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Among naturally produced polymers, bacterial cellulose is receiving enormous attention due to remarkable properties, making it suitable for a wide range of industrial applications. However, the low yield, the instability of microbial strains and the limited knowledge of the mechanisms regulating the metabolism of producer strains, limit the large-scale production of bacterial cellulose. In this study, Komagataeibacter xylinus K2G30 was adapted in mannitol based medium, a carbon source that is also available in agri-food wastes. K. xylinus K2G30 was continuously cultured by replacing glucose with mannitol (2% w/v) for 210 days. After a starting lag-phase, in which no changes were observed in the utilization of mannitol and in bacterial cellulose production (cycles 1–25), a constant improvement of the phenotypic performances was observed from cycle 26 to cycle 30, accompanied by an increase in mannitol consumption. At cycle 30, the end-point of the experiment, bacterial cellulose yield increased by 38% in comparision compared to cycle 1. Furthermore, considering the mannitol metabolic pathway, D-fructose is an intermediate in the bioconversion of mannitol to glucose. Based on this consideration, K. xylinus K2G30 was tested in fructose-based medium, obtaining the same trend of bacterial cellulose production observed in mannitol medium. The adaptive laboratory evolution approach used in this study was suitable for the phenotypic improvement of K. xylinus K2G30 in bacterial cellulose production. Metabolic versatility of the strain was confirmed by the increase in bacterial cellulose production from D-fructose-based medium. Moreover, the adaptation on mannitol did not occur at the expense of glucose, confirming the versatility of K2G30 in producing bacterial cellulose from different carbon sources. Results of this study contribute to the knowledge for designing new strategies, as an alternative to the genetic engineering approach, for bacterial cellulose production.

Among naturally produced polymers, bacterial cellulose is receiving enormous attention due to remarkable properties, making it suitable for a wide range of industrial applications. However, the low yield, the instability of microbial strains and the limited knowledge of the mechanisms regulating the metabolism of producer strains, limit the large-scale production of bacterial cellulose. In this study

The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T . By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX-loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATαP expression locus with the loxP-kanMX-loxP cassette.

Significance The ability to control damaging plant pests and vectors of animal disease is threatened by the emergence of insecticide resistance. Developing effective strategies to prevent, slow, or overcome resistance requires an understanding of the underlying genetic mechanisms. Here, we present an example of the evolution of insecticide resistance arising from transposable element–mediated disruption of a dominant insecticide-susceptible allele, resulting in expression of a recessive resistance allele and potent resistance. Our findings demonstrate how the adaptive potential of transposable elements can be revealed by environmental and genetic perturbation and that this can have profound and unexpected impacts on resistance. They also illustrate how combinations of mutations that individually confer no fitness benefit can interact to provide strong context-dependent fitness benefits., The evolution of resistance to insecticides threatens the sustainable control of many of the world’s most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.

The evolution of resistance to insecticides threatens the sustainable control of many of the world’s most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.

This study proposes a biochemical and molecular model for the interaction between the Drosophila suzukii type 1 tyramine receptor (DsTAR1) and monoterpenes. A preliminary molecular and functional characterization of DsTAR1 cDNA revealed that a 1.8 kb long ORF codes for a 600 amino acid polypeptide featuring seven transmembrane domains, as expected for a GPCR. A stable HEK 293 cell line expressing DsTAR1 was tested for responsiveness to tyramine (TA) and octopamine (OA). In intracellular calcium mobilization studies, TA led to a concentration-dependent increase in [Ca2+]i (pEC50 ~ 6.40), completely abolished by pre-incubation with the antagonist yohimbine 1 μM. Besides, in dynamic mass redistribution (DMR) studies, TA evoked a positive DMR signal in a concentration-dependent manner (pEC50 ~ 6.80). The recombinant cell line was then used to test three monoterpenes (thymol, carvacrol and α-terpineol) as putative ligands for DsTAR1. The terpenoids showed no agonist effects in both DMR and calcium mobilization assays, but they increased the potency of the endogenous ligand, TA, acting as positive allosteric modulators. Moreover, expression analysis on adults D. suzukii, exposed for 24, 72 or 120 h to a sublethal concentration of the three monoterpenes, showed a downregulation of DsTAR1. This evidence has led to hypothesize that the downregulation of DsTAR1 might be a compensatory mechanism in response to the positive allosteric modulation of the receptor induced by monoterpenes. Therefore, these findings might be useful for the development of a new generation of biopesticides against Drosophila suzukii, targeting TAR1.

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae) , resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans . No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).

Aim of the study was to investigate the performance of the new insecticide "spirotetramat" as an alternative solution of "abamectin" for the control of Cacopsylla pyri L. (Hemiptera: Psyllidae) in the context of an IPM program in European pear, Pyrus communis L.. Laboratory bioassays for the estimation of LC50 and LC90 of both insecticides were performed using four populations collected in Emilia-Romagna (Italy) orchards where different pest management strategies were used (organic, integrated, and conventional). The same populations were also analyzed for the main insecticide detoxifying activities in nymphs by spectrofluorimetric in vitro assays. The performance of the two insecticides was also tested on field on one population under integrated pest management conditions. The laboratory experiments showed that the LC90 of spirotetramat were lower than the highest field concentration allowed in Europe (172.80 mg AI liter(-1)) giving reassurance about the efficacy of the product. Concerning the abamectin, the laboratory bioassays did not show strong indications of resistance development of C. pyri populations of Emilia-Romagna. A similarity in enzyme detoxifying activity was observed in both insecticides indicating a general absence of a significant insecticide resistance. The field trial showed a high efficacy (90 %) of spirotetramat on C. pyri already after 15 d from application, and it was significantly higher from abamectin. Overall, spirotetramat is one more choice for C. pyri control, as well as abamectin in order to minimize the risks of occurrence of insecticide resistance.

Cyantraniliprole is a novel insecticide for control of multiple chewing and sucking insect pest species including the sweetpotato whitefly Bemisia tabaci (Gennadius), which is one of the most important polyphagous pests in tropical, subtropical, and Mediterranean regions. This study aims to evaluate the effects of cyantraniliprole on the probing behavior of B. tabaci on tomato. Electrical penetration graph data indicated that on plants treated with cyantraniliprole (foliar application), adult whiteflies of the genetic variant Q2 were not able to reach the phloem and consequently did not perform the activities represented by E1 and E2 waveforms, i.e., phloem salivation (during which inoculation of geminiviruses occurs) and phloem sap ingestion (during which geminiviruses are acquired by the whiteflies), respectively. The complete failure of B. tabaci biotype Q adults to feed from the phloem of tomato plants treated with cyantraniliprole could be explained by rapid cessation of ingestion because of the mode of action of this insecticide. Overall, these findings indicated that cyantraniliprole might represent a useful new tool for producers to protect tomato plants from damage by B. tabaci.

The European grapevine moth, Lobesia botrana (Denis & Schiffermuller) (Lepidoptera: Tortricidae), is the key pest of vineyard, Vitis vinifera L. In Italy, failures in field chemical pest control have been recently reported. The susceptibility to insecticides indoxacarb, methoxyfenozide, and emamectin benzoate was then evaluated in a L. botrana population collected from a vineyard in Emilia-Romagna (northeastern Italy) where pest management programs achieved unsatisfactory results. The field trial showed that the indoxacarb efficacy toward L. botrana was very low in the two timings of application (7.9 and -1.5%) in comparison with untreated control, while the efficacy of methoxyfenozide (76.1%) and emamectin benzoate (88.8%) was high. The decreased efficacy of indoxacarb was also supported by the results of the laboratory bioassay on neonate L. botrana larvae, in which the resistance ratio was 72-fold in comparison with that of the susceptible strain.

Esterase-based resistance in the peach-potato aphid, Myzus persicae (Sulzer), is generally due to one of two alternative amplified carboxylesterase genes, E4 or FE4 (fast E4). The E4 amplified form is distributed worldwide and it is correlated with a particular translocation between autosomes 1 and 3, whereas the FE4 form, which has hitherto not been found to be associated with chromosomal rearrangements, is typical of the Mediterranean regions. In this study, we present for the first time cytogenetic and molecular data on some M. persicae parthenogenetic lineages, which clearly show a chromosomal A1-3 translocation associated with esterase FE4 genes and unrelated to high levels of esterase-based resistance.

Problems with Tetranychus urticae are frequently reported in protected crops in Italy, particularly in roses where many introduced acaricides show a progressive loss of effectiveness. We have conducted bioassays to assess the response of some Italian strains of T. urticae to a number of acaricides. These include compounds that were widespread and frequently used in the past, but also some recently registered compounds. We investigated two T. urticae strains collected from rose growers where control failures were reported (SAN and PSE), together with a strain collected from unsprayed vegetables (BOSA). Adult females of the rose strains (SAN and PSE) were resistant to tebufenpyrad (Resistant Ratio-RR, RR(50) = 48.4 and 163.6) and fenpyroximate (RR(50) = 74.1 and 25.9) when compared to the susceptible BOSA strain. Lethal concentrations for these products were higher than the registered field rate. The PSE strain proved to be highly resistant to abamectin (RR(50) = 1,294.1). Variation in bifenazate susceptibility was detected amongst strains, but LC(90) values of SAN and PSE were still in the range of the registered field rate. In egg bioassays, the SAN and PSE strains exhibited high resistance levels to clofentezine (RR(50) = 66,473 and 170,714), hexythiazox (RR(50) = 70,244 and 159,493) and flufenoxuron (RR(50) = 61.9 and 117.9). But the recently introduced ovi/larvicides etoxazole and spirodiclofen exhibited high activity on all strains. The activity of detoxifying enzymes such as esterases, glutathione-S-transferases (GSTs) and cytochrome P450 monooxygenases (MFOs) was determined in these strains as a preliminary attempt to identify potential resistance mechanisms. Enzymatic assays showed that the rose strains exhibited 2.66 and 1.95-fold increased MFOs activity compared to the susceptible strain. Assays for GSTs revealed that only the SAN strain exhibited a significantly higher activity. In contrast, only the PSE strain showed a significant higher hydrolysis of 1-naphthyl acetate.

Sexual reproduction and breeding systems are driving forces for genetic diversity. The mating-type (MAT) locus represents a mutation and chromosome rearrangement hotspot in yeasts. Zygosaccharomyces rouxii complex yeasts are naturally faced with hostile low water activity (aw) environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy. Here, we investigated sex-determination system in Zygosaccharomyces sapae diploid strain ABT301T, a member of the Z. rouxii complex. We cloned three divergent mating type-like (MTL) α-idiomorph sequences and designated them as ZsMTLα copies 1, 2, and 3. They encode homologs of Z. rouxii CBS 732T MATα2 (amino acid sequence identities spanning from 67.0 to 99.5%) and MATα1 (identity range 81.5–99.5%). ABT301T possesses two divergent HO genes encoding distinct endonucleases 100% and 92.3% identical to Z. rouxii HO. Cloning of MATa-idiomorph resulted in a single ZsMTLa locus encoding two Z. rouxii-like proteins MATa1 and MATa2. To assign the cloned ZsMTLα and ZsMTLa idiomorphs as MAT, HML, and HMR cassettes, we analyzed their flanking regions. Three ZsMTLα loci exhibited the DIC1-MAT-SLA2 gene order canonical for MAT expression loci. Furthermore, four putative HML cassettes were identified, two containing the ZsMTLα copy 1 and the remaining harboring ZsMTLα copies 2 and 3. Finally, the ZsMTLa locus was 3′-flanked by SLA2, suggesting the status of MAT expression locus. In conclusion, Z. sapae ABT301T displays an aααα genotype missing of the HMR silent cassette. Our results demonstrated that mating-type switching is a hypermutagenic process in Z. rouxii complex that generates genetic diversity de novo. This error-prone mechanism could be suitable to generate progenies more rapidly adaptable to hostile environments.

Hemochromatosis is a progressive iron overload disorder that is prevalent among individuals of European descent. It is usually inherited in an autosomal-recessive pattern and associated with missense mutations in HFE, an atypical major histocompatibility class I gene. Recently, we described a large family with autosomal-dominant hemochromatosis not linked to HFE and distinguished by early iron accumulation in reticuloendothelial cells. Through analysis of a large pedigree, we have determined that this disease maps to 2q32. The gene encoding ferroportin (SLC11A3), a transmembrane iron export protein, lies within a candidate interval defined by highly significant lod scores. We show that the iron-loading phenotype in autosomal-dominant hemochromatosis is associated with a nonconservative missense mutation in the ferroportin gene. This missense mutation, converting alanine to aspartic acid at residue 77 (A77D), was not seen in samples from 100 unaffected control individuals. We propose that partial loss of ferroportin function leads to an imbalance in iron distribution and a consequent increase in tissue iron accumulation.

Background and Methods Hereditary hemochromatosis in adults is usually characterized by mutations in the hemochromatosis (HFE) gene on the short arm of chromosome 6. Most patients have a substitution of tyrosine for cysteine at position 282 (C282Y). We studied a large family from Italy that includes persons who have a hereditary iron-overload condition indistinguishable from hemochromatosis but without apparent pathogenic mutations in the HFE gene. We performed biochemical, histologic, and genetic studies of 53 living members of the family, including microsatellite analysis of chromosome 6 and direct sequencing of the HFE gene. Results Of the 53 family members, 15 had abnormal serum ferritin levels, values for transferrin saturation that were higher than 50 percent, or both. Thirteen of the 15 had elevated body iron levels, diagnosed on the basis of the clinical evaluation and liver biopsy, and underwent iron-removal therapy. The other two, both children, did not undergo liver biopsy or iron-removal thera...

An analysis of LDL-receptor gene was performed on an Italian patient with heterozygous familial hypercholesterolemia. Restriction enzyme analysis showed that the proband was heterozygous for a deletion of 4.5 kb spanning the 5' end of exon 13 (45 nucleotide residues) to intron 15. Amplification of genomic DNA, using polymerase chain reaction (PCR), followed by direct sequencing, showed that this deletion was identical to the one reported by Lehrman et al. (1986. Proc. Natl. Acad. Sci. 83: 3679-3683). As only the normal LDL-receptor mRNA was detectable in proband fibroblasts by Northern blot, we used reverse transcription-PCR to amplify the mutant mRNA using primers complementary to exon 6 (sense) and exon 18 (antisense). The amplification of control cDNA resulted in a single fragment of 1725 nucleotides containing the normal sequence. The amplification of cDNA from the proband produced the 1725-nucleotide fragment (as in the control) and three additional fragments (F1, F2, and F3) of smaller size. The direct sequence showed that in the F1 fragment exon 12 was joined to exon 16; in the F2 fragment exon 12 was joined to exon 17; and in the F3 fragment exon 11 was joined to exon 16. Thus, the deletion-bearing allele generated three mRNAs, two of which resulted from alternative splicings leading to the skipping of exons 16 and 12, respectively. It is expected that the translation of these mutant mRNAs will generate three aberrant proteins, the synthesis of which should be negligible in view of the very low content of the corresponding mRNAs.

The peach-potato aphid Myzus persicae (Sulzer) has developed a number of insecticide resistance mechanisms owing to the high selective pressure produced by world-wide insecticide treatments. Knowledge of the geographical distribution and the temporal evolution of these resistant phenotypes helps to develop suitable pest-management programs. Current understanding of the major mechanisms of resistance at the molecular level makes it possible to diagnose the presence of modified acetylcholinesterase (MACE) or knockdown resistance (kdr). This paper describes a rapid method for the identification of both resistance mechanisms in a single molecular assay by using restriction fragment length polymorphism of PCR products (RFLP-PCR) in individual as well as pooled aphids.

Recent developments in expression profile and proteomic techniques illustrated that the main oenological traits of wine yeasts are complex and influenced by several genes, each of them identified as absolutely essential. Only for monogenic properties the genetic improvement programmes of wine yeasts can be performed by alteration of individual genes. Ideally the most productive way of improving the whole-cell biocatalysts is by evolution of the entire cell genome. In this article we briefly review the main genetic improvement techniques applied in new and optimised wine strains construction, paying particular attention to blind and whole genome strategies, such as the sexual recombination and genome shuffling.

Background/Aims: The actual prevalence of the main hemochromatosis (HFE) mutations in the Italian adult population and their phenotypic expression have not yet been established. This information is key to advocate a mass-screening program. Methods: Two thousand one hundred adults were tested for the C282Y/H63D HFE gene mutations by an automated genotyping assay as well as transferrin saturation (TS) and serum ferritin levels. Results: No homozygotes for the C282Y mutation were found. Heterozygosity for the C282Y mutation was 3.1%, while heterozygosity and homozygosity for the H63D mutation were 21.5% and 2.5%, respectively. TS was significantly higher in C282Y heterozygotes and H63D homozygotes as compared to wild-type individuals (P , 0:01). Interestingly, of the HFE wild-type subjects 5.9% had a TS value above the 45% threshold. Conclusions: This study shows that (i) the predicted prevalence for C282Y homozygosity in Italy is 1:3900; (ii) the C282Y/H63D wild-type population has an increased baseline of iron parameters possibly due to genetic factors not linked to the C282Y/H63D mutations; (iii) since in the latter population the actual tissue iron burden cannot be assessed, phenotypic (TS) screening in Italy is not recommended until the true prevalence of all mutations in the HFE gene and in other hemochromatosis genes will be established. q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Abstract —The aim of this study was the characterization of mutations of the LDL receptor gene in 39 Italian patients with homozygous familial hypercholesterolemia, who were examined during the period 1994 to 1996. The age of the patients ranged from 1 to 64 years; one third of them were older than 30. Plasma LDL cholesterol level ranged from 10.8 to 25.1 mmol/L. The residual LDL receptor activity, measured in cultured fibroblasts of 32 patients, varied from r =−0.665; P

Aphids life cycle includes cyclical or obligate parthenogenesis, therefore low genetic variation is ex- pected in these insects. Genetic diversity in crop pest aphids such as Myzus persicae, is also influenced by the exten- sive use of insecticides which favoured the selection of few resistant strains. In the present work, 18 M. persicae popu- lations collected in different Italian regions, were studied by RAPD-PCR (Random Amplified Polymorphic DNA) to assess genetic variability among populations. Twelve different random primers, selected out of 24 previously inves- tigated, were employed for genomic DNA amplification. A total of 150 fragments from the aphid M. persicae popu- lations, and 31 fragments in two outgroups were identified. An UPGMA cluster analysis based on Nei and Li's ge- netic distance revealed that the M. persicae populations could be divided into two groups: aphids from Central and Southern Italy were generally located in the same cluster, while aphids from Northern Italy were more often located in the other. The presence of a positive correlation between genetic and geographical distance suggests that at least a portion of the interpopulation polymorphism evidenced could be caused by restricted gene flow.