Trichoderma spp. on various groups of plants, mostly crops. However, there is little information on the influence of these fungi on ornamental plants. Therefore, the authors of this study analyzed the influence of Trichoderma spp. on the growth, flowering, quality, and nutritional status of ornamental plants. The research showed that Trichoderma spp. in this group of plants stimulate the elongation and thickening of shoots and the formation of leaves. These fungi also stimulate or inhibit leaf elongation. They also accelerate the flowering of plants, stimulate the elongation of inflorescence shoots and inflorescences, and the development of flowers. Apart from that, Trichoderma spp. positively influence the content of chlorophyll and carotenoids in leaves, and they stimulate the uptake of micro- and macroelements.Scientists all over the world conduct research to determine the influence of  Rhizobium), and Plant Growth-Promoting Rhizobacteria (PGPR). Thus, microorganisms applied to plants can have a dual function of biocontrol agent and biostimulant [A plant biostimulant is any substance or microorganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance, and/or crop quality traits, regardless of their nutrient content. Many biostimulants improve nutrition and they do so regardless of their nutrient contents. Biofertilizers increase nutrient use efficiency and open new routes of nutrients acquisition by plants. In this sense, microbial biostimulants include mycorrhizal and non-mycorrhizal fungi, bacterial endosymbionts  in monocotyledonous and dicotyledonous plants as a result of biotic and abiotic stress. Owing to these properties, they are classified as Biological Control Agents (BCA) that are used commercially as biopesticides or biostimulants in the production of plant protection products. Trichdoderma spp. produce many biologically active compounds, such as enzymes  [Trichoderma spp. are included in microbiological formulations used to optimize the composting of materials of various origins [Fungi of the tibiosis . These fum genus ,5. Recenum genus . Howevermylases) , antibiomylases) ,8,9,10, mylases) ,11. Owin origins .Trichoderma spp. are widely described as plant growth stimulators. This trait tends to be isolate-specific rather than species-specific, whereas individual isolates show different degrees of plant specificity. Increased root and/or shoot biomass is the most common manifestation of growth stimulation; however, changes in plant morphology and development have been described too. Growth stimulation can be highly variable due to several limiting factors, such as crop type, conditions, inoculum dose, and type of formulation [Trichoderma spp. involves the recognition of molecules derived from the fungi, such as auxins and micro-organic Volatile Organic Compounds (VOCs); however, this communication is highly dependent on the environment. Contreras-Cornejo et al. [Trichoderma spp. induce growth by an auxin-dependent mechanism. They used in vitro biological tests to demonstrate that T. virens Gv29.8 and T. atroviride IMI206040 can synthesize indole-3-acetic acid (IAA) and some of its derivatives, which results in vigorous development of the root system. These authors claim that many strains of Trichoderma spp. are capable of synthesizing IAA, but only some of them can stimulate plant growth. Some researchers point out that Trichoderma spp. stimulate plant growth because they enable plants to absorb more nutrients and support the production of vitamins and growth regulators [Trichoderma bioinoculants are currently available on the market. Mixtures of strains are gaining popularity, as they ensure more consistent performance [mulation . Accordimulation , communio et al.  suggest gulators ,15,16. Aformance . The production of ornamental plants is one of the fastest growing areas in the horticultural sector. Of particular importance is the cultivation of potted ornamental plants, which has an upward trend in the international market worldwide . After tTrichoderma spp. on various groups of plants has been conducted around the world. It has been dominated by research on commercial, edible plants [Trichoderma spp. on the growth, flowering, quality, and nutritional status of ornamental plants.Research intended to determine the impact of e plants ,23,24. HTrichoderma was introduced into the literature by Persoon in 1794 [Hypocrea rufa was a teleomorph of T. viride Pers. [Trichoderma—T. viride [T. harzianum Rifai, T. viride, T. hamatum (Bonord.) Bainier, T. koningii (Oudem.) Duché & R. Heim, T. polysporum (Link) Rifai, T. piluliferum J. Webster & Rifai, T. aureoviride Rifai, T. longibrachiatum Rifai, and T. pseudokoningii Rifai. In the early 1990s, Bissett [Trichoderma. The introduction of such tools as restriction fragment length polymorphism markers (RFLP), random amplified polymorphic DNA markers (RAPD), and phylogenetic markers of coding sequence variation for the molecular identification of species had a significant impact on the development of taxonomy at that time. From the late 1990s to 2002, the number of Trichoderma species increased to 47 [Hypocrea and Trichoderma species—TrichO Key version 1.0 [Trichoderma listed in Index Fungorum [The name of the genus  in 1794 . The gen in 1794 . In 1865de Pers. . Until 1. viride . Then, i. viride  distingu Bissett ,31,32 ided to 47 . Verificed to 47 , who anasion 1.0 , has conFungorum . HoweverFungorum .Trichoderma spp. exist in conidial (imperfect) stages, which makes them unable to reproduce sexually, as well as in perfect stages, such as Hypocrea; in which case, sexual reproduction is possible. The germinating spores, or chlamydospores, develop by forming simple or branched conidiophores, the conidial spores of which are spherical or ellipsoidal in shape. Sporulation depends on the availability of nutrients and light, temperature, and competition from other microorganisms [Trichoderma spp. form circular conidial zones, formed by fungal colonies made up of bundles of conidiophores, clumped or loose. The surface of the colony resembles cotton wool. The color of the conidia varies from white-green to dark green, depending on the species  and methylamine (CH3NH2). The most frequently used source of N is ammonia (NH3); however, Trichoderma spp. also use amino acids, urea (CO(NH2)2), nitrites, and nitrates. When the concentration of N in the substrate increases, many isolates respond by forming a mass of conidial spores and chlamydospores [Trichoderma spp. is found in soils rich in C and phosphorus (P) [Trichoderma spp. is also determined by abiotic factors  and biotic factors (interactions between microorganisms). In unfavorable environmental conditions, e.g., too high temperature, the conidia of Trichoderma spp. may die, as their walls are too thin. However, the fungi can survive thanks to the formation of thick-walled chlamydospores. Such a phenomenon is observed in T. hamatum, T. harzianum, T. virens, and T. viride. Trichoderma spp. are classified as mesophilic organisms, as the optimum temperature for the growth and development of those fungi is approximately 25 °C [T. viride and T. polysporum can grow at low temperatures. Additionally, cold-tolerant strains of T. viride, T. harzianum, and T. aureoviride can become parasites of phytopathogens such as Rhizoctonia solani, Fusarium oxysporum f. sp. dianthi at low temperatures (5–10 °C) by producing enzymes characteristic of the mycoparasitism: β-glucosidase; β-1,4,-N-acetylglucosaminidase; or trypsin and chemotrypsin proteases [The carbon(C)-to-nitrogen(N) ratio (C:N) has a great impact on the development of athogens . This isdospores . The favorus (P) . The devly 25 °C . Howeverroteases .Trichoderma spp. grow very fast when the pH of the substrate is 5–5.5, but they are easily decomposed in the light, as they are sensitive to UV radiation [Trichoderma spp. also takes place in alkaline substrate, with a large amount of carbon dioxide (CO2). Das et al. [adiation . Accordiadiation , the devs et al.  indicateTrichoderma spp. [Copper (Cu) ions can also affect the growth rate, sporulation, and enzymatic activity of rma spp. .Trichoderma spp. show high resistance to many toxic compounds produced by other microorganisms, including antibiotics, as well as to terpenoid phytoalexins and peroxidases secreted by plants, and to fungicides and heavy metals. Based on molecular studies, the resistance that makes those fungi active colonizers and strong competitors may be related to the ability of Trichoderma spp. to produce ABC protein transporters [Trichoderma spp. is limited [sporters . Those psporters . As a re limited . Trichoderma spp. are currently sold in the form of biopesticdes, biofertilisers, and stimulants for growth and natural resistance. The effectiveness of these fungi can be attributed to their ability to protect plants, stimulate vegetative growth, and restrict the population of pathogens, as well as to act as substrate additives (inoculants) that improve nutrient uptake capacity. Live fungal spores (active substance) are incorporated into a variety of preparations  that are used as solutions for spraying on the leaves, on seeds, and on young plants, in post-pruning treatments in the substrate for sowing or transplanting, as well as for watering or soaking of, e.g., spore organs such as tubers, bulbs, and rhizomes. Formulations based on Trichoderma spp. are sold across the world and used to protect crops against various plant pathogens and to stimulate the growth and productivity of plants in various growing environments, such as fields, greenhouses, nurseries, and in the production of various horticultural crops, fruit crops, trees, and ornamental plants  is placed in each dish  and by Andrzejak et al. [Begonia × tuberhybrida ‘Picotee Sunburst’ . Prisa et al. [Trichoderma genus can be very high, as they proved in Limonium sinuatum (100.0%). According to Błaszczyk et al. [Trichoderma spp. colonize the external layers of the roots of herbaceous plants and trees. They also have the ability to penetrate and colonize within roots, or occur as endophytes. These authors used Triticum aestivum as an example to demonstrate that a preliminary analysis of morphological, physiological, and metabolic changes indicates that there is no clear-cut plant response to fungi of the Trichoderma genus. This may mean that changes taking place in plants depend both on the genus/strain of Trichoderma spp. and on the cultivar of the species studied. According to Souza et al. [Trichoderma stimulates root growth by producing auxins [Trichoderma spp., genes such as ASA1 and MYB77 are induced. In the root, ethylene and auxin can regulate their biosynthetic pathways [Trichoderma contributes to exogenous auxin-stimulated ethylene biosynthesis through 1-aminocyclopropane-1-carboxylicacid synthase (ACC). In this model, the activity of Trichoderma ACC desaturase (ACCD) reduces the availability of ACC necessary for ethylene biosynthesis, and the reduction of ethylene stimulates plant growth through gibberellin (GA) signaling, increasing the degradation of DELLA proteins, which are repressors of GA signaling. Moreover, GAs can control the onset of jasmonic acid (JA) and salicylic acid (SA)-dependent plant defense responses by regulating the degradation of DELLA proteins [Trichoderma.pidermis . ResearcJanowska  report ta et al.  in Freesk et al.  in Begona et al.  pointed k et al. , in the a et al. , interaca et al. . Plant ra et al. ,64. As mg auxins . During pathways . Accordipathways , IAA of proteins . Therefoproteins ,68. Accoproteins , when MYTrichoderma fungi stimulate the growth of roots, as well as growth in the length and thickness of shoots and leaf surface. However, Lorito et al. [Trichoderma genus do not affect the height and number of shoots in Begonia × tuberhybrida ‘Picotee Sunburst’, but they do stimulate leaf development in it . Moreover, plants treated with T. viride show an increased vitamin C content in leaves with greater dry mass , and they ended in longer inflorescences (by 10.0%) with more flowers developing in them (by 12.6%). However, the diameter of flowers in both years of research was not affected by Trichoderma spp. treatment. Partially similar results were obtained earlier by Sisodia et al. [Trichoderma spp. in eight cultivars of Gladiolus sp. had a positive impact on the length of the inflorescence and the duration of flowering, but had no influence on the number of flowers. Furthermore, da Cruz et al. [Trichoderma spp. applied in the cultivation of Gladiolus ‘Peter’s Pear’ had no influence on the quality of inflorescences, expressed in terms of the length of inflorescence shoot, length of the inflorescence, and the number of flowers. According to the research conducted by Andrzejak et al. [Trichoderma spp. stimulate the development of buds and flowers, and influence their size in Begonia × tuberhybrida ‘Picotee Sunburst’. The results they obtained have been confirmed by research conducted by Janowska et al. [Trichoderma spp. stimulate the development of lateral inflorescence shoots and the development of flowers in Freesia reflacta ‘Argentea’, especially in the plants provided with assimilation lighting during cultivation. According to Prisa [Trichoderma genus stimulate flowering in Pachyphytum oviferum and Crassula falcata.In some ornamental plant species,  flowers . Andrzeja et al. . The autz et al.  report tk et al. , Trichoda et al. . The autto Prisa , fungi oTrichoderma spp., occurs as a result of an increase in the number of photosynthetic pigments or the expression of genes regulating the biosynthesis of chlorophyll, proteins in the light-harvesting complex, or components of the Calvin cycle. The colonization of crop roots by Trichoderma spp. fungi causes greater regulation in genes and pigments that improve photosynthesis in plants. Plants under physiological or environmental stress lose the ability to photosynthesize, as photosystems get damaged, and many cellular processes get disrupted by Reactive Oxygen Species (ROS). Yet, some strains of Trichoderma spp. activate biochemical pathways that reduce ROS to less harmful molecules. This and other mechanisms make plants more resistant to biotic and abiotic stresses. Moreover, when the indicators of photosynthesis are increased, more carbon dioxide (CO2) gets absorbed from the atmosphere. Carotenoids, on the other hand, are responsible for the stability of lipid membranes, are involved in the accumulation of light during photosynthesis, and in the protection against photooxidation caused by the ROS formed during chlorophyll excitation during photosynthesis [Chlorophylls are a widespread group of photosynthetic pigments found in higher plants, algae, and cyanobacteria. Chlorophyll is a pigment that plays a key role in the normal course of photosynthesis, in which energy from light is converted into chemical bond energy as a result of the absorption of quanta of light in redox reactions . Therefoynthesis ,80. The ynthesis . Carotenynthesis .Trichoderma spp. on the content of chloroplast pigments in leaves refer to commercial, edible species [Trichoderma spp. applies to ornamental plants too. Andrzejak and Janowska [Gladiolus hybridus ‘Advances Red’ increased significantly following the application of Trichoderma spp. (by 66.7% for chlorophyll a+b and by 33.3% for carotenoids). The results obtained indicate that the photosynthetic capability improved in the ‘Advances Red’ cultivar. Andrzejak et al. [Trichoderma spp. stimulate the production of chlorophyll, whose content is reflected by the greenness index, in Begonia × tuberhybrida ‘Picotee Sunburst’  and calcium (Ca) by these plants. In both years of the study, the levels of P, K, and Ca found in the leaves of plants treated with Trichoderma spp. were significantly higher than in the leaves of control plants. Similar outcomes were obtained by Janowska et al. [Freesia reflacta ‘Argentea’. The authors report that Trichoderma spp. stimulate the absorption of P and Ca in non-illuminated and illuminated plants of this species. They also stimulate the absorption of K in illuminated plants . The benefits of using microorganisms to improve the uptake of nutrients present an opportunity for the latest horticultural practices, as they allow for reducing fertilizer use. The use of biological fertilizers based on microorganisms is an alternative for maintaining high productivity levels and keeping the environmental impact low ,86,87. BTrichoderma spp. impact the uptake of zinc (Zn), Fe, and boron (B) by Gladiolus hybridus ‘Advances Red’ plants [Begonia × tuberhybrida ‘Picotee Sunburst’, Trichoderma spp. also stimulated the uptake of Zn, Fe, and B , and Zn in illuminated and non-illuminated plants, whereas illumination and Trichoderma spp. stimulated the uptake of copper (Cu) .Micro-nutrients play a key role in the metabolic and physiological processes in plants. It is worth noting that they influence quality  more tha’ plants . Accordi’ plants , in the e, and B . Moreoveper (Cu) . Accordie et al. , using TTrichoderma spp. are widely described as plant growth stimulators. This trait tends to be isolate-specific rather than species-specific, and individual isolates show different degrees of plant specificity. Increased root and/or shoot biomass is the most common manifestation of growth stimulation; however, changes in plant morphology and development have been described too. Scientists all over the world conduct research to determine the influence of Trichoderma spp. on various groups of plants, mostly crops. However, there is little information on the influence of these fungi on ornamental plants. Trichoderma spp. in this group of plants is also an effective biostimulant. Trichoderma spp. are important tools in promoting the growth and flowering of ornamental plants. With them, the use of fertilizers can be reduced, thus protecting the environment. The use of Trichoderma spp. should be widespread, not only in the cultivation of edible plants, but also in ornamentals.


Vibrio pose a threat to both humans and animals, creating medical burdens and economic losses to the mariculture industry. Improvements in surveillance and diagnosis are needed to successfully manage vibriosis outbreaks. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can provide rapid diagnosis and has been widely used in the identification of Vibrio spp. The main weakness of this technology is the limited number of strains and species of Vibrio in the existing commercial database. Here, we develop a new in-house database named PVBase containing 790 main spectra projections (MSP) of ten Vibrio species that come from various regions of China and include abundant clinical and environmental strains. PVBase was validated through a blind test of 65 Vibrio strains. The identification accuracy and scoring of Vibrio strains was greatly improved through the addition of PVBase. Identification accuracy increased from 73.4 to 100%. The number of strains with identification scores above 2.2 increased from 53.1% to 96.9% and 53.1% of strains had an identification score above 2.59. Moreover, perfect discrimination was obtained when using all of the MSPs created for the Vibrio species, even for very closely related species such as V. cholerae, V. albensis, and V. mimicus or V. alginolyticus, V. parahaemolyticus, and V. harveyi. In addition, we used phyloproteomic analysis to study whether there are differences in protein fingerprints of different regions or pathogenic strains. We found that MSP characteristics of Vibrio species were not related to their region or source. With the construction of PVBase, the identification efficiency of potentially pathogenic Vibrio species has been greatly improved, which is an important advance for epidemic prevention and control, and aquaculture disease detection.The potentially pathogenic species of the genus  Vibrio is a Gram-negative bacterium that can have short rod, curve, S or spiral shapes and is widely distributed in sea water and in marine animals in estuaries, bays, and coastal waters. As of 10 December 2021, a total of 133 Vibrio species had been identified1. While not all Vibrio species are pathogenic, about 20 pathogenic Vibrio species are recognized worldwide and about 12 of these can cause disease in humans  have more than five strains in the database: V. vulnificus (11), V. parahaemolyticus (9), V. harveyi (7), and V. diazotrophicus (6). All other Vibrio species in the database are represented by fewer than five strains, and most have only one strain. This limited number of strains can greatly affect the identification accuracy of potentially pathogenic Vibrio species. In this study, we used 790 strains of Vibrio spp., collected from various regions of China and purchased from the standard bacterial library, to construct an in-house database of potential pathogenic Vibrio spp. that we call PVBase. PVBase increased the diversity of known strains in China and improved accuracy of the identification of potential pathogenic Vibrio spp. in different application scenarios by MALDI-TOF MS. To explore the genetic relationship and the characteristics of strains of Vibrio species from different regions, as well as clinical vs. environmental strains, we performed dendrogram analysis of different Vibrio species from the Bruker MBT Library and PVBase. In addition, we validated the newly created database through a blind test using 65 strains (absorbed into the database after the blind test was completed.). The establishment of PVBase enables accurate, rapid identification of a large number of Vibrio spp. and contributes to enhancing clinical diagnosis and treatment, restriction of pathogen transmission, and control of large-scale spread of diseases.At present, the latest database of MALDI BioTyper Library ver. 11.0.0.0  contains 54 species of Vibrio strains were analyzed. Briefly, most the strains were obtained from Zhejiang, Guangzhou, Guangdong, Liaoning, Taiwan, Shandong, Tianjin, Shanghai, and Beijing, and a small number of strains were shared from the Marine Culture Collection of China (MCCC) and the American Type Culture Collection (ATCC). The geographic information of the strains was shown in A total of 790 potentially pathogenic V. alginolyticus, V. harveyi, V. fluvialis, V. fischeri, V. navarrensis, V. metschnikovii, and V. vulnificus was 30°C and for V. cholerae, V. parahaemolyticus and V. mimicus growth temperature was 37°C. Type strains were incubated at the recommended temperature.Before all experiments, strains were grown on marine agar 2216 medium , and incubated for 24–48 h, at their appropriate growth temperature. After this, a single colony forming unit (CFU) was plated and incubated for 24 h, at growth temperature, to ensure purity of the isolates. The growth temperature of g for 2 min. The supernatant was discarded and the pellet was centrifuged a second time to remove ethanol residues and then dried at room temperature. Pellets were dissolved into 30 μL each of 70% formic acid (Thermo Fisher) and acetonitrile (Thermo Fisher). The solution was carefully mixed by pipetting up and down and then centrifuged at 13,000 × g for 2 min. Subsequently, 1 μL of supernatant was spotted onto an MSP 96 target polished steel BC plates in eight replicates and overlaid with 1 μL HCCA  matrix solution  after air-drying. All V. cholerae from Zhejiang were processed in the Zhejiang Centers for Disease Control laboratory according to the above-mentioned treatment methods and carried to our laboratory for the collection of the spectra.For database entry, strains were prepared with the formic acid extraction method to ensure a high quality of spectra. Fresh colonies were picked with a 1 μL inoculation loop and placed in 300 μL of LC–MS water . A volume of 900 μL pure ethanol (Thermo Fisher) was added to sterilize the bacteria and denature proteins. The tube was vortexed for 1 min and centrifuged at 13,000 × Following the manufacturer’s directions, a bacterial test standard (BTS) was used on each acquisition plate. A BTS can calibrate the instrument before each acquisition session to ensure quality of the acquisition.Spectra were acquired using the Microflex LT system (Bruker). Each spot was measured three times, resulting in 24 single spectra for each strain. The software flexControl v. 3.4 automatically acquired the spectrum of each spot through AutoXecute. Signal intensity of the highest peak in the spectra was maintained between 1,000 and 5,000 by fine-tuning the laser intensity. The linear positive ion mode was used as the acquisition mode of the instrument and parameters were set as follows: lens, 8.5 kV; ionsource1, 20 kV; ionsource2, 18.1 kV; mass range: 2,000–20,000 Da; laser frequency: 60 Hz; Shots/Spectrum: 240. The instrument was calibrated using calibrators  with molecular weights of 3,637.8 Da, 5,096.8 Da, 5,381.4 Da, 6,255.4 Da, 7,274.5 Da, 10,300.1 Da, 13,683.2 Da, and 16,952.3 Da. The masses of the measured spectrum were within the tolerance range of ± 300 ppm from nominal value.V. parahaemolyticus, V. cholerae, V. alginolyticus, V. harveyi, and V. vulnificus to observe the changes in the highest matching scores identified by different sizes of MSP present.Baseline subtraction and smoothing were performed after importing the spectra into flexAnalysis v. 3.4 (Bruker). Next, we searched the spectra set for flatline spectra, sweet spot outliers and anomalies, removing such spectra by closing out of the set. A minimum of 18 spectra were required for main spectra projections (MSP) creation; where necessary the measurement was repeated using a new sample preparation to achieve this. In the peak shift of individual masses, we selected peaks from 3,000 to 10,000 Da in steps of 1,000. The allowed peak shift between the spectra with the smallest and the largest mass was 500 ppm. Finally, we selected the remaining spectra in flexAnalysis v. 3.4 and used these data to create the MSP through MALDI BioTyper Compass Explorer v. 4.1 software. All MSPs were registered in our in-house database, PVBase. In addition, we established separate databases of different sizes for Vibrio spp. were involved in this study, 65 strains used in the blind test and 725 strains used in the PVBase database. The 65 strains for blind test were first identified using Bruker MBT Library, and then identified using a database merged Bruker MBT Library with the PVBase. Then we compared the identification results of the two databases, and analyzed the changes in the identification accuracy and accuracy scores of related strains. We also used the Moussa database  and composite correlation index (CCI) were used to analyze the differences between and within the species of Vibrio species that are potentially pathogenic in the Bruker MBT Library and our PVBase are shown in V. vulnificus, V. cholerae and V. parahaemolyticus had a wide range of sources and relatively large numbers, while the remaining Vibrio species had relatively small number of strains.In this study, a total of 790 strains were collected and included in the database. In order to select the most representative spectra and merge them into strain-specific MSP, we searched 790 spectra set for sweet spot and removed anomalies by closing out of the set by flexAnalysis ver.3.4 . All 790 MSPs were registered in an in-house database called PVBase. The numbers of MSP for the V. parahaemolyticus, V. cholerae, V. alginolyticus, V. harveyi, and V. vulnificus to observe changes in the highest matching scores with number of MSP present. Except for V. cholerae, which is not contained in Bruker MBT Library (V. parahaemolyticus, V. alginolyticus, V. harveyi, and V. vulnificus were all obtained by matching the Bruker MBT Library. In V. harveyi, the highest matching score had been on an upward trend, and had not yet reached saturation as the MSP increases. It further illustrated the necessity of increasing the number of strains in the database. For V. parahaemolyticus, V. vulnificus, V. alginolyticus, and V. cholerae the highest matching score also increased initially, but then plateaued, suggesting saturation was reached (We looked for an effect of database size on identification by establishing separate databases of different sizes of  Library , the ini reached , 3. The V. alginolyticus. The initial matching score of V. alginolyticus obtained by matching the Bruker MBT Library was 1.65–1.85, and the best matches for strains were V. mytili, V. parahaemolyticus or V. harveyi, followed by V. alginolyticus. In addition, some strains also had not been accurately identified. With the continuous increased in the number of MSPs in PVBase, the identification accuracy of V. alginolyticus had been significantly improved. When the number of MSPs was 30, all V. alginolyticus were accurately identified, and the identification score increased to approximately 2.4 (The importance of increasing database size is further clarified through in-depth consideration of results with tely 2.4 .V. cholerae were not identified, and the remaining 11 strains were all identified as V. albensis. In addition, one strain of V. harveyi was erroneously identified as V. parahaemolyticus. The remaining Vibrio species were correctly identified, and the initial identification scores of V. parahaemolyticus, V. vulnificus and V. navarrensis were highest. The test strain identifications were then obtained through PVBase and the Bruker MBT Library together (Vibrio species were correctly identified, and the identification score was generally greater than 2.2; identification scores of some strains of V. parahaemolyticus, V. vulnificus, V. cholerae, V. navarrensis and V. metschnikovii exceeded 2.59. Thus, the addition of the PVBase database greatly improved the accuracy and matching scores when identifying Vibrio species.Of the 790 strains collected in this study, 65 strains were used for the blind test, and the remaining strains were used as databases for verification The test spectra were first identified using the Bruker MBT Library . Four sttogether . After tVibrio spp., we randomly selected 77 strains of all Vibrio species, which included those in PVBase and 48 strains of all Vibrio species included in the Bruker MBT Library as representatives. We then performed a dendrogram analysis using MALDI BioTyper Compass Explorer ver.4.1 software (Vibrio including V. mimicus, V. cholerae and V. albensis, which were previously reported indistinguishable species (Vibrio species could not be clearly distinguished. The MSP of one strain of V. furnissii clustered in the group of V. fluvialis. In addition, the similarity between V. ordalii and V. gazogenes or V. mytili and V. harveyi was also high, and these species pairs cannot be distinguished easily by MSPs. However, we had few strains of these indistinguishable Vibrio species, so the relevant conclusions need to be further verified.To observe the genetic relationship between software . Accurat species . Only a Vibrio species randomly selected from PVBase (V. fluvialis and V. furnissii were relatively difficult to distinguish because of their high similarity. In addition, similarity between the Vibrio species obtained by the color depth of the CCI matrix was consistent with the dendrogram analysis.We performed a CCI analysis on the 77 strains of m PVBase . ConsistVibrio species have regional characteristics, we ran separate cluster analyses on strains of V. vulnificus and V. parahaemolyticus. V. vulnificus was collected from Taiwan, Beijing, Guangdong, and Zhejiang. Dendrogram analysis showed that using a distance level of 900, the species could be divided into three groups, but there was no obvious correlation between MSP characteristics of the strain and region (V. parahaemolyticus reached a similar conclusion. Dendrogram analysis on V. parahaemolyticus collected from Zhejiang, Liaoning and Guangzhou indicated that V. parahaemolyticus could be divided into two groups using a distance level of 950, but that group characteristics were not directly related to region (V. parahaemolyticus according to the division of north and south regions, but no statistical difference was obtained (To explore whether d region . The anao region . In addiobtained . The sumV. vulnificus and V. parahaemolyticus to explore whether either species had characteristic differences between clinical and environmental strains. Dendrogram analysis for V. vulnificus with a distance level of 900, divided the species into three groups, but there is no obvious correlation with clinical vs. environmental strain type (V. parahaemolyticus could be divided into two groups using a distance level of 950, but the groupings were not related to strain type (We clustered ain type . Likewisain type .Vibrio spp., providing high sensitivity and high throughput. MALDI-TOF MS can detect the activity of bacteria, and also has obvious advantages in large-scale detection in clinical and farm handling. Database size is a key factor affecting the accuracy of MALDI-TOF MS identification. We constructed an in-house database of 790 strains of Vibrio spp. that greatly supplemented the Bruker MBT Library with potentially pathogenic Vibrio species. Increasing the number of strains in the database improved the accuracy of identification, and increasing the number of Vibrio species expanded the scope of identification (V. parahaemolyticus, V. cholerae, V. alginolyticus, V. harveyi, and V. vulnificus, each of which have a large number of strains, as examples with which to explore the impact of increasing the number of strains in the database. With the large number of V. parahaemolyticus, V. cholerae, and V. vulnificus, the matching score gradually rose with increased number of MSPs until it tended to saturate. These values where matching score saturates represent the minimum number of main spectra required to account for intraspecific variation. Although the total number of added strains of V. alginolyticus was only 30, the identification score also appeared to become saturated. This may be due to low intra-species variability in V. alginolyticus, so that relatively few strains were required to saturate the identification score. After the establishment of PVBase, the blind test experiment showed strong improvement of identification accuracy and score for Vibrio species with the database expansion. Identification accuracy of the 65 strains in the blind test increased from 73.4 to 100%. The percentage of strains with identification scores above 2.2 increased from 53.1% to 96.9%. In addition, 53.1% of strains had identification scores above 2.59.Although isolated strains are necessary for MALDI-TOF MS identification, which is time consuming, it plays a pivotal role in the identification of fication . We tookVibrio species in the Bruker MBT Library and PVBase combined revealed good differentiation at the species level for Vibrio. V. cholerae, V. mimicus and V. albensis are closely related, as previously observed (V. cholerae and only one strain of V. albensis in the Bruker MBT Library, all V. cholerae were initially erroneously identified as V. albensis. In this study, we found that due to the small number of strains of V. furnissii and V. fluvialis, the MSP of one strain of V. furnissii we generated was clustered into the group of V. fluvialis. Similarly, the pairs V. ordalii and V. gazogenes plus V. mytili and V. harveyi also were clustered into each other’s species group. However, this does not necessarily mean that they are difficult to distinguish and require a large number of strain MSPs to support further inferences. The number of strains in the Bruker MBT Library is small, and most of them come from countries outside of China. There may be some differences in Chinese strains such that they do not adequately reflect the diversity of the Vibrio species spectra. Certainly, our findings suggest that including only one MSP in the database is not sufficient for species identification, perhaps partly due to high heterogeneity among different regions.The dendrogram analysis of observed , but theVibrio species in the latest database of the Bruker MBT Library is small. This also explains why V. alginolyticus was misidentified as V. mytili, V. parahaemolyticus or V. harveyi when identified using the Bruker MBT Library. These species are nearly indistinguishable from each other by 16S rRNA gene analysis, requiring the analysis of multiple housekeeping genes for accurate species identification (V. alginolyticus strains exhibited 56–80% similarity with V. parahaemolyticus strains. The MSPs of V. parahaemolyticus exhibited 40–45% similarity to those of V. mimicus (Vibrio species in the Bruker MBT Library and PVBase similarly indicated a close genetic relationship among V. alginolyticus, V. mytili, V. parahaemolyticus and V. harveyi that further explains why some strains of V. alginolyticus were misidentified initially. However, V. alginolyticus could be accurately identified after expanding the database. Furthermore, after adding MSPs of V. cholerae to the database, all V. cholerae were accurately identified. It is clear that augmenting the database makes it possible to discern otherwise indistinguishable species.Expanding the database could improve the identification accuracy of indistinguishable species. Although the Bruker MBT Library is the most widely used database, it lacks coverage of environmental bacterial isolates, particularly marine bacteria . Therefofication , 2007. T mimicus . It is wVibrio strains in this study came from different regions in China and included environmental and clinical strains. We analyzed the characteristics of Vibrio strains from different regions or environmental and clinical strains in order to explore the influence of the above factors on the MSP characteristics of Vibrio species. However, we did not find statistically significant patterns. Although clinical strains are mainly derived from hospital patients, patients may be infected from various regions, so this may have confounded the analysis. Previous studies showed that the characteristics of Vibrio species from different regions may differ (V. parahaemolyticus isolated from Japan, exhibit more similarities than spectra of the strains isolated from the United States (V. parahaemolyticus strains may reflect strain adaptation to a particular geographic location. However, a V. parahaemolyticus environmental strain isolated from Florida exhibited similarities to the environmental strains isolated from North Carolina (Vibrio species. Although our strains come from a wide range of regions in China, they all belong to the coastal areas connecting Oceania, and the extensive exchanges between regions may eliminate differences to a certain extent. Moreover, it may be that all strains are from China, and that domestic differences are not enough to distinguish them (Vibrio species.The y differ  and thaty differ , 2018. Fd States . DiffereCarolina . This maish them . In the The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. All main spectra of PVBase are available as btmsp file as a supplement.JLW, BZ, and WX: conceptualization and writing—review and editing. BZ, TL, and WX: methodology. LK and JL: software. TL, LK, and JX: validation. TL, JX, and WX: formal analysis. JW, SG, and JX: investigation. YL and BY: resources and data curation. TL, JX, and LK: writing—original draft preparation and visualization. YY and YL: supervision. BZ, JLW, and WX: project administration. BZ, JLW, and WX: funding acquisition. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Globally, 80% of the burdenof major depressive disorder (MDD) pertains to low- and middle-income countries. Research into genetic and environmental risk factors has the potential to uncover disease mechanisms that may contribute to better diagnosis and treatment of mental illness, yet has so far been largely limited to participants with European ancestry from high-income countries. The DIVERGE study was established to help overcome this gap and investigate genetic and environmental risk factors for MDD in Pakistan.DIVERGE aims to enrol 9000 cases and 4000 controls in hospitals across the country. Here, we provide the rationale for DIVERGE, describe the study protocol and characterise the sample using data from the first 500 cases. Exploratory data analysis is performed to describe demographics, socioeconomic status, environmental risk factors, family history of mental illness and psychopathology.Many participants had severe depression with 74% of patients who experienced multiple depressive episodes. It was a common practice to seek help for mental health struggles from faith healers and religious leaders. Socioeconomic variables reflected the local context with a large proportion of women not having access to any education and the majority of participants reporting no savings.DIVERGE is a carefully designed case–control study of MDD in Pakistan that captures diverse risk factors. As the largest genetic study in Pakistan, DIVERGE helps address the severe underrepresentation of people from South Asian countries in genetic as well as psychiatric research. Large sample size of South Asian participantsGenome-wide genotyping for all participantsComprehensive assessment of diverse risk factors, incl. population-specific onesDetailed psychopathology assessmentFocus on women’s mental health and hormonal factorsInterview by trained mental health professionalsRecontactable participantsRecruitment at multiple sites across Pakistan for better representativenessLarge proportion with severe Major Depressive DisorderQuasi matched controls to enable observational assessment of risk factorsCo-led by local researchersCommunity engagementData will be available via data access committeeDepression is a major contributor to the burden of disease affecting about 300 million people worldwide . DepressIn low- and middle-income countries (LMICs), only an estimated 4% of affected individuals receive minimally adequate treatment  is the first large comprehensive case–control study of MDD in Pakistan, and more widely in South Asia. Its aims are to investigate (a) the genetic influences of MDD, (b) nongenetic risk factors in the Pakistani population, and (c) their interactions.https://www.genes-and-mental-illness.com) that also includes the GEN-SCRIP study about the GENetics of SChizophRenia in Pakistan and the GEN-BLIP study about the GENetics of BipoLar Disorder In Pakistan.DIVERGE is a part of the Pakistan Alliance on genetic RisK factors for Health . Cases are enrolled from Psychiatry Clinics or Outpatient Psychiatry Departments of Hospitals. This ensures the recruitment of moderate-to-severe MDD cases, which is likely to be advantageous for genetic discoveries. Referral is made by treating clinicians.DIVERGE aims to enrol 9000 cases and 4000 controls for a clinical interview and a blood sample for DNA extraction. Recruitment occurs in multiple cities across Pakistan in both private and public clinics to capture ethnic diversity and ensure a wide representation of different socioeconomic strata  , 2013 anControls are recruited from nonpsychiatry outpatient departments of the same hospitals or clinics. This strategy is implemented to recruit controls from the same population to enable comparisons of risk factors .The interview aims to capture potential risk factors for depression, including well-established as well as population-specific ones, and to provide an in-depth characterisation of the disease in the cohort. Questionnaires are administered through a structured interview to enable inclusion of participants with low levels of literacy and digital technology usage . The intThe interview questions were put together in English and translated into Urdu using a back-and-forth method in case there was no previous translation . Urdu is the official language in Pakistan and is widely spoken by the majority of the population  . All individuals provided informed consent, were confirmed eligible, and their data were included in the study for exploratory data analysis. The mean age of participants was 38 years old  . Age was reported by all the participants.During the first month, the study recruited 500 participants  diagnosed with MDD in 10 data collection sites across Pakistan , which is considered early depression onset . Over a third of participants (37%) reported exposure to at least one traumatic event. This aligns well with reported prevalence of exposure to traumatic events in other LMICs (http://links.lww.com/PG/A294).Apart from depression, participants mentioned anxiety symptoms more often than other mental health problems  reported a history of taking medications for their depression, whereas the rest received their first prescription at the time of referral to the interview.Almost half  reported that they had received other forms of help for their depression, most frequently through a religious leader or a faith healer. Both terms are often used interchangeably and refer to traditional spiritual practice. This practice is widespread in Pakistan, and religious leaders and faith healers are usually the first points of contact for people who believe in the supernatural origin of mental illness and those who look for a religious cure, irrespective of their perception of the disease origin .In line with other studies in LMICs .Our study has some limitations. Patients are recruited in psychiatric hospital settings. This strategy restricts enrolment to those who have access to the healthcare system. Moreover, an observational case–control design is unable to establish temporal precedence of environmental factors with certainty and may be subject to biases, such as recall bias. Matching of controls in terms of hospital recruitment, demographic characteristics and ethnicity aims to reduce confounding when assessing risk factors. Another limitation of the study is that it restricted participation to those over 18 years old. This decision was made due to multiple reasons. First, because of the study’s cross-sectional design and inability to estimate the proportion of those whose MDD diagnosis would precede bipolar disorder onset  under the European Union’s Horizon 2020 research and innovation programme (Grant agreement No. 948561).Conceptualisation: K. Kuchenbaecker and M. Ayub. Methodology: K. Kuchenbaecker, M. Ayub, A. Hassan, G. Lewis and J. Knowles. Data collection: A. Hassan, M.I. Hussain, K. Mufti, A.A. Mufti, M. Tariq, S. Shafiq, A.R. Choudhary, M.N. Ali, A. Hussain, G. Ali, M. Rehman, S.U. Din, A.B. Mustafa, I.A. Dogar, S.A. Gill, M.K. Khan, S.T. Chaudhry, S.Q. hyder, M. Ali, N. Ilyas, P. Channar, N. Mughal, S. Channa, M. Ansari, F. Hussain, I. Khattak, M. Umar, B. Khizar, T. Nasr, S. Farooq, F. Naeem and N. Ahmad. Data analysis: M. Valkovskaya and E. Zartaloudi. Writing - original draft preparation: M. Valkovskaya, E. Zartaloudi, A. Hassan, K. Kuchenbaecker and M. Ayub. Writing - review and editing: all authors. Funding acquisition: K. Kuchenbaecker. Supervision: K. Kuchenbaecker and M. Ayub.Ethics approval: the study was performed in line with the principles of the Declaration of Helsinki. All procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation. All procedures involving human subjects/patients, questionnaire and methodology were approved by the National Bioethics Committee of Pakistan (NBC-692). Ethical approvals have also been obtained from ethics boards of University College London (14125/002) and all collaborating institutes and hospitals where recruitment takes place.Availability of data: the dataset analysed during the current study is available from the corresponding author on reasonable request. Upon completion of recruitment and genotyping, the DIVERGE data will be made available to bona fide researchers and access will be governed by a Data Access Co-ordinating Committee.


Escherichia coli from the milk of Ettawa crossbreed dairy goat at Blitar Regency, East Java, Indonesia, with the antibiotics streptomycin, sulfonamides, and trimethoprim.Antimicrobial resistance, especially antibiotic resistance, is one of the most severe public health challenges. Antibiotic resistance occurs when bacteria avoid and fight the mechanism of action of antibiotic drugs. This study aimed to determine the resistance of E. coli. Then, the E. coli isolates were tested for antibiotic resistance using the Kirby–Bauer method.A total of 34 milk samples of Ettawa crossbreed dairy goats were used in this study. The initial stages of this research included tests of the physical properties, isolation, and identification of E. coli. The physical properties of milk, namely, color, odor, flavor, and consistency, were normal. The results of the alcohol test showed normal acidity, and the specific gravity of goat milk met the criteria, with an average specific gravity of 1.0295 g/mL. The results of the antibiotic resistance test showed that 4 (12%) samples were resistant to streptomycin, 5 (15%) to sulfonamide, and 3% to trimethoprim.The results showed that all samples were positive for E. coli from Ettawa crossbreed dairy goats in Blitar Regency, East Java, Indonesia, was 100%. Furthermore, this E. coli isolate exhibited resistance to antibiotics streptomycin, sulfonamides, and trimethoprim. The use of antibiotics in the dairy goat industry in Indonesia should be controlled to prevent the spread of resistant E. coli from animals to humans through the food chain and prevent the emergence of multidrug-resistant E. coli.The prevalence of  The bacteria were allowed to stand for 10–15 min. The antibiotic  on the disk was placed on the surface, and then the plate was incubated at 37°C for 24 h. The diameter of the resulting inhibition zone was measured with a caliper and then compared with the standard from CLSI.The data were presented descriptively in percentages displayed in tables.The results of the color, odor, flavor, consistency tests, alcohol test, and specific gravity test for goat milk were normal  15, 18], 1815, 1E. coli found in the milk of the Ettawa crossbreed dairy goat at Blitar Regency, East Java, Indonesia. Thirty-four milk samples (100%) were positive for E. coli. The results of the identification test E. coli are summarized in These results indicate the percentage of E. coli resistance to antibiotics are summarized in Of the 34 milk samples, antibiotic resistance to streptomycin was 12%, sulfonamide was 15%, and trimethoprim 3%. The results of The quality of raw goat milk fit for consumption should normal, clean, white or cream color, natural flavor without any foreign matter and adulteration; furthermore, in the alcohol test, the sediment should be fine or small and specific gravity should be between 1.028 and 1.034 at 20°C . The resIn atmosphere dominated by silage or animal odors, volatile compounds may be transferred directly from the surrounding environment to the milk before, during, and after milking. Moreover, the odor of the male goat in rut is often a source of the goaty flavor in fresh goat milk. The flavor of milk from each animal is different based on genetic and physiological characteristics, feeding systems and diets, and environmental conditions . PreviouMilk viscosity is one of the important benchmarks of milk quality. Normal milk that is good for consumption is runny or liquid and does not clot. These tests are performed in accordance with the Indonesian National Standard . The visSpecific gravity, the mass of a certain quantity of material divided by its volume, is dependent on the temperature at the time of measurement, the temperature history of the material, composition of the material , and air (a complication with more viscous products). With all of this in mind, the specific gravity of milk varies from 1.027 to 1.033 g/mL at 20°C . The higFood-borne diseases in public health programs prioritize the surveillance of milk food-borne diseases by monitoring food-borne pathogens and microbial contamination in milk products. Hence, dairy farms are compromised to reduce the milk contamination source from the udder and the dairy cattle’s health status and the production environment through improved hygiene practices in cattle management and milk handling .Escherichia coli is widely found in the digestive tract and is often found in environments with poor sanitation [E. coli contamination was found in 34 samples (100%) of Ettawa crossbreed dairy goat milk (E. coli contamination was reported in 9.3% (7/75) raw goat milk samples in Luxor Governorate, Egypt [E. coli.nitation . In thisoat milk . In othee, Egypt  and 40% e, Egypt . This bae, Egypt . The watEscherichia coli is a Gram-negative commensal bacterium, commonly used as an indicator during surveillance and monitoring for antibiotic resistance [Escherichia coli was tested in this study because it is commonly found in dairy products and is often found in conjugated plasmids that are commonly transferred between enteric bacteria [E. coli ST131, quickly spread in the community and among healthcare settings. These strains often cause more severe infections and spread more easily [Escherichia coli of animal origin often show resistance to mostly older antimicrobial agents, including tetracyclines, phenicols, sulfonamides, trimethoprim, and fosfomycin [E. coli isolates from ducks in Penang, Malaysia, were resistant (100%) to vancomycin, 92.7% to tetracycline, 72.7% to ampicillin, 67.3% to streptomycin, and 67.3% to sulfamethoxazole-trimethoprim [et al. [E. coli.sistance . Escheribacteria . They ofe easily  and havee easily . Escherisfomycin . In a stethoprim . This is [et al. , who repE. coli. The study revealed that E. coli is resistant to three antibiotics. In the 34 samples studied, several samples were resistant to these antibiotics  sul3 is a more recent version of the gene associated with sulfonamide resistance.Streptomycin is widely used in the treatment of microbial infections, with the primary mechanism of action being inhibition of translation by binding to the ribosome. Moreover, streptomycin interacts with MscL and activates it, thus leading to an outward flux of Kthe drug . Sulfonathe drug  and protthe drug . Sulfonathe drug . In line [et al.  found thE. coli infections; however, its efficacy is limited given the rapidity with which trimethoprim resistance develops [Trimethoprim is widely used to treat develops  through develops .E. coli resistance to several antibiotics in Ettawa crossbreed goat milk that is consumed by the community must be accompanied by vigilance so that resistance does not spread more widely. Some resistance mechanisms emerge but do not develop after the first explosion, but others can spread around the world very quickly [The discovery of  quickly . This is quickly .E. coli (100%) in milk from Ettawa crossbreed dairy goat in Blitar Regency, East Java, Indonesia. Antibiotic resistance to streptomycin, sulfonamide, and trimethoprim was 12%, 15%, and 3%, respectively. The presence of E. coli in goat milk for human consumption can cause milk-borne diseases, and the discovery of microbial resistance can seriously impact public health. The incidence of microbial resistance leads to prolongation of treatment and recovery.In this study, we found AMR to TMA, PAW, and ML: Conceptualization and design of the study. TMA, BA, RAP, and RII: Collected samples. TMA, PAW, RII, and DKW: Performed the laboratory procedures. BA, RAP, AA, and WPL: Analyzed and interpreted the data. PAW and ML: Writing-original draft. DKW and AA: Writing-review and editing. WPL: Edited the manuscript. All authors have read and approved the final manuscript.


In the published article, there was an error in the author list, and author Maddalena Cerrone was erroneously excluded. The corrected author list appears above.The author has also been included in the Author Contributions section shown below.CR, EB, DB, KM-B, MN, BM and RW designed the study. PH, MC, AJ and EB recruited the study participants. SR, EB, MC, and CR performed the whole blood and PCF assay. EB and CR performed the flow experiments. CR analysed and interpreted the data. ASe and CLA provided critical reagents. RW, ASh and CR obtained funding to support the project. CR and EB wrote the manuscript with all authors contributing to providing critical feedback.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Phylloscopus ibericus), presumably the most ancient lineage, was the most central in the variation space, while the Siberian Chiffchaff (P. collybita tristis) was the most peripheric and also very uniform, in contrast with the Canarian Chiffchaff (P. canariensis) highly variable, as expected by the “character release hypothesis” on islands. Calls proved to be an excellent tool, especially amenable for non-biased mathematical analyses which, combined with the wide availability of records in Xeno-canto, greatly facilitates the widespread use of this methodology in a wide range of species and geographical areas.The chiffchaff complex is a group of common forest bird species, notorious for the number of cryptic taxa recently discovered, being a great example of speciation in action. Vocalizations have been crucial to unveil its hidden diversity. In this study we quantitatively analyze the acoustic characteristics of their calls with permutational analysis of variance, canonical variate analysis and a self-organizing map, to determine their variability and differences. We related these differences with the geographical and genetic distances between taxonomic groups, by means of Pearson correlations. We used recordings from Xeno-canto, an open database of bird vocalizations. Inter-taxa distances based on call traits were broadly consistent with geographic distances but not correlated with genetic distances. The Iberian Chiffchaff ( Phylloscopus warblers are a large genus of insectivorous passerines, comprising more than 70 species (sensu Phylloscopus species (Phylloscopus collybita complex) is a group of Old World Leaf Warblers with a vast breeding range that extends across nearly the entire Palaearctic. They are especially abundant in forest ecosystems, where they can comprise up to forty per cent of all birds in some sites  but, in the last decades, several cryptic species and a number of subspecies have been discovered in this group  have contributed to the revelation or confirmation of the existence of cryptic taxa in this group  . There ae.g., structure of vegetation), minimizing signal degradation and maximizing conspicuousness against background noise and interference, resulting in signal divergence across ecological gradients , Iberian Chiffchaff , Canary Islands Chiffchaff  and Mountain Chiffchaff  , breeding in Western, Central and Southern Europe, wintering in Southern Europe, North Africa and the Middle East , breeding in Northern and Eastern Europe), it winters in South Europe, North Africa, Middle East, North Arabia, Persian Gulf and Southwest Iran  (Siberian Chiffchaff), breeding in easternmost Russia, Siberia, North Kazakhstan, North Mongolia, coming into contact with P. c. abietinus in West Russia , breeding in western and northern Anatolia (Turkey); P. c. caucasicus , breeding in the Greater and Lesser Caucasus, East of P. c. brevirostris range at lower elevations south to Armenia; and P. c. menzbieri , believed to be restricted to the mountains of northeastern Iran and Turkmenistan. The Iberian Chiffchaff (P. ibericus) breeds in the Iberian Peninsula and winters in subsaharian Africa (P. canariensis) , which is believed to be extinct, and P. canariensis canariensis , from other islands of the Canary archipelago: La Palma, Hierro, Gomera, Tenerife and Gran Canaria (see P. sindianus) is restricted to mountains and includes two subspecies , from the extreme west of China (SW Xinjiang) to North Pakistan and North India and P. sindianus lorenzii , from Southwest Asia, in East Turkey to Caucasus, Transcaucasia and Northeast Iran  . In addis, 1880) : P. c. cEast see ; P. c. aIran see ; P. c. tt Russia , and wint Russia ; P. c. bn Africa   , P. canaaria see . The Moubspecies , P. sindIran see .Call recordings were obtained from Xeno-canto , an openPhylloscopus collybita collybita, P. collybita abietinus, P. collybita tristis, P. ibericus, P. sindianus lorenzii, and P. canariensis. For the latter, we sampled individuals from four of the Canary Islands . We did not include subspecies P. c. brevirostris, P. c. caucasicus and P. c. menzbieri in this study due to the scarce number of call recordings on Xeno-canto . We also did not include P. sindianus sindianus due to the lack of recordings on Xeno-canto. With the criteria above, each recording in We compiled a total of 82 recordings with 735 calls of individuals belonging to the following taxa : PhyllosP. s. lorenzii to 23 for P. canariensis  and within the groups  in regard to the bioacoustic characteristics of the calls. We also estimated their variance components, i.e., the contribution of the variation between and within groups to the overall variability of the data. Pairwise differences among groups (species and subspecies) were calculated using a multivariate analogue to the univariate t statistic, calculating pseudo- t as the square root of pseudo- F between each pair of groups. The P-values of both the pseudo- F  and the pseudo- t in the pairwise tests, were obtained using permutations. PERMANOVA analyses were performed with PRIMER 6.1.12  was used to determine differences between the R 6.1.12  with thex) the minimum value (min) and dividing by the range (max –min):(x-min)/(max–min). The canonical discriminant analysis was performed with the standardized values. In order to better understand the similarity/distance relationships between taxonomic groups, we graphed them in a canonical variates biplot that shows the spatial arrangement of the calls of the different Phylloscopus taxa studied in the canonical variates space, according to their acoustic features. We analyzed the relationship of inter-taxa CDA distances  with (i) genetic distances and (ii) geographic distances using Pearson correlations. Inter-taxa genetic distances were obtained from We used canonical discriminant analysis   with 15  the map , thus prF = 11.34, P = 0.001 between groups and Pseudo- F = 22.37, P = 0.001 within groups) when the populations in different islands within P. canariensis were combined. The variance in the characteristics of the calls accounted for by the differentiation between groups  was similar to that within groups (39.2%) The residual variance, corresponding to variation between observations not accounted for by the factor considered, in this case corresponding to calls within the sampled individuals, contributed another 18.4%. The relative contributions of the differentiation between groups and within groups varied when the islands within P. canariensis were combined, with differences between groups amounting to 36.9% of the total variance, and within groups to 44.6%. Note that the individuals sampled for each group do not coexist but were sampled from distant areas, so that the variance within groups encompasses regional, habitat and individual variance.The PERMANOVA analysis  showed tt ≥ 2.4, P ≤ 0.002) except for subspecies P. c. collybita and P. c. abietinus, whose calls were not statistically different  Despite the small sample size obtained for P. canariensis for some of the islands, some of the differences between islands were significant, specifically Gomera showed significant differences with Gran Canaria and Tenerife, and La Palma with Tenerife . See Phylloscopus group studied.Pairwise differences among species and subspecies were all significant . This percentage reached 91.7% when the loadings  in the floadings , the firPhylloscopus complex shown in the Canonical Discriminant Analysis biplot  appear in the other half of the graph . In contrast, the Siberian Chiffchaff (P. c. tristis) forms a rather compact and distinct group in the top of the biplot, with the largest average inter-group distance with all the rest and maximum variance. The greatest distances were found between P. c. tristis and the P. canariensis from the different islands sampled  and La Palma , and of La Palma  and Gran Canaria (GrC), followed by those of Gran Canaria (GrC) and Tenerife (Tnf) . Tenerife was the closest to P. c. collybita and P. c. abietinus  and examine its variance components.Phylloscopus groups analyzed in regard to the acoustic features of the calls. Their arrangement in the acoustic variation space has some resemblance to their geographical distribution, with inter-group distances broadly consistent with geographic distances between groups (r = 0.59). However, their relationship with genetic distances , as compared to the changes needed to go from any of the species or subspecies analyzed to any of the rest. This central position, however, does not match its present geographic location, which is rather peripheral in relation to the whole chiffchaff complex: In particular, P. s. lorenzi and P. c. tristis have more similar calls to P. ibericus than to P. c. abietinus and P. c. collybita, which are geographically closer. Genetic analyses also show a counter intuitive result regarding P. ibericus, as it formed the sister group of subspecies sindianus and lorenzii of P. sindianus, being closer to them than to collybita or canariensis  was occupied by  complex . This ceariensis . Again, P. ibericus, the calls of P. c. tristis were the most peripheral, and this was the subspecies showing the most differentiated calls within P. collybita, being more similar to P. s. lorenzi, a different species. The subspecies tristis is also the most distant geographically to most of the other species or subspecies, particularly to P. canariensis, with which it showed the greatest differences in call traits. The higher similarity of P. c. tristis with P. s. lorenzi than with other P. collybita subspecies is consistent with the divergence patterns shown by song and morphological (brownish plumage) characters  by the IOC World Bird List  in two islands of the Canaries, in habitats with contrasting vegetation structure, from scarcely vegetated agricultural habitats to dense forests. They found call structures differing between the two islands but also between individuals as well as microgeographic variation, but no differences between habitats, in contrast to the acoustic adaptation hypothesis  and southern(collybita) populations and, despite song similarity, playback experiments revealed that males react more strongly to males of their own subspecies than to the song of the other subspecies, which means that they distinguish the songs and react differently (collybita breeding mainly in deciduous forests while abietinus prefers coniferous forests (Phylloscopus taxa analyzed are typically forests birds, a strong effect of habitat is not to be expected, according to these previous studies.The calls of ollybita . Signififerently . There a forests . Thus, b forests . This isPhylloscopus complex. We found striking variability in the calls of P. canariensis, by far the most variable in regard to its geographical range, which agrees with the character release hypothesis associated to the reduced interspecific competition in islands. Our results are consistent with previous genetic studies in showing the distinctiveness of P. c. tristis, supporting its full species status or being in a stage of incipient speciation. Calls proved to be especially amenable for the application of non-biased mathematical analysis. This combined with the wide availability of records in an open-access repository such as Xeno-canto, greatly facilitates the widespread use of this methodology in a wide range of species and geographical areas, providing another example of the usefulness of citizen science. In conclusion, calls are a valuable tool to promote our knowledge in avian diversification processes in the near future.Our study provides the first quantitative assessment of call differentiation and patterns of variation in relation to geographic and genetic distances in the


Pseudemys peninsularis in Korea was sequenced and characterized. The mitochondrial genome is constituted of 37 genes  and a noncoding region. Phylogenetic analysis based on the 13 protein-coding gene sequences showed that P. peninsularis has closer relationship with Chrysemys picta than Trachemys scripta elegans. This is the first case for a complete mitochondrial genome from P. peninsularis, which will provide information for biogeographical studies and management plans for invasive species.The complete mitochondrial genome of  Pseudemys peninsularis Carr, 1938) is spread throughout the Florida peninsula. It ranges from north along the Atlantic coast to the mouth of the St. John’s River , Korea, and the total genomic DNA was extracted from the tail using the DNeasy Blood and Tissue kit  according to the manufacturer’s protocol. The extracted DNA sample was deposited at the Museum of Wildlife, located in Research Center of Ecomimetics, Chonnam National University, Korea . The mitochondrial genome was analyzed using Illumina HiSeq X Ten platform , which was performed by Macrogen . Raw sequence data were checked by FastQC, and adaptor trimming and quality filtering were performed by Trimmomatic , and contains 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a putative long non-coding control region. 12 protein-coding genes, 14 tRNA genes, and two rRNA genes are encoded in heavy strand, whereas one protein-coding gene (NADH dehydrogenase subunit 6) and eight tRNA genes in light strand. The nucleotide composition of the P. peninsularis mitochondrial genome  is similar to that of T. scripta elegans from Korea , Mauremys sinensis from China , M. reevesii from Korea , and Chrysemys picta from USA . The sequence of P. peninsularis has higher similarity with that of C. picta (93%) than other turtles including T. scripta elegans (90%), M. sinensis (81%), and M. reevesii (81%).The complete mitochondrial genome of P. peninsularis, the 13 protein-coding gene sequences of 14 species in Testudines were extracted from GenBank and the phylogenetic tree was constructed using MEGA X software (P. peninsularis is closer with C. picta than T. scripta elegans. But P. peninsularis is completely separated from C. picta. These data provide the information on the complete mitochondrial genome of P. peninsularis for the first time, and can contribute to further studies on biodiversity and management of P. peninsularis which is an invasive species in many countries including Korea.To investigate the phylogenetic position of software . Specifisoftware . GTR + G


Hippobosca equina. Studies have confirmed the presence of Corynebacterium pseudotuberculosis equi, Bartonella spp., and Anaplasma spp. in the organism of these insects. The frequency of anaphylactic reactions caused by H. equina attacks has been increasing. The aim of the present paper was to summarize the up-to-date knowledge of Hippobosca equina Linnaeus, 1758 due to its significance in medical and veterinary sciences as a potential vector of pathogens. Given the increasing expansion of ectoparasites, mainly related to climate change, ensuring animal welfare and human health is a priority.Arthropods of the Hippoboscoidea superfamily are parasites of animals from various systematic groups. Mass appearances of these insects and their attacks on people are increasingly being recorded. Their parasitism has a negative effect on host well-being, as it causes feelings of agitation and irritation as well as skin itching and damage. It may result in weight loss and development of diseases in the long-term perspective. Parasites can be a potential epidemiological threat for their hosts as well. One of such parasites is a cosmopolitan species of the Hippoboscinae subfamily— Arthropods of the Hippoboscoidea superfamily are ectoparasites of animals from various systematic groups. During zootechnical care or veterinary service, farmers or breeders can be attacked by these nuisance dipteran insects. People that constantly or frequently stay in the natural environment for recreational or professional reasons  are exposed to the risk of dangerous zoonotic diseases. Our observations from natural and semi-natural habitats and recent reports include the occurrence of these species in cities, which suggests their expansion towards ecosystems undergoing strong anthropogenic transformations ,2. AwareHippobosca equina Linnaeus, 1758 due to its significance in medical and veterinary sciences, and its potential role in pathogen transmission to hosts. Given the increasing expansion of ectoparasites, mainly associated with climate change, ensuring animal welfare and protection of human health is a priority.The aim of the present paper was to summarize available knowledge of Hippobosca equina is in general not a common object of research, which is evidenced by the inconsiderable number of papers focused on this species. The motivation to compile the present review was the authors’ experience of constant exposure to attacks by this ectoparasite in spring and summer 2021 and 2022 in eastern Poland. Moreover, it seems that Hippobosca equina Linnaeus, 1758 (Hippoboscidae: Hippobosca) is the earliest described species of the genus  , in which the neck, groin, back, and head were the most common sites of infestation by Hippobosca spp. [A common site of attachment of tallions . The installions ,20. Romatallions  reportedsca spp. . H. equina specimens parasitizing occasional hosts has not been fully explored to date. Apart from the study on guinea pigs mentioned previously, no information on other species is available. It has been suggested that, due to the low host specificity [Lipoptena spp. Mistakenly, as the insects cannot reproduce on this host [Capreolus capreolus L., 1758 has proved to be successful only in the western range of Fennoscandia, whereas in the eastern range, successful offspring production has been observed only once [H. equina. The final confirmation of the above-mentioned information requires a detailed analysis of the reproductive behavior of H. equina. It seems that the reproductive potential of cificity , the inshis host . For thihis host ,81 as thnly once . There aH. equina infestations in the world are rather limited. The only studies were conducted in Poland, e.g., [H. equina in horses in Poland reported the highest prevalence from mid-June to the end of July at an average air temperature of 21 °C. In turn, the highest prevalence of H. equina in Egypt was recorded from mid-June to the end of August [H. equina infestation in feral horses was 80.6%. The mean number of H. equina specimens per infested animal was 3.2 (range 1–10) [Data on the prevalence and intensity of d, e.g., ,16, and d, e.g., . Researcd, e.g.,  studyingf August . The prege 1–10) , whereasge 1–10) . Research on primitive Polish horses demonstrated that the extensiveness and intensity of infestation fluctuated between 23 and 38.5% and 0.6 and 2.2 in mares, 10 and 30% and 1.0 in sucking colts. These values were 40–80% and 2.1–9 in 1.5–2-year-old mares and 53.3–86.7% and 3.8–6.5 in stallions, respectively. This indicates that the intensity of invasion infestation in 2005 was not significant, as it did not exceed nine insects on one horse in July . In 2012H. equina was found on all examined hosts [H. equina was the only ectoparasite species found [H. equina.According to data obtained from research carried out on horses, ed hosts ,68. In ses found , which mH. equina, as well as the range expansion, probably depends on an increase in host density. Such a relationship was observed in the case of L. cervi in Finland [H. equina, climate change may affect the growth of wild host populations. In Europe, Artiodactyla and, above all, Cervus elaphus L. are the primary hosts of this species [C. elaphus and H. equina largely overlap. We frequently observed the mass occurrence of H. equina, in eastern Poland, where a large red deer population has been recorded in recent years. As shown by the data provided by the Central Statistical Office, in 2013–2019, the red deer population in Lubelskie Province alone increased by 30.26%. The constant rise in the number of red deer and their higher density increases the probability of reproductive success for H. equina [The predicted increase in the abundance of  Finland ,84. In t species . Indeed,. equina ,86.Lipoptena cervi long into late autumn. Changes in temperature and precipitation also affect many life traits of hematophagous ectoparasites, including host seeking behavior [Warm weather increases the probability of host acquisition by winged behavior . In the behavior ,89,90. Pbehavior . H. equina, we cite studies conducted by specialists from Scandinavia, on other representatives of the Hippoboscidae, and arthropod vectors with similar biology and ecology. Therefore, we presume that, factors affecting the distribution and abundance of Hippoboscidae representatives may be alike to a large extent.Due to the lack of this type of research on Variable conditions in habitats influence not only the vectors and hosts , but also transmission of vector-borne pathogens .Hippobosca equina parasitism has a negative effect on host well-being, as it causes feelings of agitation and irritation as well as skin itching and damage. The evident consequences of the species parasitism in animal hosts include anxiety manifested in intensive tail lashing and frequent scraping against posts or tree trunks, as well as skin inflammation, loss of body weight, and decreasing of the quality of host skin, hair, and fleece [d fleece ,15. In gd fleece . In the In humans, the bite of the forest fly is barely noticeable, or painless, but an increasing burning sensation with erythema may start to develop after a few hours . To dateH. equina is considered a vector of pathogens. This role is particularly important, as the insect attacks different hosts (within the same species and between different species). Unlike other hippoboscid flies, H. equina is a permanently full-winged species. Soliman et al. [H. equina and the danger of disease agents transmission between different host species. As shown by this researcher, H. equina plays such a role in both biological and mechanical transmission between different animal species. Similar conclusions were formulated by Arafa et al. [H. equina as a mechanical and biological vector of Oedematous Skin Disease (OSD) in buffaloes. This endemic disease in Egypt is characterized by diffused skin swellings in the limbs, abdominal region, and dewlap. Another symptom of the disease is the inflammation and enlargement of regional drainage lymph nodes [Hippobosca equina is involved in the horizontal transmission of Corynebacterium pseudotuberculosis equi bacteria. These pathogens were detected in 76.9% of H. equina adults feeding on animals examined and 68.5% of skin lesion samples taken from these animals [C. pseudotuberculosis equi in H. equina  [H. equina. As a result of this phenomenon, the vector can also serve as a pathogen reservoir. Hence, the forest fly was recognized as the main transmitter of the causative agent to buffaloes [H. equina activity.As demonstrated by the latest research, n et al.  highligha et al. , who indph nodes ,96. Hipp animals . In addimission) . The occuffaloes . On a gluffaloes  tested tH. equina specimens studied were positive for Bartonella spp. [H. equina collected from horses were Bartonella-positive. It was found in the same research that 13.79% of the collected H. equina specimens were infected by pathogenic bacteria from the genus Wolbachia. In turn, Halos and co-authors [Bartonella chomelii and/or Bartonella schoenbuchensis in all twelve H. equina specimens collected from cattle in different parts of France, but none of the six H. equina collected from horses was positive. Abdullah et al. [Borrelia sp. and Anaplasma sp. in H. equina specimens collected from horses in Egypt. Research conducted in Saxony showed that 82% of the lla spp. . Similarlla spp. , who fou-authors  detectedh et al.  confirmeH. equina is a vector of other pathogens, e.g., Rickettsia spp. Abdullah and co-authors [Rickettsia sp., Coxiella burnetii, and Bartonella sp. pathogens in this insect species. All 105 studied H. equina specimens were found to be free from these pathogens. Interestingly, Selmi et al. [H. equina in Tunisia exhibited a higher Anaplasma spp. infection rate than other, non-infested animals. All these bacteria cause animal and human diseases. Notably, the mere detection of a given pathogen in H. equina does not mean the arthropod is its competent vector; only that the assumption that it can serve as its reservoir is probable.To date, there is no evidence that -authors  attemptei et al.  reportedHippobosca equina feeds on the blood of a wide range of hosts, and adults of these insects take multiple blood meals during their lifespan, protection against its attacks seems to be relevant. However, the most commonly used repellents, e.g., DEET , have recently raised concerns related not only to their safety but also to the increasing insect resistance [Hippobosca equina attacks. Khater et al. demonstrated the repellent activity of camphor (Cinnamomum camphora), onion (Allium cepa), chamomile (Matricaria chamomilla), and peppermint (Mentha piperita) EOs toward dipteran ectoparasites infesting water buffaloes in Egypt [−1 b.w., respectively (discriminating dose 2.5 l). According to the study, all tested EOs repelled Hippobosca equina significantly for six days post-treatment. These results are promising because the researchers did not observe any side effects in humans or treated animals. The adverse effects usually related to the application of EOs are mild. However, severe toxic reactions have been noted, including neurotoxicity, bronchial hyperactivity, and hepatotoxicity [The potential role of the forest fly as a vector of various pathogens is also important, given the possibility of unintentional introduction of the species with the host. Since sistance ,100. Thesistance ,102,103.sistance ,100,101;in Egypt . The dostoxicity . TherefoThe analysis of the current distribution of the species worldwide carried out in this review indicates that it was also found outside the originally assumed range of two biogeographic zones , i.e., in the Afrotropical and Australasian zones. Although these were only single records , they may indicate a potential or actual spread of this species and, consequently, a threat to animals and humans in other parts of the world. The observations from the Philippines and New Caledonia date back to the 20th century (the latter was presented by Borowiec ), whereaAs shown by Boucheikhchoukh and co-authors , forest