Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.     

Susceptibility and physiological responses of Jatropha curcas accessions to broad mite infestation

The broad mite Polyphagotarsonemus latus is a key pest of physic nut (Jatropha curcas L.). The purpose of this study was to identify physic nut accessions that are less susceptible to P. latus, in support of the breeding program of J. curcas. We first evaluated population growth rate and injury symptoms of P. latus on different J. curcas accessions and then carried out physiological analyses on P. latus-infested and uninfested accessions. From the germplasm bank of the Federal University of Viçosa, 15 physic nut accessions with high seed oil content, with different genetic background, were tested. The following traits were evaluated: instantaneous population growth rate of P. latus (r _i ), injury symptoms, relative leaf water content, specific leaf area, gas exchange, photosynthetic pigments, nitrogen and biomass of the aerial part. Significant differences were observed for P. latus population growth rate and injury symptoms among accessions. A positive correlation between P. latus growth rate and injury was found. The UFVJC72 accession stood out as the more resistant, considering P. latus growth rate and injury symptoms, compared with most accessions. Physiological responses did not vary among accessions, but did between infested and uninfested plants. In P. latus-infested plants, net photosynthesis was on average 50.5 % lower than in uninfested plants, whereas stomatal conductance and transpiration decreased by 46.2 and 51.6 %, respectively.     

Genetic characterization of Dibothriocephalus latus and Dibothriocephalus dendriticus (Cestoda: Diphyllobothriidae) from Chile based on haplotype analysis using mitochondrial DNA markers

Dibothriocephalus latus and Dibothriocephalus dendriticus are found throughout the temperate and sub-arctic zones of the northern hemisphere, but they are also found in the southern core countries of South America, Chile and Argentina. Genetic characteristics of D. latus and D. dendriticus from South America have yet to be fully defined. The present study aimed to understand the genetic characteristics of D. latus and D. dendriticus from Chile by haplotype network analysis of mitochondrial cytochrome c oxidase subunit I gene (cox1) and cytochrome b gene (cob), as well as their origins. Dibothriocephalus latus and D. dendriticus plerocercoid larvae were obtained from feral and/or wild salmonids captured in Lake Llanquihue in Región de Los Lagos, and Lake Panguipulli in Región de Los Ríos, located south of central Chile. Haplotype analysis of D. latus revealed that H1 in cox1 and H2 in cob are the key haplotypes common to D. latus across the world, including Chile, and both genes exhibited limited genetic diversity in D. latus. It was assumed that D. latus was brought into South America by European and Russian immigrants in the 19th century as previously reported. In contrast, both the cox1 and cob of D. dendriticus display considerable genetic diversity, with no common haplotypes between D. dendriticus populations from Chile and the northern hemisphere. More intriguingly, two cob haplotypes (H24, H25) detected in Chilean D. dendriticus were closely linked to haplotypes (H30, H31) detected in North American D. dendriticus, strongly implying that D. dendriticus in Chile was brought by piscivorous migrating birds from North America. It has also been estimated that the D. dendriticus from Chile genetically diverged from the D. dendriticus from the northern hemisphere approximately 1.11 million years ago, long before humans migrated to the southern parts of South America.     

Characterization of pyranose oxidase variants for bioelectrocatalytic applications

Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H₂O₂. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (I_max^app/K_M^app) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.     

Potential of the predatory mite Amblydromalus zannoui to control pest mites on Jatropha curcas

Amblydromalus zannoui Sourassou, Sarmento and Moraes is a phytoseiid mite of the limonicus group described from central Brazil from leaves of physic nut, Jatropha curcas L. (Malpighiales: Euphorbiaceae), a plant potentially useful as a source of biofuel. This plant is often attacked by the mites Polyphagotarsonemus latus (Banks) (Prostigmata: Tarsonemidae) and Tetranychus bastosi Tuttle, Baker and Sales (Prostigmata: Tetranychidae). The objectives of this work were to evaluate the predation rate of A. zannoui on those phytophagous mites, to assess its life cycle on these prey and on pollen of Ricinus communis L. (Euphorbiaceae), and to determine its attraction to physic nut leaves infested by P. latus or T. bastosi. Amblydromalus zannoui attacked more nymphs and adults than eggs of P. latus, and more eggs and larvae than adults of T. bastosi. The life table parameters suggest that A. zannoui performs better on P. latus (r_m: 0.20, R_o: 18.77; λ: 1.23) and pollen (r_m: 0.18, R_o: 23.32, λ: 1.18). The predator seems to be attracted to plants with P. latus, but attraction was not clear cut for plants with T. bastosi. The results suggested that A. zannoui is a potential control agent to be used against P. latus on physic nut plants, and that R. communis pollen can be used as supplementary food to maintain the predator population in the absence of prey.

     

Suitability of the predatory mites Iphiseiodes zuluagai and Euseius concordis in controlling Polyphagotarsonemus latus and Tetranychus bastosi on Jatropha curcas plants in Brazil

One of the most promising plant species for biofuel production in Brazil is the physic nut Jatropha curcas. Major phytosanitary problems include the attack of two pest mite species, the broad mite Polyphagotarsonemus latus and the spider mite Tetranychus bastosi. Owing to pesticide-related problems, there is an increasing demand for sustainable environmental-friendly control methods such as biological control. In this study we evaluated the suitability of the predatory mite species Iphiseiodes zuluagai and Euseius concordis in controlling P. latus and T. bastosi on J. curcas. The number of T. bastosi killed by I. zuluagai was lower than the number of P. latus consumed. Euseius concordis preyed upon both T. bastosi and P. latus but the number of prey killed was always lower in comparison with I. zuluagai. However, P. latus and T. bastosi are suitable for the development of I. zuluagai and E. concordis as oviposition of both predators did not differ in relation to prey species. The preference of I. zuluagai for leaves of plants infested by either P. latus or T. bastosi, combined with the higher values for predation obtained by this predatory mite when fed on P. latus, compared to those values obtained by E. concordis, suggests that I. zuluagai can be more efficient than E. concordis in reducing populations of P. latus and T. bastosi under field conditions. Furthermore, we report here on the first record of predatory mites associated with P. latus and T. bastosi on native J. curcas plants in Brazil. In conclusion, we emphasize the crucial importance of predatory mites as agents of natural biological control of mite pests on J. curcas in small farms.     

Seasonal occurrence and food habits of larvae and juveniles of two temperate basses in the Shimanto estuary,Japan

Larvae and juveniles ofLateolabrax japonicus andL. latus occurred from January to May 1986 in the shallow waters of the Shimanto estuary.L. japonicus markedly outnumberedL. latus. Distinct ecological differences were recognized in habitats and food habits between the two species:L. japonicus mainly inhabited eelgrass beds composed ofZostera nana, whileL. latus appeared evenly in both eelgrass beds and non-eelgrass habitats; the former fed on copepods and cladocerans, while the latter fed on copepods and fish larvae. From these habitat and food habit analyses, estuaries were considered to be important as a main habitat forL. japonicus, but not forL. latus. The fact that ecological differences have occurred during the early life stages was inferred to be one of the possible keys to speculate on the speciation of the two species.     

Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes

Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A₂ (PLA₂) isozymeencoding genes, gPLA₂-o1, gPLA₂-o2 and gPLA₂-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA₂-o2) and (gPLA₂-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA₂ isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA₂isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA₂ isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA₂ isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA₂ isozyme genes appears to have been brought about by natural selection for point mutations.     

Construction of transformant Alcaligenes latus enforcing its own cloned phbC gene and characterization of poly-β-hydroxybutyrate biosynthesis

A recombined E. coli-A. latus shuttle vector plasmid pKTC32 harboring the cloned phbC gene from Alcaligenes latus was constructed, and transformed by electroporation into the parent A. latus in order to amplify the PHB synthase. The rate of PHB biosynthesis and content of PHB increased significantly after the transformation of the cloned phbC gene, plus the plasmid stability remained relatively high at around 85%. The enhanced PHB biosynthesis mechanism produced in the transformant A. latus was investigated by measuring the variations of enzyme activities related to the PHB biosynthesis.     

How promising is <Emphasis Type="Italic">Lasioseius floridensis</Emphasis> as a control agent of <Emphasis Type="Italic">Polyphagotarsonemus</Emphasis><Emphasis Type="Italic">latus</Emphasis>?

The blattisociid mite Lasioseius floridensis Berlese was found associated with the broad mite, Polyphagotarsonemus latus (Banks), on gerbera leaves in Mogi das Cruzes, State of Sao Paulo, Brazil. Blattisociid mites are not common on aerial plant parts, except under high air humidity levels. Some Lasioseius species have been mentioned as effective control agents of rice pest mites, but nothing is known about the biology of L. floridensis. The objective of this study was to evaluate whether the observed co-occurrence of L. floridensis and P. latus was just occasional or whether the latter could be important as food source for the former, assumed by laboratory evaluation of the ability of the predator to maintain itself, reproduce and develop on that prey. Biological parameters of L. floridensis were compared when exposed to P. latus and to other items as food. The study showed that mating is a pre-requisite for L. floridensis to oviposit and that oviposition rate was much higher on the soil nematode Rhabditella axei (Cobbold) (Rhabditidae) than on P. latus. Ovipositon on the acarid mite Tyrophagus putrescentiae (Schrank) was about the same as on P. latus, but it was nearly zero when the predator was fed the fungi Aspergillus flavus Link or Penicillium sp., or cattail (Typha sp.) pollen. Survivorship was higher in the presence of pollen and lower in the presence of A. flavus or Penicillium sp. than in the absence of those types of food. Life table parameters indicated that the predator performed much better on R. axei than on P. latus. To evaluate the potential effect of L. floridensis as predator of P. latus, complementary studies are warranted to determine the frequency of migration of L. floridensis to aerial plant parts, when predation on P. latus could occur.     

Culture conditions for production and purification of bioabsorbent from Alcaligenes latus B-16

Glucose and sucrose were favorable carbon sources for the production of bioabsorbent from Alcaligenes latus B-16. Yeast extract at a concentration of 0.5% was also an effective medium for growth. Biopolymers from A. latus B-16 were purified, and the biopolymers were separated into two types of biopolymers: a high-molecular weight component (acid-biopolymer) and a low-molecular weight component (neutral-biopolymer). These two types of biopolymers were shown to be homogeneous on electrophoresis and GPC (Gel Permeation Chromatography on HPLC). The high-molecular weight component was bioabsorbent, and was able to absorb water at more than 1,000 times its own weight. The total yield of acid- and neutral-biopolymers from A. latus B-16 was 20–25 g (dry weight) per liter of culture broth, and they were produced at a ratio of 20:1 (acid:neutral).     

Nematode parasites and associates of Dendroctonus spp. and Trypodendron lineatum (Coleoptera: Scolytidae), with a description of Bursaphelenchus varicauda n.sp.

Nematode parasites and associates of four bark beetle species in British Columbia were surveyed. Bursaphelenchus varicauda n.sp., Ektaphelenchus macrostylus, Panagrolaimus dentatus, and Cryptaphelenchus latus were found associated with Dendroctonus pseudotsugae. Parasitorhabditis obtusa was found in the gut and Contortylenchus reversus in the hemocoel of both D. pseudotsugae and D. rufipennis. The latter also had hemocoel infections of Sphaerulariopsis dendroetoni, which were not found concomitant with C. reversus infections. Contortylenchus brevicomi occurred in the hemocoel of D. brevicomis. The first report of a tylenchid larva parasitizing Trypodendron lineatum in North America is presented. Bursaphelenchus varicauda n.sp. was obtained from the gallery frass of D. pseudotsugae. It resembles B. corneolus and B. bestiolus but differs from the former species in female tail shape, the position of the excretory pore, spicule shape, and position of the male caudal papillae, and from the latter species in spicule shape and in the length of the esophagus and postuterine sac.     

Genetic studies of the Syrian hamster. VIII. The sex-linked gene Tortoiseshell

The occurrence of sex-linked yellow is described and designated as tortoiseshell (symbol To). The phenotype produced by the heterozygous female (To+) is a mosaic of that of ToTo and ++. Genetic inactivation of one X chromosome per cell is thus indicated. The To gene interacts phenotypically with genes Ba, Ds and Wh.     

Sampling plans for pest mites on physic nut

The starting point for generating a pest control decision-making system is a conventional sampling plan. Because the mites Polyphagotarsonemus latus and Tetranychus bastosi are among the most important pests of the physic nut (Jatropha curcas), in the present study, we aimed to establish sampling plans for these mite species on physic nut. Mite densities were monitored in 12 physic nut crops. Based on the obtained results, sampling of P. latus and T. bastosi should be performed by assessing the number of mites per cm² in 160 samples using a handheld 20× magnifying glass. The optimal sampling region for T. bastosi is the abaxial surface of the 4th most apical leaf on the branch of the middle third of the canopy. On the abaxial surface, T. bastosi should then be observed on the side parts of the middle portion of the leaf, near its edge. As for P. latus, the optimal sampling region is the abaxial surface of the 4th most apical leaf on the branch of the apical third of the canopy on the abaxial surface. Polyphagotarsonemus latus should then be assessed on the side parts of the leaf’s petiole insertion. Each sampling procedure requires 4 h and costs US$ 7.31.     

Revision ofMesorhyphus Handlirsch,Eoplecia Handlirsch andHeterorhyphus Bode (Diptera: Anisopodomorpha,Bibionomorpha) from the upper liassic of Germany

The holotypes of the Lower Jurassic DipteraMesorhyphus nanus Handlirsch 1920,M. areolatus Handlirsch 1939,M. anomalus Handlirsch 1939,Eoplecia primitiva Handlirsch 1920,Heterorhyphus latus Bode 1953,H. analivarius Bode 1953 andProtorhyphus (?)ovisimilis Bode 1953 were re-examined. The holotype ofEoplecia primitiva is the counterpart ofMesorhyphus areolatus. M. areolatus is a junior synonym ofM. nanus. Mesorrhyphoides Rohdendorf 1962 is a synonym ofHeterorhyphus Bode 1953.Heterorhyphus analivarius is a synonym ofProtorhyphus (?)ovisimilis, being mistakenly described from the counterpart of the holotype of the latter. The genusHeterorhyphus is valid and comprises onlyH. anomalus (Handlirsch 1939) n. comb.;H. latus is a synonym of the latter species. As the wing venation ofHeterorhyphus is conspicuously different from all fossil and Recent Anisopodomorpha, it is placed into a new family, Heterorhyphidae, within the Bibionomorpha.     

Review of the Acanthopagrus latus complex (Perciformes: Sparidae) with descriptions of three new species from the Indo‐West Pacific Ocean

Acanthopagrus latus, long considered a single valid Indo‐West Pacific Ocean species, characterized by having yellow pelvic, anal and caudal fins, is reviewed and separated into A. latus (east Asian shelf) and Acanthopagrus longispinnis (Bengal Bay), and three new species: Acanthopagrus morrisoni sp. nov. (north‐western Australia), Acanthopagrus arabicus sp. nov. [Middle East (except for the Red Sea) to coasts of Iran and Pakistan, and western Indian coast] and Acanthopagrus sheim sp. nov. (The Gulf). Although A. latus as redefined considerably varies in morphology and colouration, it can be recognized as a discrete east Asian endemic, with the following nominal species being junior synonyms: Chrysophrys auripes, Chrysophrys xanthopoda, Chrysophrys rubroptera and Sparus chrysopterus. Chrysophrys novaecaledoniae, known only from the holotype (type locality: Nouméa, New Caledonia), is a questionable junior synonym of A. latus, the lack of subsequent collections suggesting that the type locality is erroneous. Acanthopagrus longispinnis is differentiated from the other species in the complex by consistently having 12 dorsal‐fin spines and a much larger second anal‐fin spine, 21–26% (mean 23%) of standard length (L_S) (v. 14–24%, mean 18–21% in the other four species). Acanthopagrus morrisoni sp. nov. has the entire caudal fin yellow with a wide black posterior margin (persisting in preserved specimens) and consistently 3 ½ scale rows between the fifth dorsal‐fin spine base and the lateral line. Acanthopagrus sheim sp. nov. has the pelvic, anal and lower caudal fins vivid yellow, with two (rarely three) small black blotches on the lower inter‐radial membranes between the spinous and soft dorsal‐fin rays. Acanthopagrus arabicus sp. nov. consistently has 4 ½ scale rows between the fifth dorsal‐fin spine base and the lateral line, whereas A. latus always has black streaks proximally on the inter‐radial membranes between the yellow anal‐fin rays. A neotype and lectotye, respectively, are designated for A. latus and A. longispinnis. The p‐distance (net nucleotide substitutions per site) of partial mitochondrial 16s ribosomal RNA genes (538 bp) among the above species (except A. longispinnis) and three other congeners (Acanthopagrus berda, Acanthopagrus pacificus and Acanthopagrus bifasciatus) strongly indicates that each is a distinct species. A key is provided for the 20 species of Acanthopagrus currently known from the Indo‐West Pacific Ocean.     

The broad mite,Polyphagotarsonemus latus,as a potential prey for phytoseiid mites in California

In laboratory studies, individuals ofEuseius stipulatus Athias-Henriot,Typhlodromus rickeri Chant,T. porresi McMurtry andT. annectens DeLeon offered broad mite,Polyphagotarsonemus latus (Banks), as prey had developmental and ovipositional rates comparable to those offered a known favorable food (tetranychid mites or pollen).Euseius hibisci (Chant) had a longer developmental period and a markedly lower oviposition rate onP. latus than on the control food. Immature survival and ovipositional rates ofEuseius concordis Chant,Typhlodromus occidentalis Nesbitt,Amblyseius limonicus Garman and McGregor andIphiseius degenerans (Berlese) were very low onP. latus compared to control foods.