Identifying the scene of a crime through pollen analysis.

The forensic analysis of pollen involves the comparison of crime scene and reference pollen samples. Successful matches are frequently used to solve time- or location-related crimes. Despite its prospects in criminal investigation, forensic palynology is still underused in casework due to inherent shortcomings such as its limited evidential weighting, scarcity of skilled palynologists dedicated to forensic casework and the laborious nature of analytical procedures. To address these challenges, the current state-of-the-art in forensic palynology is transiting from the traditional light microscopic methods that dominated the early days of palynology to more contemporary approaches like Raman spectroscopy, stable isotope analysis and DNA metabarcoding. The major challenges of these methods, however, include a lack of optimisation to forensic expectations and the unavailability of robust databases to permit accurate data interpretation, and quests to resolve these problems constitute the theme of current research. While reiterating the usefulness of pollen analysis in criminal investigation, this report recommends orthogonal testing as a way of improving the evidential weighting of forensic palynology.

Description of a new species of myxobolid parasite, Henneguya pindaibensis n. sp. (Cnidaria: Myxosporea), infecting the gills of Boulengerella cuvieri (Spix and Agassiz, 1829) from Brazil.

Myxozoans are microscopic cnidarians that mainly parasitize fishes. The present study aimed to describe a new myxozoan parasite from the gills of Boulengerella cuvieri (Spix and Agassiz, 1829) by morphological and molecular analysis. The fish was collected in 2019 at the Pindaíba River, municipality of Cocalinho, Mato Grosso State, Brazil. Whitish and circular plasmodia were found in the primary gill filaments, occupying an intralamellar position, with an average of 0.5 mm in diameter. Henneguya Thélohan, 1892 myxospores found inside the plasmodia were elongated and ellipsoidal, consisting of two long and elliptical shell valves with two long, tapering caudal appendages. Morphometric measurements revealed a total spore length of 36.1 ± 2.0 µm; spore body length of 12.8 ± 0.5 µm; spore width of 4.9 ± 0.3 µm; tail length of 23.3 ± 1.6 µm; capsule length of 7.2 ± 0.4 µm; capsule width of 1.5 ± 0.2 µm; and 10 coils in the polar filament. Phylogenetic analysis showed that the isolates from this study were grouped into the main-clade of freshwater fishes, within a group of species parasitizing fishes from Brazil. Intergenotypic difference ranged from 23%-25.9% compared with other Brazilian myxozoan isolates. Using molecular and morphological characterization, this parasite was identified as a new species of the genus Henneguya.

Clustering approach for the analysis of the fluorescent bioaerosol collected by an automatic detector.

Automatically operating particle detection devices generate valuable data, but their use in routine aerobiology needs to be harmonized. The growing network of researchers using automatic pollen detectors has the challenge to develop new data processing systems, best suited for identification of pollen or spore from bioaerosol data obtained near-real-time. It is challenging to recognise all the particles in the atmospheric bioaerosol due to their diversity. In this study, we aimed to find the natural groupings of pollen data by using cluster analysis, with the intent to use these groupings for further interpretation of real-time bioaerosol measurements. The scattering and fluorescence data belonging to 29 types of pollen and spores were first acquired in the laboratory using Rapid-E automatic particle detector. Neural networks were used for primary data processing, and the resulting feature vectors were clustered for scattering and fluorescence modality. Scattering clusters results showed that pollen of the same plant taxa associates with the different clusters corresponding to particle shape and size properties. According to fluorescence clusters, pollen grouping highlighted the possibility to differentiate Dactylis and Secale genera in the Poaceae family. Fluorescent clusters played a more important role than scattering for separating unidentified fluorescent particles from tested pollen. The proposed clustering method aids in reducing the number of false-positive errors.

A New Species of ThelohanellusKudo, 1933 (Myxozoa: Bivalvulida) Infecting Skeletal Muscle of Blacktail Shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in the Chattahoochee River Basin, Georgia.

Thelohanellus magnacysta n. sp. (Bivalvulida: Myxobolidae) infects the skeletal muscle of blacktail shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in Bull Creek, Chattahoochee River Basin, eastern Georgia. Although numerous members of ThelohanellusKudo, 1933 have overlapping myxospore dimensions with the new species, it differs from all nominal congeners by polar filament coil number and polar capsule width as well as by lacking a mucous envelope, iodinophilic vacuole, and sutural markings. With the use of novel primers for Myxozoa, a phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) suggests that the new species shares a recent common ancestor with a clade of cyprinid-infecting species of Myxobolus Bütschli, 1882 (Bivalvulida: Myxobolidae) and Thelohanellus. Consistent with other published research concerning the systematics of Thelohanellus, this result suggested that Thelohanellus and Myxobolus are polyphyletic and need revision. Histological sections of infected blacktail shiners confirmed that myxospores were only found within a plasmodium and only infected skeletal muscle and that plasmodia were encapsulated by a granuloma comprising varying degrees of acute granulomatous inflammation. The new species is the fourth of Thelohanellus reported from North America and the first reported from Cyprinella, as well as the first myxozoan described from the blacktail shiner.

Temperate rainforests near the South Pole during peak Cretaceous warmth.

The mid-Cretaceous period was one of the warmest intervals of the past 140 million years1-5, driven by atmospheric carbon dioxide levels of around 1,000 parts per million by volume6. In the near absence of proximal geological records from south of the Antarctic Circle, it is disputed whether polar ice could exist under such environmental conditions. Here we use a sedimentary sequence recovered from the West Antarctic shelf-the southernmost Cretaceous record reported so far-and show that a temperate lowland rainforest environment existed at a palaeolatitude of about 82° S during the Turonian-Santonian age (92 to 83 million years ago). This record contains an intact 3-metre-long network of in situ fossil roots embedded in a mudstone matrix containing diverse pollen and spores. A climate model simulation shows that the reconstructed temperate climate at this high latitude requires a combination of both atmospheric carbon dioxide concentrations of 1,120-1,680 parts per million by volume and a vegetated land surface without major Antarctic glaciation, highlighting the important cooling effect exerted by ice albedo under high levels of atmospheric carbon dioxide.

Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy.

Many bacterial species are capable of forming long-lived dormant cells. The best characterized are heat and desiccation resistant spores produced by many Gram-positive species. Less characterized are dormant cysts produced by several Gram-negative species that are somewhat tolerant to increased temperature and very resistant to desiccation. While there is progress in understanding regulatory circuits that control spore germination, there is scarce information on how Gram-negative organisms emerges from dormancy. In this study, we show that R. centenum cysts germinate by emerging a pair of motile vegetative cells from a thick cyst cell wall coat ~ 6 hrs post induction of germination. Time-lapse transcriptomic analysis reveals that there is a defined temporal pattern of gene expression changes during R. centenum cyst germination. The first observable changes are increases in expression of genes for protein synthesis, an increase in expression of genes involved in the generation of a membrane potential and the use of this potential for ATP synthesis via ATPase expression. These early events are followed by expression changes that affect the cell wall and membrane composition, followed by expression changes that promote chromosome replication. Midway through germination, expression changes occur that promote the flow of carbon through the TCA cycle to generate reducing power and parallel synthesis of electron transfer components involved in oxidative phosphorylation. Finally, late expression changes promote the synthesis of a photosystem as well as flagellar and chemotaxis components for motility.

A rapid rice blast detection and identification method based on crop disease spores' diffraction fingerprint texture.

BACKGROUND: Rice blast fungus is a worldwide disease, and it is one of the most serious rice diseases in the north and south rice fields in China. The initial symptoms of rice blast are not obvious, and the speed of transmission is fast. Manual identification is time-consuming and laborious. At present, it is a great challenge to realize rapid and accurate early identification of rice blast. RESULTS: In this paper, an identification method based on crop disease spores' diffraction fingerprint texture for rice blast was studied; this method utilizes the light field and texture features of diffraction images. To verify the reliability of the model that we proposed, we selected two methods of manual identification and machine recognition to compare and detect rice blast spores. The experimental results show that the identification of light diffraction characteristics is not only higher than the traditional manual recognition by microscope (increased by more than 0.3%), but also faster after neural network training (increased by more than 90%). The diffraction recognition method used in this study, based on crop disease spores' diffraction fingerprint texture, can be completed in a few seconds, and its test accuracy is 97.18%. CONCLUSION: The proposed method, a rapid rice blast detection and identification method based on crop disease spores' diffraction fingerprint texture, has certain advantages compared with the existing manual identification by microscope. This method can be applied to the recognition of rice blast in agricultural research. © 2020 Society of Chemical Industry.

Are we There Yet? Understanding Interplanetary Microbial Hitchhikers using Molecular Methods.

Since the early time of space travel, planetary bodies undergoing chemical or biological evolution have been of particular interest for life detection missions. NASA's and ESA's Planetary Protection offices ensure responsible exploration of the solar system and aim at avoiding inadvertent contamination of celestial bodies with biomolecules or even living organisms. Life forms that have the potential to colonize foreign planetary bodies could be a threat to the integrity of science objectives of life detection missions. While standard requirements for assessing the cleanliness of spacecraft are still based on cultivation approaches, several molecular methods have been applied in the past to elucidate the full breadth of (micro)organisms that can be found on spacecraft and in cleanrooms, where the hardware is assembled. Here, we review molecular assays that have been applied in Planetary Protection research and list their significant advantages and disadvantages. By providing a comprehensive summary of the latest molecular methods yet to be applied in this research area, this article will not only aid in designing technological roadmaps for future Planetary Protection endeavors but also help other disciplines in environmental microbiology that deal with low biomass samples.

New data on Myxobolus imparfinis (Cnidaria, Myxosporea): host, distribution, and ultrastructural morphology.

A mass of free myxozoan spores was found in the gill filaments of specimens of Cetopsorhamdia iheringi Schubart and Gomes, 1959, popularly known as "three-barbeled catfishes" (Heptapteridae, Siluriformes) collected in streams of the Middle Paranapanema River, Upper Paraná River basin, in the state of São Paulo, Brazil. Morphological and molecular analysis identified the spores as Myxobolus imparfinis Vieira, Tagliavini, Abdallah and Azevedo, 2018. The ultrastructural morphology of this parasite is described here for the first time. Differences were observed in the number of coils of the polar filament as well as some organelles not previously described for this species. Asynchronous development was also observed, with the presence of both mature and immature spores. This is the first report of a myxozoan parasitizing C. iheringi and the first geographical record of myxozoan parasites in streams of the Middle Paranapanema River. The new data improve the original description of the species and add to the knowledge of host-parasite interactions and distribution.

Morphological and molecular characterization of Ceratomyxa batam n. sp. (Myxozoa: Ceratomyxidae) infecting the gallbladder of the cultured Trachinotus ovatus (Perciformes: Carangidae) in Batam Island, Indonesia.

A new coelozoic myxozoan species, Ceratomyxa batam n. sp., was identified in cultured carangid fish, Trachinotus ovatus (Perciformes: Carangidae), in waters off Batam Island of Indonesia. The bi- and trivalved spores were observed in the gallbladder of T. ovatus. Mature bivalved spores of C. batam n. sp. were transversely elongated and narrowly crescent in shape, 3.8 ± 0.36 (2.7-4.6) µm long and 19.2 ± 1.75 (16.2-22.0) µm thick. Two sub-spherical polar capsules were 2.3 ± 0.18 (2.0-2.8) µm long and 2.6 ± 0.16 (2.3-2.9) µm wide. Prevalence was 72.2% in 72 examined T. ovatus according to evaluations dating from November 2016. The maximum likelihood phylogenetic tree based on small subunit rDNA sequence showed similarity with Ceratomyxa robertsthomsoni and Ceratomyxa thalassomae found in Australia. This is the first report of Ceratomyxa species identified in a seawater fish at Batam Island, Indonesia.

New host records of three Kudoa spp. (K. yasunagai, K. thalassomi, and K. igami) with notable variation in the number of shell valves and polar capsules in spores.

To date, 26 Kudoa spp. (Myxozoa: Myxosporea: Multivalvulida) have been recorded in edible marine fishes in Japan. In the future, it is likely that even more marine fish multivalvulid myxosporeans will be characterized morphologically and genetically, which will aid the precise understanding of their biodiversity and biology. We examined 60 individuals of six fish species collected from the Philippine Sea off Kochi or from the border between the Philippine Sea and East China Sea around Miyako Island, Okinawa, i.e., the southern part of Japan. Newly collected parasite species included Kudoa yasunagai from the brain of Japanese meagre (Argyrosomus japonicus) and Japanese parrotfish (Calotomus japonicus), Kudoa miyakoensis n. sp. and Kudoa thalassomi from the brain and trunk muscle, respectively, of bluespine unicornfish (Naso unicornis), and Kudoa igami from the trunk muscle of Carolines parrotfish (Calotomus carolinus), African coris (Coris gaimard), and Pastel ringwrasse (Hologymnosus doliatus). With the exception of Japanese parrotfish for K. yasunagai, all these fish are new host records for each kudoid species. Notable variation in the number of shell valves (SV) and polar capsules (PC) was observed for all four kudoid species. In particular, spores with seven or eight SV/PC were prominent in K. igami isolates, despite the original Japanese parrotfish-derived description characterizing it as having spores with six, or less commonly five, SV/PC. However, molecular genetic characterization based on the ribosomal RNA gene (rDNA) and mitochondrial DNA (cytochrome c oxidase subunit 1 and ribosomal RNA small and large subunits) found no significant differences in the nucleotide sequences of isolates with different phenotypical features as far as examined in the present study. A newly erected species, K. miyakoensis n. sp., was determined to be phylogenetically closest to brain-parasitizing species, such as K. chaetodoni, K. lemniscati, and K. yasunagai based on rDNA nucleotide sequences, but differed from them morphologically.

On-line measurement of fluorescent aerosols near an industrial zone in the Yangtze River Delta region using a wideband integrated bioaerosol spectrometer.

In this work, we present on-line fluorescent aerosol measurements by the wideband integrated bioaerosol spectrometer (WIBS-4A) near an industrial zone in Nanjing, a megacity in the Yangtze-River-Delta (YRD) region. The fieldwork was conducted from April 1 to May 8, 2014. A TSI. 3321 aerosol-particle-sizer (APS) was simultaneously deployed to measure the total number size distribution of aerosol with diameter from 0.8-20 µm. Both WIBS-4A and APS reported similar number concentration and temporal profiles (R2 = 0.72). However, the daily average number of potential bioaerosols was only 0.5 ±â€¯0.2% of the total particles detected by the WIBS-4A and displayed a completely different diurnal profile from that of APS. In addition, WIBS-4A can only provide integrated fluorescent signals, which strongly limited the potential to specifically identify the bioaerosols. Accordingly, hierarchical-agglomerative-cluster-analysis (HACA) was utilized to identify and speciate the potential bioaerosols from the WIBS-4A dataset. By maximizing the total distances among all potential cluster centers, a 12-cluster solution was accepted as the optimum result. These clusters were further identified according to their fluorescent signatures, size, and morphology, i.e., non-bioaerosols, bacteria, and fungal spores and/or pollen fragments. Bacteria were the dominant bioaerosol species detected in this work. The diurnal profiles of bioaerosols correlated very well with relatively humidity (RH), reaching daily maxima around 3 AM~6 AM, indicating the presence of humidity controlled bioaerosol emission mechanism, i.e., bacteria may flourish under moderate ambient temperature, RH, and the absence of UV radiation. The size- and AF-distributions of bioaerosols indicated that bioaerosols normally varied substantially in size and assumed a rather irregular shape. Although the number concentration of bioaerosols was relatively small, most bioaerosols can efficiently serve as ice nuclei by providing rough and irregular surfaces, verified by the observation results. Therefore, WIBS-4A measurements can still be informative for investigations of bioaerosols in the atmosphere, especially when HACA method was incorporated into the data processing.

Palynological Investigation of Mummified Human Remains.

Pollen analysis was applied to a mummified homicide victim in Nebraska, U.S.A., to determine the location of death. A control sample showed the normal ambient pollen in the garage crime scene. Ambient windborne types, common in the air of the region, dominated the control. Internal samples were analyzed from the sacrum, intestine, and diaphragm. Microfossils were recovered from the rehydrated intestine lumen. The intestinal sample was dominated by Brassica (broccoli). The sacrum sample was high in dietary types but with a showing of ambient types. The pollen from the diaphragm was dominated by ambient pollen similar to the control samples. The discovery of diverse pollen spectra from within a single mummy was unexpected. They show that ingested and inhaled pollen mixed in the corpse. The data linked the decedent to a specific crime scene in her Nebraska home in the southern tier of eastern counties on the border with Kansas.

Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain.

Bacterial conjugation is a powerful tool used for DNA transfer from Escherichia coli into various bacteria including streptomycetes. In this methodology, spores are usually employed as recipient cells of the genetic information. However, some industrially important Streptomyces do not produce spores making difficult their genetic manipulation. In these strains, the use of mechanically fragmented mycelia allows DNA transfer with low efficiency. Streptomyces peucetius var. caesius is a non-sporulating bacteria which produces the antitumor compound doxorubicin. The use of aerial mycelia of this microorganism, failed to get intergeneric conjugation with E. coli. In the present work, by using young aerial mycelia of this microorganism and an excess of E. coli cells (~7×108cellsmL-1) in soybean-mannitol medium (MS) supplemented with 20mMMgCl2 resulted in a high number of exconjugant colonies (5×10-4) when compared to other reports from this genus (1.1×10-5 to 2.5×10-8). The effectiveness of these conditions was confirmed by isolating null mutants of two different glucokinases from S. peucetius var. caesius. The novelty in using young aerial mycelia as receptor cells, allowed an efficient conjugative process and opened the way for genetic manipulation of additional non-spore forming actinobacteria exhibiting natural resistance to be genetically manipulated.

Effects of postharvest oligochitosan treatment on fungal diseases and defence responses in Dongxue peach fruit.

In this study, the effects of oligochitosan treatment on controlling postharvest diseases in Dongxue peach ( Prunus Persica L. Batsch, cv Dongxuemi) were examined and the possible underlying mechanisms were discussed. Results showed that the disease incidence and lesion area in peach fruit inoculated with Monilinia fructicola and Penicillium expansum were all remarkably reduced by oligochitosan treatment. Oligochitosan treatment inhibited spore germination and mycelial growth of the two fungi in vitro. Oligochitosan treatment also induced upregulation of the salicylic acid signalling pathway-related genes (NPR1, PR1 and phenylalanine ammonia lyase) and enhanced the levels of total phenolics, flavonoids and lignin in peach. Meanwhile, enzymatic activities of superoxide dismutase, catalase, polyphenoloxidase, ascorbate peroxidase and phenylalanine ammonia lyase also increased. These findings suggest that the effects of oligochitosan on the disease control of peach fruit may be associated with its direct antimicrobial effects as well as increasing antioxidant, phenylpropanoid metabolism and accumulating antifungal compounds by activating the salicylic acid-dependent pathway.

Morphological and molecular confirmation of Myxobolus cerebralis myxospores infecting wild­caught and cultured trout in North Carolina (SE USA).

We used microscopy and molecular biology to provide the first documentation of infections of Myxobolus cerebralis (Myxozoa: Myxobolidae), the etiological agent of whirling disease, in trout (Salmonidae) from North Carolina (USA) river basins. A total of 1085 rainbow trout Oncorhynchus mykiss, 696 brown trout Salmo trutta, and 319 brook trout Salvelinus fontinalis from 43 localities across 9 river basins were screened. Myxospores were observed microscopically in pepsin-trypsin digested heads of rainbow and brown trout from the Watauga River Basin. Those infections were confirmed using the prescribed nested polymerase chain reaction (PCR; 18S rDNA), which also detected infections in rainbow, brown, and brook trout from the French Broad River Basin and the Yadkin Pee-Dee River Basin. Myxospores were 9.0-10.0 µm (mean ± SD = 9.6 ± 0.4; N = 119) long, 8.0-10.0 µm (8.8 ± 0.6; 104) wide, and 6.0-7.5 µm (6.9 ± 0.5; 15) thick and had polar capsules 4.0-6.0 µm (5.0 ± 0.5; 104) long, 2.5-3.5 µm (3.1 ± 0.3; 104) wide, and with 5 or 6 polar filament coils. Myxospores from these hosts and rivers were morphologically indistinguishable and molecularly identical, indicating conspecificity, and the resulting 18S rDNA and ITS-1 sequences derived from these myxospores were 99.5-100% and 99.3-99.8% similar, respectively, to published GenBank sequences ascribed to M. cerebralis. This report comprises the first taxonomic circumscription and molecular confirmation of M. cerebralis in the southeastern USA south of Virginia.

Myxobolus sp. and Henneguya sp. (Cnidaria: Myxobolidae) natural co-infection in the kidney of Piaractus mesopotamicus (Characiformes: Serrasalmidae).

This study evaluated the myxozoan infection and histopathology of the kidney of freshwater fish Piaractus mesopotamicus from intensive fish farming in Brazil. A total of 55 fish were examined for myxozoan infection. Infected organs were processed by usual histology and stained with hematoxylin-eosin (H&E) and Ziehl-Neelsen (ZN). From the total of 55 fish analyzed, 47 (85.45%) presented myxospores, being 9.09% (5/55) only with Myxobolus sp., 5.45% (3/55) only with Henneguya sp., and 70.91% (39/55) presenting both parasites. The presence of myxospores was associated with histological alterations in both stromal and renal parenchyma. Myxospores were found mostly in the peritubular interstitial tissue and in low intensity in the glomerulus which caused nuclear hypertrophy and loss of Bowman space. An increase in the glomerular tuft and a reduction in the lumen of the collector tubules were also observed, besides the high number of melanomacrophage cells in the glomerulus. This study reports for the first time detection of myxozoan mixed infection in one organ of pacu and discuss the possible transportation of myxospores in the circulating blood.

Ceratomyxa azevedoi n. sp. (Myxozoa: Myxosporea) parasitizing the gallbladder of Lutjanus ehrenbergii in the Arabian Gulf.

A novel myxosporean species, Ceratomyxa azevedoi sp. n. is described from the gallbladder of the blackspot snapper, Lutjanus ehrenbergii (Peters), captured from the Arabian Gulf off Saudi Arabia. A total of 45 (26.8%) out of 168 fish specimens were found to be infected with Ceratomyxa azevedoi sp. n., the highest prevalence being observed in winter (42.9%, 18/42) and the lowest in autumn (11.9%, 5/42). Mature spores appeared as crescent to slightly elliptical-shaped, measuring 5-7 (6) µm in length and 12 (10-14) µm in thickness, with spherical polar capsules containing three polar filament coils. The morphometric and morphological comparison with similar species revealed the taxonomic novelty of this form, suggesting that it should be considered as new species. The phylogenetic analysis of C. azevedoi sp. n., based on partial SSU rDNA sequences, revealed close genetic relatedness to C. buri with 91.3% homogeneity and to C. hamour, with 90.1% homogeneity.

Computer Vision for High-Throughput Quantitative Phenotyping: A Case Study of Grapevine Downy Mildew Sporulation and Leaf Trichomes.

Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × 'Horizon' [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.