticism, Dr. W. Mordue (Cambridge) for his help in correcting the language, Prof. Dr. A. Lagasse, for supplying facilities in his laboratory of EM, Mr. W. Bohyn for operating the EM and Mr. G. Maes for photography. Special thanks to Drs. G. Vrensen (Nijmegen) for the introduction in autoradiographic techniques.     

Microheterogeneity of the mammalian high mobility group (HMG) proteins 1 and 2 investigated by reverse-phase high performance liquid chromatography

Microheterogeneity within the high mobility group (HMG)-1 and HMG-2 groups of nonhistone chromatin proteins has been investigated using reverse-phase high-performance liquid chromatography (RP-HPLC) under conditions (acetonitrile elution with 0.1% trifluoroacetic acid (TFA) as the counter ion) which separate proteins primarily on the basis of differences in their overall hydrophobicity. RP-HPLC proved to be a fast and efficient means for separating multiple subspecies of both the HMG-1 and HMG-2 proteins from both crude nuclear extracts and from ion-exchange column "purified" protein samples obtained from different types of mammalian cell nuclei. In crude nuclear extracts at least eight different HMG-2 protein species (two major and six minor), but only one major HMG-1 species, could be resolved by RP-HPLC. Three of the minor HMG-2 protein species could be isolated in "pure" form from crude extracts in one RP-HPLC step whereas under the same conditions the two major HMG-2 peaks (as well as the other minor species) were contaminated with either HMG-1 or HMG-3 (a degradation product of HMG-1). In crude extracts the major HMG-1 fraction always seems to be contaminated with one of the HMG-2 subfractions. RP-HPLC analysis of apparently "pure" protein preparations isolated by ion-exchange chromatography techniques revealed that "pure" HMG-1 can be resolved into at least three different protein species and "pure" HMG-2 into at least four different species. Amino acid analyses of different resolvable forms of the HMG proteins were not inconsistent with the suggestion that at least some of these may be primary sequence variants of the individual proteins, but other possibilities also exist.     

Life cycle consumptive water use for oil shale development and implications for water supply in the Colorado River Basin

Purpose

Oil shale is an unconventional petroleum source that can be produced domestically in the USA. Oil shale resources are primarily located in Utah, Wyoming, and Colorado, within the Colorado River Basin. In this paper, we analyze the life cycle consumptive water use for oil shale production and its impacts on water resources of the Colorado River Basin.

Methods

The study is focused on life cycle consumptive water use for oil shale development. Consumptive water use is defined as “water that is evaporated, transpired, incorporated into products, or otherwise removed from the immediate water environment.” The analysis includes direct consumptive water requirements to extract, process, and refine shale oil, as well as indirect consumptive water use for generating the electricity associated with the extraction and processing. From the results, strategies for water supply certainty are discussed, and strategies for implementation are suggested. In addition, refining the shale oil outside of the oil shale region (removing the need for local water), using dry cooling systems for electricity generation, and building desalination plants in California (to replace water) are evaluated.

Results and discussion

Life cycle consumptive water use for oil shale is significant and could impact water availability for consumers in the lower Colorado River Basin. At a level of oil production of 2 million barrels per day, the life cycle consumptive water use would be significant: between 140 and 305 billion gallons (0.4 and 0.9 million acre-ft.) of water per year if surface mining and retorting is done, or between 150 and 340 billion gallons (0.5 and 1 million acre-ft.) of water per year if the Shell in situ process is used. Strategies could be implemented to provide water supply certainty including refining the shale oil outside of the region (removing some need for local water), using dry cooling systems for electricity generation, and building desalination plants in California (to replace water).

Conclusions

Water supply in the Colorado River Basin could be a primary constraint to the development of oil shale. At a level of oil production of 2 million barrels per day, the life cycle consumptive water use would be significant. Energy companies or governments may want to invest in water management and supply strategies that would eliminate the uncertainty associated with the water availability in the Colorado River Basin for oil shale development.     

Phylogenetic analysis of Buggy Creek virus: evidence for multiple clades in the Western Great Plains, United States of America

We present the first detailed phylogenetic analysis of Buggy Creek virus (BCRV), a poorly known alphavirus with transmission cycles involving a cimicid swallow bug (Oeciacus vicarius) vector and cliff swallows (Petrochelidon pyrrhonota) and house sparrows (Passer domesticus) as the principal avian hosts. Nucleotide sequences of a 2,075-bp viral envelope glycoprotein-coding region, covering the entire PE2 gene, were determined for 33 BCRV isolates taken from swallow bugs at cliff swallow colonies in Nebraska and Colorado in the summer of 2001 and were compared with the corresponding region of BCRV isolates collected from Oklahoma in the 1980s. We also analyzed isolates of the closely related Fort Morgan virus (FMV) collected from Colorado in the 1970s. Phylogenetic analysis indicated that BCRV falls into the western equine encephalomyelitis complex of alphaviruses, in agreement with antigenic results and a previous alphavirus phylogeny based on the E1 coding region. We found four distinct BCRV/FMV clades, one each unique to Nebraska, Colorado, and Oklahoma and one containing isolates from both Nebraska and Colorado. BCRV isolates within the two clades from Nebraska showed 5.7 to 6.2% nucleotide divergence and 0.7 to 1.9% amino acid divergence, and within these clades, we found multiple subclades. Nebraska subclades tended to be confined to one or a few cliff swallow colonies that were close to each other in space, although in some cases, near-identical isolates were detected at sites up to 123 km apart. Viral gene flow occurs when cliff swallows move (bugs) between colony sites, and the genetic structure of BCRV may reflect the limited dispersal abilities of its insect vector.     

Evaluation of Circulating Proteins and Hemodynamics Towards Predicting Mortality in Children with Pulmonary Arterial Hypertension

Background

Although many predictors have been evaluated, a set of strong independent prognostic mortality indicators has not been established in children with pediatric pulmonary arterial hypertension (PAH). The aim of this study was to identify a combination of clinical and molecular predictors of survival in PAH.

Methods

This single-center, retrospective cohort study was performed from children with PAH between 2001 and 2008 at Children''s Hospital Colorado. Blood samples from 83 patients (median age of 8.3 years-old) were obtained. We retrospectively analyzed 46 variables, which included 27 circulating proteins, 7 demographic variables and 12 hemodynamic and echocardiographic variables for establishing the best predictors of mortality. A data mining approach was utilized to evaluate predictor variables and to uncover complex data structures while performing variable selection in high dimensional problems.

Results

Thirteen children (16%) died during follow-up (median; 3.1 years) and survival rates from time of sample collection at 1 year, 3 years and 5 years were 95%, 85% and 79%, respectively. A subset of potentially informative predictors were identified, the top four are listed here in order of importance: Tissue inhibitors of metalloproteinases-1 (TIMP-1), apolipoprotein-AI, RV/LV diastolic dimension ratio and age at diagnosis. In univariate analysis, TIMP-1 and apolipoprotein-AI had significant association with survival time (hazard ratio [95% confidence interval]: 1.25 [1.03, 1.51] and 0.70 [0.54–0.90], respectively). Patients grouped by TIMP-1 and apolipoprotein-AI values had significantly different survival risks (p<0.01).

Conclusion

Important predictors of mortality were identified from a large number of circulating proteins and clinical markers in this cohort. If confirmed in other populations, measurement of a subset of these predictors could aid in management of pediatric PAH by identifying patients at risk for death. These findings also further support a role for the clinical utility of measuring circulating proteins.     

Serum antigen 85 levels in adjunct testing for active mycobacterial infections in orangutans

Diagnosis of active mycobacterial disease in orangutans (Pongo pygmaeus) has been impeded by high levels of non-specific intradermal skin test reactivity to mycobacterial antigens. This may be due in part to cross reactivity between antigens, tuberculin concentrations used or other species-specific factors. Antigen 85 (Ag85) complex proteins are major secretory products of actively growing mycobacteria, and measurement of serum Ag85 could provide a method for determining active mycobacterial infections that was not dependent on host immunity. Serum Ag85 was measured by dot-immunobinding assay using monoclonal anti-Ag85, purified Ag85 standard and enhanced chemiluminescence technology in coded serum samples from 14 captive orangutans from a zoo in Colorado, 15 semi-captive orangutans in Malaysia, and 19 free-ranging wild orangutans in Malaysia. Orangutans from Colorado (USA) were culture negative for Mycobacterium tuberculosis and M. avium, although all had laboratory suspicion or evidence of mycobacterial infection; median serum Ag85 was 10 microU/ml (range, <0.25-630 microU/ml). Of the semi-captive orangutans, six were skin test reactive and two were culture positive for M. avium on necropsy. Median serum Ag85 for this group was 1,880 microU/ml (0.75-7,000 microU/ml), significantly higher than that of Colorado zoo or free-ranging Malaysian orangutans. Median serum Ag85 in the latter group was 125 microU/ml (range, 0.75-2,500 microU/ml). These data suggest that suggest that additional studies using more specific reagents and more samples from animals of known status are appropriate.     

Of Travertine and Time: Otolith Chemistry and Microstructure Detect Provenance and Demography of Endangered Humpback Chub in Grand Canyon,USA

We developed a geochemical atlas of the Colorado River in Grand Canyon and in its tributary, the Little Colorado River, and used it to identify provenance and habitat use by Federally Endangered humpback chub, Gila cypha.  Carbon stable isotope ratios (δ¹³C) discriminate best between the two rivers, but fine scale analysis in otoliths requires rare, expensive instrumentation. We therefore correlated other tracers (SrSr, Ba, and Se in ratio to Ca) to δ¹³C that are easier to quantify in otoliths with other microchemical techniques. Although the Little Colorado River’s water chemistry varies with major storm events, at base flow or near base flow (conditions occurring 84% of the time in our study) its chemistry differs sufficiently from the mainstem to discriminate one from the other. Additionally, when fish egress from the natal Little Colorado River to the mainstem, they encounter cold water which causes the otolith daily growth increments to decrease in size markedly. Combining otolith growth increment analysis and microchemistry permitted estimation of size and age at first egress; size at first birthday was also estimated. Emigrants < 1 year old averaged 51.2 ± 4.4 (SE) days and 35.5 ± 3.6 mm at egress; older fish that had recruited to the population averaged 100 ± 7.8 days old and 51.0 ± 2.2 mm at egress, suggesting that larger, older emigrants recruit better. Back-calculated size at age 1 was unimodal and large (78.2 ± 3.3 mm) in Little Colorado caught fish but was bimodally distributed in Colorado mainstem caught fish (49.9 ± 3.6 and 79 ± 4.9 mm) suggesting that humpback chub can also rear in the mainstem. The study demonstrates the coupled usage of the two rivers by this fish and highlights the need to consider both rivers when making management decisions for humpback chub recovery.     

Wall-less prokaryotes from fall flowers in central United States and Maryland

Four spiroplasma strains and eleven isolates tentatively identified as acholeplasmas were obtained from fall flowers in Colorado, Nebraska, Illinois, and Maryland. Although the acholeplasma isolates were heterogeneous, all showed antigenic sharing with a group of unnamed organisms (L1 and related strains) isolated in othe studies from flowers in Florida. The W20 and W24 isolates from Nebraska were partially related to the L1 group by DNA-DNA homology and polyacrylamide gel electrophoresis (PAGE) analyses. A Colorado spiroplasma (W13) was identifed as a new strain of group IV complex. Three spiroplasma strains from flowers in Maryland old fields represent a new serovar with closest affinity to subgroup I-4 and to the LB12 and N525 serovars of group I. Widespread occurrence of acholeplasmas on flowers in this study, and on plant surfaces in general, suggests that, like spiroplasmas they probably will be found to reside in arthropods.     

NEW ALGAL SPECIES RECORD FOR COLORADO1

Colonies of the alga Nostoc parmeloides Kützing 1843, containing midge larvae, were collected from Bellows Creek in the southwestern mountains of Colorado near Creede, Mineral County, on February 27, 1970. G. W. Prescott verified the algal identification and confirmed it as a new species record for Colorado. The Nostoc-midge combination has been definitely identified in 8 other Colorado mountain streams. The streams are located in various sections of Colorado above 2333 m; distribution may be statewide.     

Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites

Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.     

Flight take-off performance of Colorado potato beetle in relation to potato phenology

The flight take-off frequency of adult Colorado potato beetles, Leptinotarsa decemlineata (Say), from potato plants, Solanum tuberosum L. 'Red Pontiac' at the bloom stage of development was 2.2-2.5-fold that of Colorado potato beetle from plants at the vegetative stage. Tests were conducted in a flight chamber over a period of 3 h. Prefeeding Colorado potato beetles for 48 h on potato plants at the bloom or at the vegetative stage before placing them into the flight chamber resulted in the same significantly higher flight take-off frequency from potato plants at the bloom stage than from plants at the vegetative stage. These results demonstrate that the factor in potato plants in bloom that stimulates the flight take-off of the Colorado potato beetle is independent of the feeding history of the beetles and begins acting only when the beetles are in the presence of the plant. According to these results, the dispersal of adult Colorado potato beetles from potato fields in bloom to younger potato fields with plants at the vegetative stage, previously reported in the literature, is at least partly explained by the effect of plant phenology on the frequency of flight take-off. Results confirm the value of planting potato fields of similar phenology over as wide an area as possible to reduce Colorado potato beetle dispersal between fields. Results also imply that staggering the planting dates of conventional potato refuge areas near Colorado potato beetle transgenic or conventionally resistant potato fields is a sound management practice, because it promotes the movement of wild beetles over to the adjacent younger resistant crops.