Effects of habitat fragmentation on genetic diversity and population differentiation of Liposcelis bostrychophila badonnel (Psocoptera: Liposcelididae) as revealed by ISSR markers

Liposcelis bostrychophila is a globally important stored product pest that is adapted to many habitats and is difficult to control due to insecticide resistance. We evaluated effects of habitat fragmentation on the genetic diversity and differentiation of L. bostrychophila populations. These genetic data were collected in Wuhu city, China using inter-simple sequence repeat (ISSR). A total of 108 discernible DNA fragments were detected, 107 were polymorphic and the percentage of polymorphic bands (PPB) was 99.1%. Shannon’s information index (I) and Nei’s gene diversity (He) from four populations were ordered (high to low) as herbstore > flourmill > bedroom > library. Population differentiation (Gst = 0.41) was average in these populations. Analysis of molecular variation (AMOVA) showed that variation existed mainly within populations (68.1%). At the same time, gene flow (Nm = 0.72) was interpreted to be moderate. Cluster analysis showed a small degree of genetic distance among different isolates and permitted grouping the isolates of L. bostrychophila into three distinct clusters. The study demonstrated clear genetic effects of habitat fragmentation on L. bostrychophila populations. Genetic diversity decreased and genetic differentiation increased between remnant populations when habitats were fragmented. Gene flow is not impeded by habitat fragmentation due to the impact of human activities. These findings are of great use for the prevention and control of this pest.     

Diagnosis of Liposcelis entomophila (Insecta: Psocodea: Liposcelididae) based on morphological characteristics and DNA barcodes

Liposcelis entomophila infests stored grain and is one of the most important psocid species worldwide. Six geographically isolated strains of L. entomophila from Asia, Europe, and United States of America (USA) were compared based on morphological attributes and by molecular methods. Decisive characters of morphological diagnosis were studied using body size measurements and Scanning Electron Microscopy (SEM). Molecular identification of the six strains was performed via identification of DNA sequence similarities and phylogenetic analyses based on a 655-bp fragment from the 5′ end of the standard mitochondrial gene cytochrome c oxidase I (COI) barcode region. The results showed that both morphological and molecular approaches were able to accurately identify this species. Kimura-2-Parameter (K2P) divergence between geographically isolated strains was on average 1.75% for the COI sequence. Phylogenetic analyses revealed that sequences of L. entomophila strains' COI barcodes formed clusters with tight cohesion that were clearly distinct from those of allied species.     

Morphological and molecular identification of Liposcelis corrodens (Heymons, 1909) (Psocodea: Liposcelididae) as the first record from China

Liposcelis corrodens (Psocodea: Liposcelididae) is recognized as one species of common stored product pests, however, it has not been reported in China. In this study, we identified Liposcelis specimens collected from a maize reserve depot in China as Liposcelis corrodens based on morphological and molecular methods. The identification of L. corrodens and keys to the stored Liposcelis species of Group IID were reported. The mitochondrial gene cytochrome c oxidase I (mtDNA COI) of the specimens was sequenced and used as a DNA barcode to conduct molecular identification and phylogenetic analyses with other Liposcelis species of Group IID from different geographical populations. This is the first record of L. corrodens in China and it will be useful for the further studies of taxonomy and management of stored booklice.     

Fungal isolates involved in biodeterioration of book-paper and their effects on substrate selection by Liposcelis bostrychophila (Badonnel) (Psocoptera: Liposcelididae)

The effects of fungi on the distribution of groups of Liposcelis bostrychophila were investigated. Two strains of Ulocladium botrytis were obtained from biodeteriorated book-paper and Eurotium amstelodami was isolated from the food used to culture L. bostrychophila. Isolated fungi were cultured on either paper sterilised with ethanol (paper) or on a malt-agar medium (medium). One strain or species was presented to groups of L. bostrychophila on a glass-fibre disc, with a blank disc as the other choice, and the number of insects on each disc and those free in the Petri dish was recorded after 12 h. In general a lower proportion of insects selected when one of the choices was either a 10,000 ppm extract of extract of U. botrytis cultured on malt-agar or one of the more dilute extracts of U. botrytis cultured on either medium. In contrast, extracts of both strains of U. botrytis (paper, 10,000 ppm) and E. amstelodami (malt-agar medium; 10,000; 1000 and 100 ppm) did not affect the proportion of insects that selected. With the exception of an extract of one of the strains of U. botrytis (paper; 10,000 ppm) significantly more selecting insects were found on the blank disc. A second set of experiments, in which extracts were applied to both discs, showed that L. bostrychophila did differentiate between the same fungus grown on paper or malt-agar medium and also between extracts of U. botrytis and E. amstelodami. It would appear that fungi grown on malt-agar medium are more repellent than fungi grown on paper and that E. amstelodami is preferred to U. botrytis. However, the selection of a substrate by L. bostrychophila very much depends on the context in which it is provided, i.e. the other choice.     

Molecular detection and identification of Rickettsia in psocids collected from herbs

The aims of this study were to assess the psocids that potentially carry Rickettsia and to characterize Rickettsia species. We collected booklice from dried fragments of Chinese medicinal herbs for retail. Species identification and Rickettsia detection were performed by using nondestructive DNA extraction method and PCR. Of the 224 booklice samples collected, the following species were identified: Liposcelis pearmani (50.89%), L. bostrychophila (25.89%) and L. entomophila (23.21%). Twenty-four samples were found to be positive for Rickettsia (10.71%, 24/224), and the number of Rickettsia-positive samples per species was as follows: L. pearmani (n = 10), L. bostrychophila (n = 8) and L. entomophila (n = 6). The phylogenetic tree revealed that the rickettsial agents found in booklice cluster along with Rickettsia felis belong to the spotted fever group (SFG). Mixed booklice contamination occurs in herbal fragments. The presence of Rickettsia-infected booklice suggests that there is a risk of louse-borne rickettsioses to humans in contact with herbs. These findings can contribute to the development of a disease control program that assists relevant practitioners and promotes public health.     

Molecular characterization of Lactobacillus species isolated from naturally fermented sausages produced in Greece,Hungary and Italy

The ecology of the lactic acid bacteria (LAB) in naturally fermented sausages produced in three European countries was investigated with the purpose of defining the geographic distribution of specific populations responsible for the fermentation and maturation of the sausages. Three-hundred and fifty eight strains belonging to the LAB group were isolated and identified by PCR-DGGE and sequencing. Three species were common to all three countries studied and were the most numerous. Lactobacillus curvatus, L. plantarum and L. sakei strains were subjected to RAPD-PCR followed by cluster analysis to obtain a genetic characterization. The distributions reflected in almost all cases the provenience of the strains, thereby distinguishing Italian, Greek and Hungarian LAB populations. The application of molecular methods for the identification and characterization of bacterial populations isolated from naturally fermented products allows a better understanding of the ecology. As a consequence measures can be taken to protect the biodiversity characterizing a specific system.     

Primers and visualization of LAMP: A rapid molecular identification method for Liposcelis entomophila (Enderlein) (Psocodea: Liposcelididae)

Liposcelis entomophila (Psocodea: Liposcelididae) is one of the most economically important booklice in the world. This species damages stored grain and represents an allergenic health risk to humans. Because of the small size of booklice, they are difficult to identify using morphological characteristics. Traditional molecular identification, such as DNA barcoding and qRT-PCR, requires specialized equipment and takes a long time, which is not appropriate for practical usage by plant health inspectors at ports. Therefore, for storage psocid identification, we suggested a simple, rapid and relatively new technique known as loop-mediated isothermal amplification (LAMP). In our study, a new rapid molecular identification method using the LAMP technique based on the mitochondrial gene cytochrome c oxidase I (COI) was developed for L. entomophila with high specificity and efficiency. Evident amplification results could be presented if the DNA concentration was 0.001 ng/μL. Visual detection was also possible with our LAMP primers using hydroxy naphthol blue (HNB), relying on the clear change in color from violet to sky blue after amplification. This method successfully determined unknown species that were newly collected from grain stores and may facilitate L. entomophila identification in plant quarantine and stored product protection.     

Food-selection by the booklouse, Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

A series of experiments, using a choice-chamber apparatus, were conducted to observe the food selection behaviour of Liposcelis bostrychophila. Following 4 days without food, groups of 50 adult insects were exposed to choices between pairs of different stored products: buckwheat, pot barley, brown rice flour, yellow millet and wheatgerm. Insects moved onto whole yellow millet in preference to whole buckwheat, whole pot barley and wheatgerm. Whole pot barley was preferred to brown rice flour. Ground grains were selected by insects in preference to whole grains. Furthermore, the distribution of insects between ground grains was different from that observed for whole grains. Carbohydrates affected the selection between foods which were equally attractive to L. bostrychophila: specifically, ground buckwheat (GBW) and ground yellow millet (GYM). Fewer booklice congregated on foodstuffs impregnated with glucose (0.5, 0.7 or 1 M). In contrast, L. bostrychophila preferred GBW with glucose (0.1 or 0.3 M) to GYM. GBW was preferred to GYM with fructose (0.5, 0.7 or 1 M), however, selection was not affected when fructose was added to GBW. Booklice preferred GBW or GYM with yeast added, to GBW or GYM without yeast. Furthermore L. bostrychophila preferred uncooked to cooked foods. These results are discussed in relation to the development of traps or lures for this increasingly prevalent domestic pest.     

Comparative residual toxicities of carbaryl, deltamethrin and permethrin as structural treatments against three liposcelidid psocid species (Psocoptera: Liposcelididae) infesting stored commodities

Residual toxicities of carbaryl, deltamethrin and permethrin were evaluated as surface treatments on concrete (porous surface) and galvanized steel (non-porous surface) panels (0.3×0.3 m²) against adults of Liposcelis bostrychophila Badonnel, Liposcelis entomophila (Enderlein), and Liposcelis paeta Pearman. Residual toxicities of these chemicals were assessed at 30±1°C, 70±2% r.h., and a photoperiod of 12 : 12 h (L:D), one day after treatment (0 wk) and thereafter at wk 1, 2, 4, 6, 8, and then every 4 wk up to wk 40. Mortality was recorded after exposure periods of 6 h, and then every 24 h until end-point was achieved. Liposcelis bostrychophila was the most susceptible species to the three chemicals tested, followed by L. paeta, and L. entomophila. Deltamethrin on concrete and permethrin on steel were the most successful chemicals, whereas permethrin on concrete and carbaryl on steel were the least successful against all three species. We conclude that for long-term protection, none of the chemicals studied is suitable against any of the three psocid species on concrete storage surfaces. On steel surfaces, only permethrin would deliver long-term protection up to 40 wk against L. bostrychophila and L. paeta infestations. This chemical however, will fail to provide long-term control against infestations where L. entomophila is present.     

Influence of autochthonous Saccharomyces cerevisiae strains on volatile profile of Negroamaro wines

Saccharomyces cerevisiae is the yeast species predominating the alcoholic fermentation of grape must. The aim of this research was to evaluate the impact of indigenous S. cerevisiae strains biodiversity on the aroma of wines from Negroamaro grapes. Grapes collected in two different Negroamaro producing micro districts in Salento (Southern Italy), were subjected to natural fermentation and two indigenous S. cerevisiae populations were isolated. Fifteen strains for each of the two populations were selected and tested by micro fermentation assay in order to evaluate their specific contribute to the volatiles composition and sensory impact of the produced wines. The aromatic profile of wines obtained by each selected strain was characterized by different contents of acetates, ethyl esters of fatty acids, higher alcohols, thus showing to be related to the strains geographical origin. The sensorial analysis of wines produced by the six best performing strains confirmed that they are good candidates as industrial starter cultures, This study indicates that the use of a “microarea-specific” starter culture is a powerful tool to enhance the peculiarity of wines deriving from specific areas.     

Multifaceted fungal characteristics determining the fungal feeding preferences of the psocid,Liposcelis bostrychophila badonnel (Psocoptera: Liposcelidae)

Psocids inhabit stored foods and cereals and cause serious problems with regard to food hygiene. They are a major target for pest control around the world, including Japan. Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelidae) is a representative species of household psocid that prefers to feed on fungi while also spreading fungi in the site it inhabits; it is therefore recognized as an indicator of fungal generation. The present study investigated the characteristics of fungi that influence the feeding behavior of this fungivorous insect using four fungus species (Aspergillus tubingensis, Aspergillus flavus, Penicillium chrysogenum, Cladosporium cladosporioides) that are representative fungi found in Japanese food factories and food storage facilities. The results of preference and feeding tests performed using an arena device (diameter: 9 cm) and a handmade olfactometer revealed that P. chrysogenum was the most attractive fungus for feeding. Based on the comparison of multiple characteristics of P. chrysogenum and the three other fungus species, the factors that were considered to evoke the feeding behavior of L. bostrychophila were a velvety appearance with hyphae that were shorter than the height of the insect as well as specific odors. The present study clarified the common characteristics of the fungi preferred by L. bostrychophila, which may be used to predict pest occurrence and develop agents for trapping and monitoring, thus leading to effective integrated pest management.     

Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l’Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of misidentified isolates. In conclusion, the 18S-ITS1-5.8S region appears to be useful in detecting genetic variability among yeast species, which is valuable for taxonomic purposes and for species identification. We have established an RFLP database for yeast species identified in milk samples using the software GelCompar II and the RFLP database constitutes an initial method for veterinary yeast identification.     

DNA barcoding,species-specific PCR for the identification of three stored-product pest species of genus Palorus (Coleoptera: Tenebrionidae)

Flour beetles of the genus Palorus (Coleoptera: Tenebrionidae) are important secondary storage pests. Correct identification of these insects is urgently required for integrated pest management. However, traditional morphological classification methods cannot identify insect fragments or immature stages and require skilled taxonomists with training and experience. In this study, three Palorus species were distinguished with two molecular techniques; DNA barcoding and species-specific PCR based on the mitochondrial DNA cytochrome oxidase subunit I (COI) gene. Forty-nine individuals of Palorus subdepressus (Wollaston), Palorus ratzeburgi (Wissman) and Palorus cerylonoides (Pascoe) were collected from 19 different locations across rice processing factory, feed factory, flour mill and bulk storages China. The COI sequences were analyzed by K2P distances and neighbor-joining (NJ) tree for DNA barcoding. The results show that these three Palorus species can be successfully identified by DNA barcoding. For the species-specific PCR study, we designed specificity primer pairs for the three Palorus species and evaluated their sensitivity. The results showed that the three Palorus species can be identified by the species-specific PCR method. Our results indicated that the two techniques can be independently used to identify the Palorus species in China for integrated pest management and quarantine. This is the first time Palorus species have been identified using two molecular techniques and the P. ratzeburgi and P. cerylonoides data have filled a gap in BOLD and GenBank.     

Isolation,identification and typification of Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius strains from orchard soil and the fruit processing environment in South Africa

Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius are thermo-acidophilic, non-pathogenic, spore-forming bacteria that can survive the typical heat processing of fruit juices and concentrates. Bacterial endospores then germinate, grow and cause spoilage of acid food products. Species of Alicyclobacillus were isolated from orchard soil and a fruit concentrate production factory in South Africa. Preliminary identification of the isolates was based on morphological, biochemical and physiological properties. Identification at species level was done by PCR amplification using genus-specific primers and 16S ribosomal RNA (rRNA) gene sequencing. The majority of isolates belonged to the species A. acidoterrestris, but A. acidocaldarius was also isolated and identified. As far as we could determine, this is the first report of the isolation of A. acidoterrestris from wash water and soil outside a fruit processing plant, as well as the isolation of A. acidocaldarius from vinigar flies. The genotypic relatedness between strains of A. acidoterrestris and between strains of A. acidocaldarius was determined by RAPD-PCR. Sixteen isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Three isolateT:/PGN/ELSEVIER/YFMIC/web/00001155/s identified as A. acidocaldarius gave three unique banding patterns.     

An application of PCR-DGGE analysis to profile the yeast populations in raw milk

Four different zones from the Friuli Venezia Giulia region, North East of Italy, were sampled for the study of the yeast bio-diversity in raw milk. Samples were analysed by traditional methods to isolate different yeast strains that were subjected to identification by sequencing the D1–D2 domains of the 26S rRNA gene. Twelve different species of yeast were identified, six of them belonging to the genera Candida and two to the genera Kluyveromyces. The identified strains were then used for the optimization of a method based on polymerase chain reaction and denaturing gradient gel electrophoresis that was used for a direct monitoring of the populations in the samples. Applying the method to the DNA extracted directly from the raw milk samples, new bands appeared in the gel underlining a different bio-diversity in respect to the traditional method. The approach described is a powerful and reliable tool to monitor directly yeast ecology in milk and milk products without the need of traditional isolation and it could be used to follow specific populations to prevent spoilage or to control contamination.     

Effects of controlled atmosphere and DDVP on population growth and resistance development by the psocid, Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

The effects of controlled atmosphere (CA) and DDVP on population growth and resistance development by the psocid, Liposcelis bostrychophila Badonnel, were studied in the laboratory. Results indicated that the population of L. bostrychophila increased rapidly under natural conditions; after 11 weeks, this population had increased 48.1-fold at 28°C, 80% relative humidity (r.h.). Exposure to CA (35% CO₂, 1% O₂) or DDVP (Dichlorvos, 0.3 mg/ml) alone failed to control the population growth. After 11 weeks, populations increased 4.3- and 9.1-fold, respectively. However, alternating exposure to CA and DDVP provided a significant increase in mortality as compared to those exposed to CA or DDVP alone. The results of bioassay showed that both populations exposed to CA and DDVP developed a low but significant resistance to CA and DDVP, respectively. After six exposures, the level of resistance to CA and DDVP increased 1.8- and 2.0-fold, respectively. Probit analysis did not show an appreciable increase in slope value of either population in spite of continuous exposures, indicating considerable heterogeneity of these psocids in response to CA or DDVP and suggesting a greater potential for the development of higher levels of resistance. It is suggested that alternating CA with insecticides could be an important management measure for psocids in storage.     

16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species

Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type.     

Validation of a Loop-Mediated Amplification/ISO 6579-Based Method for Analysing Soya Meal for the Presence of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

An alternative method for detection of Salmonella spp. in animal feed, based on the use of loop-mediated amplification (LAMP) in conjunction with a standard culturing procedure, was compared with the standard ISO 6579 as reference method, using soya meal as the test matrix. In the method comparison study, the sensitivities for both the alternative and reference methods were 100 %. The relative level of detection was 1.000. Tested against 100 Salmonella and 30 non-Salmonella strains, the LAMP-based method was 99 % inclusive and 100 % exclusive. The interlaboratory trial involved ten laboratories from eight European countries, testing eight samples at three contamination levels: 0 cfu/100 g, 1–5 cfu/100 g and 14–68 cfu/100 g. The trial specificity, or percentage correct identification of uncontaminated samples, was 96.3 % for both the reference methods and the LAMP/ISO 6579 alternative method, thus demonstrating its suitability for adoption as a procedure for rapid identification of Salmonella uncontaminated soya meal.     

Molecular study of Geotrichum strains isolated from Armada cheese

The aim of this work was the genetic characterization at the strain level of 39 presumed Geotrichum candidum isolates isolated throughout the artisanal manufacturing and ripening of Armada cheese and tentatively identified at genus and/or species level by phenotypic characteristics. The molecular identification of the strains included among others the amplification and sequencing of the D1/D2 domains of the 26S rRNA gene. A restriction fragment length polymorphism (RFLP) analysis with the ITS1-5.8S-ITS2 PCR amplicons and a randomly amplified polymorphic DNA (RAPD) analysis with five different primers were carried out. The bands pattern profile obtained through RFLP by enzymatic restriction with HinfI was the same for all the strains studied, which confirmed the classification of the strains at species level. A RAPD-PCR analysis with three different primers was applied to assess the intraspecific diversity, in this way 16 band profiles were obtained for the 39 strains studied by the combined use of primers Ari1 and Omt1. This study contributes to know the occurrence and genotypic biodiversity of G. candidum in Armada cheese.     

Phylogenetic study of Lactobacillus acidophilus group, L. casei group and L. plantarum group based on partial hsp60, pheS and tuf gene sequences

The partial nucleotide sequences encoding elongation factor Tu (tuf gene), 60-kDa heat shock protein (hsp60 gene) and phenylalanyl-tRNA synthase (pheS gene) were determined to assess the suitability as phylogenetic markers for discriminating the closely related species in Lactobacillus acidophilus group, L. casei group and L. plantarum group. A total of 234 lactobacilli were chosen from traditional fermented dairy products that were not exactly assigned to species based on biochemical tests and 16S rRNA gene sequences. The sequencing of partial tuf, hsp60 and pheS gene of all strains was performed, and then, the phylogenetic trees were constructed by neighbor-joining method. Phylogenetic tree revealed three genes provided better resolution of each Lactobacillus species than 16S rDNA, and all of strains were clearly identified as L. casei (63 strains), L. plantarum (58 strains) and L. helveticus (113 strains) by comparison of sequences with the type strains. From our results, the partial sequences of three genes had a higher discriminatory power than 16S rRNA gene sequences and were an alternative molecular tool for the taxonomical analysis of L. casei group, L. plantarum group and L. acidophilus group.