Pectobacterium parmentieri, P. brasiliense, P. atrosepticum, and Dickeya spp. Extracted plant parts enriched for BL-SRP were analyzed with TaqMan assays specific for the detection of blackleg-causing BL-SRP. In 2019, low incidences of P. parmentieri (1–6%) in leaves were found at four growing sites. At one farm, reactions were detected in TaqMan assays for D. zeae and D. chrysanthemi in leaves. In 2020, the crops of two growers were largely free from BL-SRP. At one farm, a high infection incidence (21%) was found for D. fangzhongdai in tubers. The isolated pathogen was able to cause potato blackleg. At two other farms, high infection incidences in tubers were found with P. brasiliense (35–39%) and P. parmentieri (12–19%), whereas the incidence of P. brasiliense in leaves was also high (8%). In conclusion, high infection incidences with BL-SRP in potatoes can be found in a PB1 crop at the end of the growing season. Infections in individual plants were found either in tubers or in leaves. The potential sources of initial infection are discussed.Information on the infection incidence of blackleg-causing soft rot Pectobacteriaceae (BL-SRP) in potato crops grown from minitubers (PB1-crop) and the distribution of BL-SRP in individual plants was collected during a two-year survey conducted at five potato growers located in the Netherlands. In the last weeks before haulm destruction, leaves, stems, and tubers of 100 or 200 plants were analyzed separately for the presence of  Dickeya and Pectobacterium, responsible for diseases in various important crops worldwide, including potato, tomato, maize, cabbage, and ornamental plants  wI) of infected tubers in composite samples was estimated using the statistical equation I = {1 − [(N − p)/N]1/n} × 100, in which N is the total number of subsamples tested, p is the number of subsamples tested positive, and n is the number of individuals per subsample [p = 0.05).The incidence . For tubers, the estimated infection incidence was approximately 6%. Relatively high incidences of P. brasiliense were also found in leaves at growers D and E . Interestingly, plants with leaf infections of P. brasiliense or P. parmentieri were not infected in tubers and vice versa. In 20% of all SRP-infected samples, co-infections with P. brasiliense and P. parmentieri occurred, predominantly in tubers at growers D and E. Ct-values of positive samples were low, in particular for P. brasiliense, indicating the presence of high bacterial densities after enrichment  TaqMan assay against the virulent P. brasiliense clade [P. brasiliense   . InoculaDickeya sp. strains, isolated from the tubers of grower C, strongly reacted with the Dickeya genus-specific TaqMan assay but not with TaqMan assays developed for detecting D. solani, D. dianthicola, D. chrysanthemi, D. dadantii and D. zeae, respectively. A phylogenetic analysis with gapA sequences of two isolates indicated that the tubers were infected with D. fangzhongdai, as they clustered with D. fangzhongdai strains from orchid, onion, pear, and water streams  was positive and further confirmed the identity of the strains. The D. fangzhongdai strains from potatoes were able to macerate potato tuber tissue .The P. parmentieri. Surprisingly, P. brasiliense, the dominant blackleg-causing agent in the Netherlands in the last five years, was not detected [P. parmentieri assay were weak, even though the samples were incubated under conditions favoring the growth of SRP, indicating that mainly dead cells were detected. We speculate that although haulms came in contact with airborne inoculum, conditions were unfavorable to establish an infection. As an exception, at grower D, 5% of the leaves were infected with Dickeya sp. Strong TaqMan reactions were found, indicating a successful infection. Interestingly, analysis of samples with TaqMan assays for individual Dickeya species indicated that the leaves were infected with D. chrysanthemi and D. zeae. So far, in Europe, blackleg was predominantly caused by D. dianthicola and D. solani [D. chrysanthemi has been associated with potatoes in Taiwan [D. zeae was only described in association with potatoes in Australia, where it caused soft rot but not blackleg [Dickeya species found in leaves, indicating that infections were airborne. In the Netherlands, D. chrysanthemi was isolated from begonia, chrysanthemum, dahlia and Pastinaca sp. [A two-year field survey, in which plants grown from minitubers were analyzed for infections with SRP, showed that infection prevalence was strongly dependent on location and year. In 2019, there was a low prevalence of mainly leaf infections with detected . The Taq. solani . D. chryn Taiwan  and in tn Taiwan . Recentln Taiwan . D. zeaeblackleg ,27,28. Tnaca sp. . It may D. fangzhongdai, while at growers D and E, infections with P. brasiliense and P. parmentieri were found in the PB1 crop.In our studies, minituber seed lots were not tested for BL-SRP prior to planting, but we do not expect them to be the source of infection. Between 2005 and 2008, 25–30 different seed lots of potato minitubers were tested in the Netherlands annually for the presence of BL-SRP, but they were never detected . MoreoveP. brasiliense was found after storage. The infections may be a result of low incidences of infected, rotten tubers present in the sampled crop at harvest but may also originate from other crops if machines and equipment used during harvesting and storage were not thoroughly washed and disinfected prior to use [Although BL-SRP was not detected in tubers of the sampled crop at the end of the growing season, at grower D, a high infection incidence with r to use ,30.P. brasiliense (35–38%) were found, particularly in tubers. More than 10% of the tuber samples were also infected with P. parmentieri. In addition, at growers D and E, relatively high incidences of P. brasiliense (8%) were found in leaves. P. brasiliense is a genetically and phenotypically heterogeneous group in which strains vary in their ability to cause blackleg [P. brasiliense reacted with a TaqMan assay specific for a genetically homogeneous clade of strains, from which most strains can cause blackleg [P. parmentieri strains tested previously in a field bioassay, erroneously described as a virulent group of P. carotovorum, were all able to cause blackleg, although the aggressiveness seems to be lower than of other blackleg-causing species and varies between strains [In 2020, the weather conditions during the growing period (June and July) for blackleg development were more favorable than in 2019, with a high level of precipitation. At growers A and B, low infection incidences were found, similar to 2019. However, at growers D and E, high incidences of blackleg ,31. A hiblackleg . P. parm strains ,20,32,33Plants with infected leaves did not have infected tubers and vice versa (results not shown). It is highly unlikely that the minitubers (seed potatoes) were the source of inoculum, as the seed used by growers B, C, D, and E were from the same producer, whereas only at growers D and E high infection rates were found. In addition, the infection incidence of rotten mother tubers was relatively low, 1% compared with an estimated 6% for the progeny tubers. If the minitubers had acted as an infection source, a high incidence of rotten mother tubers would have been expected.6 cfu per gram of leaf material [D. solani was transmitted during the growing season from an infected plant to the third plant in a row and to nearby plants in the next row [We hypothesize that the presence of nearby located SRP-infected potato crops of a lower class was predominantly responsible for the initial infections. As in most cases, tuber and leaf infections were not found on the same plant, we conclude that transmission pathways resulting in leaf or tuber infections were different. Leaf infections may be due to transmission of the pathogen via insects or splashing rainwater. It has been shown in glasshouse experiments that after infection, SRP populations can build up in leaves to densities of up to 10material . Tuber iD. fangzhongdai. The presence was evidenced after the isolation of the pathogen by gapA sequencing and the use of a target-specific TaqMan assay. The pathogen was not detected in leaves or stems. D. fangzhongdai was recorded for the first time in 1973 in China, where it caused a bleeding canker disease in pear [In 2020, at grower C, a relatively high percentage of the (composite) tuber samples (21%) and an estimated 5% of the tubers were infected with  in pear , and sin in pear  but neveUsing a two-step analysis, first, a multiplex assay detecting all blackleg-causing species, followed by a characterization of the causative agent using simplex TaqMan assays for individual species, worked efficiently. Most infections found in the multiplex assay could be traced back.D. zeae and D. chrysanthemi were detected, whereas, in 2020, samples were used in which P. brasiliense and D. fangzhongdai were detected by the enrichment TaqMan assay.The chance to isolate BL-SRP from latently infected plant samples positive in the enrichment TaqMan assays seems to depend on the enrichment procedure. We were more successful using extracts from vacuumed plant tissues after incubation than using incubation in a pectate enrichment broth under low oxygen conditions. The efficiency may also be dependent on the pathogen present. In 2019, attempts were made to isolate from samples in which D. zeae, D. chrysanthemi, and D. fangzhongdai, but still, they may pose a threat to seed potato production. In this study, for D. fangzhongdai, it was shown that under experimental conditions, it is highly virulent and can cause blackleg at a similar level as P. brasiliense, but up to now, it has never been isolated from diseased plants in farmers’ fields in Europe. The existence of blackleg-causing species within the genus Pectobacterium, other than P. atrosepticum, P. brasiliense, and P. parmentieri, cannot be excluded. However, in surveys in the Netherlands on blackleg-diseased plants, these three Pectobacterium species were dominant as causative agents [At this moment, only incidentally new SRP variants are found in tubers such as e agents .D. solani and P. brasiliense [The occurrence of new pathogenic variants is likely. In a recent analysis of the pangenome of SRP, it was demonstrated that the evolution of soft rot Pectobacteriaceae is a highly dynamic process, which includes gene acquisitions partly in clusters, genome rearrangements, and loss of genes . Variantsiliense ,24,36. TDickeya species in crops indicate the risks for the occurrence of new SRP variants in potato and emphasizes the need for regular surveys.In conclusion, we found that before haulms are destructed, already high infection incidences can occur in a potato crop grown from minitubers with SRP, although the risks seem to be dependent on the environmental conditions during the growing season. Data suggests that the transmission pathway of the pathogen for haulm infection is different from that of tubers. The detection of various


Silene based on optical images and image analysis has recently been described on the basis of morphological measurements of the lateral seed views. According to the outline of their silhouettes, seeds from 52 species (49 of Silene and three related species) were classified in three groups: smooth, rugose and echinate, revealing remarkable differences between these groups. This methodology has been applied here to 51 new species, making a total of 100 species of Silene analyzed so far. According to our data, a new group was described, termed papillose. The results showed morphological differences between the four mentioned seed groups, with reduced values of circularity for dorsal and lateral seed views in the papillose and echinate groups and reduced values of solidity in the papillose seeds. The method was applied to the analysis of individual as well as to average seed silhouettes and some of the differences between groups were maintained in both cases.Seed morphology is an important source of information for plant taxonomy. Nevertheless, the characters under study are diverse, and a simple, unified method is lacking in the literature. A new method for the classification of seeds of the genus  Silene L. with ca. 700 species is the largest one of the Caryophyllaceae Juss. [Silene species belong to the Northern Hemisphere, with regions of high diversity in the Mediterranean area [S. burchellii Otth. [S. undulata Aiton [S. thysanodes Fenzl. and S. mandonii (Rohrb.) Bocquet) [The genus ae Juss. ,2. An imean area ,7,8,9,10ean area ,12,13. Oean area , and eveean area . In addiii Otth.  and S. uta Aiton ), or in Bocquet) ,19.Silene has been recognized for a longtime as a valuable source of information for intrageneric classification, and seed morphological characters have been used with diverse applications for the taxonomy of this genus [The diversity in shape and ornamentation observed in the seeds of is genus ,26,27,28Silene species.The shape of lateral seed views, with the hilum oriented towards one side, has often been termed as reniform, and it could be more precisely defined as “cardioid related”, based on the quantification by comparison with models derived from the cardioid curve. A variable degree of opening of the central side, corresponding to the proximity of the hilum, revealed different shapes that can be compared with algebraically defined geometric models derived from the cardioid ,30,31,32Concerning seed coat ornamentation, there is not a complete agreement about the terminology and definition of the characters under study. For example, Arman and Jolipour  classifiSilene seeds was based on the consideration of the seed silhouettes as geometric figures. In relation to general seed shape, it allowed for comparing the seed outline with algebraically defined geometric figures providing a quantity, J index, that indicates the percentage of similarity of the seed silhouette with a given geometric figure taken as a model [Silene, one of Atocion and two of Viscaria [S. subg. Silene, while species of S. subg. Lychnis were absent. Most of the species in the group of rugose seeds belonged to S. subg. Silene, and species in the group of echinate seeds corresponded mainly to both S. subg. Behenantha and S. subg. Lychnis [The proposed description of the lateral and dorsal views of  a model ,30,31,32Viscaria . The groIn addition, previous published data indicated a relationship between the morphological groups defined based on their seed silhouettes  and the geometric models adjusting to the shape of seeds, both in the lateral and dorsal views ,32. LateSilene, making a total of 100 species of Silene analyzed so far by this method. The species Eudianthe coeli-rosa (L.) Fenzl ex Endl. was included since this taxon has also been considered as a member of the genus Silene [To explore further the relationship between morphological groups based on the geometry of the seed silhouettes and the taxonomic position of the species, we have expanded the seed morphological analysis to 51 new species of s Silene . Our aims Silene , based oSilene and Eudianthe coeli-rosa used in this study are shown in Silene seeds with the mean, minimum and maximum values of area, perimeter, circularity, roundness, aspect ratio, and solidity. Similar to other Silene species [The lateral and dorsal images of representative seeds belonging to 51 species of  species ,32, the Silene species, only four  had a smooth outline on the lateral view, since no projections appeared on their surface , with a unique sample of S. subg. Behenantha (S. baccifera).Among the studied  surface . The see surface , in contS. bupleuroides, S. caryophylloides, S. chloranta, S. chlorifolia, S. dinarica, S. foliosa, S. frivaldskyana, S. gigantea, S. hayekiana, S. hookeri, S. integripetala, S. koreana, S. linicola, S. marizii, S. multicaulis subsp. multicaulis, S. paradoxa, S. pygmaea, S. regia, S. roemeri, S. saxatilis, S. spinescens, S. squamigera subsp. vesiculifera, S. swertifolia, S. vallesia subsp. vallesia, S. villosa, S. virginica and S. waldsteinii. As with smooth seeds, most of the species with rugose seeds correspond to S. subg. Silene , while only five species belong to S. subg. Behenantha. A notable diversity about the degree of concavity around the hilum was observed, with species characterized with this region almost plane or convex  or with some degree of concavity . Regarding the dorsal seed view , while a few species were characterized with convex dorsal views without any depression .The group of rugose seeds was the largest in number, formed by 27 species : S. bupleed view , most ofS. aprica, S. chungtienensis, S. ciliata, S. dichotoma, S. fabaria, S. firma, S. fruticosa, S. jeniseensis, S. longicilia, S. multiflora, S. nana, S. petersonii, S. samojedorum, S. suksdorfii, S. viridiflora and S. yunannensis. Most of the species in this group belong to S. subg. Behenantha , while only six species belong to S. subg.Silene. In general, the lateral views of the seeds are plane around the hilum region  or present concavities smaller than the rugose seeds . A notable diversity in size and relative distribution of the spines was observed for both lateral and dorsal seed views , with a clear concavity in the upper or lower part of the seeds only observed in S. ciliata . Their dorsal silhouettes showed a rectangular shape, with the presence of marked prolongations , as well as E. coeli-rosa are shown in The mean values for all general morphological measurements, coefficients of variation and the results of the Kruskal–Wallis and post hoc tests for the comparison between the groups of p < 0.05) on the lateral views between the groups for all the measurements except aspect ratio and roundness. These measurements were excluded from further analysis. For the remaining measures, the Campbell and Skillings post-hoc test results are summarized in E. coeli-rosa populations have lower values for area and perimeter, whereas the papillose seeds have a high perimeter (p < 0.05). Regarding the circularity and solidity, there were four groups, with the lowest values in the papillose seeds, followed by echinate and E. coeli-rosa, then rugose and the highest values of the smooth seeds (p < 0.05). The obtained data for the seeds of E. coeli-rosa were, in general, similar to those for the echinate seeds in all measurements. The coefficients of variation for the lateral seed view were notably higher for the measurements of area and perimeter, intermediate in aspect ratio, circularity and roundness, and the lowest values were obtained in solidity ; and, finally, (iii) a group characterized by the highest values of roundness (p < 0.05) formed by the papillose and E. coeli-rosa seeds. The circularity results show lowest values for the papillose population, followed by echinate, and, finally, E. coeli-rosa, the rugose and smooth species with the highest values (p < 0.05). The lowest values of solidity were obtained for the papillose species, an intermediate group was formed by echinate and smooth seeds, and the highest values corresponded to the rugose population. The species E. coeli-rosa resembled different seed groups depending on the morphological feature: echinate seeds for area, perimeter and solidity; papillose seeds for aspect ratio and roundness; and rugose and smooth seeds for solidity and circularity. Finally, the coefficients of variation for the dorsal seed view revealed the highest values for the area and perimeter, the lowest values were obtained in solidity, while aspect ratio, circularity and roundness showed similar intermediate values  belong to S. Sect. Siphonomorpha. In the cases of S. subgen. Behenantha, 3 out of 8 species of the group of rugose seeds are included in S. sect. Physolychnis. Finally, the case of S. baccifera and S. littorea, of S. subgen. Behenantha are quite remarkable, since both of them are characterized by a convex shape and bright surface, and hence, they are the only species belonging to this subgenus and classified within the smooth group.The results reported here confirm and expand a certain relationship between the morphological groups obtained by the analysis of the outline of the silhouettes. A summary of them is presented in Silene and 3 related species) classified the seeds according to the outline of their silhouettes in 3 well distinguished groups: smooth, rugose and echinate [Silene classified them in the three mentioned groups plus a new group, named papillose. The four species classified as having papillose seeds  conformed a compact group well differentiated at a glance. They had distinctive morphological characteristics, such as the presence of long and broad prolongations (papillae) with mostly flat tips. The lowest values of circularity and solidity of the lateral and dorsal seed views for both individual and average seed silhouettes corresponded to this group as well.A recent report with 95 populations belonging to 52 species (49 species of echinate . In addiechinate . The datS. baccifera). In the previous report [S. littorea), this group was more numerous with a total of 12 species. Therefore, the lack of roughness of the outline of the seeds could be the main factor to support the high levels of circularity of the smooth seeds. Nevertheless, the two species S. baccifera and S. littorea would deserve further attention because they are the only species belonging to S. subg. Behenantha classified as smooth seeds. A high degree of homoplasy has been described in Silene [S. subg. Behenantha showed classification problems in phylogenetic analysis and morphological studies [Silene taxonomic work.Some differences were found between the results reported here and those given by Martín-Gómez et al. . Accordis report , althougn Silene . This me studies ,34. AccoSilene samples to check the continuity of the observed tendencies, including samples for the new seed group, papillose.The main results of the measurements of aspect ratio agreed with the results published . This siConcerning solidity, the previous work  reportedSilene regarding subgenera and can be added to other tools developed for the analysis of Silene seeds [Silene will enable, eventually, a combination with phylogenetics for a better understanding of the Silene taxonomy.The analysis by the average seed silhouettes allowed to compare between morphological properties of seeds, as independent individuals, and those of a set of images grouped together. The use of average seed silhouettes reduced the morphological variation during the process of obtention of the data, which was remarkable for rugose and echinate seeds. Despite these differences, this comparison might reaffirm the morphological tendencies found in the analysis by groups of seeds based on individual seed silhouettes. For example, the papillose seeds showed the lowest values for circularity and solidity of the lateral and dorsal seed views for both individual and average seed silhouettes. In this work, we showed robust morphological tools for large sets of seeds. These tools resulted in similar clustering than phylogenetics classifications in ne seeds ,35. The S. latifolia subsp. alba [Silene for Flora Iberica [S. latifolia  the accurate description of the characters under analysis and (2) the stability of the characters under scrutiny. The overall shape may change with aging of seeds, and also a level of variation in the shape of the surface protuberances has been reported in sp. alba . Accordi Iberica , reportstifolia d Italy  equipped with a camera Nikon DS-Fi1 of 5.24 megapixels ; lateral and dorsal views of these seeds used in the morphological analysis were taken with a camera Nikon Z6 equipped with an objective AF-S Micro NIKKOR 60 mm f/2.8G ED.https://zenodo.org/record/4478344#.YzxbmExBxD8 (accessed on 1 December 2022).The seed individual outlines and their corresponding average silhouettes for the lateral and dorsal views of the seeds in each species were obtained from 20 representative seed images by the method described ,38. A viSilene followed the previous proposed classification of the outline of the seed silhouettes [Silene seeds were classified in three groups named: smooth, rugose, and echinate. The smooth seeds were characterized by the lack of superficial projections; the rugose seeds showed rounded projections or rounded tubercles; and those seeds with most of part of the silhouette highly projected with acute tubercles or spines were classified as echinate seeds.The morphological characterization of the seed silhouettes of the different species of houettes . Based o2). The measurements of length and width were omitted from the results because the data for area and aspect ratio gave enough information concerning seed size. Circularity index is the ratio (4πA)/P2 [Measurements corresponding to the lateral and dorsal views of seeds included: area (A), perimeter (P), length of the major axis (L), width (W), aspect ratio (AR is the ratio L/W), circularity (C), roundness (R) and solidity (S). They all were obtained with ImageJ . A ruler(4πA)/P2 , while r(4πA)/P2 . Seed suMean, minimum and maximum and the standard deviation values were obtained for all the measurements indicated above . Statistical analyses were done on IBM SPSS statistics v28 (SPSS 2021) and R software v4.1.2 . As someSilene species have been classified in four well differentiated groups: smooth, rugose, echinate, and papillose. Morphological differences were found between the groups with circularity values in the lateral and dorsal seed views lower in the papillose and echinate seeds. The analysis involved the obtention of average silhouettes permitting to find those differences between groups that were more relevant. The differences found in circularity between the echinate and the smooth and rugose groups disappeared in the analysis with average silhouettes, while these between the papillose and the smooth and rugose groups were maintained after the analysis with average silhouettes. These morphological types for the lateral and dorsal views of the seeds may be related to the taxonomy of Silene based on DNA sequence analysis [Based on optical photographs and the obtention of silhouettes from the seed images, the seeds of 51 analysis ,34.


Proc. R. Soc. B283, 20152694 (Published online 25 May 2016) (https://doi.org/10.1098/rspb.2015.2694)The published article  requireshttps://doi.org/10.5281/zenodo.6565204.Second, a summary table rather than the raw dataset was uploaded to a public repository. This has been corrected, and the raw data are available at if chemical cues are used in habitat selection, rather than determining a detection threshold for this species of a concentration gradient.Additionally, we would like to clarify that the strong treatment effects found in this experiment are likely the result of a combination of factors including, but not limited to, the ecology of the focal species, the flume apparatus, the chemical comparisons being tested as well as the concentration of the chemical cues tested. The chemical cue concentration is likely higher than the organisms would experience in nature, and therefore likely a supernormal stimulus. Simply put, supernormal stimuli are bigger and more intense than normal, and elicit a larger than normal response from the animal . Here, t


Trypanosoma cruzi transmission is chemical control of vectors. This study presents a descriptive analysis of synanthropic triatomines before and after the implementation of a vector-control program in Bahia, Brazil.  Neglected tropical diseases are a growing threat to global health, and endemic Chagas disease has emerged as one of the most important health problems in America. The main strategy to prevent  Descriptive analysis and geospatial statistics were performed on triatomine data, (1) the relative abundance and (2) proportional spatial distribution, from Bahia during two periods: (A) 1957 to 1971 and (B) 2006 to 2019. Panstrongylus megistus  and Triatoma infestans , as well as an increase in the relative abundance of T. sordida  and T. pseudomaculata .  We observed a decrease in the relative abundance of P. megistus and T. infestans (last record in 2015) and an increase in the relative abundance and geographical distribution of T. sordida and T. pseudomaculata after 40 years of the vector-control program. The high frequency of other triatomine species in the municipalities of the state of Bahia and their abundance in recent years highlight the need to reinforce permanent entomological surveillance actions to prevent Chagas disease. Our results indicate a clear reduction in the occurrence of  Trypanosoma cruzi , the etiological agent of Chagas disease, also known as American trypanosomiasis. Some synanthropic triatomine species adapt to anthropic changes within their natural landscape, colonizing the household environment  and occasionally inside houses, increasing the risk of T. cruzi transmission,,,, and the environment around the householdTriatomines are blood-sucking insects (Hemiptera: Reduviidae) that are vectors of the parasite ,Triatoma infestans , which occupied household units among 12 states of Brazil and was considered the main vector involved in T. cruzi transmission in Brazil,T. infestans in 1991, a sustained decrease in domestic populations of vectors was observed-T. infestans from the Pan American Health Organization (PAHO) and the World Health Organization (WHO)T. cruzi to humans and domestic animals.In Brazil, some triatomine species have succeeded in occupying the domestic environment and expanding their spatial distribution beyond their original biomesT. cruzi was described by Carlos ChagasConorhinus megistus triatomines - syn. Panstrongylus megistus  - in the city of Mata de São João, near Salvador, the capital city of Bahia. Pirajá da Silva also identified triatomines among the cities of Feira de Santana, Candeias, São Francisco do Conde, and SalvadorP. megistus and Triatoma rubrofasciata  naturally infected by T. cruzi were found at the historic center of Salvador city, associated with human cases, which motivated a chemical control campaignP. megistus maintained T. cruzi transmission to families in the neighborhoods of SalvadorP. megistus and T. rubrofasciata were examined in Salvador, of which 16% were infected with T. cruziPirajá da Silva initially described the existence of triatomines in the state of Bahia in 1911, shortly after National campaigns focused on active vector surveillance for the identification of household infestation areas in Brazil and strategies of triatomine vector-control using chemical insecticides (BHC and pyrethroids). The 1983 Brazilian campaign was executed by the Superintendence of Public Health Campaigns (SUCAM) of Brazil’s Health Ministry. During this period, new triatomines were recorded, and new species were describedT. cruzi infection (0.77% to 2.22%) compared to the Northeast region of Brazil (0.69% to 0.88%) between 1987 and 1994T. cruzi transmissionRecent epidemiological studies indicate that Bahia had a high prevalence of human -T. cruzi vector occurrence, the use of retrospective epidemiological and entomological data has become a relevant strategy. In this study, we describe the relative abundance and proportional spatial distribution of synanthropic triatomines at the municipal level before (1957 to 1971) and after (2006 to 2019) the vector-control program implementation in the state of Bahia, which is one of the states with the greatest diversity of triatomines and highest rates of epidemiological information related to Chagas disease in northeastern Brazil.Currently, 26 triatomine species have been recognized in BahiaThe state of Bahia has 417 municipalities and is located in the southern region of northeastern Brazil, bordering eight other Brazilian states and the Atlantic Ocean in the east . DescripT. infestans: Epidemiologic Surveillance team of the State of Bahia’s Health Service (SESAB), Epidemiological Surveillance Office (DIVEP), and Central Public Health Laboratory of Bahia (LACEN/BA) Information regarding period B was obtained from the following state government databases after the certification of interruption of Chagas disease transmission by Entomological data were obtained in three different ways: (a) notification of triatomines (surveillance and community participation) performed by the population itself by taking suspicious insects to health agents; (b) notification attendance (active surveillance) performed by the health agents of each municipality, dependent on (a); and (c) active search (active surveillance), in which vectors were searched among household units around the area, regardless of any notification performed by the population. T. cruzi vector species information from both periods of the study, we evaluated: (1) the relative abundance, calculated as the proportion of each triatomine species divided by the total number of triatomines for each period; and (2) proportional spatial distribution, calculated as the proportion of municipalities that recorded a triatomine species divided by the number of sampled municipalities.To compare Spreadsheets were used to collect the following data: species, municipality, date, geographic information system (GIS) coordinates, sampling environment , and data reference. Graphs and descriptive statistics were computed using SPSS 24. In the absence of specific GIS information, the coordinates of the city's headquarters from the Brazilian Institute of Geography and Statistics (IBGE) were usedIn this study, information regarding 315 municipalities and 215597 triatomines collected during the two periods was evaluated, among which 35588 were from period A (1957-1971) and 180020 were from period B (2006-2019). During period A, data were gathered from 202/290 (71%) municipalitiesP. megistus was present in 122 (60.4%) of the sampled municipalities, followed by T. sordida (37.1%), T. pseudomaculata (19.8%), and T. infestans (12.8%). During period B, T. pseudomaculata was widely distributed, being recorded in 176 of 258 (68.2%) sampled municipalities, followed by T. sordida (61.6%), and T. brasiliensis (36.4%) ( (36.4%) .P. megistus was the most abundant species (n=22032), representing 61.9% of triatomines, followed by T. sordida (23.3%), T. brasiliensis species complex (4.3%), and T. infestans (3.6%). Conversely, in period B, T. sordida represented 81.6% of all collected triatomines, followed by T. pseudomaculata (9.2%) and T. brasiliensis species complex (6.3%).In period A, T. cruzi vector-control actions, we observed a reduction in the municipal occurrence of synanthropic populations of T. infestans and P. megistus. T. infestans was no longer detected in some municipalities in the west  and north  of Bahia  and the central region (Caatinga biome) of Bahia  and T. infestans , as well as an increase in the relative abundance of T. sordida  and T. pseudomaculata . During period B, most triatomines were captured in peridomestic habitats, with a predominance of T. sordida  and the relative abundance (%) of species in periods A and B showed a reduction for  sordida .T. cruzi vectors, initiated in 1975. Our results indicate a clear reduction in the occurrence of P. megistus and T. infestans (last record in 2015) and an increase in the relative abundance and geographical distribution of T. sordida and T. pseudomaculata after the Brazilian vector-control program.This study indicated changes in the spatial distribution and relative abundance of synanthropic triatomines in the state of Bahia, Northeast Brazil, before and after 44 years of structured control actions of Panstrongylus megistus was the predominant species between 1957 and 1971; it was found in 60% of municipalities, with a relative abundance of 62% of collected triatominesP. megistus spatial distribution has also been reduced in other Brazilian states,-P. megistus; (b) intense urbanization in these municipalities resulted in deforestation and fragmentation of the Atlantic Forest biome areas, which is the natural habitat of this species,P. megistus colonization. P. megistus is a native Brazilian species with a wide spatial distribution and high epidemiological and entomological relevance due to the high rates of T. cruzi infection and its proximity to human dwellings, as it can colonize intra-and/or peridomicile areas. In 2021, P. megistus foci were described in the metropolitan region of São Paulo, with T. cruzi-infected triatomines associated with marsupials, revealing the importance of continued surveillance of synanthropic P. megistusT. infestans were found in 12.8% of the sampled municipalities and represented 3.7% of the sampled triatomines. During period B, T. infestans was identified in only seven municipalities with residual colonies-T. infestans identification errors and the appearance of new records of this species in the study areaT. infestans in several municipalities in western and central Bahia can be explained by the T. infestans elimination plan. This plan was intensified in 2004 and included spraying households with insecticides, followed by research and capture of triatomines, surveying approximately 500000 households,T. infestans,T. infestans and other household species,.During period A, T. infestans, several native species have been recorded in a large number of municipalities. They were captured in households and frequently colonized peri domestic areas. Among them, some were infected by T. cruzi and many of them fed on domestic animals and human bloodT. sordida was recorded in 37% of the municipalities, representing 23% of the collected triatomines, while T. pseudomaculata was recorded in 19% of the municipalities, with a relative abundance of 2.5% of the gathered triatominesT. sordida was recorded in 60% of the municipalities, representing approximately 81% of the triatomines. T. sordida is the most common species in different regions of Bahia. Similarly, T. pseudomaculata was identified in the largest number of municipalities during period B (67.4%). Systematic vector-control actions had a low impact on the spatial distribution of T. sordida and T. pseudomaculata in Bahia. There was a higher occurrence of T. sordida in western and south-central Bahia, where there are areas of Cerrado, which is the original biome of the species’ natural populations,,T. pseudomaculata in western and central Bahia followed the occurrence predictions of the species based on environmental variablesT. sordida and T. pseudomaculata exceeded 90% of all triatomines collected in the state between 2006 and 2019.Although successful in controlling T. brasiliensis and similar species in Bahia, thus expanding the observations of Ribeiro-Jr et. al.T. brasiliensis species complex was registered in 10% of the municipalities of Bahia, and between 2006 and 2019, at least 94 municipalities (36%) registered this species. In the last few years, other species of the T. brasiliensis complex have been described in the state of Bahia, including T. juazeirensis, T. melanica, T. sherlocki, and T. petrocchiaeT. juazeirensis is noteworthy because it was predominantly collected inside household units. Other species, such as T. tibiamaculata, P. geniculatus, T. melanocephala, and P. lutzi, were intrusively detected inside the houses, mainly adult specimens.We observed a higher occurrence of T. tibiamaculata between the two periods. In period A, it was recorded in approximately 1% of the municipalities with a relative abundance of 0.01%T. tibiamaculata has been naturally found inside nests of marsupials, rodents, and epiphytes in forests,T. cruzi-infected T. tibiamaculata generates a transmission risk in Salvador, probably in the entire metropolitan region, and in the Atlantic Forest areasThere was a significant difference in the occurrence of T. cruzi can be classified as native/non native, wild/non wild, domestic/peridomestic to enable the definition of effective vector-control strategiesT. cruzi transmission in the domestic environment.Triatomine vectors of T. brasiliensis complex species were considered as one, and to avoid misinterpretations, data obtained in this study were evaluated at stricto sensu level,,Belminus laportei, Eratyrus mucronatus, Microtriatoma trinidadensis, Panstrongylus tupynambai, P. lignarius, Rhodnius prolixus, Triatoma maculata, T. circummaculata, and T. rubrovaria. To reduce taxonomic identification errors, a guide was developedThis study has several limitations. Since the use of reference databases does not allow a broad analysis of data, it was impossible to obtain data on triatomines within habitats (intra-and peridomicile) in period A (1957-1971). The effects of passive dispersion and seasonal changes on vector behavior were impossible to measure. In addition, the database does not allow descriptive evaluations of nymph occurrence. Moreover, not all municipalities collected data regularly during the evaluated periods, and the health surveillance service classifies municipalities among the high, medium, and low risk of transmission, emphasizing that there is no obligation to conduct regular entomological research among low-risk municipalities based on the classification presented by Brazil’s Health MinistryT. cruzi vectors P. megistus and T. infestans, respectively. In addition, we described an increase in the native species T. sordida, T. pseudomaculata, and T. brasiliensis species subcomplex and highlighted the important role of native triatomine species in T. cruzi transmission in the domiciliary environment. The observed changes in T. cruzi main vectors between the evaluated periods demonstrate the importance of reinforcing entomological surveillance actions. Furthermore, promoting and disseminating community-based scientific knowledge and health education actions on Chagas disease at a local scale will help mitigate surveillance challenges and control native triatomines.This study describes changes in the triatomine fauna of Bahia between the analyzed periods more than 40 years after the implementation of the vector-control program in the state. During this period, we observed changes in the Chagas disease surveillance policies in Brazil due to decentralization. The centralized Brazilian control program was transferred to municipalities without corresponding training. The decentralization policy of the Chagas disease vector-control program aimed to bring the actions of the government closer to the communities, but true engagement by local communities has not been achieved. After decentralization, the efficacy of some municipalities in detecting triatomines has been reduced. We observed a reduction in the relative abundance and proportional spatial distribution of domestic and domesticated species of The procedures followed the ethical standards of the Research Ethics Committee of the Gonçalo Moniz Institute . The consent form was waived since the analysis was performed using a database. However, to provide full anonymity to participants, no personal identification data were used. The research did not cause any physical, psychological, moral, intellectual, social, cultural, or religious risks to the residents or animals in the study areas. In addition, this study did not include endangered or protected species.


Proteus mirabilis, the most widespread species of all Proteus spp. bacteria, is proven to be one of the most universal pathogens in chronic wounds. In this case, a woman in her 40s consulted a physician about an asymptomatic ulceration with a stalactite appearance at the distal end of the index finger after she was exposed to a needle when vaccinating chickens. The patient did not response to ceftazidime. Physical examination revealed a well-demarcated violescent ulceration with a stalactite appearance at the distal end of the index finger. A biopsy of the lesion showed dense infiltration of multinucleated giant cells, histiocytes, and lymphocytes in the dermis. The result of metagenomics next-generation sequencing (NGS) showed 306 unique sequence reads of P. mirabilis, covering 33.49% of the nucleotide sequences. The pathogen was identified as P. mirabilis, which was resistant to ceftazidime. The patient was treated with ciprofloxacin hydrochloride and improved considerably. This case reported a distinctive cutaneous lesion of P. mirabilis on human infection and showed a successful use of NGS in P. mirabilis. Proteus mirabilis is the most widespread species of Proteus spp. bacteria , and her lesion showed substantial improvement   . The phe similar . Celluli similar . Howeverhy smell . There airabilis .P. mirabilis may be in a viable but non-culturable (VBNC) state. It was a state of bacteria where bacteria under certain conditions of starvation or stress could enter a state of no division and, therefore, become non-culturable but in the same time keep their viability and sometimes their ability to cause infection  was ineffective, and the culture was negative. nfection . Thus, Vl effect , 15.P. mirabilis, including TEM genes, VEB-1 genes, OXA genes, CTX-M genes and CIT genes, with the TEM genes being the most common resistant gene  for the publication of any potentially identifiable images or data included in this article.LZ, YD, CJ, and DC conceptualized and designed the study. LZ and YD wrote the manuscript. TG, BC, CJ, and DC revised the manuscript critically for important intellectual content. ML, QX, and TL collected clinical pictures and analyzed the data. All authors have read and approved the final 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.


Eucalyptus. These crops are the most productive in the world, but may suffer losses due to exotic pests, including Glycaspis brimblecombei Moore  found in Brazil since 2003. Interactions between Eucalyptus plants and insect pests may led to the selection of resistant genotypes. Eucalyptus species are either susceptible or resistant to this pest group, but the damage they suffer needs to be evaluated. The objective was to determine possible leaf anatomy indicators of different Eucalyptus species associated with G. brimblecombei infestations, focusing on plant resistance to this pest. The study was carried out with Eucalyptus camaldulensis, Eucalyptus grandis, Eucalyptus saligna and Eucalyptus urophylla saplings infested or not by G. brimblecombei eggs and nymphs. Eighteen anatomical characteristics of the leaves of these plants were analyzed. The number of stomata on the adaxial and abaxial sides and the glandular area in the central leaf vein are associated with greater or lesser infestation by G. brimblecombei in the Eucalyptus genotypes.The total area of forest crops in Brazil is 9.55 million hectares, of which 7.5 million hectares are  Eucalyptus. Forest plantations in Brazil are among the most productive in the world with 36.8 m³/ha year and with economic, social and environmental importance  was reported in Brazil , glands that produce essential oils, often rich in terpenoids, hardness (sclorophilia), heterophilia (differentiation between young and mature leaves) and high regrowth of enotypes .Eucalyptus plantations depends on knowledge of plant/insect interactions. The objective of this study was to determine possible indicators based on leaf anatomy of four Eucalyptus species associated with G. brimblecombei infestations. These indicators may be useful in breeding programs for plant resistance to this pest.Leaf anatomy is poorly studied and may allow us to understand pest infestations and the development of new tools for their management. Developing integrated psyllid management in Eucalyptus camaldulensis, E. grandis, E. saligna and E. urophylla were planted in 1.5 L pots with an autoclaved mixture of soil: sand: manure (2: 1: 1) and kept in a greenhouse for infestation with G. brimblecombei.The study was carried out at the Universidade Estadual Paulista (FCA/UNESP) in Botucatu, São Paulo state, Brazil. Eucalyptus species were previously classified according to their response to G. brimblecombei with E. saligna and E. urophylla being resistant, E. grandis tolerant and E. camaldulensis susceptible to damage , weekly, for 4 weeks.Eucalyptus species were used per treatment, with 10 plants (replications) infested with G. brimblecombei and another 10, as a control, free from the pest. All the plants in the control were sprayed with systemic insecticide (acephate) and the others only with water, to evaluate the effects of mechanical action of the water.Twenty seedlings of each Eucalyptus camaldulensis, E. grandis, E. saligna and E. urophylla leaves, infested or not, were analyzed. The samples were one to two leaves from the middle third of each eucalypt sapling, cut in three parts with the middle third analyzed. These samples were placed in formaldehyde + glacial acetic acid + 50% alcohol fixative solution (FAA-50) for 48 h and stored in 70% ethanol  per species of Eucalyptus infested or not by G. brimblecombei. Eighteen variables for anatomical characterization of the leaf were evaluated.The thickness and the area with the epidermal, parenchymal and vascular leaf tissues were obtained with the computer program Image Tool 3.0 (UTHSCA) to evaluate the damage by 2) (CS) in the region of the central rib, thickness of the upper (TUE) and lower (TLE) epidermis, upper (TUPP) and lower (TLPP) palisade parenchyma, spongy parenchyma (TSP), leaf (TL), mesophyll (TM), the mean area of a gland (MGA), and number of stomata/mm2 of the upper (NUS) and lower (NLS) surfaces in the internervure region (The 18 variables related to leaf anatomy were: percentages of upper (%UE) and lower (%LE) epidermis, collenchyma (%Col), phloem (%Ph), xylem (%Xy), chlorenchyma (%Chl), gland (%Gl), and total cross-sectional area   to verifG. brimblecombei was constant with low plant mortality. Eucalyptus camaldulensis was more infested than E. urophylla and E. grandis and all G. brimblecombei nymphs died in the first instars on E. saligna without development on plants of this species. Sooty mold developed on G. brimblecombei lerps. The occurrence of leaf spot from Teratosphaeria epicoccoides was observed on E. camaldulensis, E. grandis and E. urophylla and with greater damage to E. saligna.The infestation of Eucalyptus species   . The gras leaves .Eucalyptus leaves and the first two principal components (Y1 and Y2) were found to be thickness variables of the lower palisade parenchyma, mesophyll, leaf, upper palisade parenchyma, upper epidermis, as well as the number of stomata of the upper and lower surfaces. These were the main variables that served to discriminate the four Eucalyptus species, based on the first principal component (Y1)  were lower than those of the group 1 species   . The disent (Y1)  and the ent (Y1)  showed tophylla) .E. camaldulensis were higher than those for other species. The E. camaldulensis leaf profile was classified  on shoots and apical parts of Eucalyptus, which may reduce chlorenchyma, impairing plant development (E. grandis leaves in the central vein region, and phloem and xylem in the central vein than in other species may be related to the presence and production of phenolic compounds in the epidermis (G. brimblecombei.The similar percentage of epidermis in the central vein region, collenchyma and epidermis thickness in the internervural region, and thickness of spongy parenchyma for the resistant and susceptible  species , indicatelopment . The higpidermis , as a reE. camaldulensis due to stomata on the adaxial surface and a double layer of palisade parenchyma on both sides of its leaves. The single layer of palisade parenchyma was found only on the adaxial surface of the other species (Differences in the number of stomata on the upper surface, and thickness of the upper and lower palisade parenchyma on  species .Eucalyptus spp. to G. brimblecombei, because this structure is duplicated on the adaxial and abaxial surfaces of E. camaldulensis leaves and single in the adaxial surface of E. grandis, E. saligna and E. urophylla, as well as thicker, on both sides, in E. camaldulensis than in the other species. The signs of stylet insertion by G. brimblecombei nymphs through the E. camaldulensis leaf sections indicates that they passed through the parenchyma cells rather than between them. Cell-degrading proteins such as amylase, cellulase, pectinase and pectinesterase enable stylet entry into the plant tissue (E. grandis, E. saligna and E. urophylla, whereas they are present on E. camaldulensis leaf side surfaces. The total number of stomata is similar between these species, but this may explain the similar infestation on the abaxial and adaxial surfaces of E. camaldulensis compared to E. urophylla, with greater infestation on the abaxial surfaces. Stylets of G. brimblecombei nymphs penetrated the mesophyll, crossing between the guard cells of the stomata, similar to that observed for this insect in E. globulus (E. camaldulensis may confer greater susceptibility to G. brimblecombei.The palisade parenchyma probably does not confer resistance on t tissue . Stomataglobulus , 2001b aEucalyptus trees for insects include physical barriers and constitutive and inducible chemical defenses (Eucalyptus resistance to G. brimblecombei (Defense strategies of defenses . The condefenses , amountslecombei .G. brimblecombei nymph infestation and to explain E. camaldulensis susceptibility to this pest. The thinner epidermis of the adaxial surface and lower percentage of epidermal tissue on the abaxial surface of E. camaldulensis leaves are possibly related to the higher susceptibility to G. brimblecombei. This is a pioneering study evaluating anatomical foliar indicators in relation to Eucalyptus pests, and allows us to better understand pest infestation patterns, and concomitantly, the morphological characteristics that normally confer resistance, such as waxy coating, trichoids, and stomata in these plants.The proportional area and number of stomata occupying the epidermis may also be important for Eucalyptus plants to G. brimblecombei.The number of stomata in the adaxial and abaxial leaf surfaces and percentage of gland area in the central vein of the leaves are related to the resistance or susceptibility of Eucalyptus grandis, E. urophylla and E. saligna, with higher values of the leaf characteristics evaluated, may be considered resistant or moderately resistant to G. brimblecombei.10.7717/peerj.13346/supp-1Supplemental Information 1The evaluations of the quantitative anatomical variables of the different Eucalyptus genotypes.Click here for additional data file.


Metastrongylus spp. in wild boars and the earthworm intermediate host species contributing to the maintenance of the life cycle. Here, wild boars were subjected to parasitological necropsies, and lungworm species were identified morphologically, followed by confirmation using ITS-2 sequencing and a phylogenetic analysis. Earthworms were collected from wild boar habitats and investigated for the presence of larvae. The prevalence of Metastrongylus spp. in wild boars was 78.8%, and many individuals were positive for all three detected species, Metastrongylus pudendotectus, Metastrongylus salmi and Metastrongylus elongatus. The phylogenetic analysis did not clearly resolve all species, except for M. pudendotectus. Age group and season had no influence on prevalence, while intensity was significantly higher in autumn than in spring and summer . Three out of six investigated earthworm species were positive for metastrongyloid larvae , but neither their phylogenetic relationship nor ecological microhabitats were able to explain these differences. Further sequence data should be used to improve the resolution in phylogenetic trees to determine potential cryptic species in the genus, while the application of deep sequencing approaches might provide insights into species-specific epidemiology and pathology.The aim of the present study was to characterize the diversity of  Metastrongylus Molin, 1861 [The superfamily of Metastrongyloidea Lane, 1917) is composed of approximately 200 nematode species divided into eight families. Several species infecting wild and domestic animals have a worldwide distribution 917 is co,4,5,6. Ain, 1861 ,11,12,13Wild boars live in small groups, constantly migrating from one place to another. They are omnivorous, feeding on vegetative underground  and the above-ground parts of plants, as well as various fruits, berries and seeds. Animals play a significant role in the diet of the wild boar, which is dominated by earthworms, mollusks, the larvae and adults of soil insects, small reptiles, amphibians and even small rodents, and they also scavenge on carrion and dead fish. Usually, wild boars obtain food by digging up the top layers of soil .Metastrongylus have been described: Metastrongylus elongatus Dujardin, 1845 ; Metastrongylus salmi Gedoelst, 1923; Metastrongylus pudendotectus Vostokov, 1905; Metastrongylus confusus Jansen, 1964; Metastrongylus asymetricus Noda, 1973; Metastrongylus madagascariensis Chabaud and Gretillat, 1956; and Metastrongylus tschiauricus Kojawa, 1956 [M. tschiauricus from wild boars in Georgia is closely related to M. pudendotectus and possibly identical to it.To date, seven species of the genus wa, 1956  indicateMetastrongylus species in order to avoid further misclassifications of Metastrongylus species. The identification of these worms is based on the morphology of the female’s posterior body and the length of the male’s spicules [Gasso et al.  developespicules . The speMetastrongylus spp. in the bronchi and bronchioles of the lungs. However, several cases of the infection of humans with M. elongatus and M. salmi have been described, indicating that these species are at least zoonotic [Metastrongylosis is an invasive disease of wild boars and domestic pigs caused by zoonotic ,18,19. Metastrongylus  were reported in Kazakhstan [Metastrongylus occurring in Uzbekistan are fragmentary, which is reflected in the small number of publications [M. elongatus, M. salmi and M. pudendotectus, were found, but the dominant species was M. pudendotectus [Helminthological studies of wild boars in Central Asia, including Kazakhstan and Tajikistan, were carried out at the end of the 19th century and in the last century ,20,21. Tzakhstan . In the ications ,23,24. Idotectus .Metastrongylus spp. females shed fully larvated eggs that are transported up the trachea before they are swallowed and passed with feces [Metastrongylus spp., and they allow for the completion of the parasite’s life cycle. Data regarding earthworms as intermediate hosts of Metastrongylus spp. are very limited [th feces . First-sth feces . The finth feces . Various limited ,33,34,35Metastrongylus spp. found in the lungs of wild boars  to evaluate genetic similarities and to perform phylogenetic analyses [In recent years, the first and second internal transcribing spacers (ITS-1 and ITS-2) of ribosomal DNA have been proven to be useful genetic markers for pulmonary and intestinal Strongylida ,39,40,41analyses ,42,43. Metastrongylus species endemic in Uzbekistan.The current study initially focused on the identification of ITS-2, which has future diagnostic potential to identify the sympatric assemblages of boar hosts and metastrongylid parasites from Uzbekistan. The goal of our research was to study the infection of definitive and intermediate hosts and to use molecular diagnostics to unequivocally identify the Between 2012 and 2020, the Institute of Zoology of the Academy of Sciences of the Republic of Uzbekistan subjected the lungs of 33 wild boars (shot and dead) to helminthological autopsy. The lungs were delivered by hunting farms from the Bekabad district and the western spurs of the Chatkal ridge (Akhangaran district) of the Tashkent region, the Bukhara and Karakul hunting farms of the Bukhara region and the Arnasay hunting farm of the Jizzakh region of Uzbekistan. Adult worms were recovered at necropsy from boars as specified by Kontrimavichus et al. . Adult wMetastrongylus species were differentiated based on morphological features under an ML 2000 series microscope . Adult nematodes were identified based on the morphology of the female’s posterior body, a pair of massive trilobed lips and long filiform spicules and on the male’s atypical bursa. The female has a relatively long vagina. The females and males of the different species were identified based on small differences in the morphology of the vulva and the spicules, respectively [The ectively ,4,16.More than 500 specimens of earthworms were studied regarding their potential role as intermediate hosts of metastrongylid nematodes. The collection of earthworms was carried out in juniper forests near springs, tugays, reed thickets and grasslands, which are all well-known places for rookeries and the feeding of wild boars in the Tashkent, Jizzakh and Bukhara regions of Uzbekistan . For a qMetastrongylus is 0.570 mm × 0.03 mm, and the posterior end ends in a tip ; not far from the caudal end, there is a small cuticular spine; the anterior end is blunt and cut off.The species identification of earthworms (Lumbricidae) was established in accordance with the guide developed by Perel . FurtherDNA was extracted using a DNA Purification kit  and eluted twice with 100 µL of the AE buffer provided in the kit. PCR amplification used 0.25 µM of each of the primers NC1 (ACGTCTGGTTCAGGGTTGTT) and NC2 (TTAGTTTCTTTTCCTCCGCT) , 0.2 U PMetastrongylus and two sequences of Angiostrongylus vasorum, one Aleurostrongylus abstrusus sequence and one Protostrongylus hobmaieri sequence, the latter to be used as an outgroup, were downloaded. The sequences were aligned using MAFFT 6.5 [The sequences of both strands were compared and edited using BioEdit . SimilarAFFT 6.5  on an onAFFT 6.5  using thAFFT 6.5 ,53. ModeAFFT 6.5  was usedAFFT 6.5 , the ShiAFFT 6.5  and an aAFFT 6.5 . The treStatistical analyses were either conducted in GraphPad Prism 5.02 or in R 4.1.1. Prevalence with 95% confidence intervals and significant differences in prevalence were calculated with the functions binom.wilson and tab2by2.test from the epitools package 0.5-10.1. Kruskal–Wallis tests, followed by Dunn’s post hoc tests and Mann–Whitney U tests, were conducted in GraphPad. If p values were corrected for multiple testing, the Bonferroni method was used.Metastrongylus were found: M. elongatus, M. pudendotectus and M. salmi. Quantitative data on the prevalence and intensity of infection  for the three regions of Uzbekistan are shown in M. pudendotectus and either M. salmi (n = 3) or M. elongatus (n = 2). One pig was only positive for M. pudendotectus, which was also the species with the highest prevalence, but the prevalence of the different species was neither significant for the complete dataset nor for the individual regions. Moreover, the differences between the regions were not significant.As a result of the parasitological examination of the wild boars, sexually mature nematodes of three species from the genus M. podendotectus and M. salmi, as well as for all Metastrongylus spp. together. Moreover, worm counts were significantly higher in M. podendotectus than in M. salmi and M. elongatus.The intensity was significantly higher in Jizzakh than in Tashkent  for M. pIn order to confirm the morphological species identification, the ITS-2 regions were successfully amplified and sequenced from adult lung nematode specimen samples, resulting in sequences of approximately 495 bp for all three species. One ITS-2 sequence for each species was deposited in GenBank . The obtM. pudendotectus formed a very homogenous, moderately well-supported cluster clearly separated from all other species. The other species M. elongatus, M. salmi, M. confuses and M. asymetricus formed a highly supported supracluster (indicated in M. elongatus (syn. M. apri) named in M. elongatus genotype group (GG) I and II. Between the two M. elongatus GGs, two sequences were located that only differed by a two-base-pair IN/DEL, i.e., Y08009 and Y08007. While the former is labeled as M. salmi in GenBank, the latter is M. confuses. The M. salmi sequence must have been obtained from a morphological misidentified specimen since it is the only M. salmi labeled sequence within this cluster. Thus, these sequences most likely represent M. confuses. A second, poorly supported large cluster 2 (43.1% SH-LRT support) was dominated by sequences labeled as M. salmi. This cluster 2 contained three well to highly supported subclusters with different M. salmi genotypes . However, species negative for metastrongylid larvae were also sometimes endogeic (A. jassyensis) or epigeic, i.e., species dwelling on the ground and in the litter layer and feeding predominantly from plant litter [In total, six different species of earthworms were identified, and the sample size of each species was considerably high (n = 46–96). Metastrongylid larvae were found in three species, negative . Almost t litter .Metastrongylus contains several parasites of pigs that are rarely investigated. Due to the obligate intermediate host, these parasites are not relevant in industrial pig production [The genus oduction , and theoduction . Since wMetastrongylus spp. have been reported worldwide with variable frequencies [Metastrongylus. In this study, M. pudendotectus was slightly more prevalent than the other two species. Another study from Central Asia involved only 10 wild boras from two different regions of Kazakhstan. Again, the same three species were found. However, the prevalence was considerably lower, with 42.8% for M. pudendotectus and M. elongatus, but the authors did not report the prevalence of M. salmi [M. pudendotestus and M. elongatus [M. elongatus, M. pudendotectus and M. salmi [Metastrongylus spp., with M. pudendotectus being the most prevalent species, followed by M. elongatus and M. salmi [M. pudendotectus being found most frequently, followed by M. salmi, M. confusus and M. apri, with M. asymetricus being the least frequently found [M. pudendotectus, M. salmi, M. asymmetricus, M. elongatus and M. confusus. When looking into geographical regions other than Eurasia, a high prevalence of 84.5% for Metastrongylus spp. was described for wild boars in Morocco. Again, M. pudendotectus was the most prevalent species (84.5%), but with a prevalence of 72.7%, M. confusus was the second most frequently found species, followed by M. salmi (51.5%), whereas M. elongatus was not found. In Japan, wild boars showed 100% prevalence, and 64.3% were infected with all four species that were detected. The order of prevalence was M. asymmetricus, M. salmi, M. pudendotectus and M. elongatus. In feral pigs in Florida, M. apri, M. salmi and M. pudendotectus were collected during necropsies, with a prevalence of 94%, 76% and 65%, respectively [M. pudendotectus, M. elongatus and M. salmi  is widely observed, and the results from Uzbekistan are in line with many previous data from this region. quencies ,66,67,68quencies , a similM. salmi . In Eastlongatus . In a naM. salmi . In BelaM. salmi . The samM. salmi . In contM. salmi , Poland M. salmi  and SwitM. salmi . In Switly found . In PolaMetastrongylus species. This was also the case for all other studies cited above regarding the prevalence and species composition of pig lungworm. The high number of Metastrongylus species in combination with the very high overall prevalence that was described in all the studies make it clear that co-infections must be the rule rather than the exception. However, none of the studies, including the present one, has attempted to statistically analyze whether co-infections occur more frequently than one would expect according to the prevalence of each individual Metastrongylus species. The simple reason for this is that the number of animals included in these studies ranged from 5 to less than 50, and, thus, this is far too low for meaningful analyses of such dependencies.In Uzbekistan, the majority of wild boars were infected with multiple Metastrongylus spp. turned out to be highly prevalent (63.6–92.3%), with infections reaching very high intensities in the present study. While the prevalence of the nematodes was not significantly influenced by age group or the season of the year, there was a significant effect of the season on intensity, with autumn showing a higher intensity than summer and winter. However, these results should be considered with caution since the very low intensities in summer were due to only three infected wild boars from this season. The difference between summer and autumn was based on much higher animal numbers, but it was also only moderate. In contrast to the results shown here, the infestation of earthworms in spring, summer and autumn has been described to be approximately the same in Belarus [Earthworms are a major part of the feed of wild boars, and, thus,  Belarus . This suAporrectodea caliginosa, Dendrobaena octaedra, Eisenia fetida and Lumbricus terrestis [Metastrongylus spp. has been shown to be the major factor influencing infections in earthworms [A. jassyensis were all negative.Earthworms of the family Lumbricidae that serve as intermediate hosts can have a very high prevalence of almost 100%, with an intensity of tens to a few thousand larvae in one worm as described for species such as errestis . The infrthworms . Earthworthworms , which crthworms . HoweverMetastrongylus spp. with wild boars and relevant lumbricid earthworm species has rarely been investigated. The earthworms collected in the present study came from typical wild boar habitats. The fact that very closely related earthworm species, such as the two Aporectodea and the two Eisenia species, differed so strongly in the prevalence of metastrongylid larvae suggests that the members of these species pairs differ in their ecological macro- or micro-habitats. Since both are considered to be endogeic, different macrohabitats with different wild boar densities or activities might be important variables. The collection of different earthworm species with sampling sites that are georeferenced on a very small scale and with reference to the surrounding vegetation and wild boar activity might help to provide explanations for the unusual distribution pattern of metastrongyloid larvae observed here. However, such investigations are very labor-intensive since the sample site should be considered the statistical unit in many of the methods needed to analyze such data.The interaction of M. elongatus, M. pudendotectus and M. salmi. As discussed above, many of the wild boars were infected with multiple Metastrongylus species simultaneously. This makes it difficult to determine whether there are differences in the epidemiology  or pathogenicity of the different species. Molecular tools have the potential to improve the available data on individual Metastrongylus spp., e.g., tools based on non-invasive fecal samples, in the future.Based on both morphologic and molecular data, all isolated lungworms from the wild boars in Uzbekistan were M. elongatus, M. pudendotectus and M. salmi in wild boars, which allowed for the direct display of sequence variation within and among individuals representing each species.The literature data on the comparative study of the ITS-2 of other representatives of lungworms show that there are small but stable species differences that allow for the information about the structure of this site to be used as a rather effective tool for resolving the controversial issues of the taxonomy of nematodes of this group ,76. For M. pudendotectus is clearly separated from the other species. The separation of the four species M. elongatus, M. salmi, M. confusus and M. asymetricus was not completely clear. Neither the sequences of M. elongatus nor those of M. salmi formed monophyletic clusters in the phylogram since M. confusus is located in the M. elongatus and M. asymetricus in the M. salmi cluster. This could be interpreted as if M. confuses is only a special morphotype of M. elongatus and M. asymetricus a morphotype of M. salmi and that these names should be considered synonyms. However, statistical support for those clusters containing all M. elongatus (and M. confusus) and all M. salmi (and M. asymetricus) was also only poor. Alternative interpretations of the tree could be that all sequences in the supracluster belong to a single, genetically quite variable species, or that each of the genotype groups represents a different species. This would mean that M. elongatus would be split into two species, and M. salmi would be split into three species. Additional sequence data are required to determine which of these alternatives is the preferable hypothesis and to also better understand the phylogenetic history of M. asymetricus in the genus. In previous studies, it was shown that combined analyses of nuclear  and mitochondrial  genes can help to obtain both a reliable phylogenetic tree and enough resolution to discriminate between closely related, sometimes cryptic, species [M. confusus and M. asymetricus.In the phylogenetic tree,  species ,77,78,79PCR-based approaches have become an important tool for investigating parasite communities and populations but also host–parasite interactions, and they will probably also help in livestock management in the future . The abi


This allowed the nucleus to sink ((3).A 63-year-old man was referred to our hospital with a complaint of decrease in the vision of his left eye. The visual acuity of his left eye was hand motion. His intraocular pressure was 18 mmHg, with no evidence of intraocular inflammation. The lens had hypermatured as a Morgagnian cataract characterized by total liquefaction of the cortex  to sink , as show to sink . PhacoemNone.T.O. and K.M. contributed to the design and conduct of the study and the collection, management, and interpretation of data. K.M. contributed to the preparation, review, and approval of the manuscript.Not applicable.Written informed consent was obtained from the patient.


Dear Editor,Nephelium lappaceum var. lappaceum), a tropical fruit tree native to southeastern Asia, belongs to the family Sapindaceae. Rambutan is a popular table fruit and is also processed into preserves, juices, wines, and sorbets  and partially by a National Science Foundation (NSF) CAREER award [DBI-1933521], a National Institutes of Health (NIH) R01 award [R01GM140370], and a start-up grant from UNL [2019-YIN] to Y.Y. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.YY, DZ, LWM, RG, and TM conceived and designed the project. DZ, LWM, and RG collected the plant materials and generated the sequencing data. JZ performed all the data analysis under the supervision of YY. JZ and YY drafted the manuscript. All authors contributed to and approved the final manuscript.N. lappaceum cultivar R-162 have been submitted to NCBI. The BioProject ID is PRJNA766632, the BioSample ID is SAMN21855570, and the Whole Genome Shotgun accession number will be available upon publication of the paper. The genome and annotation data can also be accessed at https://bcb.unl.edu/Nla/.The raw DNA sequencing reads and the assembled genome of No conflicts of interest are declared.Horticulture Research  online.Web_Material_uhac014Click here for additional data file.


Both subjective cognitive decline (SCD) and amnestic mild cognitive impairment (aMCI) have a high risk of progression to Alzheimer's disease (AD). While most of the available evidence described changes in functional connectivity (FC) in SCD and aMCI, there was no confirmation of changes in functional connectivity density (FCD) that have not been confirmed. Therefore, the purpose of this study was to investigate the specific alterations in resting-state FCD in SCD and aMCI and further assess the extent to which these changes can distinguish the preclinical and early-stage AD.A total of 57 patients with SCD, 59 patients with aMCI, and 78 healthy controls (HC) were included. The global FCD, local FCD, and long-range FCD were calculated for each voxel to identify brain regions with significant FCD alterations. The brain regions with abnormal FCD were then used as regions of interest for FC analysis. In addition, we calculated correlations between neuroimaging alterations and cognitive function and performed receiver-operating characteristic analyses to assess the diagnostic effect of the FCD and FC alterations on SCD and aMCI.FCD mapping revealed significantly increased global FCD in the left parahippocampal gyrus (PHG.L) and increased long-range FCD in the left hippocampus for patients with SCD when compared to HCs. However, when compared to SCD, patients with aMCI showed significantly decreased global FCD and long-range FCD in the PHG.L. The follow-up FC analysis further revealed significant variations between the PHG.L and the occipital lobe in patients with SCD and aMCI. In addition, patients with SCD also presented significant changes in FC between the left hippocampus, the left cerebellum anterior lobe, and the inferior temporal gyrus. Moreover, changes in abnormal indicators in the SCD and aMCI groups were significantly associated with cognitive function. Finally, combining FCD and FC abnormalities allowed for a more precise differentiation of the clinical stages.To our knowledge, this study is the first to investigate specific alterations in FCD and FC for both patients with SCD and aMCI and confirms differential abnormalities that can serve as potential imaging markers for preclinical and early-stage Alzheimer's disease (AD). Also, it adds a new dimension of understanding to the diagnosis of SCD and aMCI as well as the evaluation of disease progression. Subjective cognitive decline (SCD) and amnestic mild cognitive impairment (aMCI) have been recognized as the preclinical and prodromal stages of Alzheimer's disease (AD), respectively , known as a non-invasive technique, is extensively applied in the AD-related spectrum for detecting functional activity in the brain by measuring various indicators such as functional connectivity (FC) and others  . According to previous studies , executive function (EF), visuospatial function (VF), and information processing speed (IPS), was conducted among the participants , with the center corresponding to the peak voxel (radius = 6 mm), and then conducted a Pearson correlation analysis between the average time courses of ROI and whole-brain voxel under the gray matter mask of the whole brain. Finally, fisher's r - z transformation was performed on all FC maps for normality .Statistical Package for Social Sciences (SPSS) software, version 22.0 , was used to perform statistical analyses that were not related to voxel computations. The χp < 0.01 with a cluster size > 150 voxels. Post-hoc comparisons, using the two-sample t-test with the results of ANOVA analyses as the mask, were conducted with age, gender, education level, and gray matter volumes as covariates. To strictly correct the results, a TFCE and the familywise error (FWE) were applied with a threshold of p < 0.05 and a cluster size > 50 voxels.Using DPABI software, a one-way ANOVA was applied to compare the differences in gFCD, lFCD, and lrFCD across three groups with age, gender, years of education, and gray matter volumes as covariates. The nonparametric permutation test (1000 permutations) was applied, and the significance level was set to For the resting-state FC analysis, it is important to note that we only performed further FC analysis between groups with significantly different FCD values. That is, if the FCD value of a seed point differed between all three groups, we would apply ANOVA and a two-sample t-test to determine the FC changes between groups. However, if the FCD value of the seed point differed between two groups, we would perform a two-sample t-test to analyze the FC changes between only these two groups. All statistical analyses included age, gender, years of education, and gray matter volume as covariates.The Pearson correlation analysis was performed to examine the correlation between the altered FCD, FCs, and cognitive domains after controlling age, gender, and education level using SPSS software. Additionally, also in SPSS software, ROC curves were analyzed based on substantially changed indicators between the patient and HC groups, such as FCD and FC, to assess their value in the distinction of pre-Alzheimer's spectrum. Each index's discriminating performance was assessed independently. Moreover, significantly altered indexes were combined using a binary logistic regression model, and the model's performance was also evaluated. Finally, we assessed the sensitivity and specificity of each biomarker individually and in combination.P = 0.3435) between the three groups but found significant differences in gender (P = 0.0252) and education level (P = 0.0402). To avoid the impact of demographic differences on subsequent analyses, all statistical analyses included gender, age, and education level as covariates. As expected, general cognitive function scores  of patients with aMCI showed significant decreases as compared to those of patients with SCD (p < 0.01) and HCs (p < 0.001). In teams of every cognitive domain, including EM, EF, IPS, and VF, patients with aMCI also had statistical deficits in comparison with HC (p < 0.05) and SCD (p < 0.05). Moreover, for the Subjective Cognitive Decline Questionnaire (SCD-Q), there were significant alterations between the three groups (p < 0.001). Details can be obtained in We found no statistical discrepancy in age  and the right temporal pole: the middle temporal gyrus. In comparison to HC, the value of gFCD of the PHG.L was significantly different between groups, with significantly higher values in patients with SCD and significantly lower values in patients with aMCI .post-hoc test, patients with SCD exhibited significantly higher lrFCD in the left hippocampus than HCs and significantly higher lrFCD in the PHG.L than patients with aMCI , and the left hippocampus. In the ith aMCI .post-hoc test were carried out. Compared with HCs, patients with SCD showed markedly increased FC in bilateral superior occipital gyrus/middle occipital gyrus (SOG/MFG) and bilateral cuneus with the peak coordinate  located in the left SOG (SOG.L). In addition, in comparison with SCD, the aMCI group presented significantly decreased FC in bilateral SOG/MFG and right calcarine with the peak coordinated  located in the right calcarine. No statistical significance was found between aMCI and HC groups , the statistical differences in FCD values of this brain area involved three groups. An ANOVA analysis and a C groups .For the left hippocampus seed , a two-sample t-test was applied between SCD and HC groups. In this comparison, the SCD group exhibited increased FC in the left cerebellum anterior lobe (CAL.L) and the ITG.L .Pearson correlation analysis showed that, in the groups consisting of aMCI and SCD, the abnormal gFCD value in the PHG.L was prominently positively correlated with EM and EF. Besides, the altered FC between the PHG.L and right calcarine was also correlated with EM positively .p < 0.001 and 0.652 with p = 0.003. FC between PHG.L and SOG.L, FC between the left hippocampus and CAL.L, and FC between the left hippocampus and ITG.L had AUC values of 0.684 with p < 0.001, 0.722 with p < 0.001, and 0.661 with p = 0.001, respectively. In addition, using a binary logistic regression model, we obtained individual predictors of the combination of multiple indexes. The AUC value of the combination of multiple indexes was 0.827 with p < 0.001, sensitivity = 87.7%, specificity = 66.7%  values of gFCD of the PHG.L and lrFCD of the left hippocampus were 0.678 with  = 66.7% .p < 0.001 and 0.771 with p < 0.001. The AUC values of gFCD of the PHG.L and lrFCD of the PHG.L were 0.741 with p < 0.001 and 0.643 with p < 0.08. The AUC value of FC between the PHG.L and right calcarine was 0.635 with p < 0.012. In addition, the AUC value of the combined index was 0.941 with p < 0.001, sensitivity = 93.2%, specificity = 80.7%  were first found in the left hemisphere. It is well-known that directional hemispheric dominance has been established for a variety of cognitive functions and that lateralization is particularly important for cognitive functions  and the VN  for the publication of any potentially identifiable images or data included in this article.XLin, JC, and YS designed the study. HW, SC, CX, YS, QY, HG, WQ, XLia, and ZY collected the data. YS, HW, and SC analyzed the data and prepared the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (No. 81701675); the Key Project supported by Medical Science and the Technology Development Foundation, Nanjing Department of Health (No. JQX18005); and the Key Research and Development Plan  Project of Jiangsu Province (No. BE2018608).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.