Chemotype variation of the weed Melaleuca quinquenervia influences the biomass and fecundity of the biological control agent Oxyops vitiosa

Host plant nutritional and non-nutritional variability can have a significant effect on herbivore populations by influencing survival, larval performance, and fecundity. The effect of chemical and physical variation of the leaves of two chemotypes of the weed Melaleuca quinquenervia was determined on the biomass and fecundity of the biological control agent Oxyops vitiosa (Coleoptera: Curculionidae). M. quinquenervia chemotypes were distinguished by the principal terpenoids E-nerolidol and viridiflorol using gas chromatography and mass spectroscopy. Not only were the terpenoid profiles of the two chemotypes different but the viridiflorol leaves had greater toughness (1.2-fold) and reduced nitrogen (0.7-fold). When the larvae and adults were fed leaves of the E-nerolidol chemotype increased adult biomass (1.1-fold) and fecundity were found (2.6- to 4.5-fold) compared with those fed leaves of the viridiflorol chemotype. Regardless of the larval diet, when adults were fed the E-nerolidol chemotype leaves they had greater egg production compared with those adults fed the viridiflorol leaves. Moreover, adult pre-oviposition period was extended (1.5-fold) when individuals were fed the viridiflorol leaves compared with those fed the E-nerolidol leaves. By rearing the O. vitiosa weevil on the more nutritious chemotype plants these results assisted in the mass production and establishment of the M. quinquenervia biological control agent.     

Field colonization,population growth,and dispersal of Boreioglycaspis melaleucae Moore,a biological control agent of the invasive tree Melaleuca quinquenervia (Cav.) Blake

Invasion of native plant communities by the Australian paperbark tree (“melaleuca”), Melaleuca quinquenervia, complicates restoration of the Florida Everglades. Biological control, within the context of a comprehensive management program, offers a means to suppress regeneration of melaleuca after removal of existing trees and a mechanism to forestall reinvasion. To meet this need, a biological control program commenced in 1997 upon the release of an Australian weevil (Oxyops vitiosa [Pascoe] [Coleoptera: Curculionidae]). Release of a second biological control agent, the melaleuca psyllid (Boreioglycaspis melaleucae Moore), followed in February 2002 at field sites containing mixed age-class melaleuca stands or coppicing stumps. Each site was inoculated with 7000–10,000 adult psyllids, with one exception where 2000 nymphs were released on seedlings the following December. Psyllid populations established everywhere irrespective of colony source, site conditions, or the quantity released, although numbers released and, to a lesser degree, colony age influenced the numbers of colonies produced. Quantity included in the release was the major determinant of the resultant number of colonies, although the duration of their tenure in quarantine culture may have also influenced this. One site, comprised mainly of coppicing stumps, contained 3.3 million psyllids per ha within 3 months after release. Less than 1% of the coppices at a similar site harbored psyllid colonies 2 months after release (May 2002), but this rose to 75% in October then to 100% by December. The census population exceeded 715,000 adults and nearly 11 million nymphs by late January 2003. Psyllid populations dispersed 2.2–10.0 km/year, with the slower rates in dense, continuous melaleuca stands and faster rates in fragmented stands. Over 1 million psyllids had been redistributed to 100 locations as of December 2005. This species now occurs throughout much of the range of melaleuca in south Florida due to natural range expansion as well as anthropogenic dissemination.     

Post-biological control invasion trajectory for Melaleuca quinquenervia in a seasonally inundated wetland

The recruitment and mortality of Melaleuca quinquenervia seedlings were evaluated over a 3-year period in a seasonally inundated wetland in the western Everglades region. The mean (±SE) density of seedlings/saplings m^?1 declined from 64.8 (±4.5) to 0.5 (±0.2) over the 3 years, a population reduction of 99.2%. Four distinct water regimes characterized this site: dry, dry to wet transition, flooded, and wet to dry transition. Seedling recruitment was highest in the dry to wet transition and lowest in the flooded water regime, while mortality was highest under flooded and dry water regimes. The mean estimate of population growth (λ) across water regimes was 0.64 ± 0.05 indicating negative population growth. Elimination of introduced insect herbivores using insecticides did not reduce mortality of recruited M. quinquenervia seedlings/saplings indicating that direct herbivory was not responsible for the decline in seedling density. On the other hand, a mean of only 0.2 (±0.03) viable seeds m^?2 d^?1 fell into the plots, an amount considerably lower than in previous studies. We submit that change in the invasion trajectory M. quinquenervia was most likely caused by reduced seed inputs from aerial seed banks depleted by insect herbivory rather than direct herbivory on seedlings. This may indicate a fundamental alteration of M. quinquenervia population dynamics ultimately resulting in a less invasive and, therefore, less ecologically damaging species.     

The attractiveness of odorous esterified fatty acids to the potential biocontrol agent,Altica cyanea

The fatty acid composition of foliar buds, young, mature, and senescent leaves, and stem parts of the rice-field weed, Ludwigia adscendens L. (Onagraceae) was analyzed by thin layer chromatography and gas chromatography flame ionization detection. The analysis of fatty acid composition revealed that saturated fatty acids (i.e., C14:0, C16:0, and C18:0) were prevailing compounds among the all weed parts except senescent leaves where C18:1 was predominant. The esterified fatty acids isolated from different weed parts over the range of 10–100 μg/ml followed by individual synthetic esterified fatty acids that were identified from the esterified extracts of different weed parts, and a mixture of synthetic esterified fatty acids except esterified eicosenoic acid and docosahexaenoic acid were applied to identify their role as a chemical cue for a potential biocontrol agent, Altica cyanea (Weber) (Coleoptera: Chrysomelidae) in a Y-tube olfactometer under laboratory conditions. In this bioassay, the esterified fatty acids from mature leaves and stem parts of this weed attracted A. cyanea at 20–100 μg/ml and at 80 μg/ml concentrations, respectively. Clear attraction was recorded by female A. cyanea insects in the mixture of synthetic esterified fatty acids at 60, 80, and 100 μg/ml concentrations. It is thus concluded that A. cyanea rely on an effective proportion of esterified fatty acids as an olfactory cue for attraction.     

Regional-scale impact of the weed biocontrol agent Mecinus janthinus on Dalmatian toadflax (Linaria dalmatica)

Dalmatian toadflax (Linaria dalmatica (L.) Mill.) is an important invasive plant on rangelands throughout western North America. In 1991, the stem-mining weevil, Mecinus janthinus Germar, was introduced into Canada from Europe as a classical biological control agent to reduce toadflax densities and improve rangelands, particularly in British Columbia. To determine if the program was a success at a regional level, this paper answers three key questions: (1) has M. janthinus spread throughout the study area, (2) is M. janthinus causing a decline in toadflax plant size or density at the regional scale, and (3) has the distribution of toadflax plants changed following M. janthinus introduction? These questions are answered by combining historical survey data and mensurative experimental data on plant and weevil densities across a 40,000 km² area in southern British Columbia. The results show that through a combination of intentional redistribution and natural dispersal weevils have spread throughout the study area. Stem densities at naturally colonized sites and historical release sites were equally low. Across weevil populations between 3 and 10 years old, weevil densities peaked in the eighth year, and there was a negative relationship between weevil density and stem length. Between 2000 and 2007, toadflax patches were found to both decrease in density and become more fragmented over time, with 15% of patches disappearing completely. These findings show that M. janthinus has had a significant negative impact on both the density and distribution of Dalmatian toadflax throughout a large part of its range in British Columbia.     

Antiparasitic lethality of sulfonamidebenzamides in kinetoplastids

A sulfonamidebenzamide series was assessed for anti-kinetoplastid parasite activity based on structural similarity to the antiparasitic drug, nifurtimox. Through structure-activity optimization, derivatives with limited mammalian cell toxicity and increased potency toward African trypanosomes and Leishmania promastigotes were developed. Compound 22 had the best potency against the trypanosome (EC₅₀ = 0.010 μM) while several compounds showed ～10-fold less potency against Leishmania promastigotes without impacting mammalian cells (EC₅₀ > 25 μM). While the chemotype originated from an unrelated optimization program aimed at selectively activating an apoptotic pathway in mammalian cancer cells, our preliminary results suggest that a distinct mechanism of action from that observed in mammalian cells is responsible for the promising activity observed in parasites.     

The importance of relative humidity and trophic resources in governing ecological niche of the invasive carabid beetle Merizodus soledadinus in the Kerguelen archipelago

Comprehensive studies to identify species-specific drivers of survival to environmental stress, reproduction, growth, and recruitment are vital to gaining a better understanding of the main ecological factors shaping species habitat distribution and dispersal routes. The present study performed a field-based assessment of habitat distribution in the invasive carabid beetle Merizodus soledadinus for the Kerguelen archipelago. The results emphasised humid habitats as a key element of the insect’s realised niche. In addition, insects faced food and water stress during dispersal events. We evaluated quantitatively how water availability and trophic resources governed the spatial distribution of this invasive predatory insect at Îles Kerguelen. Food and water stress survival durations [in 100%, 70%, and 30% relative humidity (RH) conditions] and changes in a set of primary metabolic compounds (metabolomics) were determined. Adult M. soledadinus supplied with water ad libitum were highly tolerant to prolonged starvation (LT₅₀ = 51.7 ± 6.2 d). However, food-deprived insect survival decreased rapidly in moderate (70% RH, LT₅₀ = 30.37 ± 1.39 h) and low (30% RH, LT₅₀ = 13.03 ± 0.48 h) RH conditions. Consistently, body water content decreased rapidly in insects exposed to 70% and 30% RH. Metabolic variation evidenced the effects of food deprivation in control insects (exposed to 100% RH), which exhibited a progressive decline of most glycolytic sugars and tricarboxylic acid cycle intermediates. Most metabolite levels were elevated levels during the first few hours of exposure to 30% and 70% RH. Augmented alanine and lactate levels suggested a shift to anaerobic metabolism. Simultaneously, peaks in threonine and glycolytic sugars pointed to metabolic disruption and a progressive physiological breakdown in dehydrating individuals. Overall, the results of our study indicate that the geographic distribution of M. soledadinus populations is highly dependent on habitat RH and water accessibility.     

The interaction of two potential fungal bioherbicides and a sub-lethal rate of glyphosate for the control of shattercane

Greenhouse and laboratory experiments were conducted with the potential bioherbicides Colletotrichum graminicola (Cg) and Gloeocercospora sorghi (Gs) for control of shattercane weed. Single-spray tank mixture applications containing different ratios of the two fungi resulted in additive percent weed biomass losses. Intraspecific (Cg + Cg or Gs + Gs) and interspecific (Cg + Gs or Gs + Cg) sequential applications 1- or 7-days apart indicated antagonistic interactions in percent biomass loss. Application of either fungus with, or 1–3 days prior to, a sub-lethal concentration of glyphosate resulted in an antagonistic percent biomass loss; while application of glyphosate prior to either potential bioherbicide resulted in a synergistic weed disease response. Conidia germination studies conducted both in vitro on agar plates and with leaf impression peels suggest that antagonistic interactions observed in weed disease severity are probably due to the host–pathogen response following infection.     

<Emphasis Type="Italic">Melaleuca quinquenervia</Emphasis> dominated forests in Florida: analyses of natural-enemy impacts on stand dynamics

Melaleuca quinquenervia (melaleuca) is a native of Australia but has become an invasive plant in Florida, USA. We conducted a long-term demographic study of melaleuca in three sections (central, transitional, and peripheral) of monoculture stands located in Florida, and quantified absolute density, diameter at breast height and basal area of trees by section at three sites. Additionally, we monitored the impacts of natural enemy (insects and fungi) on melaleuca populations which became apparent after 2001. Both absolute density and basal area, from before (1997–2001) and after noticeable natural-enemy impact (2002–2005), were compared. Prior to the natural-enemy impact, absolute density of melaleuca trees declined primarily due to self-thinning and associated losses of small trees, but diameter at breast height increased, as did the basal area. Later during the period when natural enemies prevailed, absolute density declined at a significantly greater rate across all sections but was highest at the periphery. The decrease in mean absolute density and basal area/ha of melaleuca during the natural-enemy impacted period coincided with the increased incidence of the populations of plant-feeding insects and fungi. The mean diameter at breast height continued to increase in all sections of the stands throughout the study period. An increasing trend in basal area prior to natural-enemy impact was reversed after increase in natural-enemy abundance and noticeable impact in all three sections of the stands. These findings lend support to a growing body of literature that implicates natural enemies as increasingly important density-independent regulators of M. quinquenervia populations. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged     

Modifying orchard planting sites conserves entomopathogenic nematodes,reduces weevil herbivory and increases citrus tree growth,survival and fruit yield

In Florida, a root weevil pest of citrus, Diaprepes abbreviatus, is more damaging and attains higher population density in some orchards on fine textured, poorly drained “flatwoods” soils than in those on the deep, coarse sandy soils of the central ridge. Previous research revealed that sentinel weevil larvae were killed by indigenous entomopathogenic nematodes (EPNs) at significantly higher rates in an orchard on the central ridge, compared to one in the flatwoods. We hypothesized that filling tree planting holes in a flatwoods orchard with sandy soil from the central ridge would provide a more suitable habitat for EPNs, thereby reducing weevil numbers and root herbivory. Fifty trees were planted in oversized planting holes filled with coarse sand and 50 trees were planted in native soil in a split plot design where whole plots were species of introduced EPNs and split plots were soil type. Each of Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, or no EPNs were introduced into the rhizospheres in 10 plots of each soil type. During four years, EPN numbers in soil samples and the relative abundance of seven species of nematophagous fungi associated with nematodes were measured three times using real-time PCR. The efficacy of EPNs against sentinel weevil larvae was also measured three times by burying caged weevils in situ. EPN species richness (P = 0.001) and diversity (P = 0.01) were always higher in sand than native soil. Soil type had no effect on numbers of EPNs in samples, but EPNs were detected more frequently (P = 0.01) in plots of sandy soil than native soil in 2011. Two nematophagous fungi species, Paecilomyces lilacinus and Catenaria sp. were significantly more abundant in nematode samples from sandy soil on all three sampling dates. Efficacy of EPNs against weevil larvae was greater in sandy soil inoculated with S. diaprepesi (P = 0.03) in June 2010 and in all treatments in sandy soil in May 2011 (P = 0.03). Sixty-eight percent more adult weevils (P = 0.01) were trapped emerging from native soil during two years than from sandy soil. By May 2011, the cumulative number of weevils emerging from each plot was inversely related (P = 0.01) to the numbers of EPNs detected in plots and to EPN efficacy against sentinels. Three trees in sandy soil died as a result of root herbivory compared to 21 trees in native soil. Surviving trees in sandy soil had trunk diameters that were 60% larger (P = 0.001) and produced 85% more fruit (P = 0.001) than those in native soil. Although it is not possible to characterize all of the mechanisms by which the two soil treatments affected weevils and trees, substitution of sand for native soil was an effective means of conserving EPNs and shows promise as a cultural practice to manage D. abbreviatus in flatwoods citrus orchards with a history of weevil damage to trees.     

Associations of soil iron with citrus tree decline and variability of sand,soil water,pH, magnesium and Diaprepes abbreviatus root weevil: Two-site study

The hypothesis of associations of environmental soil heterogeneity with citrus tree decline and Diaprepes abbreviatus (L.) root weevil variability was tested in two flatwoods fields of ‘Hamlin’ orange trees (Citrus sinensis (L.) Osb.). Studies were conducted on a loamy, poorly drained Mollisol in Osceola County, central Florida in 2002, and on a sandy, poorly drained Spodosol in DeSoto County, south-west Florida during 2001–2003. Adult weevils were monitored using 50 Tedders traps arranged in a 34 m × 25 m grid at the Osceola site, and using 100 identical traps in a 30 m × 15 m grid at the DeSoto site. Soil water content (SWC), texture, pH, Ca, Mg, Fe, Cu and other nutrients were measured at each trap. Soil was strongly acidic (pH 4.9 ± 0.4) at the Osceola site but near neutral (pH 6.6 ± 0.4) at the DeSoto site. The Mehlich-I extractable soil Mg and Ca were correlated to soil pH and SWC in both soils, and extractable Fe was related to pH, SWC and Mg in the Spodosol (0.30 < R² < 0.65, P < 0.01). The weevil density was high in areas low in soil Mg and Ca in the acidic Mollisol, but high in areas with high soil pH, and Mg and low sand content in the near neutral Spodosol (P < 0.05). Tree decline was associated with soil Fe concentrations >40 mg kg⁻¹ in the Mollisol (P < 0.01). Weevil density was low at a soil pH between 5.7 and 6.2. The range of spatial dependence of weevil population, soil pH, SWC, Fe, Mg and sand varied between 60 and 100 m in the Mollisol and the Spodosol. Soil-weevil-tree simple and multivariate linear models were established to put into practices for predicting and controlling the weevil population and tree decline in the future. Differences in site characteristics suggested the need for site-specific weevil and citrus tree management.     

Effect of Norwegian entomopathogenic fungal isolates against Otiorhynchus sulcatus larvae at low temperatures and persistence in strawberry rhizospheres

Biological control of belowground stages of the black vine weevil Otiorhynchus sulcatus F. (Coleoptera: Curculionidae) in strawberries in cool temperate regions using entomopathogens is challenged by low temperatures during the periods when larvae are vulnerable to infections. In a laboratory study we tested six indigenous Norwegian isolates of entomopathogenic fungi (one Beauveria bassiana, three Beauveria pseudobassiana, and two Metarhizium brunneum; Ascomycota: Hypocreales) for their efficacy against O. sulcatus larvae at 6, 12, and 18 °C. At the lowest temperature only Beauveria spp. affected survival of O. sulcatus while all three fungal species reduced larval survival compared to the control treatment at 12 and 18 °C. Two of the Norwegian isolates, one B. pseudobassiana and one M. brunneum, were then evaluated for long-term persistence (>1 year) in the bulk soil and the rhizosphere soil of strawberries in a semi-field experiment. An exotic isolate of M. brunneum sharing origin with a widespread commercial biocontrol agent (F52/Met52 (Novozymes)) was included for comparison. All three isolates showed significantly higher abundances in the rhizosphere soil compared to bulk soil at 153, 366, and 471 days after inoculation, thus indicating rhizosphere competence for B. pseudobassiana. Notably, CFU levels for both Norwegian isolates were much higher than for the exotic M. brunneum isolate. Selection of locally adapted isolates may therefore be of importance when considering biocontrol strategies of belowground pests in strawberry production.     

Monitored releases of Rhinoncomimus latipes (Coleoptera: Curculionidae), a biological control agent of mile-a-minute weed (Persicaria perfoliata), 2004–2008

Mile-a-minute weed, Persicaria perfoliata (L.) H. Gross, is an invasive annual vine of Asian origin that has developed extensive monocultures, especially in disturbed open areas in the Mid-Atlantic region of the United States. A host-specific Asian weevil, Rhinoncomimus latipes Korotyaev, was approved for release in North America in 2004, and weevils have been reared at the New Jersey Department of Agriculture Beneficial Insect Laboratory since then. By the end of 2007 more than 53,000 weevils had been reared and released, mostly in New Jersey, but also in Delaware, Maryland, Pennsylvania, and West Virginia. The beetles established at 63 out of 65 sites (96.9%) where they were released between 2004 and 2007, with successful releases consisting of as few as 200 weevils. Weevils were recorded at 30 additional non-release sites in New Jersey, where they had dispersed at an average rate of 4.3 km/year. Standardized monitoring of fixed quadrats was conducted in paired release and control sites at eight locations. Significant differences in mile-a-minute weed populations in the presence and absence of weevils were found at three locations, with reduction in spring densities to 25% or less of what they had been at the start within 2–3 years at release sites, while weed densities at control sites were largely unchanged. Mile-a-minute weed populations at a fourth site were similarly reduced at the release site, but without control data for comparison due to rapid colonization of the paired control site. At the other four locations, all on islands, mile-a-minute weed populations were reduced at both release and control sites without large weevil populations developing, apparently due to environmental conditions such as late frost and extreme drought.     

Identification of novel antitubercular compounds through hybrid virtual screening approach

Growing resistance of prevalent antitubercular (antiTB) agents in clinical isolates of Mycobacterium tuberculosis (MTB) provoked an urgent need to discover novel antiTB agents. Enoyl acyl carrier protein (ACP) reductase (InhA) from Mtb is a well known and thoroughly studied as antitubucular therapy target. Here we have reported the discovery of potent antiTB agents through ligand and structure based approaches using computational tools. Initially compounds with more than 0.500 Tanimoto similarity coefficient index using functional class fingerprints (FCFP_4) to the reference chemotype were mined from the chemdiv database. Further, the molecular docking was performed to select the compounds on the basis of their binding energies, binding modes, and tendencies to form reasonable interactions with InhA (PDB ID = 2NSD) protein. Eighty compounds were evaluated for antitubercular activity against H37RV M. tuberculosis strain, out of which one compound showed MIC of 5.70 μM and another showed MIC of 13.85 μM. We believe that these two new scaffolds might be the good starting point from hit to lead optimization for new antitubercular agents.     

Dieckol,a SARS-CoV 3CLpro inhibitor,isolated from the edible brown algae Ecklonia cava

SARS-CoV 3CL^pro plays an important role in viral replication. In this study, we performed a biological evaluation on nine phlorotannins isolated from the edible brown algae Ecklonia cava. The nine isolated phlorotannins (1–9), except phloroglucinol (1), possessed SARS-CoV 3CL^pro inhibitory activities in a dose-dependently and competitive manner. Of these phlorotannins (1–9), two eckol groups with a diphenyl ether linked dieckol (8) showed the most potent SARS-CoV 3CL^pro trans/cis-cleavage inhibitory effects (IC₅₀s = 2.7 and 68.1 μM, respectively). This is the first report of a (8) phlorotannin chemotype significantly blocking the cleavage of SARS-CoV 3CL^pro in a cell-based assay with no toxicity. Furthermore, dieckol (8) exhibited a high association rate in the SPR sensorgram and formed extremely strong hydrogen bonds to the catalytic dyad (Cys145 and His41) of the SARS-CoV 3CL^pro.     

Field host-specificity of the mile-a-minute weevil,Rhinoncomimus latipes Korotyaev (Coleoptera: Curculionidae)

The safe practice of biological control relies, in part, on an accurate evaluation of a potential agent’s host-specificity via testing through a “filter of safety”. The results of laboratory tests may differ from those obtained in open field host-specificity tests, where agents are able to use their full range of host-selection behaviors. It was hypothesized that Rhinoncomimus latipes (Coleoptera: Curculionidae), the biological control agent released against mile-a-minute weed, Persicaria perfoliata (Polygonaceae), would not feed or oviposit on nontarget plants in a two-phase, open field setting. Ten weevils were placed at the base of each of 13 test plant species in a randomized complete block design with six replicates. Weevils placed at the base of mile-a-minute weed were marked with yellow fluorescent dust, and yellow weevils were subsequently found only on mile-a-minute. Weevils placed at the base of nontarget plants (marked with red fluorescent dust) rapidly colonized mile-a-minute weed. Three hours after release, the number of R. latipes found on mile-a-minute weed was significantly higher than predicted by a random distribution of weevils on all test plants. The likelihood of finding more weevils on mile-a-minute compared to nontarget plant species was 31.0% at 3 h and increased to 96.5% at 44 h after release. Whereas prerelease studies showed feeding at low levels on 9 of the 13 plant species tested here, under open field conditions R. latipes did not feed on any nontarget plant species and dispersed from these plants. In an open field setting, where the weevil was able to use its full range of host-selection behaviors, there was no observed risk of nontarget effects for any species tested.     

Arsenic reduced scale-insect infestation on arsenic hyperaccumulator Pteris vittata L.

Arsenic hyperaccumulation by Pteris vittata L. (Chinese brake fern) may serve as a defense mechanism against herbivore attack. This study examined the effects of arsenic exposure (0, 5, 15 and 30 mg kg^?1) on scale insect (Saissetia neglecta) infestation of P. vittata. Scale insects were counted as a percentage fallen from the plant to the total number of insects after 1 week of As-treatment. The arsenic concentrations in the fronds ranged from 5.40 to 812 mg kg^?1. Greater arsenic concentrations resulted in higher percentage of fallen-scale insects (17.2–55.0%). Lower arsenic concentrations (≤5 mg kg^?1) showed significantly lower effect on the population compared to 15–30 mg kg^?1 (p < 0.05). Arsenic content in the fallen-scale insects was as high as 194 mg kg^?1, which indicated that arsenic has been ingested by the scale insects via plant sap. This study is consistent with the hypothesis that arsenic may help P. vittata defend against herbivore's attack.     

A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria)

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow K_cat (low V_max) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g⁻¹ dry cell weight (dcw) versus the 0.03 mg g⁻¹ dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms.     

The Hohenheimer Box – A new way to rear and release Lariophagus distinguendus to control stored product pest insects

To improve the biological control of stored product pests, the present paper reports on the development of a rearing box for parasitoids of pest insects. The box contains breeding substrate and populations of hosts and parasitoids and is placed in storage sites, where parasitoids are released continuously over several months. The box was developed to rear Lariophagus distinguendus (Förster) (Hymenoptera: Pteromalidae) to control the granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae). Due to sanitary reasons, the bean weevil Acanthoscelides obtectus Say (Coleoptera: Bruchidae) was chosen as an alternative host. Rearing experiments revealed that the cowpea Vigna unguiculata unguiculata (L.) Walp. is most suitable as host substrate. For the outlet of the rearing device, a wire gauze mesh size of 0.8–1.0 mm was found suitable to release wasps while holding back the bean weevils. The size of the starting populations of hosts and parasitoids was determined experimentally in a storage building. An amount of 5 ml weevils plus 21–60 adult parasitoids on 2 kg of cowpeas produced an average of 56 and 62 wasps per week respectively, from June to September. Wasps reared in the boxes had the same number of offspring on granary weevils as wasps from regular lab-cultures. This study demonstrates the feasibility of a rearing box for parasitoids of stored product pests that releases large numbers of wasps over several months. We consider our study as a guideline for the development of similar rearing boxes also for other parasitoid-pest systems in stored products protection throughout the world.     

Synthesis,anti-HIV activity,integrase enzyme inhibition and molecular modeling of catechol,hydroquinone and quinol labdane analogs

Labdane analogs with o-quinol, catechol and hydroquinone moiety have been synthesized using Diels–Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate, 3,4-dioxocyclohexa-1,5-diene-carboxylic acid and 3,6-dioxocyclohexa-1,4-dienecarboxylic acid with mono terpene 1,3-dienes, namely ocimene and myrcene. The resulting molecules and their derivatives were evaluated for their anti-HIV-1 activity using TZM-bl cell based virus infectivity assay. Two molecules 13 and 18 showed anti-HIV activity with IC₅₀ values 5.0 (TI = 11) and 4.6 (TI = 46) μM, respectively. The compounds 17, 18 and 20 showed efficacy against HIV-1 integrase activity and showed inhibition with IC₅₀ 13.4, 11.1 and 11.5 μM, respectively. The HIV-1 integrase inhibition activity of these synthetic molecules was comparable with integric acid, the natural fungal metabolite. Molecular modeling studies for the HIV-1 integrase inhibition of these active synthetic molecules indicated the binding to the active site residues of the enzyme.