Origin, evolution, phylogeny and taxonomy of Pulex irritans.

The human flea Pulex irritans Linnaeus, 1758 (Siphonaptera: Pulicidae) is one of the most studied species together with the cat flea Ctenocephalides felis Bouché, 1835, because they have a cosmopolitan distribution and are closely related to humans. The present study aimed to carry out a comparative morphometric and molecular study of two different populations of P. irritans (Spain and Argentina). Accordingly, internal transcribed spacer (ITS)1 and ITS2 of rDNA and the partial cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) mtDNA genes of these taxa were sequenced. Furthermore, the taxonomy, origin, evolution and phylogeny of P. irritans was assessed. The morphometric data obtained did not show significant differences between P. irritans specimens from Spain and Argentina, even when these two populations were collected from different hosts; however, there was a considerable degree of molecular divergence between both populations based on nuclear and mitochondrial markers. Thus, it is proposed that P. irritans, in contrast with other generalist fleas, maintains a certain degree of morphological similarity, at least between Western Palearctic and Neotropical areas. Furthermore, two well defined geographical genetic lineages within the P. irritans species are indicated, suggesting the existence of two cryptic species that could be discriminated by a polymerase chain reaction-linked restriction fragment length polymorphism.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a useful tool for the rapid identification of wild flea vectors preserved in alcohol.

Flea identification is a significant issue because some species are considered as important vectors of several human pathogens that have emerged or re-emerged recently, such as Bartonella henselae (Rhizobiales: Bartonellaceae) and Rickettsia felis (Rickettsiales: Rickettsiaceae). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated in recent years for the identification of multicellular organisms, including arthropods. A preliminary study corroborated the usefulness of this technique for the rapid identification of fleas, creating a preliminary database containing the spectra of five species of flea. However, longterm flea preservation in ethanol did not appear to be an adequate method of storage in the context of specimen identification by MALDI-TOF MS profiling. The goal of the present work was to assess the performance of MALDI-TOF MS in the identification of seven flea species [Ctenocephalides felis (Siphonaptera: Pulicidae), Ctenocephalides canis, Pulex irritans (Siphonaptera: Pulicidae), Archaeopsylla erinacei (Siphonaptera: Pulicidae), Leptopsylla taschenbergi (Siphonaptera: Ceratophyllidae), Stenoponia tripectinata (Siphonaptera: Stenoponiidae) and Nosopsyllus fasciatus (Siphonaptera: Ceratophyllidae)] collected in the field and stored in ethanol for different periods of time. The results confirmed that MALDI-TOF MS can be used for the identification of wild fleas stored in ethanol. Furthermore, this technique was able to discriminate not only different flea genera, but also the two congeneric species C. felis and C. canis.

Morphological, biometrical and molecular characterization of Archaeopsylla erinacei (Bouché, 1835).

In the present work, we carried out a morphological, biometrical and molecular study of the species Archaeopsylla erinacei (Bouché, 1835) and their subspecies: Archaeopsylla erinacei erinacei (Bouché, 1835) and Archaeopsylla erinacei maura (Jordan & Rothschild, 1912) isolated from hedgehogs (Erinaceus europaeus) from different geographical regions (Seville and Corse). We have found morphological differences in females of A. erinacei from the same geographical origin that did not correspond with molecular differences. We suggest that some morphological characters traditionally used to discriminate females of both subspecies should be revised as well as we set the total length of the spermatheca as a valid criterion in order to discriminate between both subspecies. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and partial 18S rRNA gene, and partial cytochrome c-oxidase 1 (cox1) and cytochrome b (cytb) mtDNA gene sequences were determined to clarify the taxonomic status of these taxa and to assess intra-specific and intra-population similarity. In addition, a phylogenetic analysis with other species of fleas using Bayesian and Maximum Likelihood analysis was performed. All molecular markers used, except 18S, showed molecular differences between populations corresponding with geographical origins. Thus, based on the phylogenetic and molecular study of two nuclear markers (ITS1, ITS2) and two mitochondrial markers (cox1 and cytb), as well as concatenated sequences of both subspecies, we reported the existence of two geographical genetic lineages in A. erinacei corresponding with two different subspecies: A. e. erinacei (Corse, France) and A. e. maura (Seville, Spain), that could be discriminated by polymerase chain reaction-linked random-fragment-length polymorphism.

Monitoring volatile compounds production throughout fermentation by Saccharomyces and non-Saccharomyces strains using headspace sorptive extraction.

Currently, there is a growing interest in the use of non-Saccharomyces yeast to enhance the aromatic quality of wine, with pure or mixed cultures, as well as sequential inoculation. Volatile components of wines were closely related to their sensory quality. Hence, to study the evolution of volatile compounds during fermentation was of great interest. For this, sampling methods that did not alter the volume of fermentation media were the most suitable. This work reports the usefulness of headspace sorptive extraction as non-invasive method to monitor the changes in volatile compounds during fermentation. This method allowed monitoring of 141 compounds throughout the process of fermentation by Saccharomyces cerevisiae and Lachancea thermotolerans strains. Both strains showed a similar ability to ferment a must with high sugar content. The S. cerevisiae strain produced higher amount of volatile compounds especially esters that constitutes fruity aroma than L. thermotorelans.

Molecular study of Stenoponia tripectinata tripectinata (Siphonaptera: Ctenophthalmidae: Stenoponiinae) from the Canary Islands: taxonomy and phylogeny.

In the present work, we carried out a comparative molecular study of Stenoponia tripectinata tripectinata isolated from Mus musculus from the Canary Islands, Spain. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and 18S ribosomal RNA partial gene and cytochrome c-oxidase 1 (cox1) mitochondrial DNA partial gene sequences of this subspecies were determined to clarify the taxonomic status of this subspecies and to assess inter-population variation and inter-specific sequence differences. In addition, we have carried out a comparative phylogenetic study with other species of fleas using Bayesian, Maximum Parsimony, Maximum Likelihood and Neighbor-Joining analysis. A geographical signal was detected between the cox1 partial gene sequences of S. t. tripectinata isolated from M. musculus from different islands and those isolated from Apodemus sylvaticus from the Iberian Peninsula. Our results assess the monophyletic origin of Stenoponiinae and a different genetic lineage from Ctenophthalmidae. Thus, the elevation of subfamily Stenoponiinae to family level (Stenoponiidae) is suggested.

Morphological, biometrical, and molecular characterization of Ctenocephalides felis and Ctenocephalides canis isolated from dogs from different geographical regions.

In the present work, a comparative morphological, biometrical and molecular study of Ctenocephalides spp. isolated from dogs (Canis lupus familiaris) from different geographical regions (Spain, Iran, and South Africa) has been carried out. The internal transcribed spacer 1 (ITS1) sequences of Ctenocephalides felis and Ctenocephalides canis collected from dogs from different geographical regions have been determined to clarify the taxonomic status of these species and to assess intraspecific variation and interspecific sequence differences. In addition, a phylogenetic analysis based on ITS1 sequences has been performed. Four different morphological populations were observed in the individuals of C. felis collected from dogs from different geographical locations. Nevertheless, the comparative study of the ITS1 sequences of the different morphological populations observed in C. felis did not show molecular differences. The results showed clear molecular differences between C. felis and C. canis and some specific recognition sites for endonucleases were detected between both species. Thus, BfrBI and DraI sites have diagnostic value for specific determination in C. felis. The phylogenetic tree based on the ITS1 sequences of C. felis and C. canis revealed that all the populations of C. felis from different geographical regions clustered together and separated, with high bootstrap values, from C. canis. We conclude that ITS1 region is a useful tool to approach different taxonomic and phylogenetic questions in Ctenocephalides species.

A non-destructive sampling method for food authentication using gas chromatography coupled to mass spectrometry or ion mobility spectrometry.

The study of volatile compounds obtained by gas chromatography (GC) coupled to mass spectrometry (MS) or ion mobility spectrometry (IMS) may be very useful to protect food quality, especially when using a non-destructive sampling method. In this work, the authentication of the highly appreciated dry-cured Iberian ham by those techniques was studied and compared. The results obtained show the suitability of a non-destructive sampling method coupled to headspace sampling (HS)-GC-IMS or HS-GC-MS to determine volatile markers in the feeding Iberian pig regime. Although both methods were suitable to differentiate the ham categories, HS-GC-IMS was more sensitive detecting a higher number of compounds than HS-GC-MS, which provided accurate qualitative results. The results of principal component analysis showed that ethanol, 2-propanol and 3-methylbutanol, identified by HS-GC-IMS, and 3-methylbutanal and heptane, identified by HS-GC-MS, could be considered potential markers to identify ham from different feeding regimes.

Influence of the ripening chamber's geographical location on dry-cured Iberian ham's key odorants.

Olfactometric and sensory analyses have been applied to study the possible influence of the ripening chamber's geographical location on the aroma sensory profiles and key odorants of Iberian ham. Dry-cured Iberian ham was obtained from 3 acorn-fed pigs and, for the first time, both of the participating production facilities, located in two different Andalusian municipalities with different altitudes above mean sea level, processed one of the two hind legs from each pig. The descriptive sensory profile of orthonasal and retronasal odours was determined by trained panellists, while odour-active compounds were determined by gas chromatography/mass spectrometry-olfactometry (GC/MS-O). The results obtained showed that, separately, both techniques enable Iberian ham samples to be differentiated by their ripening chamber's geographical location. For sensory analysis, retronasal sensory analysis appeared to be the most suitable for this goal, highlighting the "meat broth odour" and "roasted nuts odour" descriptors which presented significant differences between geographical locations for samples from all pigs. Moreover, ripening chamber's geographical location characteristics and the initial composition of the raw material seemed to influence the content of some odour-active compounds. The odour-active compound identified as octane/acetone and isobutanol were conditioned by the ripening chamber's geographical location, while decanal/2-ethyl-1-hexanol, 1-undecanol, 2-furanmethanol and cis-2-nonenal were also influenced by the individual pig itself. This study showed that slight climatological differences due to the location of the ripening chamber seem to have somewhat of an influence on the aromatic profile.

Sensory and spectroscopic characterization of Argentinean wine and balsamic vinegars: A comparative study with European vinegars.

In Argentina, vinegars are cheap agro-food products without exhaustive regulation and the production of high-quality vinegars has not been exploited yet. In fact, Argentinean vinegars have not been studied. In this context, a first study of Argentinean balsamic and wine vinegars was carried out by a sensory and spectroscopic characterization and by a comparison with well-recognized European vinegars. For that, ultraviolet-visible and fluorescence spectroscopies were applied together with principal component analysis (PCA) and parallel factor analysis (PARAFAC) performed on each data set, respectively. Results showed differences between acetification processes, origin countries and a wide variability within Argentinean production. The sensory characterization on Argentinean wine vinegars was performed by triangular and ordering preference tests showing statistically significant preferences toward the traditional and the rapid vinegars. This work highlights the effect of production on quality in order to provide added value to the Argentinean vinegars.

Assessment of UV-visible spectroscopy as a useful tool for determining grape-must caramel in high-quality wine and balsamic vinegars.

The addition of E-150d caramel, known as grape-must caramel in vinegars, is a legal but without limitation practice in Spanish wine vinegars, as those with a protected designation of origin (PDO), for unifying the final colour of different batches and barrels. It is also used in the production of 'Balsamic vinegar of Modena' vinegars, with a maximum addition fixed at 2% v/v by law. Although its quantification in vinegars was studied by other techniques, there is still not any official method for it. Therefore, UV-vis spectroscopy was assessed as a quick analytical method able to quantify grape-must caramel in Balsamic vinegars of Modena and Spanish PDO wine vinegars. Univariate and multivariate calibrations were assessed for this quantification. Results demonstrated the ability of UV-vis spectroscopy coupled with multivariate calibration in the quantification of grape-must caramel, predicting caramel amounts under 2% v/v in both Balsamic and PDO wine vinegars.

Differentiation of Trichuris species using a morphometric approach.

Trichuris trichiura is a nematode considered as the whipworm present in humans and primates. The systematics of the genus Trichuris is complex. Morphological studies of Trichuris isolated from primates and humans conclude that the species infecting these hosts is the same. Furthermore, numerous molecular studies have been carried out so far to discriminate parasite species from humans and Non-Human Primates using molecular techniques, but these studies were not performed in combination with a parallel morphological study. The hypothesised existence of more species of Trichuris in primates opens the possibility to revise the zoonotic potential and host specificity of T. trichiura and other putative new species of whipworms. In the present work, a study of Trichuris Roederer, 1761 (Nematoda:Trichuridae) parasitizing C. g. kikuyensis, P. ursinus, Macaca sylvanus, Pan troglodytes, and Sus scrofa domestica has been carried out using modern morphometric techniques in order to differentiate populations of Trichuris isolated from four species of captive NHP from different geographical regions, and swine, respectively. The results obtained revealed strong support for geometrical morphometrics as a useful tool to differentiate male Trichuris populations. Therefore, morphometrics in combination with other techniques, such as molecular biology analyses, ought to be applied to further the differentiation of male populations. On the other hand, morphometrics applied to female Trichuris species does not seem to contribute new information as all the measurements combinations of obtained from females always showed similar results.

Optimization and validation of headspace sorptive extraction for the analysis of volatile compounds in wine vinegars.

Quantification of aroma compounds in wine vinegars is challenging due to the complexity of the matrix and the low concentrations expected. A method for the determination of volatile compounds in wine vinegars employing headspace sorptive extraction-thermal desorption-gas chromatography-mass spectrometry (HSSE-TD-GC-MS) was developed. A central composite design was used to optimize the sampling condition. The proposed method was successfully validated and low detection and quantification limits was obtained. The application of the proposed methodology allows the determination of 53 compounds in different wine vinegars (red, Sherry). Five of them have been detected in wine vinegars for the first time.

Comparative assessment of software for non-targeted data analysis in the study of volatile fingerprint changes during storage of a strawberry beverage.

Five free software packages were compared to assess their utility for the non-targeted study of changes in the volatile profile during the storage of a novel strawberry beverage. AMDIS coupled to Gavin software turned out to be easy to use, required the minimum handling for subsequent data treatment and its results were the most similar to those obtained by manual integration. However, AMDIS coupled to SpectConnect software provided more information for the study of volatile profile changes during the storage of strawberry beverage. During storage, volatile profile changed producing the differentiation among the strawberry beverage stored at different temperatures, and this difference increases as time passes; these results were also supported by PCA. As expected, it seems that cold temperature is the best way of preservation for this product during long time storage. Variable Importance in the Projection (VIP) and correlation scores pointed out four volatile compounds as potential markers for shelf-life of our strawberry beverage: 2-phenylethyl acetate, decanoic acid, γ-decalactone and furfural.

Volatile profile characterisation of Chilean sparkling wines produced by traditional and Charmat methods via sequential stir bar sorptive extraction.

The volatile compositions of Charmat and traditional Chilean sparkling wines were studied for the first time. For this purpose, EG-Silicone and PDMS polymeric phases were compared and, afterwards, the most adequate was selected. The best extraction method turned out to be a sequential extraction in the headspace and by immersion using two PDMS twisters. A total of 130 compounds were determined. In traditional Chilean sparkling wines, ethyl esters were significantly higher, while acetic esters and ketones were predominant in the Charmat wines. PCA and LDA confirmed the differences in the volatile profiles between the production methods (traditional vs. Charmat).

Recent developments in the analysis of musty odour compounds in water and wine: A review.

One of the most common taints in foods is a musty or earthy odour, which is commonly associated with the activity of microorganisms. Liquid foods, particularly wine and water, can be affected by this defect due to the presence of certain aromatic organic compounds at very low concentrations (ng/L) consistent with human threshold perception levels. The volatile compounds responsible for a mouldy off-aroma include approximately 20 compounds, namely, haloanisoles, geosmin, 2-methylisoborneol, several alkyl-methoxypyrazines, 1-octen-3-ol, 1-octen-3-one, trans-octenol, 3-octanone, fenchol and fenchone. Methods for determining these very low concentrations of odour compounds must be extremely sensitive and selective with efficient preconcentration treatments. A number of extraction techniques based on LLME (liquid-liquid microextraction), SPME (solid-phase microextraction) or SBSE (stir-bar sorptive extraction) can be applied and should be selected on a case-by-case basis. Moreover, recent developments in GC instrumentation coupled to different detection systems can effectively increase the selectivity and sensitivity of the analysis of target compounds.

Taxonomy and phylogeny of Trichuris globulosa Von Linstow, 1901 from camels. A review of Trichuris species parasitizing herbivorous.

At the present work, we carried out a morph-biometrical and molecular study of Trichuris species isolated from Camelus dromedarius from Iran and from Ovis aries from South Africa comparatively with other species of Trichuris from different herbivorous hosts and geographical regions. The population from camels from Iran was identified as Trichuris globulosa. Two different morphometrically populations of Trichuris sp. from sheep from South Africa were identified: Trichuris ovis and Trichuris skrjabini. Ribosomal data did not reveal significate differences in the ITS2 sequences between T. ovis and T. globulosa to assess a specific determination. The mitochondrial data suggest that T. globulosa constitute a different genetic lineage to T. ovis. Cytochrome c-oxidase and cytochrome b partial gene sequences corroborated the existence of a different genetic lineage of T. ovis from sheep of South Africa that would be closely related to the populations of T. globulosa from camels from Iran. The cytochrome c-oxidase and cytochrome b partial gene sequences of T. globulosa have been reported for the first time.

Impact of gluconic fermentation of strawberry using acetic acid bacteria on amino acids and biogenic amines profile.

This paper studies the amino acid profile of beverages obtained through the fermentation of strawberry purée by a surface culture using three strains belonging to different acetic acid bacteria species (one of Gluconobacter japonicus, one of Gluconobacter oxydans and one of Acetobacter malorum). An HPLC-UV method involving diethyl ethoxymethylenemalonate (DEEMM) was adapted and validated. From the entire set of 21 amino acids, multiple linear regressions showed that glutamine, alanine, arginine, tryptophan, GABA and proline were significantly related to the fermentation process. Furthermore, linear discriminant analysis classified 100% of the samples correctly in accordance with the microorganism involved. G. japonicus consumed glucose most quickly and achieved the greatest decrease in amino acid concentration. None of the 8 biogenic amines were detected in the final products, which could serve as a safety guarantee for these strawberry gluconic fermentation beverages, in this regard.

A survey of biogenic amines in vinegars.

This paper reports the determination of biogenic amines by high-performance liquid chromatography (HPLC) and fluorescence detection after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in balsamic, apple, and red, white, and Sherry wine vinegars. A solid-phase extraction (SPE) with mixed-mode resins method was used before analysis. The method was successfully validated obtaining adequate values of selectivity, response linearity, precision, accuracy, and low detection and quantification limits. The total content of biogenic amines in vinegars ranged from 23.35 to 1445.2 µg/L, being lower than those reported in wines. Putrescine was the amine that showed the highest concentrations in most samples. Methylamine and phenylethylamine were not determined in any vinegar. Balsamic and "Pedro Ximénez" Sherry vinegars reached the highest amounts of biogenic amines, while apple, white and Sherry wine vinegars had the lowest concentrations. Principal component analysis using the biogenic amines as variables, allowed to separate the different kind of vinegars, excepting red vinegars.

16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations.

Comparative morphological, biometrical and molecular studies of Trichuris discolor isolated from Bos taurus from Spain and Iran was carried out. Furthermore, Trichuris ovis isolated from B. taurus and Capra hircus from Spain has been, molecularly, analyzed. Morphological studies revealed clear differences between T. ovis and T. discolor isolated from B. taurus but differences were not observed between populations of T. discolor isolated from different geographical regions. Nevertheless, the molecular studies based on the amplification and sequencing of the internal transcribed spacers 1 and 2 ribosomal DNA and 16S partial gene mitochondrial DNA showed clear differences between both populations of T. discolor from Spain and Iran suggesting two cryptic species. Phylogenetic studies corroborated these data. Thus, phylogenetic trees based on ITS1, ITS2 and 16S partial gene sequences showed that individuals of T. discolor from B. taurus from Iran clustered together and separated, with high bootstrap values, of T. discolor isolated from B. taurus from Spain, while populations of T. ovis from B. taurus and C. hircus from Spain clustered together but separated with high bootstrap values of both populations of T. discolor. Furthermore, a comparative phylogenetic study has been carried out with the ITS1and ITS2 sequences of Trichuris species from different hosts. Three clades were observed: the first clustered all the species of Trichuris parasitizing herbivores (T. discolor, T. ovis, Trichuris leporis and Trichuris skrjabini), the second clustered all the species of Trichuris parasitizing omnivores (Trichuris trichiura and Trichuris suis) and finally, the third clustered species of Trichuris parasitizing carnivores (Trichuris muris, Trichuris arvicolae and Trichuris vulpis).

Validation of an analytical method for the determination of ethyl carbamate in vinegars.

A solid phase extraction method (SPE) using Isolute ENV+ cartridges was validated for the determination of ethyl carbamate (EC) in different kinds of vinegars. The method proved to be quite sensitive, precise and accurate, improving the recovery and LQD of other existing methods for the same purpose. For the optimization of the method, different pH values of the sample were tested, resulting 5.5 the most adequate. Among the 14 samples analysed, only 5 of them had contents of EC above the quantification limits, ranging between 6.73 µg/L and 56.4 µg/L. The highest value was found in red wine vinegar. Taking into account the amount of vinegar consumed in a meal and the limits established for alcoholic beverages in some countries, the levels of ethyl carbamate in the vinegars tested in this work were acceptable.